Detection of the High-Level Aminoglycoside Resistance Gene aph(2")-Ib in Enterococcus faecium

PubMed Central

Kao, Susan J.; You, Il; Clewell, Don B.; Donabedian, Susan M.; Zervos, Marcus J.; Petrin, Joanne; Shaw, Karen J.; Chow, Joseph W.

2000-01-01

A new high-level gentamicin resistance gene, designated aph(2")-Ib, was cloned from Enterococcus faecium SF11770. The deduced amino acid sequence of the 897-bp open reading frame of aph(2")-Ib shares homology with the aminoglycoside-modifying enzymes AAC(6′)-APH(2"), APH(2")-Ic, and APH(2")-Id. The observed phosphotransferase activity is designated APH(2")-Ib. PMID:10991878

Long-term clonal dynamics of Enterococcus faecium strains causing bloodstream infections (1995-2015) in Spain.

PubMed

Tedim, Ana P; Ruíz-Garbajosa, Patricia; Rodríguez, Maria Concepción; Rodríguez-Baños, Mercedes; Lanza, Val F; Derdoy, Laura; Cárdenas Zurita, Gonzalo; Loza, Elena; Cantón, Rafael; Baquero, Fernando; Coque, Teresa M

2017-01-01

To investigate the population structure of Enterococcus faecium causing bloodstream infections (BSIs) in a tertiary Spanish hospital with low glycopeptide resistance, and to enhance our knowledge of the dynamics of emergence and spread of high-risk clonal complexes. All available E. faecium causing BSIs (n = 413) in our hospital (January 1995-May 2015) were analysed for antibiotic susceptibility (CLSI), putative virulence traits (PCR, esp, hyl Efm ) and clonal relationship (SmaI-PFGE, MLST evaluated by goeBURST and BAPS). The increased incidence of BSIs caused by enterococci [2.3‰ of attended patients (inpatients and outpatients) in 1996 to 3.0‰ in 2014] significantly correlated with the increase in BSIs caused by E. faecium (0.33‰ of attended patients in 1996 to 1.3‰ in 2014). The BSIs Enterococcus faecalis:E. faecium ratio changed from 5:1 in 1996 to 1:1 in 2014. During the last decade an increase in E. faecium BSIs episodes in cancer patients (10.9% in 1995-2005 and 37.1% in 2006-15) was detected. Ampicillin-susceptible E. faecium (ASEfm; different STs/BAPS) and ampicillin-resistant E. faecium (AREfm; ST18/ST17-BAPS 3.3a) isolates were recovered throughout the study. Successive waves of BAPS 2.1a-AREfm (ST117, ST203 and ST80) partially replaced ASEfm and ST18-AREfm since 2006. Different AREfm clones (belonging to BAPS 2.1a and BAPS 3.3a) consistently isolated during the last decade from BSIs might be explained by a continuous and dense colonization (favouring both invasion and cross-transmission) of hospitalized patients. High-density colonization by these clones is probably enhanced in elderly patients by heavy and prolonged antibiotic exposure, particularly in oncological patients. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Comparative Analysis of the First Complete Enterococcus faecium Genome

PubMed Central

Lam, Margaret M. C.; Seemann, Torsten; Bulach, Dieter M.; Gladman, Simon L.; Chen, Honglei; Haring, Volker; Moore, Robert J.; Ballard, Susan; Grayson, M. Lindsay; Johnson, Paul D. R.; Howden, Benjamin P.

2012-01-01

Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infections in health care facilities around the globe. In particular, infections caused by vancomycin-resistant Enterococcus faecium are becoming increasingly common. Comparative and functional genomic studies of E. faecium isolates have so far been limited owing to the lack of a fully assembled E. faecium genome sequence. Here we address this issue and report the complete 3.0-Mb genome sequence of the multilocus sequence type 17 vancomycin-resistant Enterococcus faecium strain Aus0004, isolated from the bloodstream of a patient in Melbourne, Australia, in 1998. The genome comprises a 2.9-Mb circular chromosome and three circular plasmids. The chromosome harbors putative E. faecium virulence factors such as enterococcal surface protein, hemolysin, and collagen-binding adhesin. Aus0004 has a very large accessory genome (38%) that includes three prophage and two genomic islands absent among 22 other E. faecium genomes. One of the prophage was present as inverted 50-kb repeats that appear to have facilitated a 683-kb chromosomal inversion across the replication terminus, resulting in a striking replichore imbalance. Other distinctive features include 76 insertion sequence elements and a single chromosomal copy of Tn1549 containing the vanB vancomycin resistance element. A complete E. faecium genome will be a useful resource to assist our understanding of this emerging nosocomial pathogen. PMID:22366422

Horizontal transfer of antibiotic resistance from Enterococcus faecium of fermented meat origin to clinical isolates of E. faecium and Enterococcus faecalis.

PubMed

Jahan, Musarrat; Zhanel, George G; Sparling, Richard; Holley, Richard A

2015-04-16

Enterococcus species are part of the normal intestinal flora of a large number of mammals including humans and consequently, they can be used as indicators of faecal contamination in food and water for human consumption. Their presence in large numbers in foods may indicate a lapse in sanitation and their ability to serve as a genetic reservoir of transferable antibiotic resistance is of concern. In the present study, Enterococcus spp., isolated from commercially fermented meat and human clinical specimen were studied to determine genetic relationships. SmaI pulsed-field gel electrophoresis (PFGE) patterns exhibited genomic heterogeneity within and between both groups of isolates. However, in spite of this heterogeneity there were still substantial phenotypic similarities which suggested that food might be a potential vehicle for distribution of resistant bacteria among humans. In vitro conjugation experiments demonstrated transfer of the tetracycline resistant determinant, tet(M), from Enterococcus faecium S27 isolated from fermented sausage to clinical isolates of both E. faecium and Enterococcus faecalis. The streptomycin resistance of E. faecium S27 was also transferred to a clinical strain, E. faecalis 82916, which was confirmed by the presence of the streptomycin resistance gene, aadA, in the donor and transconjugant strains. Since the aadA gene is associated with a class 1 integron, results also suggested that resistance transfer might have occurred via an integron. It appears this is the first identification of a class 1 integron in E. faecium isolated from food. The importance of food enterococci as a reservoir of antibiotic resistance genes and the potential for their genetic transfer to human strains following consumption of uncooked or undercooked contaminated meat is underlined by this work. Copyright © 2015 Elsevier B.V. All rights reserved.

Wide distribution of virulence genes among Enterococcus faecium and Enterococcus faecalis clinical isolates.

PubMed

Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasanthakumari; Sadeghifard, Nourkhoda; Ramli, Ramliza; Hamat, Rukman Awang

2014-01-01

Enterococcus, a Gram-positive facultative anaerobic cocci belonging to the lactic acid bacteria of the phylum Firmicutes, is known to be able to resist a wide range of hostile conditions such as different pH levels, high concentration of NaCl (6.5%), and the extended temperatures between 5(°)C and 65(°)C. Despite being the third most common nosocomial pathogen, our understanding on its virulence factors is still poorly understood. The current study was aimed to determine the prevalence of different virulence genes in Enterococcus faecalis and Enterococcus faecium. For this purpose, 79 clinical isolates of Malaysian enterococci were evaluated for the presence of virulence genes. pilB, fms8, efaAfm, and sgrA genes are prevalent in all clinical isolates. In conclusion, the pathogenicity of E. faecalis and E. faecium could be associated with different virulence factors and these genes are widely distributed among the enterococcal species.

Wide Distribution of Virulence Genes among Enterococcus faecium and Enterococcus faecalis Clinical Isolates

PubMed Central

Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasanthakumari; Sadeghifard, Nourkhoda; Ramli, Ramliza; Hamat, Rukman Awang

2014-01-01

Enterococcus, a Gram-positive facultative anaerobic cocci belonging to the lactic acid bacteria of the phylum Firmicutes, is known to be able to resist a wide range of hostile conditions such as different pH levels, high concentration of NaCl (6.5%), and the extended temperatures between 5°C and 65°C. Despite being the third most common nosocomial pathogen, our understanding on its virulence factors is still poorly understood. The current study was aimed to determine the prevalence of different virulence genes in Enterococcus faecalis and Enterococcus faecium. For this purpose, 79 clinical isolates of Malaysian enterococci were evaluated for the presence of virulence genes. pilB, fms8, efaAfm, and sgrA genes are prevalent in all clinical isolates. In conclusion, the pathogenicity of E. faecalis and E. faecium could be associated with different virulence factors and these genes are widely distributed among the enterococcal species. PMID:25147855

Draft genome sequence of Enterococcus faecium strain LMG 8148.

PubMed

Michiels, Joran E; Van den Bergh, Bram; Fauvart, Maarten; Michiels, Jan

2016-01-01

Enterococcus faecium, traditionally considered a harmless gut commensal, is emerging as an important nosocomial pathogen showing increasing rates of multidrug resistance. We report the draft genome sequence of E. faecium strain LMG 8148, isolated in 1968 from a human in Gothenburg, Sweden. The draft genome has a total length of 2,697,490 bp, a GC-content of 38.3 %, and 2,402 predicted protein-coding sequences. The isolation of this strain predates the emergence of E. faecium as a nosocomial pathogen. Consequently, its genome can be useful in comparative genomic studies investigating the evolution of E. faecium as a pathogen.

Co-diversification of Enterococcus faecium Core Genomes and PBP5: Evidences of pbp5 Horizontal Transfer

PubMed Central

Novais, Carla; Tedim, Ana P.; Lanza, Val F.; Freitas, Ana R.; Silveira, Eduarda; Escada, Ricardo; Roberts, Adam P.; Al-Haroni, Mohammed; Baquero, Fernando; Peixe, Luísa; Coque, Teresa M.

2016-01-01

Ampicillin resistance has greatly contributed to the recent dramatic increase of a cluster of human adapted Enterococcus faecium lineages (ST17, ST18, and ST78) in hospital-based infections. Changes in the chromosomal pbp5 gene have been associated with different levels of ampicillin susceptibility, leading to protein variants (designated as PBP5 C-types to keep the nomenclature used in previous works) with diverse degrees of reduction in penicillin affinity. Our goal was to use a comparative genomics approach to evaluate the relationship between the diversity of PBP5 among E. faecium isolates of different phylogenomic groups as well as to assess the pbp5 transferability among isolates of disparate clonal lineages. The analyses of 78 selected E. faecium strains as well as published E. faecium genomes, suggested that the diversity of pbp5 mirrors the phylogenomic diversification of E. faecium. The presence of identical PBP5 C-types as well as similar pbp5 genetic environments in different E. faecium lineages and clones from quite different geographical and environmental origin was also documented and would indicate their horizontal gene transfer among E. faecium populations. This was supported by experimental assays showing transfer of large (≈180–280 kb) chromosomal genetic platforms containing pbp5 alleles, ponA (transglycosilase) and other metabolic and adaptive features, from E. faecium donor isolates to suitable E. faecium recipient strains. Mutation profile analysis of PBP5 from available genomes and strains from this study suggests that the spread of PBP5 C-types might have occurred even in the absence of a significant ampicillin resistance phenotype. In summary, genetic platforms containing pbp5 sequences were stably maintained in particular E. faecium lineages, but were also able to be transferred among E. faecium clones of different origins, emphasizing the growing risk of further spread of ampicillin resistance in this nosocomial pathogen. PMID

Enterococcus faecium isolated from honey synthesized bacteriocin-like substances active against different Listeria monocytogenes strains.

PubMed

Ibarguren, Carolina; Raya, Raúl R; Apella, María C; Audisio, M Carina

2010-02-01

Four Enterococcus faecium strains, isolated from honeycombs (C1 and M2d strains) and feral combs (Mori1 and M1b strains) secreted antimicrobial substances active against fourteen different Listeria spp. strains. The antimicrobial compound(s) present in the cell free supernatant were highly thermostable (121 degrees C for 15 min) and inactivated by proteolytic enzymes, but not by alpha-amylase and lipase, thus suggesting a peptidic nature. Since the structural bacteriocin gene determinants of enterocins A and B were PCR amplified from the four E. faecium isolates, only the bacteriocin produced by strain C1 was further characterized: it showed a broad band of approximately 4.0-7.0 kDa in SDS-PAGE and was bactericidal (4 log decrease) against L. monocytogenes 99/287. L. monocytogenes 99/287R, a clone spontaneously resistant to the enterocin produced by E. avium DSMZ17511 (ex PA1), was not inhibited by the enterocin-like compounds produced by strain C1. However, it was inhibited in mixed culture fermentations by E. faecium C1 and a bacteriostatic effect was observed. The bacteriocin-producer Enterococcus strains were not haemolytic; gelatinase negative and sensitive to vancomycin and other clinically relevant antibiotics.

Incidence of virulence determinants in clinical Enterococcus faecalis and Enterococcus faecium isolates collected in Bulgaria.

PubMed

Strateva, Tanya; Atanasova, Daniela; Savov, Encho; Petrova, Guergana; Mitov, Ivan

2016-01-01

To evaluate the prevalence of some virulence genes among 510 clinical Enterococcus spp. isolates and to assess the association of those genes with the species, infection site, and patient group (inpatients/outpatients). Adhesins genes (aggregation substances agg and asa1 of Enterococcus faecalis and Enterococcus faecium, respectively), enterococcal surface protein (esp), endocarditis-specific antigen A (efaA), collagen-binding proteins (ace/acm)); invasins (hyaluronidase (hyl) and gelatinase (gelE)); cytotoxines (activation of cytolysin (cylA) in E. faecalis); and modulators of the host immunity and inflammation (enhanced expression pheromone (eep) in E. faecalis) were detected by polymerase chain reaction. The overall prevalence was: esp - 44.3%, agg/asa1 - 38.4%, ace/acm - 64.3%, efaA - 85.9%, eep - 69.4%, gelE - 64.3%, hyl - 25.1%, and cylA - 47.1%. E. faecalis isolates had significantly higher frequency of adhesin genes (esp and agg/asa1) and gelatinase in comparison to E. faecium. Multiple virulence genes in E. faecalis were significantly more prevalent than in E. faecium isolates. Domination of E. faecium with or without only one gene compared to the isolates of E. faecalis were found. Enterococcus spp. isolates obtained from outpatients compared to inpatients isolates had significantly higher frequency of agg/asa1, eep, gelE and cylA. Some adhesins genes (esp, agg/asa1 and efaA) had higher prevalence among the non-invasive Enterococcus spp. isolates compared to those causing invasive bacteremia, while ace/acm revealed higher dissemination in isolates causing invasive infections compared to non-invasive isolates. Most E. faecalis attaches to abiotic surfaces in hospital environment, which correlates with higher prevalence of gene encoding for virulence factors involved in biofilm formation, such as enterococcal surface protein, aggregation substance, and gelatinase. The intestinal tract is an important reservoir for opportunistic enterococcal pathogens and

Characterization of Tn1546 in vancomycin-resistant Enterococcus faecium isolated from canine urinary tract infections: evidence of gene exchange between human and animal enterococci.

PubMed

Simjee, S; White, D G; McDermott, P F; Wagner, D D; Zervos, M J; Donabedian, S M; English, L L; Hayes, J R; Walker, R D

2002-12-01

Thirty-five enterococcal isolates were recovered from dogs diagnosed with urinary tract infections at the Michigan State University Veterinary Teaching Hospital over a 2-year period (1996 to 1998). Isolated species included Enterococcus faecium (n = 13), Enterococcus faecalis (n = 7), Enterococcus gallinarum (n = 11), and Enterococcus casseliflavus (n = 4). Antimicrobial susceptibility testing revealed several different resistance phenotypes, with the majority of the enterococcal isolates exhibiting resistance to three or more antibiotics. One E. faecium isolate, CVM1869, displayed high-level resistance to vancomycin (MIC > 32 micro g/ml) and gentamicin (MIC > 2,048 micro g/ml). Molecular analysis of this isolate revealed the presence of Tn1546 (vanA), responsible for high-level vancomycin resistance, and Tn5281 carrying aac6'-aph2", conferring high-level aminoglycoside resistance. Pulsed-field gel electrophoresis analysis revealed that CVM1869 was a canine E. faecium clone that had acquired Tn1546, perhaps from a human vancomycin-resistant E. faecium. Transposons Tn5281 and Tn1546 were located on two different conjugative plasmids. Sequence analysis revealed that in Tn1546, ORF1 had an 889-bp deletion and an IS1216V insertion at the 5' end and an IS1251 insertion between vanS and vanH. To date, this particular form of Tn1546 has only been described in human clinical vancomycin-resistant enterococcus isolates unique to the United States. Additionally, this is the first report of a vancomycin-resistant E. faecium isolated from a companion animal in the United States.

Enterocin TW21, a novel bacteriocin from dochi-isolated Enterococcus faecium D081821.

PubMed

Chang, S-Y; Chen, Y-S; Pan, S-F; Lee, Y-S; Chang, C-H; Chang, C-H; Yu, B; Wu, H-C

2013-09-01

Purification and characterization of a novel bacteriocin produced by strain Enterococcus faecium D081821. Enterococcus faecium D081821, isolated from the traditional Taiwanese fermented food dochi (fermented black beans), was previously found to produce a bacteriocin against Listeria monocytogenes and some Gram-positive bacteria. This bacteriocin, termed enterocin TW21, was purified from culture supernatant by ammonium sulfate precipitation, Sep-Pak C18 cartridge, ion-exchange and gel filtration chromatography. Mass spectrometry analysis showed the mass of the peptide to be approximately 5300·6 Da. The N-terminal amino acid sequencing yielded a partial sequence NH2 -ATYYGNGVYxNTQK by Edman degradation, and it contains the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The open reading frame (ORF) encoding the bacteriocin was identified from the draft genome sequence of Enterococcus faecium D081821, and sequence analysis of this peptide indicated that enterocin TW21 is a novel bacteriocin. Enterococcus faecium D081821 produced a bacteriocin named enterocin TW21, the molecular weight and amino acid sequence both revealed it to be a novel bacteriocin. A new member of class IIa bacteriocin was identified. This bacteriocin shows great inhibitory ability against L. monocytogenes and could be applied as a natural food preservative. © 2013 The Society for Applied Microbiology.

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates.

PubMed

Rathnayake, I U; Hargreaves, M; Huygens, F

2012-07-01

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk. Copyright © 2012 Elsevier GmbH. All rights reserved.

Complete Genome Sequences of Isolates of Enterococcus faecium Sequence Type 117, a Globally Disseminated Multidrug-Resistant Clone

PubMed Central

Tedim, Ana P.; Lanza, Val F.; Manrique, Marina; Pareja, Eduardo; Ruiz-Garbajosa, Patricia; Cantón, Rafael; Baquero, Fernando; Tobes, Raquel

2017-01-01

ABSTRACT The emergence of nosocomial infections by multidrug-resistant sequence type 117 (ST117) Enterococcus faecium has been reported in several European countries. ST117 has been detected in Spanish hospitals as one of the main causes of bloodstream infections. We analyzed genome variations of ST117 strains isolated in Madrid and describe the first ST117 closed genome sequences. PMID:28360174

Restricted Gene Flow among Hospital Subpopulations of Enterococcus faecium

PubMed Central

Willems, Rob J. L.; Top, Janetta; van Schaik, Willem; Leavis, Helen; Bonten, Marc; Sirén, Jukka; Hanage, William P.; Corander, Jukka

2012-01-01

ABSTRACT Enterococcus faecium has recently emerged as an important multiresistant nosocomial pathogen. Defining population structure in this species is required to provide insight into the existence, distribution, and dynamics of specific multiresistant or pathogenic lineages in particular environments, like the hospital. Here, we probe the population structure of E. faecium using Bayesian-based population genetic modeling implemented in Bayesian Analysis of Population Structure (BAPS) software. The analysis involved 1,720 isolates belonging to 519 sequence types (STs) (491 for E. faecium and 28 for Enterococcus faecalis). E. faecium isolates grouped into 13 BAPS (sub)groups, but the large majority (80%) of nosocomial isolates clustered in two subgroups (2-1 and 3-3). Phylogenetic and eBURST analysis of BAPS groups 2 and 3 confirmed the existence of three separate hospital lineages (17, 18, and 78), highlighting different evolutionary trajectories for BAPS 2-1 (lineage 78) and 3-3 (lineage 17 and lineage 18) isolates. Phylogenomic analysis of 29 E. faecium isolates showed agreement between BAPS assignment of STs and their relative positions in the phylogenetic tree. Odds ratio calculation confirmed the significant association between hospital isolates with BAPS 3-3 and lineages 17, 18, and 78. Admixture analysis showed a scarce number of recombination events between the different BAPS groups. For the E. faecium hospital population, we propose an evolutionary model in which strains with a high propensity to colonize and infect hospitalized patients arise through horizontal gene transfer. Once adapted to the distinct hospital niche, this subpopulation becomes isolated, and recombination with other populations declines. PMID:22807567

Selection of potential probiotic Enterococcus faecium isolated from Portuguese fermented food.

PubMed

Barbosa, Joana; Borges, Sandra; Teixeira, Paula

2014-11-17

Four Enterococcus faecium strains isolated from fermented products were evaluated for potential use as probiotic strains. In addition to efaAfm gene, commonly found in E. faecium food isolates, none of the isolates possessed virulence genes and none had positive reactions for the production of tyramine, histamine, putrescine and cadaverine in the screening medium used. All of these four isolates proved to be resistant to 65 °C. E. faecium 119 did not show antimicrobial activity against any of the target bacteria investigated. E. faecium 85 and 101 inhibited Listeria innocua and E. faecium DSMZ 13590. The strain E. faecium 120 inhibited seven target bacteria (Listeria monocytogenes 7946, L. monocytogenes 7947, L. innocua 2030c, L. innocua NCTC 11286, E. faecium DSMZ 13590, Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213) and was chosen as the representative to assess the ability to survive gastrointestinal tract passage simulation, as well as the protective role of two food matrices (skim milk and Alheira) during its passage. For both matrices used, no significant differences (p<0.05) were obtained between the types of digestion - quick and slow passage simulation. In the skim milk matrix the isolate was reduced to values below the detection limit of the enumeration technique by the end of the two digestions, in contrast to the Alheira matrix, for which isolate 120 showed a reduction of only ca. 1 log CFU/ml. The E. faecium strain 120 was shown to be a potential candidate for further investigations as a potential probiotic culture. Copyright © 2014. Published by Elsevier B.V.

Dissemination of Enterococcus faecalis and Enterococcus faecium in a ricotta processing plant and evaluation of pathogenic and antibiotic resistance profiles.

PubMed

Fernandes, Meg da Silva; Fujimoto, Graciela; de Souza, Leandro Pio; Kabuki, Dirce Yorika; da Silva, Márcio José; Kuaye, Arnaldo Yoshiteru

2015-04-01

In this work, the sources of contamination by Enterococcus spp. in a ricotta processing line were evaluated. The isolated strains were tested for virulence genes (gelE, cylA,B, M, esp, agg, ace, efaA, vanB), expression of virulence factors (hemolysin and gelatinase), and the resistance to 10 different antibiotics. Enterococcus faecium and Enterococcus faecalis were subjected to discriminatory identification by intergenic spacer region (ITS)-polymerase chain reaction and sequencing of the ITS region. The results showed that Enterococcus spp. was detected in the raw materials, environment samples and the final product. None of the 107 Enterococcus isolates were completely free from all virulence genes considered. A fraction of 21.5% of the isolates containing all of the genes of the cylA, B, M operon also expressed β-hemolysis. Most of the isolates showed the gelE gene, but only 9.3% were able to hydrolyze gelatin. In addition, 23.5% of the observed Enterococcus isolates had the vanB gene but were susceptible to vancomycin in vitro. The dissemination of antibiotic-resistant enterococci was revealed in this study: 19.3% of the E. faecium samples and 78.0% of the E. faecalis samples were resistant to at least one of the antibiotics tested. Sequencing of region discriminated 5 and 7 distinct groups among E. faecalis and E. faecium, respectively. Although some similarity was observed among some of the isolates, all E. faecalis and E. faecium isolates had genetic differences both in the ITS region and in the virulence profile, which makes them different from each other. © 2015 Institute of Food Technologists®

Subinhibitory Concentrations of Ciprofloxacin Enhance Antimicrobial Resistance and Pathogenicity of Enterococcus faecium

PubMed Central

Sinel, Clara; Cacaci, Margherita; Meignen, Pierrick; Guérin, François; Davies, Bryan W.; Sanguinetti, Maurizio; Giard, Jean-Christophe

2017-01-01

ABSTRACT Enterococcus faecium has emerged as a major opportunistic pathogen for 2 decades with the spread of hospital-adapted multidrug-resistant clones. As members of the intestinal microbiota, they are subjected to numerous bacterial stresses, including antibiotics at subinhibitory concentrations (SICs). Since fluoroquinolones are extensively prescribed, SICs are very likely to occur in vivo, with potential effects on bacterial metabolism with subsequent modulation of opportunistic traits. The aim of this study was to evaluate globally the impact of SICs of ciprofloxacin on antimicrobial resistance and pathogenicity of E. faecium. Transcriptomic analysis was performed by RNA sequencing (RNA-seq) (HiSeq 2500; Illumina) using the vanB-positive reference strain E. faecium Aus0004 in the absence or presence of ciprofloxacin SIC (0.38 mg/liter, i.e., 1/8 of the MIC). Several genetic and phenotypic tests were used for validation. In the presence of ciprofloxacin SIC, 196 genes were significantly induced, whereas 286 genes were significantly repressed, meaning that 16.8% of the E. faecium genome was altered. Among upregulated genes, EFAU004_02294 (fold change, 14.3) encoded a protein (Qnr of E. faecium [EfmQnr]) homologue of Qnr proteins involved in quinolone resistance in Gram-negative bacilli. Its implication in intrinsic and adaptive fluoroquinolone (FQ) resistance in E. faecium was experimentally ascertained. Moreover, EFAU004_02292, coding for the collagen adhesin Acm, was also induced by the SIC of ciprofloxacin (fold change, 8.2), and higher adhesion capabilities were demonstrated phenotypically. Both EfmQnr and Acm determinants may play an important role in the transition from a commensal to a pathogenic state of E. faecium that resides in the gut of patients receiving fluoroquinolone therapy. PMID:28193670

Subinhibitory Concentrations of Ciprofloxacin Enhance Antimicrobial Resistance and Pathogenicity of Enterococcus faecium.

PubMed

Sinel, Clara; Cacaci, Margherita; Meignen, Pierrick; Guérin, François; Davies, Bryan W; Sanguinetti, Maurizio; Giard, Jean-Christophe; Cattoir, Vincent

2017-05-01

Enterococcus faecium has emerged as a major opportunistic pathogen for 2 decades with the spread of hospital-adapted multidrug-resistant clones. As members of the intestinal microbiota, they are subjected to numerous bacterial stresses, including antibiotics at subinhibitory concentrations (SICs). Since fluoroquinolones are extensively prescribed, SICs are very likely to occur in vivo , with potential effects on bacterial metabolism with subsequent modulation of opportunistic traits. The aim of this study was to evaluate globally the impact of SICs of ciprofloxacin on antimicrobial resistance and pathogenicity of E. faecium Transcriptomic analysis was performed by RNA sequencing (RNA-seq) (HiSeq 2500; Illumina) using the vanB -positive reference strain E. faecium Aus0004 in the absence or presence of ciprofloxacin SIC (0.38 mg/liter, i.e., 1/8 of the MIC). Several genetic and phenotypic tests were used for validation. In the presence of ciprofloxacin SIC, 196 genes were significantly induced, whereas 286 genes were significantly repressed, meaning that 16.8% of the E. faecium genome was altered. Among upregulated genes, EFAU004_02294 (fold change, 14.3) encoded a protein (Qnr of E. faecium [EfmQnr]) homologue of Qnr proteins involved in quinolone resistance in Gram-negative bacilli. Its implication in intrinsic and adaptive fluoroquinolone (FQ) resistance in E. faecium was experimentally ascertained. Moreover, EFAU004_02292, coding for the collagen adhesin Acm, was also induced by the SIC of ciprofloxacin (fold change, 8.2), and higher adhesion capabilities were demonstrated phenotypically. Both EfmQnr and Acm determinants may play an important role in the transition from a commensal to a pathogenic state of E. faecium that resides in the gut of patients receiving fluoroquinolone therapy. Copyright © 2017 American Society for Microbiology.

Nonclinical and Clinical Enterococcus faecium Strains, but Not Enterococcus faecalis Strains, Have Distinct Structural and Functional Genomic Features

PubMed Central

Kim, Eun Bae

2014-01-01

Certain strains of Enterococcus faecium and Enterococcus faecalis contribute beneficially to animal health and food production, while others are associated with nosocomial infections. To determine whether there are structural and functional genomic features that are distinct between nonclinical (NC) and clinical (CL) strains of those species, we analyzed the genomes of 31 E. faecium and 38 E. faecalis strains. Hierarchical clustering of 7,017 orthologs found in the E. faecium pangenome revealed that NC strains clustered into two clades and are distinct from CL strains. NC E. faecium genomes are significantly smaller than CL genomes, and this difference was partly explained by significantly fewer mobile genetic elements (ME), virulence factors (VF), and antibiotic resistance (AR) genes. E. faecium ortholog comparisons identified 68 and 153 genes that are enriched for NC and CL strains, respectively. Proximity analysis showed that CL-enriched loci, and not NC-enriched loci, are more frequently colocalized on the genome with ME. In CL genomes, AR genes are also colocalized with ME, and VF are more frequently associated with CL-enriched loci. Genes in 23 functional groups are also differentially enriched between NC and CL E. faecium genomes. In contrast, differences were not observed between NC and CL E. faecalis genomes despite their having larger genomes than E. faecium. Our findings show that unlike E. faecalis, NC and CL E. faecium strains are equipped with distinct structural and functional genomic features indicative of adaptation to different environments. PMID:24141120

Phenotypic and molecular antibiotic resistance profile of Enterococcus faecalis and Enterococcus faecium isolated from different traditional fermented foods.

PubMed

Sánchez Valenzuela, Antonio; Lavilla Lerma, Leyre; Benomar, Nabil; Gálvez, Antonio; Pérez Pulido, Rubén; Abriouel, Hikmate

2013-02-01

A collection of 55 enterococci (41 Enterococcus faecium and 14 E. faecalis strains) isolated from various traditional fermented foodstuffs of both animal and vegetable origins, and water was evaluated for resistance against 15 antibiotics. Lower incidence of resistance was observed with gentamicin, ampicillin, penicillin and teicoplanin. However, a high incidence of antibiotic resistance was detected for rifampicin (12 out of 14 of isolates), ciprofloxacin (9/14), and quinupristin/dalfopristin (8/14) in E. faecalis strains. Enterococcus faecium isolates were resistant to rifampicin (25/41), ciprofloxacin (23/41), erythromycin (18/41), levofloxacin (16/41), and nitrofurantoin (15/41). One Enterococcus faecalis and two E. faecium strains were resistant to vancomycin (MIC>16 μg/mL). Among 55 isolates, 27 (19 E. faecium and eight E. faecalis) were resistant to at least three antibiotics. High level of multidrug resistance to clinically important antibiotics was detected in E. faecalis strains (57% of E. faecalis versus 46% of E. faecium), which showed resistance to six to seven antibiotics, especially those isolated from foods of animal origin. So, it is necessary to re-evaluate the use of therapeutic antibiotics in stock farms at both regional and international levels due to the high number of multiple resistant (MR) bacteria. Fifty-six MR E. faecalis and E. faecium strains selected from this and previous studies (Valenzuela et al., 2008, 2010) were screened by polymerase chain reaction for antibiotic resistance genes, revealing the presence of tet(L), tet(M), ermB, cat, efrA, efrB, mphA, or msrA/B genes. The ABC Multidrug Efflux Pump EfrAB was detected in 96% of E. faecalis strains and also in 13% of E. faecium strains; this is the first report describing EfrAB in this enterococcal species. The efflux pump-associated msrA/B gene was detected in 66.66% of E. faecium strains, but not in E. faecalis strains.

Mature biofilms of Enterococcus faecalis and Enterococcus faecium are highly resistant to antibiotics.

PubMed

Holmberg, Anna; Rasmussen, Magnus

2016-01-01

Enterococcus faecalis and Enterococcus faecium are important nosocomial pathogens that form biofilms on implanted materials. We compare the antibiotic sensitivity of bacteria in new (established during 24 hours) and mature (established during 120 hours) enterococcal biofilms. Mature biofilms contained more bacteria and were much more tolerant to antibiotics, including rifampicin-containing combinations, as judged by determination of minimal biofilm eradication concentrations and by time-kill experiments of bacteria in biofilms formed on beads of bone cement. Copyright © 2016 Elsevier Inc. All rights reserved.

Multidrug-resistant enterococci in animal meat and faeces and co-transfer of resistance from an Enterococcus durans to a human Enterococcus faecium.

PubMed

Vignaroli, Carla; Zandri, Giada; Aquilanti, Lucia; Pasquaroli, Sonia; Biavasco, Francesca

2011-05-01

Forty-eight isolates resistant to at least two antibiotics were selected from 53 antibiotic-resistant enterococci from chicken and pig meat and faeces and analysed for specific resistance determinants. Of the 48 multidrug-resistant (MDR) strains, 31 were resistant to two antibiotics (29 to erythromycin and tetracycline, 1 to erythromycin and vancomycin, 1 to vancomycin and tetracycline), 14 to three (erythromycin, tetracycline and vancomycin or ampicillin) and 3 to four (erythromycin, vancomycin, ampicillin and gentamicin). erm(B), tet(M), vanA and aac (6')-Ie aph (2'')-Ia were the antibiotic resistance genes most frequently detected. All 48 MDR enterococci were susceptible to linezolid and daptomycin. Enterococcus faecalis (16), Enterococcus faecium (8), Enterococcus mundtii (2) and Enterococcus gallinarum (1) were identified in meat, and E. faecium (13) and Enterococcus durans (13) in faeces. Clonal spread was not detected, suggesting a large role of gene transfer in the dissemination of antibiotic resistance. Conjugative transfer of resistance genes was more successful when donors were enterococcal strains isolated from faeces; co-transfer of vanA and erm(B) to a human E. faecium occurred from both E. faecium and E. durans pig faecal strains. These data show that multidrug resistance can be found in food and animal species other than E. faecium and E. faecalis, and that these species can efficiently transfer antibiotic resistance to human strains in inter-specific matings. In particular, the occurrence of MDR E. durans in the animal reservoir could have a role in the emergence of human enterococcal infections difficult to eradicate with antibiotics.

Fitness costs of various mobile genetic elements in Enterococcus faecium and Enterococcus faecalis

PubMed Central

Starikova, Irina; Al-Haroni, Mohammed; Werner, Guido; Roberts, Adam P.; Sørum, Vidar; Nielsen, Kaare M.; Johnsen, Pål J.

2013-01-01

Objectives To determine the fitness effects of various mobile genetic elements (MGEs) in Enterococcus faecium and Enterococcus faecalis when newly acquired. We also tested the hypothesis that the biological cost of vancomycin resistance plasmids could be mitigated during continuous growth in the laboratory. Methods Different MGEs, including two conjugative transposons (CTns) of the Tn916 family (18 and 33 kb), a pathogenicity island (PAI) of 200 kb and vancomycin-resistance (vanA) plasmids (80–200 kb) of various origins and classes, were transferred into common ancestral E. faecium and E. faecalis strains by conjugation assays and experimentally evolved (vanA plasmids only). Transconjugants were characterized by PFGE, S1 nuclease assays and Southern blotting hybridization analyses. Single specific primer PCR was performed to determine the target sites for the insertion of the CTns. The fitness costs of various MGEs in E. faecium and E. faecalis were estimated in head-to-head competition experiments, and evolved populations were generated in serial transfer assays. Results The biological cost of a newly acquired PAI and two CTns were both host- and insertion-locus-dependent. Newly acquired vanA plasmids may severely reduce host fitness (25%–27%), but these costs were rapidly mitigated after only 400 generations of continuous growth in the absence of antibiotic selection. Conclusions Newly acquired MGEs may impose an immediate biological cost in E. faecium. However, as demonstrated for vanA plasmids, the initial costs of MGE carriage may be mitigated during growth and beneficial plasmid–host association can rapidly emerge. PMID:23833178

Safety, beneficial and technological properties of Enterococcus faecium isolated from Brazilian cheeses

PubMed Central

dos Santos, Karina Maria Olbrich; Vieira, Antônio Diogo Silva; Salles, Hévila Oliveira; Oliveira, Jacqueline da Silva; Rocha, Cíntia Renata Costa; Borges, Maria de Fátima; Bruno, Laura Maria; Franco, Bernadette Dora Gombossy de Melo; Todorov, Svetoslav Dimitrov

2015-01-01

This study aimed to characterize the safety and technological properties of Enterococcus faecium strains isolated from Brazilian Coalho cheeses. High levels of co-aggregation were observed between Enterococcus faecium strains EM485 and EM925 and both Escherichia coli and Clostridium perfringens . Both strains presented low levels of hydrophobicity. E. faecium EM485 and EM925 were both able to grow in the presence of 0.5% of the sodium salts of taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), and glycodeoxycholic acid (GDC), although they showed the ability to deconjugate only GDC and TDC. Both strains showed good survival when exposed to conditions simulating the gastro intestinal tract (GIT). When tested for the presence of virulence genes, only tyrosine decarboxylase and vancomycin B generated positive PCR results. PMID:26221113

Safety, beneficial and technological properties of Enterococcus faecium isolated from Brazilian cheeses.

PubMed

Dos Santos, Karina Maria Olbrich; Vieira, Antônio Diogo Silva; Salles, Hévila Oliveira; Oliveira, Jacqueline da Silva; Rocha, Cíntia Renata Costa; Borges, Maria de Fátima; Bruno, Laura Maria; Franco, Bernadette Dora Gombossy de Melo; Todorov, Svetoslav Dimitrov

2015-03-01

This study aimed to characterize the safety and technological properties of Enterococcus faecium strains isolated from Brazilian Coalho cheeses. High levels of co-aggregation were observed between Enterococcus faecium strains EM485 and EM925 and both Escherichia coli and Clostridium perfringens . Both strains presented low levels of hydrophobicity. E. faecium EM485 and EM925 were both able to grow in the presence of 0.5% of the sodium salts of taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), and glycodeoxycholic acid (GDC), although they showed the ability to deconjugate only GDC and TDC. Both strains showed good survival when exposed to conditions simulating the gastro intestinal tract (GIT). When tested for the presence of virulence genes, only tyrosine decarboxylase and vancomycin B generated positive PCR results.

A novel gene, optrA, that confers transferable resistance to oxazolidinones and phenicols and its presence in Enterococcus faecalis and Enterococcus faecium of human and animal origin.

PubMed

Wang, Yang; Lv, Yuan; Cai, Jiachang; Schwarz, Stefan; Cui, Lanqing; Hu, Zhidong; Zhang, Rong; Li, Jun; Zhao, Qin; He, Tao; Wang, Dacheng; Wang, Zheng; Shen, Yingbo; Li, Yun; Feßler, Andrea T; Wu, Congming; Yu, Hao; Deng, Xuming; Xia, Xi; Shen, Jianzhong

2015-08-01

The oxazolidinone-resistant Enterococcus faecalis E349 from a human patient tested negative for the cfr gene and 23S rRNA mutations. Here we report the identification of a novel oxazolidinone resistance gene, optrA, and a first investigation of the extent to which this gene was present in E. faecalis and Enterococcus faecium from humans and food-producing animals. The resistance gene optrA was identified by whole-plasmid sequencing and subsequent cloning and expression in a susceptible Enterococcus host. Transformation and conjugation assays served to investigate the transferability of optrA. All optrA-positive E. faecalis and E. faecium isolates of human and animal origin were analysed for their MICs and their genotype, as well as the location of optrA. The novel plasmid-borne ABC transporter gene optrA from E. faecalis E349 conferred combined resistance or elevated MICs (when no clinical breakpoints were available) to oxazolidinones (linezolid and tedizolid) and phenicols (chloramphenicol and florfenicol). The corresponding conjugative plasmid pE349, on which optrA was located, had a size of 36 331 bp and also carried the phenicol exporter gene fexA. The optrA gene was functionally expressed in E. faecalis, E. faecium and Staphylococcus aureus. It was detected more frequently in E. faecalis and E. faecium from food-producing animals (20.3% and 5.7%, respectively) than from humans (4.2% and 0.6%, respectively). Enterococci with elevated MICs of linezolid and tedizolid should be tested not only for 23S rRNA mutations and the gene cfr, but also for the novel resistance gene optrA. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Global Spread of the hylEfm Colonization-Virulence Gene in Megaplasmids of the Enterococcus faecium CC17 Polyclonal Subcluster▿

PubMed Central

Freitas, Ana R.; Tedim, Ana P.; Novais, Carla; Ruiz-Garbajosa, Patricia; Werner, Guido; Laverde-Gomez, Jenny A.; Cantón, Rafael; Peixe, Luísa; Baquero, Fernando; Coque, Teresa M.

2010-01-01

Enterococcus faecium has increasingly been reported as a nosocomial pathogen since the early 1990s, presumptively associated with the expansion of a human-associated Enterococcus faecium polyclonal subcluster known as clonal complex 17 (CC17) that has progressively acquired different antibiotic resistance (ampicillin and vancomycin) and virulence (espEfm, hylEfm, and fms) traits. We analyzed the presence and the location of a putative glycoside hydrolase hylEfm gene among E. faecium strains obtained from hospitalized patients (255 patients; outbreak, bacteremic, and/or disseminated isolates from 23 countries and five continents; 1986 to 2009) and from nonclinical origins (isolates obtained from healthy humans [25 isolates], poultry [30], swine [90], and the environment [55]; 1999 to 2007). Clonal relatedness was established by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmid analysis included determination of content and size (S1-PFGE), transferability (filter mating), screening of Rep initiator proteins (PCR), and location of vanA, vanB, ermB, and hylEfm genes (S1/I-CeuI hybridization). Most E. faecium isolates contained large plasmids (>150 kb) and showed variable contents of van, hylEfm, or espEfm. The hylEfm gene was associated with megaplasmids (170 to 375 kb) of worldwide spread (ST16, ST17, and ST18) or locally predominant (ST192, ST203, ST280, and ST412) ampicillin-resistant CC17 clones collected in the five continents since the early 1990s. All but one hylEfm-positive isolate belonged to the CC17 polyclonal subcluster. The presence of hylEfm megaplasmids among CC17 from Europe, Australia, Asia, and Africa since at least the mid-1990s was documented. This study further demonstrates the pandemic expansion of particular CC17 clones before acquisition of vancomycin resistance and putative virulence traits and describes the presence of megaplasmids in most of the contemporary E. faecium isolates with different origins. PMID

Novel type of VanB2 teicoplanin-resistant hospital-associated Enterococcus faecium.

PubMed

Santona, Antonella; Paglietti, Bianca; Al-Qahtani, Ahmed A; Bohol, Marie Fe F; Senok, Abiola; Deligios, Massimo; Rubino, Salvatore; Al-Ahdal, Mohammed N

2014-08-01

Seven high-risk clones of vancomycin-resistant Enterococcus faecium (VREF) belonging to clonal complex 17 were identified using multilocus sequence typing (MLST) among clinical isolates from Saudi Arabia. Among these isolates, a new hospital-associated sequence type (ST795), VanB(2)-type teicoplanin-resistant strain was detected. Its unusual phenotype resulted from a new combination of mutations in the ddl, vanS and vanW genes, which confirmed the trend of evolution in VanB-type resistance. Furthermore, characteristics of adaptation and persistence in the hospital environment of ST795 were emphasised by the presence of genes and clusters recognised to be specific for hospital-associated VREF. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Growth condition-dependent cell surface proteome analysis of Enterococcus faecium.

PubMed

Sinnige, Jan C; de Been, Mark; Zhou, Miaomiao; Bonten, Marc J M; Willems, Rob J L; Top, Janetta

2015-11-01

The last 30 years Enterococcus faecium has become an important nosocomial pathogen in hospitals worldwide. The aim of this study was to obtain insight in the cell surface proteome of E. faecium when grown in laboratory and clinically relevant conditions. Enterococcus faecium E1162, a clinical blood stream isolate, was grown until mid-log phase in brain heart infusion medium (BHI) with, or without 0.02% bile salts, Tryptic Soy Broth with 1% glucose (TSBg) and urine, and its cell surface was "shaved" using immobilized trypsin. Peptides were identified using MS/MS. Mapping against the translated E1162 whole genome sequence identified 67 proteins that were differentially detected in different conditions. In urine, 14 proteins were significantly more and nine proteins less abundant relative to the other conditions. Growth in BHI-bile and TSBg, revealed four and six proteins, respectively, which were uniquely present in these conditions while two proteins were uniquely present in both conditions. Thus, proteolytic shaving of E. faecium cells identified differentially surface exposed proteins in different growth conditions. These proteins are of special interest as they provide more insight in the adaptive mechanisms and may serve as targets for the development of novel therapeutics against this multi-resistant emerging pathogen. All MS data have been deposited in the ProteomeXchange with identifier PXD002497 (http://proteomecentral.proteomexchange.org/dataset/PXD002497). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enterococcus faecium and Enterococcus durans isolated from cheese: Survival in the presence of medications under simulated gastrointestinal conditions and adhesion properties.

PubMed

Amaral, Daniel M F; Silva, Luana F; Casarotti, Sabrina N; Nascimento, Liane Caroline Sousa; Penna, Ana Lúcia B

2017-02-01

In this study, we evaluated the survival of Enterococcus faecium and Enterococcus durans, isolated from cheese, in the presence of medications and under simulated in vitro gastrointestinal conditions. The presence of genes encoding virulence factors, the susceptibility to antimicrobial agents, and adhesion properties were also assessed. Enterococcus faecium and E. durans both exhibited resistance to most of the tested medications but showed a large sensitivity to analgesics and antihypertensives; they also showed wide susceptibility to antimicrobial agents. Enterococcus durans SJRP29 had greater resistance to the presence of medications in comparison with the probiotic Lactobacillus acidophilus La-5. The strains, except for E. durans SJRP05, did not harbor virulence genes. Enterococcus durans SJRP14, SJRP17, and SJRP26 were sensitive to all tested antimicrobial agents. Enterococcus faecium was more stable during the simulation of gastrointestinal tract and showed greater viability. At the end of the assay, except for E. durans SJRP17, all strains showed high viability (>7 log cfu/mL). Enterococcus durans SJRP29 stood out from the other strains and was selected for further evaluation; it tolerated up to 3.0% NaCl at 30 and 37°C, besides having good adhesion properties (high values of auto-aggregation, co-aggregation, and hydrophobicity). Additionally, the microorganism did not show bile salt hydrolase activity or mucin degradation. These results encourage carrying out additional tests to evaluate the probiotic features by using in vitro dynamic models and in vivo tests before applying these strains to a food system. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The rise of ampicillin-resistant Enterococcus faecium high-risk clones as a frequent intestinal colonizer in oncohaematological neutropenic patients on levofloxacin prophylaxis: a risk for bacteraemia?

PubMed

Sánchez-Díaz, A M; Cuartero, C; Rodríguez, J D; Lozano, S; Alonso, J M; Rodríguez-Domínguez, M; Tedim, A P; Del Campo, R; López, J; Cantón, R; Ruiz-Garbajosa, P

2016-01-01

Levofloxacin extended prophylaxis (LEP), recommended in oncohaematological neutropenic patients to reduce infections, might select resistant bacteria in the intestine acting as a source of endogenous infection. In a prospective observational study we evaluated intestinal emergence and persistence of ampicillin-resistant Enterococcus faecium (AREfm), a marker of hospital adapted high-risk clones. AREfm was recovered from the faeces of 52 patients with prolonged neutropenia after chemotherapy, at admission (Basal), during LEP, and twice weekly until discharge (Pos-LEP). Antibiotic susceptibility, virulence traits and population structure (pulsed-field gel electrophoresis and multilocus sequence typing) were determined and compared with bacteraemic isolates. Gut enterococcal population was monitored using a quantitative PCR quantification approach. AREfm colonized 61.4% of patients (194/482 faecal samples). Sequential AREfm acquisition (25% Basal, 36.5% LEP, 50% Pos-LEP) and high persistent colonization rates (76.9-89.5%) associated with a decrease in clonal diversity were demonstrated. Isolates were clustered into 24 PFGE-patterns within 13 sequence types, 95.8% of them belonging to hospital-associated Bayesian analysis of population structure subgroups 2.1a and 3.3a. Levofloxacin resistance and high-level streptomycin resistance were a common trait of these high-risk clones. AREfm-ST117, the most persistent clone, was dominant (60.0% isolates, 32.6% patients). It presented esp gene and caused 18.2% of all bacteraemia episodes in 21% of patients previously colonized by this clone. In AREfm-colonized patients, intestinal enrichment in the E. faecium population with a decline in total bacterial load was observed. AREfm intestinal colonization increases during hospital stay and coincides with enterococci population enrichment in the gut. Dominance and intestinal persistence of the ST117 clone might increase the risk of bacteraemia. Copyright © 2015 European Society of

Studies on the drug resistance profile of Enterococcus faecium distributed from poultry retailers to hospitals.

PubMed

Limayem, Alya; Donofrio, Robert Scott; Zhang, Chao; Haller, Edward; Johnson, Michael G

2015-01-01

The multidrug resistant Enterococcus faecium (MEF) strains originating from farm animals are proliferating at a substantial pace to impact downstream food chains and could reach hospitals. This study was conducted to elucidate the drug susceptibility profile of MEF strains collected from poultry products in Ann Arbor, MI area and clinical settings from Michigan State Lab and Moffitt Cancer Center (MCC) in Florida. Presumptive positive Enterococcus isolates at species level were identified by Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) analysis. The antibiotic susceptibility profile for both poultry and clinical strains was determined by the Thermo Scientific's Sensititre conform to the National Committee for Clinical Laboratory Standards (NCCLS) and validated via quantitative real-time PCR (qPCR) methods. Out of 50 poultry samples (Turkey: n = 30; Chicken: n = 20), 36 samples were positive for Enterococcus species from which 20.83% were identified as E. faecium. All the E. faecium isolates were multidrug resistant and displayed resistance to the last alternative drug, quinupristin/dalfopristin (QD) used to treat vancomycin resistant E. faecium (VRE) in hospitals. Results indicate the presence of MEF strains in food animals and clinical settings that are also resistant to QD.

Gastrointestinal Tract Colonization Dynamics by Different Enterococcus faecium Clades

PubMed Central

Montealegre, Maria Camila; Singh, Kavindra V.; Murray, Barbara E.

2016-01-01

Colonization of the gastrointestinal tract (GIT) generally precedes infection with antibiotic-resistant Enterococcus faecium. We used a mouse GIT colonization model to test differences in the colonization levels by strains from different E. faecium lineages: clade B, part of the healthy human microbiota; subclade A1, associated with infections; and subclade A2, primarily associated with animals. After mono-inoculation, there was no significant difference in colonization (measured as the geometric mean number of colony-forming units per gram) by the E. faecium clades at any time point (P > .05). However, in competition assays, with 6 of the 7 pairs, clade B strains outcompeted clade A strains in their ability to persist in the GIT; this difference was significant in some pairs by day 2 and in all pairs by day 14 (P < .0008–.0283). This observation may explain the predominance of clade B in the community and why antibiotic-resistant hospital-associated E. faecium are often replaced by clade B strains once patients leave the hospital. PMID:26671890

Comparative genome analysis of multiple vancomycin-resistant Enterococcus faecium isolated from two fatal cases.

PubMed

Lim, Shu Yong; Yap, Kien-Pong; Teh, Cindy Shuan Ju; Jabar, Kartini Abdul; Thong, Kwai Lin

2017-04-01

Enterococcus faecium is both a commensal of the human intestinal tract and an opportunistic pathogen. The increasing incidence of enterococcal infections is mainly due to the ability of this organism to develop resistance to multiple antibiotics, including vancomycin. The aim of this study was to perform comparative genome analyses on four vancomycin-resistant Enterococcus faecium (VRE fm ) strains isolated from two fatal cases in a tertiary hospital in Malaysia. Two sequence types, ST80 and ST203, were identified which belong to the clinically important clonal complex (CC) 17. This is the first report on the emergence of ST80 strains in Malaysia. Three of the studied strains (VREr5, VREr6, VREr7) were each isolated from different body sites of a single patient (patient Y) and had different PFGE patterns. While VREr6 and VREr7 were phenotypically and genotypically similar, the initial isolate, VREr5, was found to be more similar to VRE2 isolated from another patient (patient X), in terms of the genome contents, sequence types and phylogenomic relationship. Both the clinical records and genome sequence data suggested that patient Y was infected by multiple strains from different clones and the strain that infected patient Y could have derived from the same clone from patient X. These multidrug resistant strains harbored a number of virulence genes such as the epa locus and pilus-associated genes which could enhance their persistence. Apart from that, a homolog of E. faecalis bee locus was identified in VREr5 which might be involved in biofilm formation. Overall, our comparative genomic analyses had provided insight into the genetic relatedness, as well as the virulence potential, of the four clinical strains. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of Class IIa Bacteriocin Resistance in Enterococcus faecium

PubMed Central

Geldart, Kathryn

2017-01-01

ABSTRACT Vancomycin-resistant enterococci, particularly resistant Enterococcus faecium, pose an escalating threat in nosocomial environments because of their innate resistance to many antibiotics, including vancomycin, a treatment of last resort. Many class IIa bacteriocins strongly target these enterococci and may offer a potential alternative for the management of this pathogen. However, E. faecium's resistance to these peptides remains relatively uncharacterized. Here, we explored the development of resistance of E. faecium to a cocktail of three class IIa bacteriocins: enterocin A, enterocin P, and hiracin JM79. We started by quantifying the frequency of resistance to these peptides in four clinical isolates of E. faecium. We then investigated the levels of resistance of E. faecium 6E6 mutants as well as their fitness in different carbon sources. In order to elucidate the mechanism of resistance of E. faecium to class IIa bacteriocins, we completed whole-genome sequencing of resistant mutants and performed reverse transcription-quantitative PCR (qRT-PCR) of a suspected target mannose phosphotransferase (ManPTS). We then verified this ManPTS's role in bacteriocin susceptibility by showing that expression of the ManPTS in Lactococcus lactis results in susceptibility to the peptide cocktail. Based on the evidence found from these studies, we conclude that, in accord with other studies in E. faecalis and Listeria monocytogenes, resistance to class IIa bacteriocins in E. faecium 6E6 is likely caused by the disruption of a particular ManPTS, which we believe we have identified. PMID:28115354

Enterocin P Selectively Dissipates the Membrane Potential of Enterococcus faecium T136

PubMed Central

Herranz, C.; Chen, Y.; Chung, H.-J.; Cintas, L. M.; Hernández, P. E.; Montville, T. J.; Chikindas, M. L.

2001-01-01

Enterocin P is a pediocin-like, broad-spectrum bacteriocin which displays a strong inhibitory activity against Listeria monocytogenes. The bacteriocin was purified from the culture supernatant of Enterococcus faecium P13, and its molecular mechanism of action against the sensitive strain E. faecium T136 was evaluated. Although enterocin P caused significant reduction of the membrane potential (ΔΨ) and the intracellular ATP pool of the indicator organism, the pH gradient (ΔpH) component of the proton motive force (Δp) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from E. faecium T136-derived liposomes. PMID:11282622

Prevalence, outcome and risk factor associated with vancomycin-resistant Enterococcus faecalis and Enterococcus faecium at a Tertiary Care Hospital in Northern India.

PubMed

Tripathi, A; Shukla, S K; Singh, A; Prasad, K N

2016-01-01

To determine the prevalence, genotype, risk factors and mortality in patients having vancomycin-resistant Enterococcus faecalis (VR E. faecalis) and Enterococcus faecium (VR E. faecium) infection or colonisation. A total of 1488 clinical isolates of E. faecalis and E. faecium were tested for vancomycin resistance by phenotypic (disk diffusion, E-test and broth micro-dilution test) and genotypic polymerase chain reaction methods. Records of all 1488 patients who had E. faecalis or E. faecium infection or colonisation were reviewed for the identification of host, hospital and medication related risk factors associated with VR E. faecalis and VR E. faecium. Of 1488 isolates, 118 (7.9%) were vancomycin-resistant and their distributions were as follows: E. faecalis=72 (61%) and E. faecium=46 (39%). All 118 vancomycin-resistant isolates were vanA genotype (minimum inhibitory concentration [MIC] to vancomycin ≥64 μg/ml and MIC to teicoplanin≥32 μg/ml) and none of the isolates was vanB genotype. Multivariate logistic regression analysis identified ventilator support and hospital stay for ≥48 h as independent risk factors associated with VR E. faecalis and VR E. faecium infection or colonisation. Hospital stay≥48 h was the only independent risk factor for mortality in patients infected with vancomycin-resistant enterococci. Strategies to limit the nosocomial infection especially in patients on ventilator support can reduce VRE incidence and related mortality.

Characterization of Class IIa Bacteriocin Resistance in Enterococcus faecium.

PubMed

Geldart, Kathryn; Kaznessis, Yiannis N

2017-04-01

Vancomycin-resistant enterococci, particularly resistant Enterococcus faecium , pose an escalating threat in nosocomial environments because of their innate resistance to many antibiotics, including vancomycin, a treatment of last resort. Many class IIa bacteriocins strongly target these enterococci and may offer a potential alternative for the management of this pathogen. However, E. faecium 's resistance to these peptides remains relatively uncharacterized. Here, we explored the development of resistance of E. faecium to a cocktail of three class IIa bacteriocins: enterocin A, enterocin P, and hiracin JM79. We started by quantifying the frequency of resistance to these peptides in four clinical isolates of E. faecium We then investigated the levels of resistance of E. faecium 6E6 mutants as well as their fitness in different carbon sources. In order to elucidate the mechanism of resistance of E. faecium to class IIa bacteriocins, we completed whole-genome sequencing of resistant mutants and performed reverse transcription-quantitative PCR (qRT-PCR) of a suspected target mannose phosphotransferase (ManPTS). We then verified this ManPTS's role in bacteriocin susceptibility by showing that expression of the ManPTS in Lactococcus lactis results in susceptibility to the peptide cocktail. Based on the evidence found from these studies, we conclude that, in accord with other studies in E. faecalis and Listeria monocytogenes , resistance to class IIa bacteriocins in E. faecium 6E6 is likely caused by the disruption of a particular ManPTS, which we believe we have identified. Copyright © 2017 American Society for Microbiology.

Role of Combination Antimicrobial Therapy for Vancomycin-Resistant Enterococcus faecium Infections: Review of the Current Evidence.

PubMed

Yim, Juwon; Smith, Jordan R; Rybak, Michael J

2017-05-01

Enterococcus species are the second most common cause of nosocomial infections in the United States and are particularly concerning in critically ill patients with preexisting comorbid conditions. Rising resistance to antimicrobials that were historically used as front-line agents for treatment of enterococcal infections, such as ampicillin, vancomycin, and aminoglycosides, further complicates the treatment of these infections. Of particular concern are Enterococcus faecium strains that are associated with the highest rate of vancomycin resistance. The introduction of antimicrobial agents with specific activity against vancomycin-resistant Enterococcus (VRE) faecium including daptomycin, linezolid, quinupristin-dalfopristin, and tigecycline did not completely resolve this clinical dilemma. In this review, the mechanisms of action and resistance to currently available anti-VRE antimicrobial agents including newer agents such as oritavancin and dalbavancin will be presented. In addition, novel combination therapies including β-lactams and fosfomycin, and the promising results from in vitro, animal studies, and clinical experience in the treatment of VRE faecium will be discussed. © 2017 Pharmacotherapy Publications, Inc.

Gastrointestinal Tract Colonization Dynamics by Different Enterococcus faecium Clades.

PubMed

Montealegre, Maria Camila; Singh, Kavindra V; Murray, Barbara E

2016-06-15

Colonization of the gastrointestinal tract (GIT) generally precedes infection with antibiotic-resistant Enterococcus faecium We used a mouse GIT colonization model to test differences in the colonization levels by strains from different E. faecium lineages: clade B, part of the healthy human microbiota; subclade A1, associated with infections; and subclade A2, primarily associated with animals. After mono-inoculation, there was no significant difference in colonization (measured as the geometric mean number of colony-forming units per gram) by the E. faecium clades at any time point (P > .05). However, in competition assays, with 6 of the 7 pairs, clade B strains outcompeted clade A strains in their ability to persist in the GIT; this difference was significant in some pairs by day 2 and in all pairs by day 14 (P < .0008-.0283). This observation may explain the predominance of clade B in the community and why antibiotic-resistant hospital-associated E. faecium are often replaced by clade B strains once patients leave the hospital. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

High-Level Fosfomycin Resistance in Vancomycin-Resistant Enterococcus faecium

PubMed Central

Guo, Yan; Tomich, Adam D.; McElheny, Christi L.; Cooper, Vaughn S.; Tait-Kamradt, Amelia; Wang, Minggui; Hu, Fupin; Rice, Louis B.; Sluis-Cremer, Nicolas

2017-01-01

Of 890 vancomycin-resistant Enterococcus faecium isolates obtained by rectal screening from patients in Pittsburgh, Pennsylvania, USA, 4 had MICs >1,024 μg/mL for fosfomycin. These isolates had a Cys119Asp substitution in the active site of UDP-N-acetylglucosamine enolpyruvyl transferase. This substitution increased the fosfomycin MIC >4-fold and rendered this drug inactive in biochemical assays. PMID:29048285

Drug-resistant and hospital-associated Enterococcus faecium from wastewater, riverine estuary and anthropogenically impacted marine catchment basin.

PubMed

Sadowy, Ewa; Luczkiewicz, Aneta

2014-03-14

Enterococci, ubiquitous colonizers of humans and other animals, play an increasingly important role in health-care associated infections (HAIs). It is believed that the recent evolution of two clinically relevant species, Enterococcus faecalis and Enterococcus faecium occurred in a big part in a hospital environment, leading to formation of high-risk enterococcal clonal complexes (HiRECCs), which combine multidrug resistance with increased pathogenicity and epidemicity. The aim of this study was to establish the species composition in wastewater, its marine recipient as well as a river estuary and to investigate the antimicrobial susceptibility of collected isolates. Molecular methods were additionally applied to test the presence of HiRRECC-related E. faecium. Two wastewater treatment plants (WWTPs), their marine outfalls and Vistula river that influence significantly the quality of waters in Gulf of Gdansk were sampled to investigate the presence of Enterococcus spp. Four-hundred-twenty-eight isolates were obtained, including E. faecium (244 isolates, 57.0%), E. hirae (113 isolates, 26.4%) and E. faecalis (63 isolates, 14.7%); other species (E. gallinarum/casseliflavus, E. durans and E. avium) accounted for 1.9%. Antimicrobial susceptibility testing revealed the presence of isolates resistant to erythromycin, tetracycline, amipicillin, fluoroquinolones and aminoglycosides (high-level resistance), especially among E. faecium, where such isolates were usually characterized by multilocus sequence types associated with nosocomial lineages 17, 18 and 78 of this species representing HiRECC, formerly called CC17. These isolates not only carried several resistance determinants but were also enriched in genes encoding pathogenicity factors (Esp, pili) and genes associated with mobile genetic elements (MGE), a feature also typical for nosocomial HiRECC. Our data show that WWTPs constitute an important source of enterococcal strains carrying antimicrobial resistance

Drug-resistant and hospital-associated Enterococcus faecium from wastewater, riverine estuary and anthropogenically impacted marine catchment basin

PubMed Central

2014-01-01

Background Enterococci, ubiquitous colonizers of humans and other animals, play an increasingly important role in health-care associated infections (HAIs). It is believed that the recent evolution of two clinically relevant species, Enterococcus faecalis and Enterococcus faecium occurred in a big part in a hospital environment, leading to formation of high-risk enterococcal clonal complexes (HiRECCs), which combine multidrug resistance with increased pathogenicity and epidemicity. The aim of this study was to establish the species composition in wastewater, its marine recipient as well as a river estuary and to investigate the antimicrobial susceptibility of collected isolates. Molecular methods were additionally applied to test the presence of HiRRECC-related E. faecium. Results Two wastewater treatment plants (WWTPs), their marine outfalls and Vistula river that influence significantly the quality of waters in Gulf of Gdansk were sampled to investigate the presence of Enterococcus spp. Four-hundred-twenty-eight isolates were obtained, including E. faecium (244 isolates, 57.0%), E. hirae (113 isolates, 26.4%) and E. faecalis (63 isolates, 14.7%); other species (E. gallinarum/casseliflavus, E. durans and E. avium) accounted for 1.9%. Antimicrobial susceptibility testing revealed the presence of isolates resistant to erythromycin, tetracycline, amipicillin, fluoroquinolones and aminoglycosides (high-level resistance), especially among E. faecium, where such isolates were usually characterized by multilocus sequence types associated with nosocomial lineages 17, 18 and 78 of this species representing HiRECC, formerly called CC17. These isolates not only carried several resistance determinants but were also enriched in genes encoding pathogenicity factors (Esp, pili) and genes associated with mobile genetic elements (MGE), a feature also typical for nosocomial HiRECC. Conclusions Our data show that WWTPs constitute an important source of enterococcal strains carrying

Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium

PubMed Central

Zhang, Xinglin; Paganelli, Fernanda L.; Bierschenk, Damien; Kuipers, Annemarie; Bonten, Marc J. M.; Willems, Rob J. L.; van Schaik, Willem

2012-01-01

Enterococcus faecium has become a nosocomial pathogen of major importance, causing infections that are difficult to treat owing to its multi-drug resistance. In particular, resistance to the β-lactam antibiotic ampicillin has become ubiquitous among clinical isolates. Mutations in the low-affinity penicillin binding protein PBP5 have previously been shown to be important for ampicillin resistance in E. faecium, but the existence of additional resistance determinants has been suggested. Here, we constructed a high-density transposon mutant library in E. faecium and developed a transposon mutant tracking approach termed Microarray-based Transposon Mapping (M-TraM), leading to the identification of a compendium of E. faecium genes that contribute to ampicillin resistance. These genes are part of the core genome of E. faecium, indicating a high potential for E. faecium to evolve towards β-lactam resistance. To validate the M-TraM results, we adapted a Cre-lox recombination system to construct targeted, markerless mutants in E. faecium. We confirmed the role of four genes in ampicillin resistance by the generation of targeted mutants and further characterized these mutants regarding their resistance to lysozyme. The results revealed that ddcP, a gene predicted to encode a low-molecular-weight penicillin binding protein with D-alanyl-D-alanine carboxypeptidase activity, was essential for high-level ampicillin resistance. Furthermore, deletion of ddcP sensitized E. faecium to lysozyme and abolished membrane-associated D,D-carboxypeptidase activity. This study has led to the development of a broadly applicable platform for functional genomic-based studies in E. faecium, and it provides a new perspective on the genetic basis of ampicillin resistance in this organism. PMID:22761597

High-level aminoglycoside resistance in Enterococcus faecalis and Enterococcus faecium causing invasive infection: Twelve-year surveillance in the Minami Ibaraki Area.

PubMed

Osuka, Hanako; Nakajima, Jun; Oishi, Tsuyoshi; Funayama, Yasunori; Ebihara, Tsugio; Ishikawa, Hiroichi; Saito, Kazuto; Koganemaru, Hiroshi; Hitomi, Shigemi

2016-01-01

We examined prevalence of high-level aminoglycoside resistance (HLAR) in Enterococcus faecalis and Enterococcus faecium causing invasive infection in the Minami Ibaraki Area. Ten strains of both species each, recovered from the blood or the cerebrospinal fluid between 2003 and 2014, were randomly selected every year. High-level resistance to gentamicin (HLR-GM) and streptomycin (HLR-SM) was detected in 34% (41 of 120 strains) and 18% (21) of E. faecalis and 9% (11) and 39% (48) of E. faecium, respectively. In comparisons of the proportions among three four-year periods, HLR-SM among E. faecium was significantly lower in the 2011-2014 period. All strains with HLR-GM were positive for the aac(6')-Ie-aph(2″)-Ia gene. The ant(6')-Ia gene was detected in all with HLR-SM except for one E. faecalis strain. The present study showed that prevalence of HLR-GM among E. faecalis and E. faecium causing invasive infection in this area was nearly equivalent to that described in previous studies in Japan and that proportions of strains with HLAR did not vary during the study period except for that of HLR-SM among E. faecium. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Probiotic Potential of Enterococcus faecium Isolated from Chicken Cecum with Immunomodulating Activity and Promoting Longevity in Caenorhabditis elegans.

PubMed

Sim, Insuk; Park, Keun-Tae; Kwon, Gayeung; Koh, Jong-Ho; Lim, Young-Hee

2018-04-12

Probiotics including Enterococcus faecium confer a health benefit on the host. An Enterococcus strain was isolated from healthy chicken cecum, identified as E. faeciumby 16S rDNA gene sequence analysis, and designatedas E. faecium L11.Toevaluate the potential of E. faecium L11 as a probiotics, the gastrointestinal tolerance, immunomodulatory activity, and lifespan extension properties of the strain were assayed. E.faecium L11 showed >66% and >62% survival in artificial gastric juices (0.3% pepsin, pH 2.5) and simulated smallintestinal juice(0.5% bile salt and 0.1%pancreatin), respectively. Heat-killed E.faecium L11significantly ( p < 0.05) increased immune cell proliferation compared with controls, and stimulated the production of cytokines(IL-6 and TNF-α) by activated macrophages obtained from ICRmice. In addition, E.faecium L11 showed a protective effect against Salmonella Typhimurium infection in Caenorhabditis elegans . In addition, feeding E.faecium L11 significantly ( p < 0.05) extended the lifespan of C. elegans compared with the control. Furthermore, genes related to aging and host defense were upregulated in E.faecium L11-fed worms. In conclusion, E. faecium L11 which prolongs the lifespan of C. elegans may be a potent probiotic supplement for livestock.

Antimicrobial resistance profiles of Enterococcus faecalis and Enterococcus faecium isolated from artisanal food of animal origin in Argentina.

PubMed

Delpech, Gastón; Pourcel, Gisela; Schell, Celia; De Luca, María; Basualdo, Juan; Bernstein, Judith; Grenovero, Silvia; Sparo, Mónica

2012-10-01

Enterococci are part of the indigenous microbiota of human gastrointestinal tract and food of animal origin. Enterococci inhabiting non-human reservoirs play a critical role in the acquisition and dissemination of antimicrobial resistance determinants. The aim of this work was to investigate the antimicrobial resistance in Enterococcus faecalis and Enterococcus faecium strains recovered from artisanal food of animal origin. Samples of goat cheese (n = 42), cow cheese (n = 40), artisanal salami (n = 30), and minced meat for the manufacture of hamburgers (n = 60) were analyzed. Phenotypic and genotypic tests for species-level identification of the recovered isolates were carried out. Minimum inhibitory concentration (MIC) study for in vitro quantitative antimicrobial resistance assessment was performed, and 71 E. faecalis and 22 E. faecium were isolated. The recovered enterococci showed different multi-drug resistance patterns that included tretracycline, erythromycin, ciprofloxacin, linezolid, penicillin, ampicillin, vancomycin, teicoplanin, gentamicin (high-level resistance), and streptomycin (high-level resistance). VanA-type E. faecium were detected. β-lactamase activity was not observed. Artisanal foods of animal origin act as a non-human reservoir of E. faecalis and E. faecuim strains, expressing multi-resistance to antimicrobials. In conclusion, the implementation of a continuous antimicrobial resistance surveillance in enterococci isolated from artisanal food of animal origin is important.

Characterization of an anti-listerial enterocin from wheat silage based Enterococcus faecium.

PubMed

Bal, Emel Banu Buyukunal; Isevi, Taner; Bal, Mehmet Ali

2012-10-01

Two Enterococcus faecium and one E. faecalis strains isolated and identified from wheat silage were characterized based on plasmid content, hemolytic activity, antibiotic resistance patterns, bacteriocin production potential, and presence of enterocin structural genes (entA, entB, entP, entL50B). Among the isolates, only the E. faecium U7 strain exhibited bacteriocin activity against Listeria monocytogenes ATCC 7644, and vancomycin resistant Enterococcus spp. (VRE). A combination of three structural genes (entA, entB, and entP) was detected in E. faecium U7. A relationship between the presence of enterocin structural genes, and bacteriocin activity was detected in E. faecium U7; therefore partially purified enterocin (PPE) was further investigated from the isolate. Several bands of different molecular weights were expressed from PPE extracts following tricine SDS-PAGE analysis. However, the only band showing bacteriocin activity was in an approximate 4-kDa region. PPE treatment with proteinase K, lysozyme, and α -amylase caused complete loss of bacteriocin activity. PPE heat treatment at various temperatures resulted in a notable reduction in bacteriocin expression. Enterocin U7 was relatively heat stable, and presumably exhibits a glucoprotein nature with distinct inhibitory properties. Specific bacterial inhibitory activity of enterocin U7, and the producer strain absence of β -hemolysis and vancomycin susceptibility features deserves further investigation to evaluate its potential application in silage inoculation and food preservation. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The acute-phase response impairs host defence against Enterococcus faecium peritonitis

PubMed Central

Leendertse, Masja; Willems, Rob J L; Giebelen, Ida A J; van den Pangaart, Petra S; Bonten, Marc J M; van der Poll, Tom

2009-01-01

Enterococcus faecium is an emerging pathogen that causes infections in hospitalized patients with various co-morbid diseases. These underlying diseases are often associated with an acute-phase response that renders patients vulnerable to nosocomial infections. To study the influence of the acute-phase response induced by sterile tissue injury on host defence against E. faecium, mice were injected subcutaneously with either turpentine or casein 1 day before intraperitoneal infection with E. faecium. Control mice were subcutaneously injected with saline or sodium bicarbonate, respectively. Turpentine and casein induced an acute-phase response as reflected by increases in the plasma concentrations of interleukin-6, serum amyloid P and C3. A pre-existent acute-phase response in mice was associated with a strongly reduced capacity to clear E. faecium, resulting in prolonged bacteraemia for several days. The inflammatory response to E. faecium was impaired in mice with an acute-phase response, as shown by reduced capacity to mount a neutrophilic leucocytosis in peripheral blood and by decreased local cytokine concentrations. These data indicate that the acute-phase response impairs host defence against E. faecium, suggesting that this condition may contribute to the increased vulnerability of critically ill patients to enterococcal infections. PMID:19175794

Isolation of Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512 as novel probiotics with immunomodulatory properties.

PubMed

Mansour, Nahla M; Heine, Holger; Abdou, Sania M; Shenana, Mohamed E; Zakaria, Mohamed K; El-Diwany, Ahmed

2014-10-01

Probiotics, defined as living bacteria that are beneficial for human health, mainly function through their immunomodulatory abilities. Hence, these microorganisms have proven successful for treating diseases resulting from immune deregulation. The aim of this study was to find novel candidates to improve on and complement current probiotic treatment strategies. Of 60 lactic acid bacterial strains that were isolated from fecal samples of healthy, full-term, breast-fed infants, three were chosen because of their ability to activate human immune cells. These candidates were then tested with regard to immunomodulatory properties, antimicrobial effects on pathogens, required pharmacological properties and their safety profiles. To identify the immunomodulatory structures of the selected isolates, activation of specific innate immune receptors was studied. The three candidates for probiotic treatment were assigned Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512. Compared with the established allergy-protective strain Lactococcus lactis G121, these isolates induced release of similar amounts of IL-12, a potent inducer of T helper 1 cells. In addition, all three neonatal isolates had antimicrobial activity against pathogens. Analysis of pharmacological suitability showed high tolerance of low pH, bile salts and pancreatic enzymes. In terms of safe application in humans, the isolates were sensitive to three antibiotics (chloramphenicol, tetracycline and erythromycin). In addition, the Enterococcus isolates were free from the four major virulence genes (cylA, agg, efaAfs and ccf). Moreover, the isolates strongly activated Toll-like receptor 2, which suggests lipopeptides as their active immunomodulatory structure. Thus, three novel bacterial strains with great potential as probiotic candidates and promising immunomodulatory properties have here been identified and characterized. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

Enterocin P Causes Potassium Ion Efflux from Enterococcus faecium T136 Cells

PubMed Central

Herranz, Carmen; Cintas, Luis M.; Hernández, Pablo E.; Moll, Gert N.; Driessen, Arnold J. M.

2001-01-01

Enterocin P is a bacteriocin produced by Enterococcus faecium P13. We studied the mechanism of its bactericidal action using enterocin-P-sensitive E. faecium T136 cells. The bacteriocin is incapable of dissipating the transmembrane pH gradient. On the other hand, depending on the buffer used, enterocin P dissipates the transmembrane potential. Enterocin P efficiently elicits efflux of potassium ions, but not of intracellularly accumulated anions like phosphate and glutamate. Taken together, these data demonstrate that enterocin P forms specific, potassium ion-conducting pores in the cytoplasmic membrane of target cells. PMID:11181377

Antimicrobial Susceptibility Patterns of Enterococcus faecalis and Enterococcus faecium Isolated from Poultry Flocks in Germany.

PubMed

Maasjost, J; Mühldorfer, K; Cortez de Jäckel S; Hafez, H M

2015-03-01

Between 2010 and 2011, 145 Enterococcus isolates (Enterococcus faecalis, n = 127; Enterococcus faecium, n = 18) were collected during routine bacteriologic diagnostics from broilers, layers, and fattening turkeys in Germany showing various clinical signs. The susceptibility to 24 antimicrobial agents was investigated by broth microdilution test to determine minimum inhibitory concentrations (MICs). All E. faecalis isolates (n = 127) were susceptible to the beta-lactam antibiotics ampicillin, amoxicillin-clavulanic acid, and penicillin. Corresponding MIC with 50% inhibition (MIC50) and MIC with 90% inhibition (MIC90) values of these antimicrobial agents were at the lower end of the test range (≤ 4 μg/ml). In addition, no vancomycin-resistant enterococci (VRE) were found. High resistance rates were identified in both Enterococcus species for lincomycin (72%-99%) and tetracycline (67%-82%). Half or more than half of Enterococcus isolates were resistant to gentamicin (54%-72%) and the macrolide antibiotics erythromycin (44%-61%) and tylosin-tartate (44%-56%). Enterococcus faecalis isolated from fattening turkeys showed the highest prevalence of antimicrobial resistance compared to other poultry production systems. Eighty-nine out of 145 Enterococcus isolates were resistant to three or more antimicrobial classes. Again, turkeys stood out with 42 (8 1%) multiresistant isolates. The most-frequent resistance patterns of E. faecalis were gentamicin, lincomycin, and tetracycline in all poultry production systems.

The mazEF toxin-antitoxin system as an attractive target in clinical isolates of Enterococcus faecium and Enterococcus faecalis.

PubMed

Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasantha Kumari; Sadeghifard, Nourkhoda; Taherikalani, Morovat; Khosravi, Afra; Ramli, Ramliza; Hamat, Rukman Awang

2015-01-01

The toxin-antitoxin (TA) system is a regulatory system where two sets of genes encode the toxin and its corresponding antitoxin. In this study, the prevalence of TA systems in independently isolated clinical isolates of Enterococcus faecium and Enterococcus faecalis was determined, the dominant TA system was identified, different virulence genes in E. faecium and E. faecalis were surveyed, the level of expression of the virulence and TA genes in normal and stress conditions was determined, and finally their associations with the TA genes were defined. Remarkably, the analysis demonstrated higBA and mazEF in all clinical isolates, and their locations were on chromosomes and plasmids, respectively. On the other hand, a quantitative analysis of TA and virulence genes revealed that the expression level in both genes is different under normal and stress conditions. The results obtained by anti-mazF peptide nucleic acids demonstrated that the expression level of virulence genes had decreased. These findings demonstrate an association between TA systems and virulence factors. The mazEF on the plasmids and the higBA TA genes on the chromosomes of all E. faecium and E. faecalis strains were dominant. Additionally, there was a decrease in the expression of virulence genes in the presence of anti-mazF peptide nucleic acids. Therefore, it is suggested that mazEF TA systems are potent and sensitive targets in all E. faecium and E. faecalis strains.

The mazEF toxin–antitoxin system as an attractive target in clinical isolates of Enterococcus faecium and Enterococcus faecalis

PubMed Central

Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasantha Kumari; Sadeghifard, Nourkhoda; Taherikalani, Morovat; Khosravi, Afra; Ramli, Ramliza; Hamat, Rukman Awang

2015-01-01

The toxin–antitoxin (TA) system is a regulatory system where two sets of genes encode the toxin and its corresponding antitoxin. In this study, the prevalence of TA systems in independently isolated clinical isolates of Enterococcus faecium and Enterococcus faecalis was determined, the dominant TA system was identified, different virulence genes in E. faecium and E. faecalis were surveyed, the level of expression of the virulence and TA genes in normal and stress conditions was determined, and finally their associations with the TA genes were defined. Remarkably, the analysis demonstrated higBA and mazEF in all clinical isolates, and their locations were on chromosomes and plasmids, respectively. On the other hand, a quantitative analysis of TA and virulence genes revealed that the expression level in both genes is different under normal and stress conditions. The results obtained by anti-mazF peptide nucleic acids demonstrated that the expression level of virulence genes had decreased. These findings demonstrate an association between TA systems and virulence factors. The mazEF on the plasmids and the higBA TA genes on the chromosomes of all E. faecium and E. faecalis strains were dominant. Additionally, there was a decrease in the expression of virulence genes in the presence of anti-mazF peptide nucleic acids. Therefore, it is suggested that mazEF TA systems are potent and sensitive targets in all E. faecium and E. faecalis strains. PMID:26005332

Draft Genome Sequence of Enterococcus faecium Strain J19, Isolated from Cabbage

PubMed Central

2018-01-01

ABSTRACT Herein, we report the draft genome sequence of a newly discovered probiotic strain, Enterococcus faecium J19, which was isolated from cabbage. Strain J19 has shown antagonistic effects against the human foodborne pathogen Listeria monocytogenes in coculture and in different food matrices. PMID:29622613

Emergence of endemic MLST non-typeable vancomycin-resistant Enterococcus faecium.

PubMed

Carter, Glen P; Buultjens, Andrew H; Ballard, Susan A; Baines, Sarah L; Tomita, Takehiro; Strachan, Janet; Johnson, Paul D R; Ferguson, John K; Seemann, Torsten; Stinear, Timothy P; Howden, Benjamin P

2016-12-01

Enterococcus faecium is a major nosocomial pathogen causing significant morbidity and mortality worldwide. Assessment of E. faecium using MLST to understand the spread of this organism is an important component of hospital infection control measures. Recent studies, however, suggest that MLST might be inadequate for E. faecium surveillance. To use WGS to characterize recently identified vancomycin-resistant E. faecium (VREfm) isolates non-typeable by MLST that appear to be causing a multi-jurisdictional outbreak in Australia. Illumina NextSeq and Pacific Biosciences SMRT sequencing platforms were used to determine the genome sequences of 66 non-typeable E. faecium (NTEfm) isolates. Phylogenetic and bioinformatics analyses were subsequently performed using a number of in silico tools. Sixty-six E. faecium isolates were identified by WGS from multiple health jurisdictions in Australia that could not be typed by MLST due to a missing pstS allele. SMRT sequencing and complete genome assembly revealed a large chromosomal rearrangement in representative strain DMG1500801, which likely facilitated the deletion of the pstS region. Phylogenomic analysis of this population suggests that deletion of pstS within E. faecium has arisen independently on at least three occasions. Importantly, the majority of these isolates displayed a vancomycin-resistant genotype. We have identified NTEfm isolates that appear to be causing a multi-jurisdictional outbreak in Australia. Identification of these isolates has important implications for MLST-based typing activities designed to monitor the spread of VREfm and provides further evidence supporting the use of WGS for hospital surveillance of E. faecium. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Genomic Features and Niche-Adaptation of Enterococcus faecium Strains from Korean Soybean-Fermented Foods.

PubMed

Kim, Eun Bae; Jin, Gwi-Deuk; Lee, Jun-Yeong; Choi, Yun-Jaie

2016-01-01

Certain strains of Enterococcus faecium contribute beneficially to human health and food fermentation. However, other E. faecium strains are opportunistic pathogens due to the acquisition of virulence factors and antibiotic resistance determinants. To characterize E. faecium from soybean fermentation, we sequenced the genomes of 10 E. faecium strains from Korean soybean-fermented foods and analyzed their genomes by comparing them with 51 clinical and 52 non-clinical strains of different origins. Hierarchical clustering based on 13,820 orthologous genes from all E. faecium genomes showed that the 10 strains are distinguished from most of the clinical strains. Like non-clinical strains, their genomes are significantly smaller than clinical strains due to fewer accessory genes associated with antibiotic resistance, virulence, and mobile genetic elements. Moreover, we identified niche-associated gene gain and loss from the soybean strains. Thus, we conclude that soybean E. faecium strains might have evolved to have distinctive genomic features that may contribute to its ability to thrive during soybean fermentation.

Genomic Features and Niche-Adaptation of Enterococcus faecium Strains from Korean Soybean-Fermented Foods

PubMed Central

Kim, Eun Bae; Jin, Gwi-Deuk; Lee, Jun-Yeong; Choi, Yun-Jaie

2016-01-01

Certain strains of Enterococcus faecium contribute beneficially to human health and food fermentation. However, other E. faecium strains are opportunistic pathogens due to the acquisition of virulence factors and antibiotic resistance determinants. To characterize E. faecium from soybean fermentation, we sequenced the genomes of 10 E. faecium strains from Korean soybean-fermented foods and analyzed their genomes by comparing them with 51 clinical and 52 non-clinical strains of different origins. Hierarchical clustering based on 13,820 orthologous genes from all E. faecium genomes showed that the 10 strains are distinguished from most of the clinical strains. Like non-clinical strains, their genomes are significantly smaller than clinical strains due to fewer accessory genes associated with antibiotic resistance, virulence, and mobile genetic elements. Moreover, we identified niche-associated gene gain and loss from the soybean strains. Thus, we conclude that soybean E. faecium strains might have evolved to have distinctive genomic features that may contribute to its ability to thrive during soybean fermentation. PMID:27070419

Biocide and antibiotic resistance of Enterococcus faecalis and Enterococcus faecium isolated from the swine meat chain.

PubMed

Rizzotti, Lucia; Rossi, Franca; Torriani, Sandra

2016-12-01

In this study nine strains of Enterococcus faecalis and 12 strains of Enterococcus faecium, isolated from different sample types in the swine meat chain and previously characterized for the presence of antibiotic resistance genes, were examined for phenotypic tolerance to seven biocides (chlorexidine, benzalkonium chloride, triclosan, sodium hypochlorite, 2-propanol, formaldehyde and hydrogen peroxide) and resistance to nine antibiotics (ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol). Moreover, the presence of efflux system encoding genes qacA/B, qacC, qacE, qacEΔ1, emeA, and stress response genes, sigV and gsp65, involved in the tolerance to biocides, was analysed. Most strains were not tolerant to the biocides, but showed minimum inhibitory concentrations (MICs) higher than the recommended cut-off values for all the antibiotics tested, except for vancomycin and chloramphenicol. Only weak correlations, if any, were found between biocide and antibiotic resistance data. One E. faecalis strain was tolerant to triclosan and one E. faecium strain, with higher tolerance to chlorexidine than the other strains tested, was found to carry a qacA/B gene. Our results indicated that phenotypic resistance to antibiotics is very frequent in enterococcal isolates from the swine meat chain, but phenotypic tolerance to biocides is not common. On the other hand, the gene qacA/B was found for the first time in the species E. faecium, an indication of the necessity to adopt measures suitable to control the spread of biocide resistance determinants among enterococci. Copyright © 2016 Elsevier Ltd. All rights reserved.

Susceptibility of vancomycin-resistant and -sensitive Enterococcus faecium obtained from Danish hospitals to benzalkonium chloride, chlorhexidine and hydrogen peroxide biocides.

PubMed

Alotaibi, Sulaiman M I; Ayibiekea, Alafate; Pedersen, Annemette Frøling; Jakobsen, Lotte; Pinholt, Mette; Gumpert, Heidi; Hammerum, Anette M; Westh, Henrik; Ingmer, Hanne

2017-12-01

In Danish hospitals, the number of infections caused by vancomycin-resistant Enterococcus faecium (VRE faecium) has dramatically increased in recent years. Hospital disinfectants are essential in eliminating pathogenic microorganisms, and reduced susceptibility may contribute to hospital-associated infections. We have addressed whether clinical VRE faecium display decreased biocide susceptibility when compared to vancomycin-sensitive Enterococcus faecium (VSE faecium) isolates. In total 12 VSE faecium and 37 VRE faecium isolates obtained from Danish hospitals over an extended time period were tested for susceptibility towards three commonly applied biocides, namely benzalkonium chloride, chlorhexidine and hydrogen peroxide. For benzalkonium chloride, 89 % of VRE faecium strains had a minimal inhibitory concentration (MIC) of 8 mg l -1 , whereas for VSE faecium, only 25 % of the strains had an MIC of 8 mg l -1 . For chlorhexidine, the MIC of 95 % of VRE faecium strains was 4 mg l -1 or higher, while only 33 % of VSE faecium strains displayed MIC values at the same level. In contrast, both VRE and VSE faecium displayed equal susceptibility to hydrogen peroxide, but a higher minimal bactericidal concentration (MBC) was found for the former. The efflux activity was also assessed, and this was generally higher for the VRE faecium strains compared to VSE faecium. VRE faecium from Danish hospitals demonstrated decreased susceptibility towards benzalkonium chloride and chlorhexidine compared to VSE faecium, where the use of chlorhexidine is particularly heavy in the hospital environment. These findings suggest that biocide tolerance may characterize VRE faecium isolated in Danish hospitals.

Detection of a cfr(B) Variant in German Enterococcus faecium Clinical Isolates and the Impact on Linezolid Resistance in Enterococcus spp.

PubMed

Bender, Jennifer K; Fleige, Carola; Klare, Ingo; Fiedler, Stefan; Mischnik, Alexander; Mutters, Nico T; Dingle, Kate E; Werner, Guido

2016-01-01

The National Reference Centre for Staphylococci and Enterococci in Germany has received an increasing number of clinical linezolid-resistant E. faecium isolates in recent years. Five isolates harbored a cfr(B) variant gene locus the product of which is capable of conferring linezolid resistance. The cfr(B)-like methyltransferase gene was also detected in Clostridium difficile. Antimicrobial susceptibility was determined for cfr(B)-positive and linezolid-resistant E. faecium isolates and two isogenic C. difficile strains. All strains were subjected to whole genome sequencing and analyzed with respect to mutations in the 23S rDNA, rplC, rplD and rplV genes and integration sites of the cfr(B) variant locus. To evaluate methyltransferase function, the cfr(B) variant of Enterococcus and Clostridium was expressed in both E. coli and Enterococcus spp. Ribosomal target site mutations were detected in E. faecium strains but absent in clostridia. Sequencing revealed 99.9% identity between cfr(B) of Enterococcus and cfr of Clostridium. The methyltransferase gene is encoded by transposon Tn6218 which was present in C. difficile Ox3196, truncated in some E. faecium and absent in C. difficile Ox3206. The latter finding explains the lack of linezolid and chloramphenicol resistance in C. difficile Ox3206 and demonstrates for the first time a direct correlation of elevated linezolid MICs in C. difficile upon cfr acquisition. Tn6218 insertion sites revealed novel target loci for integration, both within the bacterial chromosome and as an integral part of plasmids. Importantly, the very first plasmid-association of a cfr(B) variant was observed. Although we failed to measure cfr(B)-mediated resistance in transformed laboratory strains the occurrence of the multidrug resistance gene cfr on putatively highly mobile and/or extrachromosomal DNA in clinical isolates is worrisome with respect to dissemination of antibiotic resistances.

Detection of a cfr(B) Variant in German Enterococcus faecium Clinical Isolates and the Impact on Linezolid Resistance in Enterococcus spp.

PubMed Central

Fleige, Carola; Klare, Ingo; Fiedler, Stefan; Mischnik, Alexander; Mutters, Nico T.; Dingle, Kate E.; Werner, Guido

2016-01-01

The National Reference Centre for Staphylococci and Enterococci in Germany has received an increasing number of clinical linezolid-resistant E. faecium isolates in recent years. Five isolates harbored a cfr(B) variant gene locus the product of which is capable of conferring linezolid resistance. The cfr(B)-like methyltransferase gene was also detected in Clostridium difficile. Antimicrobial susceptibility was determined for cfr(B)-positive and linezolid-resistant E. faecium isolates and two isogenic C. difficile strains. All strains were subjected to whole genome sequencing and analyzed with respect to mutations in the 23S rDNA, rplC, rplD and rplV genes and integration sites of the cfr(B) variant locus. To evaluate methyltransferase function, the cfr(B) variant of Enterococcus and Clostridium was expressed in both E. coli and Enterococcus spp. Ribosomal target site mutations were detected in E. faecium strains but absent in clostridia. Sequencing revealed 99.9% identity between cfr(B) of Enterococcus and cfr of Clostridium. The methyltransferase gene is encoded by transposon Tn6218 which was present in C. difficile Ox3196, truncated in some E. faecium and absent in C. difficile Ox3206. The latter finding explains the lack of linezolid and chloramphenicol resistance in C. difficile Ox3206 and demonstrates for the first time a direct correlation of elevated linezolid MICs in C. difficile upon cfr acquisition. Tn6218 insertion sites revealed novel target loci for integration, both within the bacterial chromosome and as an integral part of plasmids. Importantly, the very first plasmid-association of a cfr(B) variant was observed. Although we failed to measure cfr(B)-mediated resistance in transformed laboratory strains the occurrence of the multidrug resistance gene cfr on putatively highly mobile and/or extrachromosomal DNA in clinical isolates is worrisome with respect to dissemination of antibiotic resistances. PMID:27893790

Phenotypic and genotypic characterization of vancomycin-resistant Enterococcus faecium clinical isolates from two hospitals in Mexico: First detection of VanB phenotype-vanA genotype.

PubMed

Bocanegra-Ibarias, Paola; Flores-Treviño, Samantha; Camacho-Ortiz, Adrián; Morfin-Otero, Rayo; Villarreal-Treviño, Licet; Llaca-Díaz, Jorge; Martínez-Landeros, Erik Alan; Rodríguez-Noriega, Eduardo; Calzada-Güereca, Andrés; Maldonado-Garza, Héctor Jesús; Garza-González, Elvira

2016-01-01

Enterococcus faecium has emerged as a multidrug-resistant nosocomial pathogen involved in outbreaks worldwide. Our aim was to determine the antimicrobial susceptibility, biofilm production, and clonal relatedness of vancomycin-resistant E. faecium (VREF) clinical isolates from two hospitals in Mexico. Consecutive clinical isolates (n=56) were collected in two tertiary care hospitals in Mexico from 2011 to 2014. VREF isolates were characterized by phenotypic and molecular methods including pulsed-field gel electrophoresis (PFGE). VREF isolates were highly resistant to vancomycin, erythromycin, norfloxacin, high-level streptomycin, and teicoplanin, and showed lower resistance to tetracycline, nitrofurantoin and quinupristin-dalfopristin. None of the isolates were resistant to linezolid. The vanA gene was detected in all isolates. Two VanB phenotype-vanA genotype isolates, highly resistant to vancomycin and susceptible to teicoplanin, were detected. Furthermore, 17.9% of the isolates were classified as biofilm producers, and the espfm gene was found in 98.2% of the isolates. A total of 37 distinct PFGE patterns and 6 clones (25% of the isolates as clone A, 5.4% as clone B, and 3.6% each as clone C, D, E, and F) were detected. Clone A was detected in 5 different wards of the same hospital during 14 months of surveillance. The high resistance to most antimicrobial agents and the moderate cross-transmission of VREF detected accentuates the need for continuous surveillance of E. faecium in the hospital setting. This is also the first reported incidence of the E. faecium VanB phenotype-vanA genotype in the Americas. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Aroma compounds generation in citrate metabolism of Enterococcus faecium: Genetic characterization of type I citrate gene cluster.

PubMed

Martino, Gabriela P; Quintana, Ingrid M; Espariz, Martín; Blancato, Victor S; Magni, Christian

2016-02-02

Enterococcus is one of the most controversial genera belonging to Lactic Acid Bacteria. Research involving this microorganism reflects its dual behavior as regards its safety. Although it has also been associated to nosocomial infections, natural occurrence of Enterococcus faecium in food contributes to the final quality of cheese. This bacterium is capable of fermenting citrate, which is metabolized to pyruvate and finally derives in the production of the aroma compounds diacetyl, acetoin and 2,3 butanediol. Citrate metabolism was studied in E. faecium but no data about genes related to these pathways have been described. A bioinformatic approach allowed us to differentiate cit(-) (no citrate metabolism genes) from cit(+) strains in E. faecium. Furthermore, we could classify them according to genes encoding for the transcriptional regulator, the oxaloacetate decarboxylase and the citrate transporter. Thus we defined type I organization having CitI regulator (DeoR family), CitM cytoplasmic soluble oxaloacetate decarboxylase (Malic Enzyme family) and CitP citrate transporter (2-hydroxy-carboxylate transporter family) and type II organization with CitO regulator (GntR family), OAD membrane oxaloacetate decarboxylase complex (Na(+)-transport decarboxylase enzyme family) and CitH citrate transporter (CitMHS family). We isolated and identified 17 E. faecium strains from regional cheeses. PCR analyses allowed us to classify them as cit(-) or cit(+). Within the latter classification we could differentiate type I but no type II organization. Remarkably, we came upon E. faecium GM75 strain which carries the insertion sequence IS256, involved in adaptative and evolution processes of bacteria related to Staphylococcus and Enterococcus genera. In this work we describe the differential behavior in citrate transport, metabolism and aroma generation of three strains and we present results that link citrate metabolism and genetic organizations in E. faecium for the first time

SNP diversity of Enterococcus faecalis and Enterococcus faecium in a South East Queensland waterway, Australia, and associated antibiotic resistance gene profiles

PubMed Central

2011-01-01

Background Enterococcus faecalis and Enterococcus faecium are associated with faecal pollution of water, linked to swimmer-associated gastroenteritis and demonstrate a wide range of antibiotic resistance. The Coomera River is a main water source for the Pimpama-Coomera watershed and is located in South East Queensland, Australia, which is used intensively for agriculture and recreational purposes. This study investigated the diversity of E. faecalis and E. faecium using Single Nucleotide Polymorphisms (SNPs) and associated antibiotic resistance profiles. Results Total enterococcal counts (cfu/ml) for three/six sampling sites were above the United States Environmental Protection Agency (USEPA) recommended level during rainfall periods and fall into categories B and C of the Australian National Health and Medical Research Council (NHMRC) guidelines (with a 1-10% gastrointestinal illness risk). E. faecalis and E. faecium isolates were grouped into 29 and 23 SNP profiles (validated by MLST analysis) respectively. This study showed the high diversity of E. faecalis and E. faecium over a period of two years and both human-related and human-specific SNP profiles were identified. 81.8% of E. faecalis and 70.21% of E. faecium SNP profiles were associated with genotypic and phenotypic antibiotic resistance. Gentamicin resistance was higher in E. faecalis (47% resistant) and harboured the aac(6')-aph(2') gene. Ciprofloxacin resistance was more common in E. faecium (12.7% resistant) and gyrA gene mutations were detected in these isolates. Tetracycline resistance was less common in both species while tet(L) and tet(M) genes were more prevalent. Ampicillin resistance was only found in E. faecium isolates with mutations in the pbp5 gene. Vancomycin resistance was not detected in any of the isolates. We found that antibiotic resistance profiles further sub-divided the SNP profiles of both E. faecalis and E. faecium. Conclusions The distribution of E. faecalis and E. faecium

Emergence of a daptomycin-non-susceptible Enterococcus faecium strain that encodes mutations in DNA repair genes after high-dose daptomycin therapy.

PubMed

Matono, Takashi; Hayakawa, Kayoko; Hirai, Risen; Tanimura, Akira; Yamamoto, Kei; Fujiya, Yoshihiro; Mawatari, Momoko; Kutsuna, Satoshi; Takeshita, Nozomi; Mezaki, Kazuhisa; Ohmagari, Norio; Miyoshi-Akiyama, Tohru

2016-04-01

An increasing number of reports have documented the emergence of daptomycin-nonsusceptible Enterococcus in patients during daptomycin therapy. Even though several mechanisms for daptomycin-nonsusceptibility have been suggested, the potential genetic mutations which might contribute to the daptomycin-nonsusceptibility are not fully understood. We isolated a vancomycin-susceptible, daptomycin nonsusceptible Enterococcus faecium strain from a patient with acute lymphocytic leukemia who received high-dose daptomycin therapy for E. faecium endocarditis. Whole-genome sequencing analysis revealed mutations within genes encoding DNA repair proteins MutL and RecJ of the daptomycin-nonsusceptible Enterococcus strain which might have facilitated its emergence. We identified the mutations of DNA mismatch repair genes in a clinical isolate of daptomycin nonsusceptible E. faecium which emerged in spite of high-dose daptomycin therapy. The finding implicates the possible association of DNA repair mechanism and daptomycin resistance. Careful monitoring is necessary to avoid the emergence of daptomycin non-susceptible isolates of E. faecium and particularly in cases of long-term daptomycin use or in immunocompromised patients.

Isolation and characterization of large spectrum and multiple bacteriocin-producing Enterococcus faecium strain from raw bovine milk.

PubMed

Gaaloul, N; ben Braiek, O; Hani, K; Volski, A; Chikindas, M L; Ghrairi, T

2015-02-01

To assess the antimicrobial properties of lactic acid bacteria from Tunisian raw bovine milk. A bacteriocin-producing Enterococcus faecium strain was isolated from raw cow milk with activity against Gram-positive and Gram-negative bacteria. Antimicrobial substances produced by this strain were sensitive to proteolytic enzymes and were thermostable and resistant to a broad range of pH (2-10). Mode of action of antimicrobial substances was determined as bactericidal. Maximum activity was reached at the end of the exponential growth phase when checked against Listeria ivanovii BUG 496 (2366.62 AU ml(-1)). However, maximum antimicrobial activity against Pseudomonas aeruginosa 28753 was recorded at the beginning of the exponential growth phase. Enterococcus faecium GGN7 was characterized as free from virulence factors and was susceptible to tested antibiotics. PCR analysis of the micro-organism's genome revealed the presence of genes coding for enterocins A and B. Mass spectrometry analysis of RP-HPLC active fractions showed molecular masses corresponding to enterocins A (4835.77 Da) and B (5471.56 Da), and a peptide with a molecular mass of 3215.5 Da active only against Gram-negative indicator strains. The latter was unique in the databases. Enterococcus faecium GGN7 produces three bacteriocins with different inhibitory spectra. Based on its antimicrobial properties and safety, Ent. faecium GGN7 is potentially useful for food biopreservation. The results suggest the bacteriocins from GGN7 strain could be useful for food biopreservation. © 2014 The Society for Applied Microbiology.

Identification of a new peptide deformylase gene from enterococcus faecium and establishment of a new screening model targeted on PDF for novel antibiotics.

PubMed

Tang, Xian-Bing; Si, Shu-Yi; Zhang, Yue-Qin

2004-09-01

To identify a new peptide deformylase (PDF) gene (Genebank Accession AY238515) from Enterococcus faecium and to establish a new screening model targeted on PDF. A new PDF gene was identified by BLAST analysis and PCR and was subsequently over-expressed in the prokaryotic expression host E. coli B121(DE3). Over-expressed protein was purified for enzymatic assay by metal affinity chromatography and a new screening model was established for novel antibiotics. A new PDF gene of Enterococcus faecium was identified successfully. Ten positive samples were picked up from 8000 compound library and the microbial fermentation broth samples. A new PDF of gene Enterococcus faecium was first identified and the model had a high efficacy. Positive samples screened may be antibacterial agents of broad spectrum.

Effect of Enterococcus faecium SF68 on serum cobalamin and folate concentrations in healthy dogs.

PubMed

Lucena, R; Olmedilla, A B; Blanco, B; Novales, M; Ginel, P J

2018-04-17

To study the effect of a 14-day administration of the probiotic Enterococcus faecium SF68 on serum concentrations of cobalamin and folate in healthy dogs. Thirty-six healthy dogs were randomly allocated between probiotic and control groups. Enterococcus faecium SF68 was administered to the probiotic group for 14 days whereas the control group did not receive any product. A blood sample was taken from all dogs when starting the administration (day 1), when the administration ended (day 14) and 14 days later (day 28). Serum cobalamin and folate concentrations and the canine inflammatory bowel disease activity index scores were determined at each time point. There was a progressive reduction of mean serum cobalamin in the probiotic group during the 28-day study, with significantly lower concentration at day 28 compared to baseline and day 14 concentrations. Moderate hypocobalaminaemia was observed in eight dogs at day 28. Probiotic administration was associated with a non-significant increase in mean serum folate concentration at day 14, and a significant decrease at day 28 compared with day 1. The canine inflammatory bowel disease activity index score remained unaltered during the study. Short-term Enterococcus faecium SF68 administration caused a significant reduction of mean cobalamin concentration and moderate hypocobolaminaemia in eight of 18 dogs. Monitoring serum folate appears unnecessary because the probiotic caused a non-significant increase that returned to baseline values after administration was discontinued. © 2018 British Small Animal Veterinary Association.

Recent Recombination Events in the Core Genome Are Associated with Adaptive Evolution in Enterococcus faecium

PubMed Central

de Been, Mark; van Schaik, Willem; Cheng, Lu; Corander, Jukka; Willems, Rob J.

2013-01-01

Reasons for the rising clinical impact of the bacterium Enterococcus faecium include the species’ rapid acquisition of adaptive genetic elements. Here, we focused on the impact of recombination on the evolution of E. faecium. We used the recently developed BratNextGen algorithm to detect recombinant regions in the core genome of 34 E. faecium strains, including three newly sequenced clinical strains. Recombination was found to have a significant impact on the E. faecium genome: of the original 1.2 million positions in the core genome, 0.5 million were predicted to have been affected by recombination in at least one strain. Importantly, strains in one of the two major E. faecium clades (clade B), which contains most of the E. faecium human gut commensals, formed the most important reservoir for donating foreign DNA to the second major E. faecium clade (clade A), which contains most of the clinical isolates. Also, several genomic regions were found to mainly recombine in specific hospital-associated E. faecium strains. One of these regions (the epa-like locus) likely encodes the biosynthesis of cell wall polysaccharides. These findings suggest a crucial role for recombination in the emergence of E. faecium as a successful hospital-associated pathogen. PMID:23882129

Beneficial effects of probiotic cholesterol-lowering strain of Enterococcus faecium WEFA23 from infants on diet-induced metabolic syndrome in rats.

PubMed

Zhang, Fen; Qiu, Liang; Xu, Xiongpeng; Liu, Zhengqi; Zhan, Hui; Tao, Xueying; Shah, Nagendra P; Wei, Hua

2017-03-01

The aim of this study was to select probiotic Enterococcus strains that have the potential to improve metabolic syndrome (MS). Ten Enterococcus strains isolated from healthy infants were evaluated for their probiotic properties in vitro, and Enterococcus faecium WEFA23 was selected due to its cholesterol removal ability (1.89 ± 0.07 mg/10 10 cfu), highest glycodeoxycholic acid-hydrolase activity (1.86 ± 0.01 U/mg), and strong adhesion capacity to Caco-2 cells (17.90 ± 0.19%). The safety of E. faecium WEFA23 was verified by acute oral administration in mice, and it was found to have no adverse effects on general health status, bacterial translocation, and gut mucosal histology. Moreover, the beneficial effects of E. faecium WEFA23 on high-fat diet-induced MS in rats were investigated, and we found WEFA23 significantly decreased body weight, serum lipid levels (total cholesterol, triacylglycerols, and low-density lipoprotein cholesterol), blood glucose level, and insulin resistance in rats fed with a high-fat diet. This indicated that administration of E. faecium WEFA23 improved almost all key markers of MS, including obesity, hyperlipidemia, hyperglycemia, and insulin resistance. Our results supported E. faecium WEFA23 as a candidate for cholesterol-lowering dairy products and improvement of MS. Our research provided novel insights on Enterococcus as a strategy to combat MS. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Enterococcus faecium ST17 from Coastal Marine Sediment Carrying Transferable Multidrug Resistance Plasmids.

PubMed

Morroni, Gianluca; Di Cesare, Andrea; Di Sante, Laura; Brenciani, Andrea; Vignaroli, Carla; Pasquaroli, Sonia; Giovanetti, Eleonora; Sabatino, Raffaella; Rossi, Luigia; Magnani, Mauro; Biavasco, Francesca

2016-10-01

The multidrug-resistant Enterococcus faecium 17i48, sequence type 17, from marine sediment, carrying erm(B), tet(M), and tet(L) genes, was analyzed for the presence of antibiotic resistance plasmids and for the ability to transfer resistance genes. The strain was found to harbor the replicon type (repA) of pRE25, pRUM, pHTβ, and the axe-txe toxin-antitoxin (TA) system. In mating experiments, tet(M) and tet(L) were cotransferred with the repA pRE25 , whereas erm(B) was consistently cotransferred with the axe-txe and repA pRUM , suggesting that tetracycline and erythromycin resistance genes were carried on different elements both transferable by conjugation, likely via pHTβ-mediated mobilization. Hybridization and PCR mapping demonstrated that tet(M) and tet(L) were located in tandem on a pDO1-like plasmid that also carried the repA pRE25 , whereas erm(B) was carried by a pRUM-like plasmid. Sequencing of the latter plasmid showed a high nucleotide identity with pRUM and the presence of cat, aadE, sat4, and a complete aphA resistance genes. These findings show that the genetic features of E. faecium 17i48 are consistent with a hospital-adapted clone and suggest that antibiotic resistance may spread in the environment, also in the absence of antibiotic pressure, due to TA system plasmid maintenance.

Prolonged linezolid use is associated with the development of linezolid-resistant Enterococcus faecium.

PubMed

Smith, Tiffeny T; Tamma, Pranita D; Do, Tiffany B; Dzintars, Kathryn E; Zhao, Yuan; Cosgrove, Sara E; Avdic, Edina

2018-06-01

We assessed risk factors for and outcomes of linezolid-resistant vancomycin-resistant Enterococcus faecium (LRVREF) bacteremia over 7 years. Thirty-four LRVREF cases were matched to 68 linezolid-susceptible VREF controls. The odds of bacteremia with LRVREF increased by 7% for each additional day of prior linezolid exposure. Copyright © 2018 Elsevier Inc. All rights reserved.

Characteristic of Enterococcus faecium clinical isolates with quinupristin/dalfopristin resistance in China.

PubMed

Wang, Shanshan; Guo, Yinjuan; Lv, Jingnan; Qi, Xiuqin; Li, Dan; Chen, Zengqiang; Zhang, Xueqing; Wang, Liangxing; Yu, Fangyou

2016-10-21

Quinupristin/dalfopristin (Q/D) is a valuable alternative antibiotic to vancomycin for the treatment of multi-drug resistant Enterococcus faecium infections. However, resistance to Q/D in E. faecium clinical isolates and nosocomial dissemination of Q/D-resistant E. faecium have been reported in several countries and should be of concern. From January 2012 to December 2015, 911 E. faecium clinical isolates were isolated from various specimens of inpatients at the first Affiliated Hospital of Wenzhou Medical University located in Wenzhou, east China. Of 911 E. faecium clinical isolates, 9 (1.0 %, 9/911) were resistant to Q/D, with the Q/D MIC values of 64 mg/L(1), 32 mg/L(1), 16 mg/L(3), 8 mg/L(1) and 4 mg/L(3) determined by broth microdilution. All Q/D-resistant isolates were susceptible to vancomycin, tigecycline and teicoplanin but resistant to penicillin, ampicillin and erythromycin. vatE was only found in one Q/D-resistant E. faecium isolate while vatD was not detected in any of the isolates tested. 8 of 9 Q/D-resistant E. faecium isolates were found be positive for both ermB and msrC. The combinations of Q/D resistance determinants were ermB-msrC (7 isolates) and ermB-msrC-vatE (one isolate). ST78, ST761, ST94, ST21 and ST323 accounted for 4, 2, 1, 1 and 1 isolate, respectively, among which ST78 was the prevalent ST. Q/D-resistant E. faecium clinical isolates were first described in China. Carriage of vatE, ermB and msrC was responsible for Q/D resistance.

Requirement of the CroRS Two-Component System for Resistance to Cell Wall-Targeting Antimicrobials in Enterococcus faecium.

PubMed

Kellogg, Stephanie L; Little, Jaime L; Hoff, Jessica S; Kristich, Christopher J

2017-05-01

Enterococci are serious opportunistic pathogens that are resistant to many cell wall-targeting antibiotics. The CroRS two-component signaling system responds to antibiotic-mediated cell wall stress and is critical for resistance to cell wall-targeting antibiotics in Enterococcus faecalis Here, we identify and characterize an orthologous two-component system found in Enterococcus faecium that is functionally equivalent to the CroRS system of E. faecalis Deletion of croRS in E. faecium resulted in marked susceptibility to cell wall-targeting agents including cephalosporins and bacitracin, as well as moderate susceptibility to ampicillin and vancomycin. As in E. faecalis , exposure to bacitracin and vancomycin stimulates signaling through the CroRS system in E. faecium Moreover, the CroRS system is critical in E. faecium for enhanced beta-lactam resistance mediated by overexpression of Pbp5. Expression of a Pbp5 variant that confers enhanced beta-lactam resistance cannot overcome the requirement for CroRS function. Thus, the CroRS system is a conserved signaling system that responds to cell wall stress to promote intrinsic resistance to important cell wall-targeting antibiotics in clinically relevant enterococci. Copyright © 2017 American Society for Microbiology.

Requirement of the CroRS Two-Component System for Resistance to Cell Wall-Targeting Antimicrobials in Enterococcus faecium

PubMed Central

Kellogg, Stephanie L.; Little, Jaime L.; Hoff, Jessica S.

2017-01-01

ABSTRACT Enterococci are serious opportunistic pathogens that are resistant to many cell wall-targeting antibiotics. The CroRS two-component signaling system responds to antibiotic-mediated cell wall stress and is critical for resistance to cell wall-targeting antibiotics in Enterococcus faecalis. Here, we identify and characterize an orthologous two-component system found in Enterococcus faecium that is functionally equivalent to the CroRS system of E. faecalis. Deletion of croRS in E. faecium resulted in marked susceptibility to cell wall-targeting agents including cephalosporins and bacitracin, as well as moderate susceptibility to ampicillin and vancomycin. As in E. faecalis, exposure to bacitracin and vancomycin stimulates signaling through the CroRS system in E. faecium. Moreover, the CroRS system is critical in E. faecium for enhanced beta-lactam resistance mediated by overexpression of Pbp5. Expression of a Pbp5 variant that confers enhanced beta-lactam resistance cannot overcome the requirement for CroRS function. Thus, the CroRS system is a conserved signaling system that responds to cell wall stress to promote intrinsic resistance to important cell wall-targeting antibiotics in clinically relevant enterococci. PMID:28223383

The first report of the vanC₁ gene in Enterococcus faecium isolated from a human clinical specimen.

PubMed

Sun, Mingyue; Wang, Yue; Chen, Zhongju; Zhu, Xuhui; Tian, Lei; Sun, Ziyong

2014-09-01

The vanC₁ gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC₁gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC₁ and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC₁ gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC₁gene. However, this study is the first to report the presence of the vanC₁gene in E. faecium of human origin. Additionally, our research showed the vanC₁gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC₁gene from different species.

A polyclonal outbreak of bloodstream infections by Enterococcus faecium in patients with hematologic malignancies.

PubMed

Alatorre-Fernández, Pamela; Mayoral-Terán, Claudia; Velázquez-Acosta, Consuelo; Franco-Rodríguez, Cecilia; Flores-Moreno, Karen; Cevallos, Miguel Ángel; López-Vidal, Yolanda; Volkow-Fernández, Patricia

2017-03-01

Enterococcus faecium causes bloodstream infection (BSI) in patients with hematologic malignancies (HMs). We studied the clinical features and outcomes of patients with HM with vancomycin-sensitive E faecium (VSE) and vancomycin-resistant E faecium (VRE) BSI and determined the genetic relatedness of isolates and circumstances associated with the upsurge of E faecium BSI. Case-control study of patients with HM and E faecium-positive blood culture from January 2008-December 2012; cases were patients with VRE and controls were VSE isolates. The strains were tested for Van genes by polymerase chain reaction amplification and we performed pulsed-field gel electrophoresis to determine genetic relatedness. Fifty-eight episodes of E faecium BSI occurred: 35 sensitive and 23 resistant to vancomycin. Mortality was 46% and 57%, attributable 17% and 40%, respectively. Early stage HM was associated with VSE (P = .044), whereas an episode of BSI within the 3 months before the event (P = .039), prophylactic antibiotics (P = .013), and vancomycin therapy during the previous 3 months (P = .001) was associated with VRE. The VanA gene was identified in 97% of isolates studied. E faecium isolates were not clonal. E faecium BSI was associated with high mortality. This outbreak of VRE was not clonal; it was associated with antibiotic-use pressure and highly myelosuppressive chemotherapy. Copyright © 2017 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Characterization of functional properties of Enterococcus faecium strains isolated from human gut.

PubMed

İspirli, Hümeyra; Demirbaş, Fatmanur; Dertli, Enes

2015-11-01

The aim of this work was to characterize the functional properties of Enterococcus faecium strains identified after isolation from human faeces. Of these isolates, strain R13 showed the best resistance to low pH, bile salts, and survival in the simulated in vitro digestion assay, and demonstrated an important level of adhesion to hexadecane as a potential probiotic candidate. Analysis of the antibiotic resistance of E. faecium strains indicated that in general these isolates were sensitive to the tested antibiotics and no strain appeared to be resistant to vancomycin. Examination of the virulence determinants for E. faecium strains demonstrated that all strains contained the virulence genes common in gut- and food-originated enterococci, and strain R13 harboured the lowest number of virulence genes. Additionally, no strain contained the genes related to cytolysin metabolism and showed hemolytic activity. The antimicrobial role of E. faecium strains was tested against several pathogens, in which different levels of inhibitory effects were observed, and strain R13 was inhibitory to all tested pathogens. PCR screening of genes encoding enterocin A and B indicated the presence of these genes in E. faecium strains. Preliminary characterization of bacteriocins revealed that their activity was lost after proteolytic enzyme treatments, but no alteration in antimicrobial activity was observed at different pHs (3.5 to 9.5) and after heat treatments. In conclusion, this study revealed the functional characteristics of E. faecium R13 as a gut isolate, and this strain could be developed as a new probiotic after further tests.

Lipoteichoic acid synthesis inhibition in combination with antibiotics abrogates growth of multidrug-resistant Enterococcus faecium.

PubMed

Paganelli, Fernanda L; van de Kamer, Tim; Brouwer, Ellen C; Leavis, Helen L; Woodford, Neil; Bonten, Marc J M; Willems, Rob J L; Hendrickx, Antoni P A

2017-03-01

Enterococcus faecium is a multidrug-resistant (MDR) nosocomial pathogen causing significant morbidity in debilitated patients. New antimicrobials are needed to treat antibiotic-resistant E. faecium infections in hospitalised patients. E. faecium incorporates lipoteichoic acid (LTA) (1,3-polyglycerol-phosphate linked to glycolipid) in its cell wall. The small-molecule inhibitor 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] specifically blocks the activity of Staphylococcus aureus LtaS synthase, which polymerises 1,3-glycerolphosphate into LTA polymers. Here we characterised the effects of the small-molecule inhibitor 1771 on the growth of E. faecium isolates, alone (28 strains) or in combination with the antibiotics vancomycin, daptomycin, ampicillin, gentamicin or linezolid (15 strains), and on biofilm formation (16 strains). Inhibition of LTA synthesis at the surface of the cell by compound 1771 in combination with current antibiotic therapy abrogates enterococcal growth in vitro but does not affect mature E. faecium biofilms. Targeting LTA synthesis may provide new possibilities to treat MDR E. faecium infections. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

High-level penicillin resistance and penicillin-gentamicin synergy in Enterococcus faecium.

PubMed Central

Torres, C; Tenorio, C; Lantero, M; Gastañares, M J; Baquero, F

1993-01-01

Thirty-seven Enterococcus faecium strains with different levels of penicillin susceptibility were studied in time-kill experiments with a fixed concentration (5 micrograms/ml) of gentamicin combined with different penicillin concentrations (6 to 600 micrograms/ml). Synergy was defined as a relative decrease in counts of greater than 2 log10 CFU per milliliter after 24 h of incubation when the combination of the antibiotics was compared with its most active component alone. The minimal synergistic penicillin concentrations found were 6 micrograms/ml for 16 of 16 strains for which penicillin MICs were < or = 25 micrograms/ml, 20 to 100 micrograms/ml for 14 of 17 strains for which penicillin MICs were 50 to 200 micrograms/ml, and 200 to 500 micrograms/ml for 4 of 4 strains for which MICs penicillin were > 200 micrograms/ml. Penicillin-gentamicin synergy was observed even in high-level penicillin-resistant E. faecium strains at penicillin concentrations close to one-half the penicillin MIC. The possibility of treating infections caused by high-level penicillin-resistant E. faecium strains with penicillin-gentamicin combinations in particular cases may depend on the penicillin levels attainable in vivo. PMID:8285628

Antibiotic resistance patterns and genetic relatedness of Enterococcus faecalis and Enterococcus faecium isolated from military working dogs in Korea.

PubMed

Bang, Kiman; An, Jae-Uk; Kim, Woohyun; Dong, Hee-Jin; Kim, Junhyung; Cho, Seongbeom

2017-06-30

Enterococcus spp. are normally present in the gastrointestinal tracts of animals and humans, but can cause opportunistic infections that can be transmitted to other animals or humans with integrated antibiotic resistance. To investigate if this is a potential risk in military working dogs (MWDs), we analyzed antibiotic resistance patterns and genetic relatedness of Enterococcus spp. isolated from fecal samples of MWDs of four different age groups. Isolation rates of Enterococcus spp., Enterococcus ( E. ) faecalis , and E. faecium , were 87.7% (57/65), 59.6% (34/57), and 56.1% (32/57), respectively, as determined by bacterial culture and multiplex PCR. The isolation rate of E. faecalis gradually decreased with age (puppy, 100%; adolescent, 91.7%; adult, 36.4%; and senior, 14.3%). Rates of resistance to the antibiotics ciprofloxacin, gentamicin, streptomycin, sulfamethoxazole/trimethoprim, imipenem, and kanamycin among Enterococcus spp. increased in adolescents and adults and decreased in senior dogs, with some isolates having three different antibiotic resistance patterns. There were indistinguishable pulsed-field gel electrophoresis patterns among the age groups. The results suggest that Enterococcus is horizontally transferred, regardless of age. As such, periodic surveillance studies should be undertaken to monitor changes in antibiotic resistance, which may necessitate modification of antibiotic regimens to manage antibiotic resistance transmission.

Effects of the probiotic Enterococcus faecium NCIMB 10415 on selected lactic acid bacteria and enterobacteria in co-culture.

PubMed

Starke, I C; Zentek, J; Vahjen, W

2015-01-01

Enterococcus faecium NCIMB 10415 is used as a probiotic for piglets and has been shown to modify the porcine intestinal microbiota. However, the mode of action of this probiotic modification is still unclear. One possible explanation is the direct growth inhibiting or stimulating effect of the probiotic on other indigenous bacteria. Therefore, the aim of the present study was to examine the growth interactions of the probiotic with different indigenous porcine bacteria in vitro. Reference strains were cultivated with the probiotic E. faecium strain NCIMB10415 (SF68) in a checkerboard assay with 102 to 105 cells/ml inoculum per strain. Growth kinetics were recorded for 8 h and used to determine specific growth of the co-cultures. Additionally, total DNA was extracted from the co-cultures at the end of the incubation to verify which strain in the co-culture was affected. Co-cultivation with eight Enterococcus spp. tester strains showed strain-specific growth differences. Three of four E. faecium strains were not influenced by the probiotic strain. PCR results showed reduced growth of the probiotic strain in co-culture with E. faecium DSM 6177. Three of four Enterococcus faecalis strains showed reduced specific growth in co-culture with the probiotic strain. However, E. faecalis DSM 20478 impaired growth of the probiotic E. faecium strain. The growth of Lactobacillus johnsonii DSM 10533 and Lactobacillus reuteri DSM 20016 was enhanced in co-culture with the probiotic strain, but co-cultivations with Lactobacillus mucosae DSM13345 or Lactobacillus amylovorus DSM10533 showed no differences. Co-cultures with the probiotic E. faecium showed no impact on the growth rate of four different enterobacterial reference strains (2 strains of Salmonella enterica and 2 strains of Escherichia coli), but PCR results showed reduced cell numbers for a pathogenic E. coli isolate at higher concentration of the probiotic strain. As the in vitro effect of the probiotic E. faecium on

Inhibitory Effect of Enterococcus faecium WB2000 on Volatile Sulfur Compound Production by Porphyromonas gingivalis

PubMed Central

Higuchi, Takuya; Nakajima, Masato; Fujimoto, Akie; Hanioka, Takashi; Hirofuji, Takao

2016-01-01

Volatile sulfur compounds (VSCs) produced by oral anaerobes are the major compounds responsible for oral malodor. Enterococcus faecium WB2000 is recognized as an antiplaque probiotic bacterium. In this study, the effect of E. faecium WB2000 on VSC production by Porphyromonas gingivalis was evaluated, and the mechanism of inhibition of oral malodor was investigated. P. gingivalis ATCC 33277 was cultured in the presence of four lactic acid bacteria, including E. faecium WB2000. Subsequently, P. gingivalis ATCC 33277, W50, W83, and two clinical isolates were cultured in the presence or absence of E. faecium WB2000, and the emission of VSCs from spent culture medium was measured by gas chromatography. The number of P. gingivalis ATCC 33277 in mixed culture with E. faecium WB2000 decreased at 6 h, and the rate of decrease was higher than that in mixed cultures with the other lactic acid bacteria. The numbers of five P. gingivalis strains decreased at similar rates in mixed culture with E. faecium WB2000. The concentration of methyl mercaptan was lower in spent culture medium from P. gingivalis and E. faecium WB2000 cultures compared with that from P. gingivalis alone. Therefore, E. faecium WB2000 may reduce oral malodor by inhibiting the growth of P. gingivalis and neutralizing methyl mercaptan. PMID:27799940

[BIOLOGICAL ACTIVITY OF ANTIMICROBIAL PEPTIDES OF ENTEROCOCCUS FAECIUM].

PubMed

Vasilchenko, A S; Rogozhin, E A; Valyshev, A V

2015-01-01

Isolate bacteriocins from Enterococcus faecium metabolites and characterize their effect on cells of Gram positive (Listeria monocytogenes) and Gram negative (Escherichia coli) bacteria. Methods of solid-phase extraction, ion-exchange and reversed phase chromatography were applied for isolation of bacteriocins from cultural medium of bacteria MALDI time-of-flight mass-spectrometry was used for characterization of the obtained preparations. The mechanism of biological effect of peptides was evaluated using DNA-tropic dyes (SYTO 9 and PI) with subsequent registration of fluorescence spectra: Atomic-force microscopy (AFM) was used for characterization of morpho-functional reaction of target cells. Peptide fractions with mass of 1.0 - 3.0 kDa were isolated from enterococci metabolites, that inhibit the growth of indicator microorganisms. E. faecium strain exoproducts were shown to increase membrane permeability during interaction with L. monocytogenes, that results in subsequent detectable disturbance of normal cell morphology of listeria. Alterations of E. coli surface during the effect of purified peptide fraction was detected using AFM. The studies carried out have revealed the effect of bacteriocins of enterococci on microorganisms with various types of cell wall composition and have confirmed the importance of bacterial barrier structure permeability disturbance in the mechanism of antimicrobial effect of enterocins.

Assessment of virulence factors, antibiotic resistance and amino-decarboxylase activity in Enterococcus faecium MXVK29 isolated from Mexican chorizo.

PubMed

Alvarez-Cisneros, Y M; Fernández, F J; Sainz-Espuñez, T; Ponce-Alquicira, E

2017-02-01

Enterococcus faecium MXVK29 has the ability to produce an antimicrobial compound that belongs to Class IIa of the Klaenhammer classification, and could be used as part of a biopreservation technology through direct inoculation of the strain as a starter or protective culture. However, Enterococcus is considered as an opportunistic pathogen, hence, the purpose of this work was to study the food safety determinants of E. faecium MXVK29. The strain was sensitive to all of the antibiotics tested (penicillin, tetracycline, vancomycin, erythromycin, chloramphenicol, gentamicin, neomycin, kanamycin and netilmicin) and did not demonstrate histamine, cadaverine or putrescine formation. Furthermore, tyrosine-decarboxylase activity was detected by qualitative assays and PCR. Among the virulence factors analysed for the strain, only the genes encoding the sexual pheromone cCF10 precursor lipoprotein (ccf) and cell-wall adhesion (efaA fm ) were amplified. The presence of these genes has low impact on pathogenesis, as there are no other genes encoding for virulence factors, such as aggregation proteins. Therefore, Enterococcus faecium could be employed as part of a bioconservation method, because it does not produce risk factors for consumer's health; in addition, it could be used as part of the hurdle technology in foods. The use of molecular techniques has allowed, in recent years, to detect pathogenicity genes present in the genome of starter cultures used in food processing and preservation. The presence of these genes is undesirable, because horizontal transfer may occur with the natural biota of consumers. For this reason, it is important to analyse the presence of pathogenicity genes in such cultures. In this work, virulence factors and antibiotic resistance of Enterococcus faecium strain MXVK29, producing an antimicrobial compound with high antilisterial activity, were analysed. The results indicate that the strain is safe to be used in food processing as starter

Draft Genome Sequences of Four Enterococcus faecium Strains Isolated from Argentine Cheese

PubMed Central

Martino, Gabriela P.; Quintana, Ingrid M.; Espariz, Martín; Blancato, Victor S.; Gallina Nizo, Gabriel; Esteban, Luis

2016-01-01

We report the draft genome sequences of four Enterococcus faecium strains isolated from Argentine regional cheeses. These strains were selected based on their technological properties, i.e., their ability to produce aroma compounds (diacetyl, acetoin, and 2,3-butanediol) from citrate. The goal of our study is to provide further genetic evidence for the rational selection of enterococci strains based on their pheno- and genotype in order to be used in cheese production. PMID:26847907

Investigating the mobilome in clinically important lineages of Enterococcus faecium and Enterococcus faecalis.

PubMed

Mikalsen, Theresa; Pedersen, Torunn; Willems, Rob; Coque, Teresa M; Werner, Guido; Sadowy, Ewa; van Schaik, Willem; Jensen, Lars Bogø; Sundsfjord, Arnfinn; Hegstad, Kristin

2015-04-10

The success of Enterococcus faecium and E. faecalis evolving as multi-resistant nosocomial pathogens is associated with their ability to acquire and share adaptive traits, including antimicrobial resistance genes encoded by mobile genetic elements (MGEs). Here, we investigate this mobilome in successful hospital associated genetic lineages, E. faecium sequence type (ST)17 (n=10) and ST78 (n=10), E. faecalis ST6 (n=10) and ST40 (n=10) by DNA microarray analyses. The hybridization patterns of 272 representative targets including plasmid backbones (n=85), transposable elements (n=85), resistance determinants (n=67), prophages (n=29) and clustered regularly interspaced short palindromic repeats (CRISPR)-cas sequences (n=6) separated the strains according to species, and for E. faecalis also according to STs. RCR-, Rep_3-, RepA_N- and Inc18-family plasmids were highly prevalent and with the exception of Rep_3, evenly distributed between the species. There was a considerable difference in the replicon profile, with rep 17/pRUM , rep 2/pRE25 , rep 14/EFNP1 and rep 20/pLG1 dominating in E. faecium and rep 9/pCF10 , rep 2/pRE25 and rep 7 in E. faecalis strains. We observed an overall high correlation between the presence and absence of genes coding for resistance towards antibiotics, metals, biocides and their corresponding MGEs as well as their phenotypic antimicrobial susceptibility pattern. Although most IS families were represented in both E. faecalis and E. faecium, specific IS elements within these families were distributed in only one species. The prevalence of IS256-, IS3-, ISL3-, IS200/IS605-, IS110-, IS982- and IS4-transposases was significantly higher in E. faecium than E. faecalis, and that of IS110-, IS982- and IS1182-transposases in E. faecalis ST6 compared to ST40. Notably, the transposases of IS981, ISEfm1 and IS1678 that have only been reported in few enterococcal isolates were well represented in the E. faecium strains. E. faecalis ST40 strains harboured

Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation

PubMed Central

Paganelli, F. L.; de Been, M.; Braat, J. C.; Hoogenboezem, T.; Vink, C.; Bayjanov, J.; Rogers, M. R. C.; Huebner, J.; Bonten, M. J. M.; Willems, R. J. L.

2015-01-01

Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms. PMID:26209668

Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation.

PubMed

Paganelli, F L; de Been, M; Braat, J C; Hoogenboezem, T; Vink, C; Bayjanov, J; Rogers, M R C; Huebner, J; Bonten, M J M; Willems, R J L; Leavis, H L

2015-10-01

Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Production of enterocin A by Enterococcus faecium MMRA isolated from 'Rayeb', a traditional Tunisian dairy beverage.

PubMed

Rehaiem, A; Martínez, B; Manai, M; Rodríguez, A

2010-05-01

Characterization and purification of a bacteriocin produced by a wild Enterococcus faecium strain, isolated from a Tunisian traditional fermented milk. Enterococcus faecium MMRA was selected on the basis of its strong anti-Listeria activity. The antibacterial activity was sensitive to proteases, confirming its proteinaceous nature. It was extremely heat stable (15 min at 121 degrees C), remained active over a wide pH range (2-12), and also after treatment with lipase, amylase, organic solvents, detergents, lyophilisation and long-term storage at -20 degrees C. Production of the bacteriocin occurred throughout the logarithmic growth phase, it did not adhere to the surface of the producer cells and the mode of action was bactericidal. After partial purification of the active supernatants, a 4-kDa band with antibacterial activity was revealed by SDS-PAGE electrophoresis and bioassay. Tryptic digestion followed by MALDI-TOF mass spectrometry identified the peptide as enterocin A. The inhibitory activity of Ent. faecium MMRA, a wild strain isolated from the artisan dairy beverage 'Rayeb', is due to the synthesis of an enterocin A. Traditional fresh Tunisian fermented dairy products are generally manufactured with raw milk that can be used as a source of uncharacterized wild lactic acid bacteria strains. To our knowledge, this is the first report on the isolation of an enterocin A producing Ent. faecium from 'Rayeb'. This bacteriocin or the producing strain might have a promising potential in biopreservation to enhance the hygienic quality of this dairy product.

Genome Sequence of Enterococcus faecium Strain ICIS 96 Demonstrating Intermicrobial Antagonism Associated with Bacteriocin Production.

PubMed

Pashkova, Tatiana M; Vasilchenko, Alexey S; Khlopko, Yuriy A; Kochkina, Elena E; Kartashova, Olga L; Sycheva, Maria V

2018-03-08

We report here the complete genome sequence of Enterococcus faecium strain ICIS 96, which was isolated from the feces of a horse. Bacteriological characterization of strain ICIS 96 revealed the absence of pathogenicity factors, while its spectrum of antagonistic activity was found to be broad, having activities associated with both Gram-positive and Gram-negative bacteria. Analysis of the E. faecium ICIS 96 genome revealed five genes associated with antimicrobial activity (enterocin [ent] A, ent B, lactobin A/cerein 7b, and ent L50 A/B). No genes that correlate with human pathogenicity were identified. Copyright © 2018 Pashkova et al.

Genome Sequence of Enterococcus faecium Strain ICIS 96 Demonstrating Intermicrobial Antagonism Associated with Bacteriocin Production

PubMed Central

Pashkova, Tatiana M.; Vasilchenko, Alexey S.; Khlopko, Yuriy A.; Kochkina, Elena E.; Kartashova, Olga L.

2018-01-01

ABSTRACT We report here the complete genome sequence of Enterococcus faecium strain ICIS 96, which was isolated from the feces of a horse. Bacteriological characterization of strain ICIS 96 revealed the absence of pathogenicity factors, while its spectrum of antagonistic activity was found to be broad, having activities associated with both Gram-positive and Gram-negative bacteria. Analysis of the E. faecium ICIS 96 genome revealed five genes associated with antimicrobial activity (enterocin [ent] A, ent B, lactobin A/cerein 7b, and ent L50 A/B). No genes that correlate with human pathogenicity were identified. PMID:29519833

[Emergence of glycopeptide resistant Enterococcus faecium in Algeria: a case report].

PubMed

Hamidi, Moufida; Ammari, Houria; Ghaffor, Mohamed; Benamrouche, Nabila; Tali-Maamar, Hassiba; Tala-Khir, Farida; Younsi, Mokhtar; Rahal, Kheira

2013-01-01

A glycopeptide-resistant Enterococcus faecium (EFRG) was isolated from a wound in a patient hospitalized in a university hospital in Algiers. This strain was resistant to several antibiotics. This patient was carrying this strain in the digestive tract which may partly explain its origin. Genotypic comparison of the two strains by pulsed field gel electrophoresis showed that it was the same strain. Glycopeptide resistance was due to the presence of the vanA gene. Vigilance is required facing the emergence of strains of EFRG in our hospitals.

Molecular Epidemiology of Vancomycin-Resistant Enterococcus faecalis and Enterococcus faecium Isolated from Clinical Specimens in the Northwest of Iran.

PubMed

Jahansepas, Ali; Ahangarzadeh Rezaee, Mohammad; Hasani, Alka; Sharifi, Yaeghob; Rahnamaye Farzami, Marjan; Dolatyar, Alireza; Aghazadeh, Mohammad

2018-04-30

This study was conducted to investigate the phenotypic and genotypic characteristics of vancomycin-resistant Enterococcus faecalis and Enterococcus faecium. Antibiotic resistance and virulence genes in the aforementioned resistant isolates were studied using the epsilometer (E)-test and polymerase chain reaction (PCR). These isolates were subjected to typing by pulsed-field gel electrophoresis (PFGE). Thirty vancomycin-resistant enterococci (VRE; 18.75%) were isolated from a total of 160 various clinical specimens cultured for any bacterial growth. Of these, 11 (36.7%) isolates were identified as E. faecalis and 19 (63.3%) as E. faecium. Minimum inhibitory concentrations (MICs) of vancomycin, teicoplanin, and three alternative therapeutic options (linezolid, daptomycin, and quinupristin/dalfopristin) were determined using the E-test. Multiplex PCR was done for confirming species, identification of the resistant genotypes, and the detection of the virulence genes. Finally, the clonal relationship of all VRE strains was studied by PFGE. All VRE strains showed vancomycin MIC ≥256 μg/mL, and 27 (90%) isolates carried the vanA gene, whereas none of the isolates carried vanB. The most common resistance antibiotic pattern observed was toward rifampicin (n = 30 [100%]). Among all virulence genes studied, gelE (n = 28 [93.33%]) was found as the most prevalent virulent gene. VRE isolates exhibited 90%, 46.67%, 100%, and 66.67% resistance to teicoplanin, linezolid, quinupristin/dalfopristin, and daptomycin, respectively. Molecular typing demonstrated 16 PFGE types of VRE isolates (A-P). Although vanA was carried by most of the isolates, PFGE displayed small clonal dissemination among VR E. faecium and VR E. faecalis species.

The Two-Component System ChtRS Contributes to Chlorhexidine Tolerance in Enterococcus faecium.

PubMed

Guzmán Prieto, Ana M; Wijngaarden, Jessica; Braat, Johanna C; Rogers, Malbert R C; Majoor, Eline; Brouwer, Ellen C; Zhang, Xinglin; Bayjanov, Jumamurat R; Bonten, Marc J M; Willems, Rob J L; van Schaik, Willem

2017-05-01

Enterococcus faecium is one of the primary causes of nosocomial infections. Disinfectants are commonly used to prevent infections with multidrug-resistant E. faecium in hospitals. Worryingly, E. faecium strains that exhibit tolerance to disinfectants have already been described. We aimed to identify and characterize E. faecium genes that contribute to tolerance to the disinfectant chlorhexidine (CHX). We used a transposon mutant library, constructed in a multidrug-resistant E. faecium bloodstream isolate, to perform a genome-wide screen to identify genetic determinants involved in tolerance to CHX. We identified a putative two-component system (2CS), composed of a putative sensor histidine kinase (ChtS) and a cognate DNA-binding response regulator (ChtR), which contributed to CHX tolerance in E. faecium Targeted chtR and chtS deletion mutants exhibited compromised growth in the presence of CHX. Growth of the chtR and chtS mutants was also affected in the presence of the antibiotic bacitracin. The CHX- and bacitracin-tolerant phenotype of E. faecium E1162 was linked to a unique, nonsynonymous single nucleotide polymorphism in chtR Transmission electron microscopy showed that upon challenge with CHX, the Δ chtR and Δ chtS mutants failed to divide properly and formed long chains. Normal growth and cell morphology were restored when the mutations were complemented in trans Morphological abnormalities were also observed upon exposure of the Δ chtR and Δ chtS mutants to bacitracin. The tolerance to both chlorhexidine and bacitracin provided by ChtRS in E. faecium highlights the overlap between responses to disinfectants and antibiotics and the potential for the development of cross-tolerance for these classes of antimicrobials. Copyright © 2017 American Society for Microbiology.

Inhibition of Enterococcus faecium adherence to collagen by antibodies against high-affinity binding subdomains of Acm.

PubMed

Nallapareddy, Sreedhar R; Sillanpää, Jouko; Ganesh, Vannakambadi K; Höök, Magnus; Murray, Barbara E

2007-06-01

Strains of Enterococcus faecium express a cell wall-anchored protein, Acm, which mediates adherence to collagen. Here, we (i) identify the minimal and high-affinity binding subsegments of Acm and (ii) show that anti-Acm immunoglobulin Gs (IgGs) purified against these subsegments reduced E. faecium TX2535 strain collagen adherence up to 73 and 50%, respectively, significantly more than the total IgGs against the full-length Acm A domain (28%) (P < 0.0001). Blocking Acm adherence with functional subsegment-specific antibodies raises the possibility of their use as therapeutic or prophylactic agents.

pHTβ-promoted mobilization of non-conjugative resistance plasmids from Enterococcus faecium to Enterococcus faecalis.

PubMed

Di Sante, Laura; Morroni, Gianluca; Brenciani, Andrea; Vignaroli, Carla; Antonelli, Alberto; D'Andrea, Marco Maria; Di Cesare, Andrea; Giovanetti, Eleonora; Varaldo, Pietro E; Rossolini, Gian Maria; Biavasco, Francesca

2017-09-01

To analyse the recombination events associated with conjugal mobilization of two multiresistance plasmids, pRUM17i48 and pLAG (formerly named pDO1-like), from Enterococcus faecium 17i48 to Enterococcus faecalis JH2-2. The plasmids from two E. faecalis transconjugants (JH-4T, tetracycline resistant, and JH-8E, erythromycin resistant) and from the E. faecium donor (also carrying a pHTβ-like conjugative plasmid, named pHTβ17i48) were investigated by several methods, including PCR mapping and sequencing, S1-PFGE followed by Southern blotting and hybridization, and WGS. Two locations of repApHTβ were detected in both transconjugants, one on a ∼50 kb plasmid (as in the donor) and the other on plasmids of larger sizes. In JH-4T, WGS disclosed an 88.6 kb plasmid resulting from the recombination of pHTβ17i48 (∼50 kb) and a new plasmid, named pLAG (35.3 kb), carrying the tet(M), tet(L), lsa(E), lnu(B), spw and aadE resistance genes. In JH-8E, a 75 kb plasmid resulting from the recombination of pHTβ17i48 and pRUM17i48 was observed. In both cases, the cointegrates were apparently derived from replicative transposition of an IS1216 present in each of the multiresistance plasmids into pHTβ17i48. The cointegrates could resolve to yield the multiresistance plasmids and a pHTβ17i48 derivative carrying an IS1216 (unlike the pHTβ17i48 of the donor). Our results completed the characterization of the multiresistance plasmids carried by the E. faecium 17i48, confirming the role of pHT plasmids in the mobilization of non-conjugative antibiotic resistance elements among enterococci. Results also revealed that mobilization to E. faecalis was associated with the generation of cointegrate plasmids promoted by IS1216-mediated transposition. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Antibiotic-Driven Dysbiosis Mediates Intraluminal Agglutination and Alternative Segregation of Enterococcus faecium from the Intestinal Epithelium.

PubMed

Hendrickx, Antoni P A; Top, Janetta; Bayjanov, Jumamurat R; Kemperman, Hans; Rogers, Malbert R C; Paganelli, Fernanda L; Bonten, Marc J M; Willems, Rob J L

2015-11-10

The microbiota of the mammalian gastrointestinal tract is a complex ecosystem of bacterial communities that continuously interact with the mucosal immune system. In a healthy host, the mucosal immune system maintains homeostasis in the intestine and prevents invasion of pathogenic bacteria, a phenomenon termed colonization resistance. Antibiotics create dysbiosis of microbiota, thereby decreasing colonization resistance and facilitating infections caused by antibiotic-resistant bacteria. Here we describe how cephalosporin antibiotics create dysbiosis in the mouse large intestine, allowing intestinal outgrowth of antimicrobial-resistant Enterococcus faecium. This is accompanied by a reduction of the mucus-associated gut microbiota layer, colon wall, and Muc-2 mucus layer. E. faecium agglutinates intraluminally in an extracellular matrix consisting of secretory IgA (sIgA), polymeric immunoglobulin receptor (pIgR), and epithelial cadherin (E-cadherin) proteins, thereby maintaining spatial segregation of E. faecium from the intestinal wall. Addition of recombinant E-cadherin and pIgR proteins or purified IgA to enterococci in vitro mimics agglutination of E. faecium in vivo. Also, the Ca(2+) levels temporarily increased by 75% in feces of antibiotic-treated mice, which led to deformation of E-cadherin adherens junctions between colonic intestinal epithelial cells and release of E-cadherin as an extracellular matrix entrapping E. faecium. These findings indicate that during antibiotic-induced dysbiosis, the intestinal epithelium stays separated from an invading pathogen through an extracellular matrix in which sIgA, pIgR, and E-cadherin are colocalized. Future mucosal vaccination strategies to control E. faecium or other opportunistic pathogens may prevent multidrug-resistant infections, hospital transmission, and outbreaks. Infections with antibiotic-resistant enterococci are an emerging worldwide problem because enterococci are resistant to most of the

Draft Genome Sequences of Four Enterococcus faecium Strains Isolated from Argentine Cheese.

PubMed

Martino, Gabriela P; Quintana, Ingrid M; Espariz, Martín; Blancato, Victor S; Gallina Nizo, Gabriel; Esteban, Luis; Magni, Christian

2016-02-04

We report the draft genome sequences of four Enterococcus faecium strains isolated from Argentine regional cheeses. These strains were selected based on their technological properties, i.e., their ability to produce aroma compounds (diacetyl, acetoin, and 2,3-butanediol) from citrate. The goal of our study is to provide further genetic evidence for the rational selection of enterococci strains based on their pheno- and genotype in order to be used in cheese production. Copyright © 2016 Martino et al.

Characterization of an Enterococcus faecium small-colony variant isolated from blood culture.

PubMed

Gröbner, Sabine; Beck, Julia; Schaller, Martin; Autenrieth, Ingo B; Schulte, Berit

2012-01-01

Small-colony variants (SCVs) of bacteria are slow-growing subpopulations which can cause latent or recurrent infections due to better intracellular survival compared to their wild-type counterparts. Atypical colony morphology and altered biochemical profile may lead to failure in identification of SCV strains. We here report for the first time the isolation of an Enterococcus faecium SCV phenotype. The case of a 65-year-old woman with acute myeloid leukaemia who developed symptoms of sepsis during induction chemotherapy is presented. E. faecium with normal and SCV phenotype was isolated from blood cultures. At the same time urine culture was positive with E. faecium suggesting that bacteraemia originated from the urinary tract. The SCV phenotype was characterized by atypical growth behaviour. Electron microscopic analyses revealed perturbation of the separation of daughter cells and the accumulation of cell wall material. Accordingly, the SCV variant showed a dysfunction or lack of spontaneous autolysis whereas the normal phenotype did not. In contrast to conventional identification systems based on biochemical characteristics, the E. faecium SCV was precisely identified by MALDI-TOF MS analysis implemented in our laboratory. Hence, the increasing use of MALDI-TOF MS analysis for the identification of bacteria might be an appropriate tool for the detection of SCV variants, the diagnosis of which is of importance for the clinical outcome and the antibiotic treatment. Copyright © 2011. Published by Elsevier GmbH.

Failure of daptomycin β-Lactam combination therapy to prevent resistance emergence in Enterococcus faecium.

PubMed

Menon, Vidthiya; Davis, Rebecca; Shackel, Nick; Espedido, Bjorn A; Beukers, Alicia G; Jensen, Slade O; van Hal, Sebastiaan J

2018-02-01

Daptomycin β-Lactam combination therapy offers "protection" against daptomycin non-susceptibility (DNS) development in Enterococcus faecium. We report failure of this strategy and the importance of source control. Mutations were detected in the LiaF and cls genes in DNS isolates. A single DNS isolate contained an unrecognized mutation, which requires confirmation. Copyright © 2017 Elsevier Inc. All rights reserved.

High Rate of Resistance to Quinupristin-Dalfopristin in Enterococcus faecium Clinical Isolates from Korea

PubMed Central

Oh, Won Sup; Ko, Kwan Soo; Song, Jae-Hoon; Lee, Mi Young; Park, Sulhee; Peck, Kyong Ran; Lee, Nam Yong; Kim, Choon-Kwan; Lee, Hyuck; Kim, Shin-Woo; Chang, Hyun-Ha; Kim, Yeon-Sook; Jung, Sook-In; Son, Jun Seong; Yeom, Joon-Sup; Ki, Hyun Kyun; Woo, Gun-Jo

2005-01-01

We tested the in vitro susceptibilities of 603 enterococcal isolates from eight tertiary-care hospitals in Korea. The quinupristin-dalfopristin resistance rate in Enterococcus faecium was very high (25 isolates, 10.0%). It was suggested that both clonal spread and the sporadic emergence of quinupristin-dalfopristin-resistant isolates may explain the high prevalence of quinupristin-dalfopristin resistance in Korea. PMID:16304198

Influence of isolate origin and presence of various genes on biofilm formation by Enterococcus faecium.

PubMed

Almohamad, Sam; Somarajan, Sudha R; Singh, Kavindra V; Nallapareddy, Sreedhar R; Murray, Barbara E

2014-04-01

Enterococcus faecium, a major cause of nosocomial infections, is often isolated from conditions where biofilm is considered to be important in the establishment of infections. We investigated biofilm formation among E. faecium isolates from diverse sources and found that the occurrence and amount of biofilm formation were significantly greater in clinical isolates than fecal isolates from community volunteers. We also found that the presence of the empfm (E. faecium pilus) operon was associated with the amount of biofilm formation. Furthermore, we analyzed the possible association between the distribution of 16 putative virulence genes and the occurrence of biofilm production. Even though the prevalence of these virulence genes was significantly higher in clinical isolates, we did not observe any correlation with the occurrence of biofilm formation. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Characterization of antimicrobial resistance and quinolone resistance factors in high-level ciprofloxacin-resistant Enterococcus faecalis and Enterococcus faecium isolates obtained from fresh produce and fecal samples of patients.

PubMed

Kim, Min-Chan; Woo, Gun-Jo

2017-07-01

The emergence of fluoroquinolone-resistant enterococci is worldwide. Antimicrobial resistance was characterized and the effect of quinolone-resistance factors was analyzed in high-level ciprofloxacin-resistant (HLCR) Enterococcus faecalis and Enterococcus faecium isolated from fresh produce and fecal samples of patients. Among the 81 ciprofloxacin-resistant Enterococcus isolates, 46 showed high levels of ciprofloxacin resistance, resistance to other quinolone antibiotics, and multidrug resistance profiles. The virulence factors esp and hyl were identified in 27 (58.7%) and 25 (54.3%) of isolates, respectively. Sequence type analysis showed that 35 strains of HLCR E. faecium were clonal complex 17. Eleven strains of HLCR E. faecalis were confirmed as sequence type (ST) 28, ST 64 and ST 125. Quinolone resistance-determining region mutation was identified in HLCR Enterococcus isolates; with serine being changed in gyrA83, gyrA87 and parC80. This result shows that gyrA and parC mutations could be important factors for high-level resistance to fluoroquinolones. No significant differences were observed in antimicrobial resistance patterns and genetic characteristics among the isolates from fresh produce and fecal samples. Therefore, good agricultural practices in farming and continuous monitoring of patients, food and the environment for Enterococcus spp. should be performed to prevent antimicrobial resistance and enable reduction of resistance rates. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

PubMed

Nallapareddy, Sreedhar R; Singh, Kavindra V; Murray, Barbara E

2006-01-01

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the

Enterococcus faecium PBP5-S/R, the missing link between PBP5-S and PBP5-R.

PubMed

Pietta, Ester; Montealegre, Maria Camila; Roh, Jung Hyeob; Cocconcelli, Pier Sandro; Murray, Barbara E

2014-11-01

During a study to investigate the evolution of ampicillin resistance in Enterococcus faecium, we observed that a number of E. faecium strains, mainly from the recently described subclade A2, showed PBP5 sequences in between PBP5-S and PBP5-R. These hybrid PBP5-S/R patterns reveal a progression of amino acid changes from the S form to the R form of this protein; however, these changes do not strictly correlate with changes in ampicillin MICs. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Resistance mechanisms of linezolid-nonsusceptible enterococci in Korea: low rate of 23S rRNA mutations in Enterococcus faecium.

PubMed

Lee, Sae-Mi; Huh, Hee Jae; Song, Dong Joon; Shim, Hyang Jin; Park, Kyung Sun; Kang, Cheol-In; Ki, Chang-Seok; Lee, Nam Yong

2017-12-01

To investigate linezolid-resistance mechanisms in linezolid-nonsusceptible enterococci (LNSE) isolated from a tertiary hospital in Korea. Enterococcal isolates exhibiting linezolid MICs ≥4 mg l -1 that were isolated between December 2011 and May 2016 were investigated by PCR and sequencing for mutations in 23S rRNA or ribosomal proteins (L3, L4 and L22) and for the presence of cfr, cfr(B) and optrA genes.Results/Key findings. Among 135 LNSE (87 Enterococcus faecium and 48 Enterococcus faecalis isolates), 39.1 % (34/87) of E. faecium and 18.8 % (9/48) of E. faecalis isolates were linezolid-resistant. The optrA carriage was the dominant mechanism in E. faecalis: 13 isolates, including 10 E. faecalis [70 % (7/10) linezolid-resistant and 30 % (3/10) linezolid-intermediate] and three E. faecium [33.3 % (1/3) linezolid-resistant and 66.7 % (2/3) linezolid-intermediate], contained the optrA gene. G2576T mutations in the 23S rRNA gene were detected only in E. faecium [14 isolates; 71.4 % (10/14) linezolid-resistant and 28.6 % (4/14) linezolid-intermediate]. One linezolid-intermediate E. faecium harboured a L22 protein alteration (Ser77Thr). No isolates contained cfr or cfr(B) genes and any L3 or L4 protein alterations. No genetic mechanism of resistance was identified for 67.6 % (23/34) of linezolid-resistant E. faecium. A low rate of 23S rRNA mutations and the absence of known linezolid-resistance mechanisms in the majority of E. faecium isolates suggest regional differences in the mechanisms of linezolid resistance and the possibility of additional mechanisms.

Biofilms of Enterococcus faecalis and Enterococcus faecium isolated from the processing of ricotta and the control of these pathogens through cleaning and sanitization procedures.

PubMed

da Silva Fernandes, Meg; Kabuki, Dirce Yorika; Kuaye, Arnaldo Yoshiteru

2015-05-04

The biofilm formation of Enterococcus faecalis and Enterococcus faecium isolated from the processing of ricotta on stainless steel coupons was evaluated, and the effect of cleaning and sanitization procedures in the control of these biofilms was determined. The formation of biofilms was observed while varying the incubation temperature (7, 25 and 39°C) and time (0, 1, 2, 4, 6 and 8 days). At 7°C, the counts of E. faecalis and E. faecium were below 2 log10 CFU/cm(2). For the temperatures of 25 and 39°C, after 1 day, the counts of E. faecalis and E. faecium were 5.75 and 6.07 log10 CFU/cm(2), respectively, which is characteristic of biofilm formation. The tested sanitation procedures a) acid-anionic tensioactive cleaning, b) anionic tensioactive cleaning+sanitizer and c) acid-anionic tensioactive cleaning+sanitizer were effective in removing the biofilms, reducing the counts to levels below 0.4 log10 CFU/cm(2). The sanitizer biguanide was the least effective, and peracetic acid was the most effective. These studies revealed the ability of enterococci to form biofilms and the importance of the cleaning step and the type of sanitizer used in sanitation processes for the effective removal of biofilms. Copyright © 2015 Elsevier B.V. All rights reserved.

Core Genome Multilocus Sequence Typing Scheme for High- Resolution Typing of Enterococcus faecium.

PubMed

de Been, Mark; Pinholt, Mette; Top, Janetta; Bletz, Stefan; Mellmann, Alexander; van Schaik, Willem; Brouwer, Ellen; Rogers, Malbert; Kraat, Yvette; Bonten, Marc; Corander, Jukka; Westh, Henrik; Harmsen, Dag; Willems, Rob J L

2015-12-01

Enterococcus faecium, a common inhabitant of the human gut, has emerged in the last 2 decades as an important multidrug-resistant nosocomial pathogen. Since the start of the 21st century, multilocus sequence typing (MLST) has been used to study the molecular epidemiology of E. faecium. However, due to the use of a small number of genes, the resolution of MLST is limited. Whole-genome sequencing (WGS) now allows for high-resolution tracing of outbreaks, but current WGS-based approaches lack standardization, rendering them less suitable for interlaboratory prospective surveillance. To overcome this limitation, we developed a core genome MLST (cgMLST) scheme for E. faecium. cgMLST transfers genome-wide single nucleotide polymorphism(SNP) diversity into a standardized and portable allele numbering system that is far less computationally intensive than SNP-based analysis of WGS data. The E. faecium cgMLST scheme was built using 40 genome sequences that represented the diversity of the species. The scheme consists of 1,423 cgMLST target genes. To test the performance of the scheme, we performed WGS analysis of 103 outbreak isolates from five different hospitals in the Netherlands, Denmark, and Germany. The cgMLST scheme performed well in distinguishing between epidemiologically related and unrelated isolates, even between those that had the same sequence type (ST), which denotes the higher discriminatory power of this cgMLST scheme over that of conventional MLST. We also show that in terms of resolution, the performance of the E. faecium cgMLST scheme is equivalent to that of an SNP-based approach. In conclusion, the cgMLST scheme developed in this study facilitates rapid, standardized, and high-resolution tracing of E. faecium outbreaks.

Bacteriocinogenic potential and safety evaluation of non-starter Enterococcus faecium strains isolated from home made white brine cheese.

PubMed

Favaro, Lorenzo; Basaglia, Marina; Casella, Sergio; Hue, Isabelle; Dousset, Xavier; Gombossy de Melo Franco, Bernadette Dora; Todorov, Svetoslav Dimitrov

2014-04-01

Four LAB strains, isolated from Bulgarian home made white brine cheese, were selected for their effective inhibition against Listeria monocytogenes. According to their biochemical and physiological characteristics, the strains were classified as members of Enterococcus genus, and then identified as Enterococcus faecium by 16S rDNA sequencing. Their bacteriocin production and inhibitory spectrum were evaluated together with the occurrence of several bacteriocin genes (entA, entB, entP, entL50B). Their virulence potential and safety was assessed both using PCR targeted to the genes gelE, hyl, asa1, esp, cylA, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc and by phenotypical tests for antibiotic resistance, gelatinase, lipase, DNAse and α- and β-haemolysis. The E. faecium strains harboured at least one enterocin gene while the occurrence of virulence, antibiotic resistance and biogenic amines genes was limited. Considering their strong antimicrobial activity against L. monocytogenes strains, the four E. faecium strains exhibited promising potential as bio-preservatives cultures for fermented food productions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Changes in antimicrobial susceptibility of native Enterococcus faecium in chickens fed virginiamycin.

PubMed

McDermott, Patrick F; Cullen, Patti; Hubert, Susannah K; McDermott, Shawn D; Bartholomew, Mary; Simjee, Shabbir; Wagner, David D

2005-09-01

The extent of transfer of antimicrobial resistance from agricultural environments to humans is controversial. To assess the potential hazard posed by streptogramin use in food animals, this study evaluated the effect of virginiamycin exposure on antimicrobial resistance in Enterococcus faecium recovered from treated broilers. Four consecutive broiler feeding trials were conducted using animals raised on common litter. In the first three trials, one group of birds was fed virginiamycin continuously in feed at 20 g/ton, and a second group served as the nontreated control. In the fourth trial, antimicrobial-free feed was given to both groups. Fecal samples were cultured 1 day after chickens hatched and then at 1, 3, 5, and 7 weeks of age. Isolates from each time point were tested for susceptibility to a panel of different antimicrobials. Quinupristin/dalfopristin-resistant E. faecium appeared after 5 weeks of treatment in trial 1 and within 7 days of trials 2 to 4. Following removal of virginiamycin in trial 4, no resistant isolates were detected after 5 weeks. PCR failed to detect vat, vgb, or erm(B) in any of the streptogramin-resistant E. faecium isolates, whereas the msr(C) gene was detected in 97% of resistant isolates. In an experimental setting using broiler chickens, continuous virginiamycin exposure was required to maintain a stable streptogramin-resistant population of E. faecium in the animals. The bases of resistance could not be explained by known genetic determinants.

Atypical Genetic Locus Associated with Constitutive Production of Enterocin B by Enterococcus faecium BFE 900

PubMed Central

Franz, Charles M. A. P.; Worobo, Randy W.; Quadri, Luis E. N.; Schillinger, Ulrich; Holzapfel, Wilhelm H.; Vederas, John C.; Stiles, Michael E.

1999-01-01

A purified bacteriocin produced by Enterococcus faecium BFE 900 isolated from black olives was shown by Edman degradation and mass spectrometric analyses to be identical to enterocin B produced by E. faecium T136 from meat (P. Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernández, and H. Holo, Microbiology 143:2287–2294, 1997). The structural gene was located on a 2.2-kb HindIII fragment and a 12.0-kb EcoRI chromosomal fragment. The genetic characteristics and production of EntB by E. faecium BFE 900 differed from that described so far by the presence of a conserved sequence like a regulatory box upstream of the EntB gene, and its production was constitutive and not regulated. The 2.2-kb chromosomal fragment contained the hitherto undetected immunity gene for EntB in an atypical orientation that is the reverse of that of the structural gene. Typical transport and other genes associated with bacteriocin production were not detected on the 12.0-kb chromosomal fragment containing the EntB structural gene. This makes the EntB genetic system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of Carnobacterium piscicola LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal peptide, and expressed by the general secretory pathway in Enterococcus faecalis ATCC 19433. PMID:10224016

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis.

PubMed

de Sousa, Marylane; Manzo, Ricardo M; García, José L; Mammarella, Enrique J; Gonçalves, Luciana R B; Pessela, Benevides C

2017-12-06

l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N -His-l-AI and C -His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C -His-l-AI was preferentially hexameric in solution, whereas N -His-l-AI was mainly monomeric. The specific activity of the N -His-l-AI at acidic pH was higher than that of C -His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg -1 , respectively. However, C -His-l-AI was more active and stable at alkaline pH than N -His-l-AI. N -His-l-AI follows a Michaelis-Menten kinetic, whereas C -His-l-AI fitted to a sigmoidal saturation curve.

Evaluation of Enterococcus faecium NRRL B-2354 as a Surrogate for Salmonella During Extrusion of Low-Moisture Food.

PubMed

Verma, Tushar; Wei, Xinyao; Lau, Soon Kiat; Bianchini, Andreia; Eskridge, Kent M; Subbiah, Jeyamkondan

2018-04-01

Salmonella in low-moisture foods is an emerging challenge due to numerous food product recalls and foodborne illness outbreaks. Identification of suitable surrogate is critical for process validation at industry level due to implementation of new Food Safety Modernization Act of 2011. The objective of this study was to evaluate Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella during the extrusion of low-moisture food. Oat flour, a low-moisture food, was adjusted to different moisture (14% to 26% wet basis) and fat (5% to 15% w/w) contents and was inoculated with E. faecium NRRL B-2354. Inoculated material was then extruded in a lab-scale single-screw extruder running at different screw speeds (75 to 225 rpm) and different temperatures (75, 85, and 95 °C). A split-plot central composite 2nd order response surface design was used, with the central point replicated six times. The data from the selective media (m-Enterococcus agar) was used to build the response surface model for inactivation of E. faecium NRRL B-2354. Results indicated that E. faecium NRRL B-2354 always had higher heat resistance compared to Salmonella at all conditions evaluated in this study. However, the patterns of contour plots showing the effect of various product and process parameters on inactivation of E. faecium NRRL B-2354 was different from that of Salmonella. Although E. faecium NRRL B-2354 may be an acceptable surrogate for extrusion of low-moisture products due to higher resistance than Salmonella, another surrogate with similar inactivation behavior may be preferred and needs to be identified. Food Safety Modernization Act requires the food industry to validate processing interventions. This study validated extrusion processing and demonstrated that E. faecium NRRL B-2354 is an acceptable surrogate for extrusion of low-moisture products. The developed response surface model allows the industry to identify process conditions to achieve a desired lethality for their

Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family.

PubMed

Nallapareddy, Sreedhar R; Weinstock, George M; Murray, Barbara E

2003-03-01

A collagen-binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen-binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin-resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI-binding vancomycin-resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non-CI-binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration-dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm-specific IgGs purified from anti-recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen-binding clinical isolates of E. faecium tested, but in none of the strains with a non-functional pseudo acm gene. Introduction of a functional acm gene into two non-CI-binding natural acm mutant strains conferred a CI-binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection-derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the

Core Genome Multilocus Sequence Typing Scheme for High-Resolution Typing of Enterococcus faecium

PubMed Central

de Been, Mark; Pinholt, Mette; Top, Janetta; Bletz, Stefan; van Schaik, Willem; Brouwer, Ellen; Rogers, Malbert; Kraat, Yvette; Bonten, Marc; Corander, Jukka; Westh, Henrik; Harmsen, Dag

2015-01-01

Enterococcus faecium, a common inhabitant of the human gut, has emerged in the last 2 decades as an important multidrug-resistant nosocomial pathogen. Since the start of the 21st century, multilocus sequence typing (MLST) has been used to study the molecular epidemiology of E. faecium. However, due to the use of a small number of genes, the resolution of MLST is limited. Whole-genome sequencing (WGS) now allows for high-resolution tracing of outbreaks, but current WGS-based approaches lack standardization, rendering them less suitable for interlaboratory prospective surveillance. To overcome this limitation, we developed a core genome MLST (cgMLST) scheme for E. faecium. cgMLST transfers genome-wide single nucleotide polymorphism (SNP) diversity into a standardized and portable allele numbering system that is far less computationally intensive than SNP-based analysis of WGS data. The E. faecium cgMLST scheme was built using 40 genome sequences that represented the diversity of the species. The scheme consists of 1,423 cgMLST target genes. To test the performance of the scheme, we performed WGS analysis of 103 outbreak isolates from five different hospitals in the Netherlands, Denmark, and Germany. The cgMLST scheme performed well in distinguishing between epidemiologically related and unrelated isolates, even between those that had the same sequence type (ST), which denotes the higher discriminatory power of this cgMLST scheme over that of conventional MLST. We also show that in terms of resolution, the performance of the E. faecium cgMLST scheme is equivalent to that of an SNP-based approach. In conclusion, the cgMLST scheme developed in this study facilitates rapid, standardized, and high-resolution tracing of E. faecium outbreaks. PMID:26400782

A decade of genomic history for healthcare-associated Enterococcus faecium in the United Kingdom and Ireland

PubMed Central

Raven, Kathy E.; Reuter, Sandra; Reynolds, Rosy; Brodrick, Hayley J.; Russell, Julie E.; Török, M. Estée; Parkhill, Julian; Peacock, Sharon J.

2016-01-01

Vancomycin-resistant Enterococcus faecium (VREfm) is an important cause of healthcare-associated infections worldwide. We undertook whole-genome sequencing (WGS) of 495 E. faecium bloodstream isolates from 2001–2011 in the United Kingdom and Ireland (UK&I) and 11 E. faecium isolates from a reference collection. Comparison between WGS and multilocus sequence typing (MLST) identified major discrepancies for 17% of isolates, with multiple instances of the same sequence type (ST) being located in genetically distant positions in the WGS tree. This confirms that WGS is superior to MLST for evolutionary analyses and is more accurate than current typing methods used during outbreak investigations. E. faecium has been categorized as belonging to three clades (Clades A1, hospital-associated; A2, animal-associated; and B, community-associated). Phylogenetic analysis of our isolates replicated the distinction between Clade A (97% of isolates) and Clade B but did not support the subdivision of Clade A into Clade A1 and A2. Phylogeographic analyses revealed that Clade A had been introduced multiple times into each hospital referral network or country, indicating frequent movement of E. faecium between regions that rarely share hospital patients. Numerous genetic clusters contained highly related vanA-positive and -negative E. faecium, which implies that control of vancomycin-resistant enterococci (VRE) in hospitals also requires consideration of vancomycin-susceptible E. faecium. Our findings reveal the evolution and dissemination of hospital-associated E. faecium in the UK&I and provide evidence for WGS as an instrument for infection control. PMID:27527616

A decade of genomic history for healthcare-associated Enterococcus faecium in the United Kingdom and Ireland.

PubMed

Raven, Kathy E; Reuter, Sandra; Reynolds, Rosy; Brodrick, Hayley J; Russell, Julie E; Török, M Estée; Parkhill, Julian; Peacock, Sharon J

2016-10-01

Vancomycin-resistant Enterococcus faecium (VREfm) is an important cause of healthcare-associated infections worldwide. We undertook whole-genome sequencing (WGS) of 495 E. faecium bloodstream isolates from 2001-2011 in the United Kingdom and Ireland (UK&I) and 11 E. faecium isolates from a reference collection. Comparison between WGS and multilocus sequence typing (MLST) identified major discrepancies for 17% of isolates, with multiple instances of the same sequence type (ST) being located in genetically distant positions in the WGS tree. This confirms that WGS is superior to MLST for evolutionary analyses and is more accurate than current typing methods used during outbreak investigations. E. faecium has been categorized as belonging to three clades (Clades A1, hospital-associated; A2, animal-associated; and B, community-associated). Phylogenetic analysis of our isolates replicated the distinction between Clade A (97% of isolates) and Clade B but did not support the subdivision of Clade A into Clade A1 and A2. Phylogeographic analyses revealed that Clade A had been introduced multiple times into each hospital referral network or country, indicating frequent movement of E. faecium between regions that rarely share hospital patients. Numerous genetic clusters contained highly related vanA-positive and -negative E. faecium, which implies that control of vancomycin-resistant enterococci (VRE) in hospitals also requires consideration of vancomycin-susceptible E. faecium Our findings reveal the evolution and dissemination of hospital-associated E. faecium in the UK&I and provide evidence for WGS as an instrument for infection control. © 2016 Raven et al.; Published by Cold Spring Harbor Laboratory Press.

Effectiveness of automated ultraviolet-C light for decontamination of textiles inoculated with Enterococcus faecium.

PubMed

Smolle, C; Huss, F; Lindblad, M; Reischies, F; Tano, E

2018-01-01

Healthcare textiles are increasingly recognized as potential vehicles for transmission of hospital-acquired infections. This study tested the ability of an automated ultraviolet-C (UV-C) room disinfection device (Tru-D Smart UV-C) to decontaminate textiles inoculated with Enterococcus faecium in a clinical setting. Contaminated polycotton (50/50 polyester/cotton) swatches were distributed to predefined locations in a ward room and exposed to UV-C light. UV-C decontamination reduced E. faecium counts by a mean log 10 reduction factor of 1.37 (all P = 0.005, Wilcoxon signed rank test). UV-C decontamination may be a feasible adjunctive measure to conventional laundering to preserve the cleanliness of healthcare textiles in ward rooms. Copyright © 2017 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Evaluation of technological properties of Enterococcus faecium CECT 8849, a strain isolated from human milk, for the dairy industry.

PubMed

Cárdenas, Nivia; Arroyo, Rebeca; Calzada, Javier; Peirotén, Ángela; Medina, Margarita; Rodríguez, Juan Miguel; Fernández, Leonides

2016-09-01

In this work, a variety of biochemical properties of Enterococcus faecium CECT 8849, which had been isolated from breast milk, were analyzed. Its acidifying capacity and proteolytic activity were low but, in contrast, remarkable peptidase and esterase activities were observed. Ethanol and 3-hydroxy-2-butanone were the most abundant volatile compounds found in experimental model cheese manufactured with E. faecium CECT 8849. This strain inhibited the growth of several Listeria monocytogenes and Listeria innocua strains in vitro. Enterocin A and B structural genes were detected in E. faecium CECT 8849. Model fermented milk and cheeses were manufactured from milk inoculated or not with L. innocua CECT 8848 (2.5-3 log10 colony forming units mL(-1)) using E. faecium CECT 8849 or Lactococcus lactis ESI 153 as starter cultures. Although E. faecium CECT 8849 controlled Listeria growth in both dairy models, it led to lower reduction in Listeria counts when compared with L. lactis ESI 153.

Effects of probiotic Enterococcus faecium and Saccharomyces cerevisiae on the faecal microflora of pet rabbits.

PubMed

Benato, L; Hastie, P; O'Shaughnessy, P; Murray, J-A; Meredith, A

2014-09-01

Probiotics are frequently used in the treatment of gastrointestinal diseases in pet rabbits based largely on anecdotal evidence of a beneficial effect. However, there has been little work performed to assess any such benefit in health or disease. The aim of this study was to determine the effect of probiotics on faecal levels of four important candidate gastrointestinal bacteria (Bacteroides species, Enterococcus faecium, Fibrobacter succinogenes and Clostridium spiroforme) in pet rabbits. Additional aims were to evaluate the effect of probiotics on bodyweight and faecal weight and diameter. Double-blind triple cross-over study in six healthy rabbits orally administered two probiotic strains, Saccharomyces cerevisiae NCYC Sc47 and E. faecium NCIMB 30183. Levels of bacteria in faecal pellets were subsequently determined by real-time quantitative polymerase chain reaction. Oral administration of probiotic E. faecium NCIMB 30183 was associated with a significant (P = 0 · 042) increase in faecal levels of E. faecium. However, probiotic treatment did not affect faecal levels of Bacteroides species, F. succinogenes or C. spiroforme, bodyweight, or faecal weight and diameter. The inclusion of dietary probiotic supplementation using E. faecium NCIMB 30183 can increase faecal levels of certain bacterial flora of healthy adult rabbits. Further work is required to investigate the effects of probiotics in animals affected with gastrointestinal disease. © 2014 British Small Animal Veterinary Association.

Contribution of the collagen adhesin Acm to pathogenesis of Enterococcus faecium in experimental endocarditis.

PubMed

Nallapareddy, Sreedhar R; Singh, Kavindra V; Murray, Barbara E

2008-09-01

Enterococcus faecium is a multidrug-resistant opportunist causing difficult-to-treat nosocomial infections, including endocarditis, but there are no reports experimentally demonstrating E. faecium virulence determinants. Our previous studies showed that some clinical E. faecium isolates produce a cell wall-anchored collagen adhesin, Acm, and that an isogenic acm deletion mutant of the endocarditis-derived strain TX0082 lost collagen adherence. In this study, we show with a rat endocarditis model that TX0082 Deltaacm::cat is highly attenuated versus wild-type TX0082, both in established (72 h) vegetations (P < 0.0001) and for valve colonization 1 and 3 hours after infection (P faecium pathogenesis. In contrast, no mortality differences were observed in a mouse peritonitis model. While 5 of 17 endocarditis isolates were Acm nonproducers and failed to adhere to collagen in vitro, all had an intact, highly conserved acm locus. Highly reduced acm mRNA levels (>or=50-fold reduction relative to an Acm producer) were found in three of these five nonadherent isolates, including the sequenced strain TX0016, by quantitative reverse transcription-PCR, indicating that acm transcription is downregulated in vitro in these isolates. However, examination of TX0016 cells obtained directly from infected rat vegetations by flow cytometry showed that Acm was present on 40% of cells grown during infection. Finally, we demonstrated a significant reduction in E. faecium collagen adherence by affinity-purified anti-Acm antibodies from E. faecium endocarditis patient sera, suggesting that Acm may be a potential immunotarget for strategies to control this emerging pathogen.

Purification and characterization of enterocin MC13 produced by a potential aquaculture probiont Enterococcus faecium MC13 isolated from the gut of Mugil cephalus.

PubMed

Satish Kumar, R; Kanmani, P; Yuvaraj, N; Paari, K A; Pattukumar, V; Arul, V

2011-12-01

A bacteriocin producer strain MC13 was isolated from the gut of Mugil cephalus (grey mullet) and identified as Enterococcus faecium. The bacteriocin of E. faecium MC13 was purified to homogeneity, as confirmed by Tricine sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE). Reverse-phase high-performance liquid chromatography (HPLC) analysis showed a single active fraction eluted at 26 min, and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry analysis showed the molecular mass to be 2.148 kDa. The clear zone in native PAGE corresponding to enterocin MC13 band further substantiated its molecular mass. A dialyzed sample (semicrude preparation) of enterocin MC13 was broad spectrum in its action and inhibited important seafood-borne pathogens: Listeria monocytogenes , Vibrio parahaemolyticus, and Vibrio vulnificus. This antibacterial substance was sensitive to proteolytic enzymes: trypsin, protease, and chymotrypsin but insensitive to catalase and lipase, confirming that inhibition was due to the proteinaceous molecule, i.e., bacteriocin, and not due to hydrogen peroxide. Enterocin MC13 tolerated heat treatment (up to 90 °C for 20 min). Enterococcus faecium MC13 was effective in bile salt tolerance, acid tolerance, and adhesion to the HT-29 cell line. These properties reveal the potential of E. faecium MC13 to be a probiotic bacterium. Enterococcus faecium MC13 could be used as potential fish probiotic against pathogens such as V. parahaemolyticus, Vibrio harveyi, and Aeromonas hydrophila in fisheries. Also, this could be a valuable seafood biopreservative against L. monocytogenes.

Bacteriocin-Producing Enterococcus faecium LCW 44: A High Potential Probiotic Candidate from Raw Camel Milk

PubMed Central

Vimont, Allison; Fernandez, Benoît; Hammami, Riadh; Ababsa, Ahlem; Daba, Hocine; Fliss, Ismaïl

2017-01-01

Bacterial isolates from raw camel milk were screened for antibacterial activity using the agar diffusion assay. Ten isolates selected for their inhibition of Gram-positive bacteria were identified by 16S sequencing as Enterococcus faecium or durans. An isolate named E. faecium LCW 44 exhibited the broadest antibacterial spectrum with an inhibitory activity against several Gram-positive strains belonging to the genera Clostridium, Listeria, Staphylococcus, and Lactobacillus. E. faecium LCW 44 was shown to produce N-formylated enterocins L50A and L50B, as revealed by mass spectrometry and PCR analyses. This isolate did not harbor any of the virulence factors tested and was shown to be sensitive to all tested antibiotics. It showed high resistance to gastric and intestinal conditions (78 ± 4% survival). Its adhesion index was evaluated at 176 ± 86 and 24 ± 86 on Caco-2 cells and HT-29 cells, respectively, and it significantly reduced adhesion of Listeria monocytogenes by 65 and 49%, respectively. In Macfarlane broth (simulating the nutrient content of the colon), counts of L. monocytogenes were reduced by 2 log10 cycles after 24 h in co-culture with E. faecium LCW 44, compared to the increase of 4 log10 cycles when cultured alone. Comparison with a bacteriocin-non-producing mutant of E. faecium LCW 44 strongly suggests that inhibition of L. monocytogenes was due to bacteriocin production. Altogether, E. faecium LCW 44 thus has potential for use as a probiotic for humans and veterinary medicine. PMID:28572793

Global Emergence and Dissemination of Enterococci as Nosocomial Pathogens: Attack of the Clones?

PubMed Central

Guzman Prieto, Ana M.; van Schaik, Willem; Rogers, Malbert R. C.; Coque, Teresa M.; Baquero, Fernando; Corander, Jukka; Willems, Rob J. L.

2016-01-01

Enterococci are Gram-positive bacteria that are found in plants, soil and as commensals of the gastrointestinal tract of humans, mammals, and insects. Despite their commensal nature, they have also become globally important nosocomial pathogens. Within the genus Enterococcus, Enterococcus faecium, and Enterococcus faecalis are clinically most relevant. In this review, we will discuss how E. faecium and E. faecalis have evolved to become a globally disseminated nosocomial pathogen. E. faecium has a defined sub-population that is associated with hospitalized patients and is rarely encountered in community settings. These hospital-associated clones are characterized by the acquisition of adaptive genetic elements, including genes involved in metabolism, biofilm formation, and antibiotic resistance. In contrast to E. faecium, clones of E. faecalis isolated from hospitalized patients, including strains causing clinical infections, are not exclusively found in hospitals but are also present in healthy individuals and animals. This observation suggests that the division between commensals and hospital-adapted lineages is less clear for E. faecalis than for E. faecium. In addition, genes that are reported to be associated with virulence of E. faecalis are often not unique to clinical isolates, but are also found in strains that originate from commensal niches. As a reflection of more ancient association of E. faecalis with different hosts, these determinants Thus, they may not represent genuine virulence genes but may act as host-adaptive functions that are useful in a variety of intestinal environments. The scope of the review is to summarize recent trends in the emergence of antibiotic resistance and explore recent developments in the molecular epidemiology, population structure and mechanisms of adaptation of E. faecium and E. faecalis. PMID:27303380

Hypocholesterolemic and bioactive potential of exopolysaccharide from a probiotic Enterococcus faecium K1 isolated from kalarei.

PubMed

Bhat, Bilqeesa; Bajaj, Bijender Kumar

2018-04-01

Bioprospecting of novel probiotic strains especially from unexplored eco-niches has been a continuous practice. Enterococcus faecium K1, an isolate from indigenously fermented milk product kalarei possesses numerous desirable functional attributes. In current study, E. faecium K1 has been used for EPS production, and it yielded 355 ± 0.019 mg/L EPS. EPS demonstrates remarkable hypocholesterolemic, antioxidant, antibiofilm, and emulsification characteristics. EPS is constituted of mannose, glucose and galactose. SEM analysis reveals flake like compact structure of EPS while TEM and X-ray diffractogram confirms the amorphous structure of EPS. FTIR substantiates the functional groups/bonds typical of polysaccharides. Thermal analysis indicates adequate stability of EPS at 237 °C with average weight loss of 22%. E. faecium K1 EPS possesses unique functional bioactivities and physicochemical characteristics, and may potentially be explored for applications in food/pharmaceutical industries. Copyright © 2018 Elsevier Ltd. All rights reserved.

Evolutionary dynamics of Enterococcus faecium reveals complex genomic relationships between isolates with independent emergence of vancomycin resistance

PubMed Central

Ip, Camilla L. C.; Ansari, M. Azim; Wilson, Daniel J.; Espedido, Bjorn A.; Jensen, Slade O.; Bowden, Rory

2016-01-01

Enterococcus faecium, a major cause of hospital-acquired infections, remains problematic because of its propensity to acquire resistance to vancomycin, which currently is considered first-line therapy. Here, we assess the evolution and resistance acquisition dynamics of E. faecium in a clinical context using a series of 132 bloodstream infection isolates from a single hospital. All isolates, of which 49 (37 %) were vancomycin-resistant, underwent whole-genome sequencing. E. faecium was found to be subject to high rates of recombination with little evidence of sequence importation from outside the local E. faecium population. Apart from disrupting phylogenetic reconstruction, recombination was frequent enough to invalidate MLST typing in the identification of clonal expansion and transmission events, suggesting that, where available, whole-genome sequencing should be used in tracing the epidemiology of E. faecium nosocomial infections and establishing routes of transmission. Several forms of the Tn1549-like element–vanB gene cluster, which was exclusively responsible for vancomycin resistance, appeared and spread within the hospital during the study period. Several transposon gains and losses and instances of in situ evolution were inferred and, although usually chromosomal, the resistance element was also observed on a plasmid background. There was qualitative evidence for clonal expansions of both vancomycin-resistant and vancomycin-susceptible E. faecium with evidence of hospital-specific subclonal expansion. Our data are consistent with continuing evolution of this established hospital pathogen and confirm hospital vancomycin-susceptible and vancomycin-resistant E. faecium patient transmission events, underlining the need for careful consideration before modifying current E. faecium infection control strategies. PMID:27713836

Evolutionary dynamics of Enterococcus faecium reveals complex genomic relationships between isolates with independent emergence of vancomycin resistance.

PubMed

van Hal, Sebastiaan J; Ip, Camilla L C; Ansari, M Azim; Wilson, Daniel J; Espedido, Bjorn A; Jensen, Slade O; Bowden, Rory

2016-01-19

Enterococcus faecium , a major cause of hospital-acquired infections, remains problematic because of its propensity to acquire resistance to vancomycin, which currently is considered first-line therapy. Here, we assess the evolution and resistance acquisition dynamics of E. faecium in a clinical context using a series of 132 bloodstream infection isolates from a single hospital. All isolates, of which 49 (37 %) were vancomycin-resistant, underwent whole-genome sequencing. E. faecium was found to be subject to high rates of recombination with little evidence of sequence importation from outside the local E. faecium population. Apart from disrupting phylogenetic reconstruction, recombination was frequent enough to invalidate MLST typing in the identification of clonal expansion and transmission events, suggesting that, where available, whole-genome sequencing should be used in tracing the epidemiology of E. faecium nosocomial infections and establishing routes of transmission. Several forms of the Tn 1549 -like element- vanB gene cluster, which was exclusively responsible for vancomycin resistance, appeared and spread within the hospital during the study period. Several transposon gains and losses and instances of in situ evolution were inferred and, although usually chromosomal, the resistance element was also observed on a plasmid background. There was qualitative evidence for clonal expansions of both vancomycin-resistant and vancomycin-susceptible E. faecium with evidence of hospital-specific subclonal expansion. Our data are consistent with continuing evolution of this established hospital pathogen and confirm hospital vancomycin-susceptible and vancomycin-resistant E. faecium patient transmission events, underlining the need for careful consideration before modifying current E. faecium infection control strategies.

Characterization of Enterococcus faecium bacteriophage IME-EFm5 and its endolysin LysEFm5.

PubMed

Gong, Pengjuan; Cheng, Mengjun; Li, Xinwei; Jiang, Haiyan; Yu, Chuang; Kahaer, Nadire; Li, Juecheng; Zhang, Lei; Xia, Feifei; Hu, Liyuan; Sun, Changjiang; Feng, Xin; Lei, Liancheng; Han, Wenyu; Gu, Jingmin

2016-05-01

Due to the worldwide prevalence of antibiotic resistant strains, phages therapy has been revitalized recently. In this study, an Enterococcus faecium phage named IME-EFm5 was isolated from hospital sewage. Whole genomic sequence analysis demonstrated that IME-EFm5 belong to the Siphoviridae family, and has a double-stranded genome of 42,265bp (with a 35.51% G+C content) which contains 70 putative coding sequences. LysEFm5, the endolysin of IME-EFm5, contains an amidase domain in its N-terminal and has a wider bactericidal spectrum than its parental phage IME-EFm5, including 7 strains of vancomycin-resistant E. faecium. The mutagenesis analysis revealed that the zinc ion binding residues (H27, H132, and C140), E90, and T138 are required for the catalysis of LysEFm5. However, the antibacterial activity of LysEFm5 is zinc ion independent, which is inconsistent with most of other amidase members. The phage lysin LysEFm5 might be an alternative treatment strategy for infections caused by multidrug-resistant E. faecium. Copyright © 2016 Elsevier Inc. All rights reserved.

Complex Routes of Nosocomial Vancomycin-Resistant Enterococcus faecium Transmission Revealed by Genome Sequencing.

PubMed

Raven, Kathy E; Gouliouris, Theodore; Brodrick, Hayley; Coll, Francesc; Brown, Nicholas M; Reynolds, Rosy; Reuter, Sandra; Török, M Estée; Parkhill, Julian; Peacock, Sharon J

2017-04-01

Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of nosocomial infection. Here, we describe the utility of whole-genome sequencing in defining nosocomial VREfm transmission. A retrospective study at a single hospital in the United Kingdom identified 342 patients with E. faecium bloodstream infection over 7 years. Of these, 293 patients had a stored isolate and formed the basis for the study. The first stored isolate from each case was sequenced (200 VREfm [197 vanA, 2 vanB, and 1 isolate containing both vanA and vanB], 93 vancomycin-susceptible E. faecium) and epidemiological data were collected. Genomes were also available for E. faecium associated with bloodstream infections in 15 patients in neighboring hospitals, and 456 patients across the United Kingdom and Ireland. The majority of infections in the 293 patients were hospital-acquired (n = 249) or healthcare-associated (n = 42). Phylogenetic analysis showed that 291 of 293 isolates resided in a hospital-associated clade that contained numerous discrete clusters of closely related isolates, indicative of multiple introductions into the hospital followed by clonal expansion associated with transmission. Fine-scale analysis of 6 exemplar phylogenetic clusters containing isolates from 93 patients (32%) identified complex transmission routes that spanned numerous wards and years, extending beyond the detection of conventional infection control. These contained both vancomycin-resistant and -susceptible isolates. We also identified closely related isolates from patients at Cambridge University Hospitals NHS Foundation Trust and regional and national hospitals, suggesting interhospital transmission. These findings provide important insights for infection control practice and signpost areas for interventions. We conclude that sequencing represents a powerful tool for the enhanced surveillance and control of nosocomial E. faecium transmission and infection. © The Author 2017. Published by Oxford

1,2,4-Oxadiazole antimicrobials act synergistically with daptomycin and display rapid kill kinetics against MDR Enterococcus faecium.

PubMed

Carter, Glen P; Harjani, Jitendra R; Li, Lucy; Pitcher, Noel P; Nong, Yi; Riley, Thomas V; Williamson, Deborah A; Stinear, Timothy P; Baell, Jonathan B; Howden, Benjamin P

2018-06-01

Enterococcus faecium is an important nosocomial pathogen. It has a high propensity for horizontal gene transfer, which has resulted in the emergence of MDR strains that are difficult to treat. The most notorious of these, vancomycin-resistant E. faecium, are usually treated with linezolid or daptomycin. Resistance has, however, been reported, meaning that new therapeutics are urgently needed. The 1,2,4-oxadiazoles are a recently discovered family of antimicrobials that are active against Gram-positive pathogens and therefore have therapeutic potential for treating E. faecium. However, only limited data are available on the activity of these antimicrobials against E. faecium. To determine whether the 1,2,4-oxadiazole antimicrobials are active against MDR and daptomycin-non-susceptible E. faecium. The activity of the 1,2,4-oxadiazole antimicrobials against vancomycin-susceptible, vancomycin-resistant and daptomycin-non-susceptible E. faecium was determined using susceptibility testing, time-kill assays and synergy assays. Toxicity was also evaluated against human cells by XTT and haemolysis assays. The 1,2,4-oxadiazoles are active against a range of MDR E. faecium, including isolates that display non-susceptibility to vancomycin and daptomycin. This class of antimicrobial displays rapid bactericidal activity and demonstrates superior killing of E. faecium compared with daptomycin. Finally, the 1,2,4-oxadiazoles act synergistically with daptomycin against E. faecium, with subinhibitory concentrations reducing the MIC of daptomycin for non-susceptible isolates to a level below the clinical breakpoint. The 1,2,4-oxadiazoles are active against MDR and daptomycin-non-susceptible E. faecium and hold great promise as future therapeutics for treating infections caused by these difficult-to-treat isolates.

Enterococcus faecium FC-K Derived from Kimchi Is a Probiotic Strain That Shows Anti-Allergic Activity.

PubMed

Rho, Man-Kwang; Kim, Young-Eun; Rho, Hyun-In; Kim, Tae-Rahk; Kim, Yoon-Bum; Sung, Won-Kyung; Kim, Taw-Woo; Kim, Dae-Ok; Kang, Hee

2017-06-28

A rise in the occurrence of allergic diseases is attributed to the dysregulated balance of type 1/type 2 immunity, where type 2 T-helper (Th2) cells predominate over type 1 T-helper (Th1) cells, leading to an abnormally increased production of IgE in response to unharmful antigens. Kimchi, a traditional Korean fermented food, is a rich source of beneficial lactic acid bacteria. In this study, we investigated the ability of Enterococcus faecium FC-K derived from kimchi to induce type I immunity in the presence of Th2 polarizing conditions in vitro and in vivo. Stimulation of mouse peritoneal macrophages with E. faecium FC-K induced the production of tumor necrosis factor alpha, interleukin (IL)-6, and IL-12. Under the in vitro Th2 conditions in which splenic T cells were activated in the presence of IL-4, E. faecium FC-K enhanced the ability of T cells to produce interferon (IFN)-γ. Using the ovalbumin (OVA)-induced allergy model, male BALB/c mice receiving E. faecium FC-K reduced the serum level of total IgE, but not that of OVA-specific IgE. Furthermore, the population of activated splenic B cells during OVA immunization was decreased in E. faecium FC-K-treated mice, accounting for a reduction of total IgE in the serum. Restimulating splenocytes from OVA-immunized mice with OVA ex vivo resulted in an increased production of IFN-γ, with no effect on IL-4, in E. faecium FC-Ktreated mice. These observations provide the evidence that E. faecium FC-K can be a beneficial probiotic strain that can modulate the Th2-mediated pathologic response.

Application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry in the screening of vanA-positive Enterococcus faecium.

PubMed

Wang, Li-jun; Lu, Xin-xin; Wu, Wei; Sui, Wen-jun; Zhang, Gui

2014-01-01

In order to evaluate a rapid matrix-assisted laser desorption ionization-time of flight mass spectrometry (MAIDI-TOF MS) assay in screening vancomycin-resistant Enterococcus faecium, a total of 150 E. faecium clinical strains were studied, including 60 vancomycin-resistant E. faecium (VREF) isolates and 90 vancomycin-susceptible (VSEF) strains. Vancomycin resistance genes were detected by sequencing. E. faecium were identified by MALDI-TOF MS. A genetic algorithm model with ClinProTools software was generated using spectra of 30 VREF isolates and 30 VSEF isolates. Using this model, 90 test isolates were discriminated between VREF and VSEF. The results showed that all sixty VREF isolates carried the vanA gene. The performance of VREF detection by the genetic algorithm model of MALDI-TOF MS compared to the sequencing method was sensitivity = 80%, specificity = 90%, false positive rate =10%, false negative rate =10%, positive predictive value = 80%, negative predictive value= 90%. MALDI-TOF MS can be used as a screening test for discrimination between vanA-positive E. faecium and vanA-negative E. faecium.

Comparison of Enterococcus faecium and Enterococcus faecalis Strains Isolated from Water and Clinical Samples: Antimicrobial Susceptibility and Genetic Relationships

PubMed Central

Castillo-Rojas, Gonzalo; Mazari-Hiríart, Marisa; Ponce de León, Sergio; Amieva-Fernández, Rosa I.; Agis-Juárez, Raúl A.; Huebner, Johannes; López-Vidal, Yolanda

2013-01-01

Enterococci are part of the normal intestinal flora in a large number of mammals, and these microbes are currently used as indicators of fecal contamination in water and food for human consumption. These organisms are considered one of the primary causes of nosocomial and environmental infections due to their ability to survive in the environment and to their intrinsic resistance to antimicrobials. The aims of this study were to determine the biochemical patterns and antimicrobial susceptibilities of Enterococcus faecalis and E. faecium isolates from clinical samples and from water (groundwater, water from the Xochimilco wetland, and treated water from the Mexico City Metropolitan Area) and to determine the genetic relationships among these isolates. A total of 121 enterococcus strains were studied; 31 and 90 strains were isolated from clinical samples and water (groundwater, water from the Xochimilco wetland, and water for agricultural irrigation), respectively. Identification to the species level was performed using a multiplex PCR assay, and antimicrobial profiles were obtained using a commercial kit. Twenty-eight strains were analyzed by pulsed-field gel electrophoresis (PFGE). E. faecium strains isolated from water showed an atypical biochemical pattern. The clinical isolates showed higher resistance to antibiotics than those from water. Both the enterococci isolated from humans, and those isolated from water showed high genetic diversity according to the PFGE analysis, although some strains seemed to be closely related. In conclusion, enterococci isolated from humans and water are genetically different. However, water represents a potential route of transmission to the community and a source of antimicrobial resistance genes that may be readily transmitted to other, different bacterial species. PMID:23560050

Comparison of Enterococcus faecium and Enterococcus faecalis Strains isolated from water and clinical samples: antimicrobial susceptibility and genetic relationships.

PubMed

Castillo-Rojas, Gonzalo; Mazari-Hiríart, Marisa; Ponce de León, Sergio; Amieva-Fernández, Rosa I; Agis-Juárez, Raúl A; Huebner, Johannes; López-Vidal, Yolanda

2013-01-01

Enterococci are part of the normal intestinal flora in a large number of mammals, and these microbes are currently used as indicators of fecal contamination in water and food for human consumption. These organisms are considered one of the primary causes of nosocomial and environmental infections due to their ability to survive in the environment and to their intrinsic resistance to antimicrobials. The aims of this study were to determine the biochemical patterns and antimicrobial susceptibilities of Enterococcus faecalis and E. faecium isolates from clinical samples and from water (groundwater, water from the Xochimilco wetland, and treated water from the Mexico City Metropolitan Area) and to determine the genetic relationships among these isolates. A total of 121 enterococcus strains were studied; 31 and 90 strains were isolated from clinical samples and water (groundwater, water from the Xochimilco wetland, and water for agricultural irrigation), respectively. Identification to the species level was performed using a multiplex PCR assay, and antimicrobial profiles were obtained using a commercial kit. Twenty-eight strains were analyzed by pulsed-field gel electrophoresis (PFGE). E. faecium strains isolated from water showed an atypical biochemical pattern. The clinical isolates showed higher resistance to antibiotics than those from water. Both the enterococci isolated from humans, and those isolated from water showed high genetic diversity according to the PFGE analysis, although some strains seemed to be closely related. In conclusion, enterococci isolated from humans and water are genetically different. However, water represents a potential route of transmission to the community and a source of antimicrobial resistance genes that may be readily transmitted to other, different bacterial species.

Purification and characterization of enterocin FH 99 produced by a faecal isolate Enterococcus faecium FH 99.

PubMed

Gupta, H; Malik, R K; Bhardwaj, A; Kaur, G; De, S; Kaushik, J K

2010-06-01

Enterococcus faecium FH 99 was isolated from human faeces and selected because of its broad spectrum of inhibitory activity against several Gram-positive foodborne spoilage and pathogenic bacteria. Ent. faecium FH 99 accumulates enterocin in large number in early stationary phase of the growth. The enterocin FH 99 was stable over a wide pH range (2-10) and recovered activity even after treatment at high temperatures (10 min at 100°C). The enterocin was subjected to different purification techniques viz., gel filteration, cation exchange chromatography and reverse-phase high-performance liquid chromatography. The activity was eluted as one individual active fraction. SDSPAGE revealed a molecular weight of less than 6.5 kDa. Studies carried out to identify the genetic determinants for bacteriocin production showed that this trait may be plasmid encoded as loss in both of the plasmids (size>chromosomal DNA) led to loss in bacteriocin production by Ent. faecium FH 99. Ent. faecium strain FH 99 is a newly discovered high bacteriocin producer with Activity Units 1.8 × 10(5) AU ml(-1) and its characteristics indicate that it may have strong potential for application as a protective agent against pathogens and spoilage bacteria in foods.

Single nucleotide polymorphism analysis of the enterocin P structural gene of Enterococcus faecium strains isolated from nonfermented animal foods.

PubMed

Arlindo, Samuel; Calo, Pilar; Franco, Carlos; Prado, Marta; Cepeda, Alberto; Barros-Velázquez, Jorge

2006-12-01

The bacteriocins produced by two lactic acid bacteria isolated from nonfermented fresh meat and fish, respectively, and exhibiting a remarkable antilisterial activity, were characterized. Bacteriocinogenic strains were identified as Enterococcus faecium and the maximum bacteriocin production by both strains was detected in the stationary phase of growth. The activity against Listeria monocytogenes was maintained in pH range of 3-7 and was stable in both strains after heating at 100 or 121 degrees C. The genes coding for enterocin P were detected, isolated, and sequenced in both E. faecium strains. They exhibited DNA/DNA homology in the 87.1-97.2% range with respect to the other four enterocin P genes reported so far. Three single nucleotide polymorphism events, silent at the amino acid level, were detected at nucleotide positions 45 (G/A), 75 (A/G), and 90 (T/C) in E. faecium LHICA 28-4 and may explain the differences reported for those loci in other enterocin P-producing E. faecium strains. This work provides the first description of enterocin P-producing E. faecium strains in nonfermented foodstuffs and, in the case of E. faecium LHICA 51, the first report of an enterocin P-producing strain isolated from fish so far.

Molecular Epidemiology of Vancomycin-Resistant Enterococcus faecium: a Prospective, Multicenter Study in South American Hospitals▿

PubMed Central

Panesso, Diana; Reyes, Jinnethe; Rincón, Sandra; Díaz, Lorena; Galloway-Peña, Jessica; Zurita, Jeannete; Carrillo, Carlos; Merentes, Altagracia; Guzmán, Manuel; Adachi, Javier A.; Murray, Barbara E.; Arias, Cesar A.

2010-01-01

Enterococcus faecium has emerged as an important nosocomial pathogen worldwide, and this trend has been associated with the dissemination of a genetic lineage designated clonal cluster 17 (CC17). Enterococcal isolates were collected prospectively (2006 to 2008) from 32 hospitals in Colombia, Ecuador, Perú, and Venezuela and subjected to antimicrobial susceptibility testing. Genotyping was performed with all vancomycin-resistant E. faecium (VREfm) isolates by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. All VREfm isolates were evaluated for the presence of 16 putative virulence genes (14 fms genes, the esp gene of E. faecium [espEfm], and the hyl gene of E. faecium [hylEfm]) and plasmids carrying the fms20-fms21 (pilA), hylEfm, and vanA genes. Of 723 enterococcal isolates recovered, E. faecalis was the most common (78%). Vancomycin resistance was detected in 6% of the isolates (74% of which were E. faecium). Eleven distinct PFGE types were found among the VREfm isolates, with most belonging to sequence types 412 and 18. The ebpAEfm-ebpBEfm-ebpCEfm (pilB) and fms11-fms19-fms16 clusters were detected in all VREfm isolates from the region, whereas espEfm and hylEfm were detected in 69% and 23% of the isolates, respectively. The fms20-fms21 (pilA) cluster, which encodes a putative pilus-like protein, was found on plasmids from almost all VREfm isolates and was sometimes found to coexist with hylEfm and the vanA gene cluster. The population genetics of VREfm in South America appear to resemble those of such strains in the United States in the early years of the CC17 epidemic. The overwhelming presence of plasmids encoding putative virulence factors and vanA genes suggests that E. faecium from the CC17 genogroup may disseminate in the region in the coming years. PMID:20220167

Enterococcus faecium biofilm formation: identification of major autolysin AtlAEfm, associated Acm surface localization, and AtlAEfm-independent extracellular DNA Release.

PubMed

Paganelli, Fernanda L; Willems, Rob J L; Jansen, Pamela; Hendrickx, Antoni; Zhang, Xinglin; Bonten, Marc J M; Leavis, Helen L

2013-04-16

Enterococcus faecium is an important multidrug-resistant nosocomial pathogen causing biofilm-mediated infections in patients with medical devices. Insight into E. faecium biofilm pathogenesis is pivotal for the development of new strategies to prevent and treat these infections. In several bacteria, a major autolysin is essential for extracellular DNA (eDNA) release in the biofilm matrix, contributing to biofilm attachment and stability. In this study, we identified and functionally characterized the major autolysin of E. faecium E1162 by a bioinformatic genome screen followed by insertional gene disruption of six putative autolysin genes. Insertional inactivation of locus tag EfmE1162_2692 resulted in resistance to lysis, reduced eDNA release, deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and significant chaining compared to that of the wild type. Therefore, locus tag EfmE1162_2692 was considered the major autolysin in E. faecium and renamed atlAEfm. In addition, AtlAEfm was implicated in cell surface exposure of Acm, a virulence factor in E. faecium, and thereby facilitates binding to collagen types I and IV. This is a novel feature of enterococcal autolysins not described previously. Furthermore, we identified (and localized) autolysin-independent DNA release in E. faecium that contributes to cell-cell interactions in the atlAEfm mutant and is important for cell separation. In conclusion, AtlAEfm is the major autolysin in E. faecium and contributes to biofilm stability and Acm localization, making AtlAEfm a promising target for treatment of E. faecium biofilm-mediated infections. IMPORTANCE Nosocomial infections caused by Enterococcus faecium have rapidly increased, and treatment options have become more limited. This is due not only to increasing resistance to antibiotics but also to biofilm-associated infections. DNA is released in biofilm matrix via cell lysis, caused by autolysin, and acts as a matrix stabilizer. In this study

Enterocin HZ produced by a wild Enterococcus faecium strain isolated from a traditional, starter-free pickled cheese.

PubMed

Yildirim, Zeliha; Bilgin, Harun; Isleroglu, Hilal; Tokatli, Kader; Sahingil, Didem; Yildirim, Metin

2014-05-01

Bacteriogenic Enterococcus faecium HZ was identified by using biochemical (Strep-API 20, API-50 CHL, fatty acid profile) and 16S rRNA analysis (99·99 %). Ent. faecium HZ was sensitive to clinically important antibiotics such as vancomycin, and did not have gelatinase and haemolysis activities. Enterocin HZ, a bacteriocin from Ent. faecium HZ, was sensitive to papain and tyripsin, but resistant to pepsin, lipase, catalase, α-amylase, organic solvents, detergents, ß-mercaptoethanol, and heat treatment (90 °C/30 min). It was biologically active at pH 2·0-9·0 and synthesised at the highest level in MRS or M17 broth at 32 or 37 °C with an inoculum amount of 0·1-0·5 % and an initial pH of 6·0-7·0. Enterocin HZ production reached maximum level at middle and late logarithmic phase and its molecular weight was ∼4·5 kDa. It was active against some Gram-positive foodborne bacteria. Ent. faecium HZ or its bacteriocin enterocin HZ is a good candidate to be studied as a food biopreservative since enterocin HZ showed strong bactericidal activity against Listeria monocytogenes in UHT milk and also Ent. faecium HZ grew very well in milk and produced enterocin HZ at maximum level.

Enterococcal surface protein Esp is not essential for cell adhesion and intestinal colonization of Enterococcus faecium in mice

PubMed Central

2009-01-01

Background Enterococcus faecium has globally emerged as a cause of hospital-acquired infections with high colonization rates in hospitalized patients. The enterococcal surface protein Esp, identified as a potential virulence factor, is specifically linked to nosocomial clonal lineages that are genetically distinct from indigenous E. faecium strains. To investigate whether Esp facilitates bacterial adherence and intestinal colonization of E. faecium, we used human colorectal adenocarcinoma cells (Caco-2 cells) and an experimental colonization model in mice. Results No differences in adherence to Caco-2 cells were found between an Esp expressing strain of E. faecium (E1162) and its isogenic Esp-deficient mutant (E1162Δesp). Mice, kept under ceftriaxone treatment, were inoculated orally with either E1162, E1162Δesp or both strains simultaneously. Both E1162 and E1162Δesp were able to colonize the murine intestines with high and comparable numbers. No differences were found in the contents of cecum and colon. Both E1162 and E1162Δesp were able to translocate to the mesenteric lymph nodes. Conclusion These results suggest that Esp is not essential for Caco-2 cell adherence and intestinal colonization or translocation of E. faecium in mice. PMID:19178704

Inhibitory Influence of Enterococcus faecium on the Propagation of Swine Influenza A Virus In Vitro

PubMed Central

Wang, Zhenya; Chai, Weidong; Burwinkel, Michael; Twardziok, Sven; Wrede, Paul; Palissa, Christiane; Esch, Bettina; Schmidt, Michael F. G.

2013-01-01

The control of infectious diseases such as swine influenza viruses (SwIV) plays an important role in food production both from the animal health and from the public health point of view. Probiotic microorganisms and other health improving food supplements have been given increasing attention in recent years, but, no information on the effects of probiotics on swine influenza virus is available. Here we address this question by assessing the inhibitory potential of the probiotic Enterococcus faecium NCIMB 10415 (E. faecium) on the replication of two porcine strains of influenza virus (H1N1 and H3N2 strain) in a continuous porcine macrophage cell line (3D4/21) and in MDBK cells. Cell cultures were treated with E. faecium at the non-toxic concentration of 1×106 CFU/ml in growth medium for 60 to 90 min before, during and after SwIV infection. After further incubation of cultures in probiotic-free growth medium, cell viability and virus propagation were determined at 48 h or 96 h post infection. The results obtained reveal an almost complete recovery of viability of SwIV infected cells and an inhibition of virus multiplication by up to four log units in the E. faecium treated cells. In both 3D4/21- and MDBK-cells a 60 min treatment with E. faecium stimulated nitric oxide (NO) release which is in line with published evidence for an antiviral function of NO. Furthermore, E. faecium caused a modified cellular expression of selected mediators of defence in 3D4-cells: while the expression of TNF-α, TLR-3 and IL-6 were decreased in the SwIV-infected and probiotic treated cells, IL-10 was found to be increased. Since we obtained experimental evidence for the direct adsorptive trapping of SwIV through E. faecium, this probiotic microorganism inhibits influenza viruses by at least two mechanisms, direct physical interaction and strengthening of innate defence at the cellular level. PMID:23308134

Safety of the Surrogate Microorganism Enterococcus faecium NRRL B-2354 for Use in Thermal Process Validation

PubMed Central

Kopit, Lauren M.; Kim, Eun Bae; Siezen, Roland J.; Harris, Linda J.

2014-01-01

Enterococcus faecium NRRL B-2354 is a surrogate microorganism used in place of pathogens for validation of thermal processing technologies and systems. We evaluated the safety of strain NRRL B-2354 based on its genomic and functional characteristics. The genome of E. faecium NRRL B-2354 was sequenced and found to comprise a 2,635,572-bp chromosome and a 214,319-bp megaplasmid. A total of 2,639 coding sequences were identified, including 45 genes unique to this strain. Hierarchical clustering of the NRRL B-2354 genome with 126 other E. faecium genomes as well as pbp5 locus comparisons and multilocus sequence typing (MLST) showed that the genotype of this strain is most similar to commensal, or community-associated, strains of this species. E. faecium NRRL B-2354 lacks antibiotic resistance genes, and both NRRL B-2354 and its clonal relative ATCC 8459 are sensitive to clinically relevant antibiotics. This organism also lacks, or contains nonfunctional copies of, enterococcal virulence genes including acm, cyl, the ebp operon, esp, gelE, hyl, IS16, and associated phenotypes. It does contain scm, sagA, efaA, and pilA, although either these genes were not expressed or their roles in enterococcal virulence are not well understood. Compared with the clinical strains TX0082 and 1,231,502, E. faecium NRRL B-2354 was more resistant to acidic conditions (pH 2.4) and high temperatures (60°C) and was able to grow in 8% ethanol. These findings support the continued use of E. faecium NRRL B-2354 in thermal process validation of food products. PMID:24413604

Antibiotic-Driven Dysbiosis Mediates Intraluminal Agglutination and Alternative Segregation of Enterococcus faecium from the Intestinal Epithelium

PubMed Central

Top, Janetta; Bayjanov, Jumamurat R.; Kemperman, Hans; Rogers, Malbert R. C.; Paganelli, Fernanda L.; Bonten, Marc J. M.; Willems, Rob J. L.

2015-01-01

ABSTRACT The microbiota of the mammalian gastrointestinal tract is a complex ecosystem of bacterial communities that continuously interact with the mucosal immune system. In a healthy host, the mucosal immune system maintains homeostasis in the intestine and prevents invasion of pathogenic bacteria, a phenomenon termed colonization resistance. Antibiotics create dysbiosis of microbiota, thereby decreasing colonization resistance and facilitating infections caused by antibiotic-resistant bacteria. Here we describe how cephalosporin antibiotics create dysbiosis in the mouse large intestine, allowing intestinal outgrowth of antimicrobial-resistant Enterococcus faecium. This is accompanied by a reduction of the mucus-associated gut microbiota layer, colon wall, and Muc-2 mucus layer. E. faecium agglutinates intraluminally in an extracellular matrix consisting of secretory IgA (sIgA), polymeric immunoglobulin receptor (pIgR), and epithelial cadherin (E-cadherin) proteins, thereby maintaining spatial segregation of E. faecium from the intestinal wall. Addition of recombinant E-cadherin and pIgR proteins or purified IgA to enterococci in vitro mimics agglutination of E. faecium in vivo. Also, the Ca2+ levels temporarily increased by 75% in feces of antibiotic-treated mice, which led to deformation of E-cadherin adherens junctions between colonic intestinal epithelial cells and release of E-cadherin as an extracellular matrix entrapping E. faecium. These findings indicate that during antibiotic-induced dysbiosis, the intestinal epithelium stays separated from an invading pathogen through an extracellular matrix in which sIgA, pIgR, and E-cadherin are colocalized. Future mucosal vaccination strategies to control E. faecium or other opportunistic pathogens may prevent multidrug-resistant infections, hospital transmission, and outbreaks. PMID:26556272

Enterococcus faecium QU 50: a novel thermophilic lactic acid bacterium for high-yield l-lactic acid production from xylose.

PubMed

Abdel-Rahman, Mohamed Ali; Tashiro, Yukihiro; Zendo, Takeshi; Sakai, Kenji; Sonomoto, Kenji

2015-01-01

Production of optically pure lactic acid from lignocellulosic material for commercial purposes is hampered by several difficulties, including heterofermentation of pentose sugars and high energy consumption by mesophilic lactic acid bacteria. Here, we report a novel lactic acid bacterium, strain QU 50, that has the potential to produce optically pure l-lactic acid (≥99.2%) in a homofermentative manner from xylose under thermophilic conditions. Strain QU 50 was isolated from Egyptian fertile soil and identified as Enterococcus faecium QU 50 by analyzing its sugar fermentation pattern and 16S rRNA gene sequence. Enterococcus faecium QU 50 fermented xylose efficiently to produce lactic acid over wide pH (6.0-10.0) and temperature ranges (30-52°C), with a pH of 6.5 and temperature of 50°C being optimal. To our knowledge, this is the first report of homofermentative lactic acid production from xylose by a thermophilic lactic acid bacterium. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Enterocin X, a novel two-peptide bacteriocin from Enterococcus faecium KU-B5, has an antibacterial spectrum entirely different from those of its component peptides.

PubMed

Hu, Chih-Bo; Malaphan, Wanna; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

2010-07-01

Enterocin X, composed of two antibacterial peptides (Xalpha and Xbeta), is a novel class IIb bacteriocin from Enterococcus faecium KU-B5. When combined, Xalpha and Xbeta display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known.

Development and evaluation of a Quadruplex Taq Man real-time PCR assay for simultaneous detection of clinical isolates of Enterococcus faecalis, Enterococcus faecium and their vanA and vanB genotypes.

PubMed

Naserpour Farivar, Taghi; Najafipour, Reza; Johari, Pouran; Aslanimehr, Masoumeh; Peymani, Amir; Jahani Hashemi, Hoasan; Mirzaui, Baman

2014-10-01

We developed and evaluated the utility of a quadruplex Taqman real-time PCR assay that allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. The specificity of the assay was tested using reference strains of vancomycin-resistant and susceptible enterococci. In total, 193 clinical isolates were identified and subsequently genotyped using a Quadruplex Taqman real-time PCR assay and melting curve analysis. Representative Quadruplex Taqman real-time PCR amplification curve were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis. Phenotypic and genotypic analysis of the isolates gave same results for 82 enterococcal isolates, while in 5 isolates, they were inconsistent. We had three mixed strains, which were detected by the TaqMan real-time PCR assay and could not be identified correctly using phenotypic methods. Vancomycin resistant enterococci (VRE) genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using TaqMan real-time multiplex real-time PCR assay.

Comparative genomics of Enterococcus spp. isolated from bovine feces.

PubMed

Beukers, Alicia G; Zaheer, Rahat; Goji, Noriko; Amoako, Kingsley K; Chaves, Alexandre V; Ward, Michael P; McAllister, Tim A

2017-03-08

Enterococcus is ubiquitous in nature and is a commensal of both the bovine and human gastrointestinal (GI) tract. It is also associated with clinical infections in humans. Subtherapeutic administration of antibiotics to cattle selects for antibiotic resistant enterococci in the bovine GI tract. Antibiotic resistance genes (ARGs) may be present in enterococci following antibiotic use in cattle. If located on mobile genetic elements (MGEs) their dissemination between Enterococcus species and to pathogenic bacteria may be promoted, reducing the efficacy of antibiotics. We present a comparative genomic analysis of twenty-one Enterococcus spp. isolated from bovine feces including Enterococcus hirae (n = 10), Enterococcus faecium (n = 3), Enterococcus villorum (n = 2), Enterococcus casseliflavus (n = 2), Enterococcus faecalis (n = 1), Enterococcus durans (n = 1), Enterococcus gallinarum (n = 1) and Enterococcus thailandicus (n = 1). The analysis revealed E. faecium and E. faecalis from bovine feces share features with human clinical isolates, including virulence factors. The Tn917 transposon conferring macrolide-lincosamide-streptogramin B resistance was identified in both E. faecium and E. hirae, suggesting dissemination of ARGs on MGEs may occur in the bovine GI tract. An E. faecium isolate was also identified with two integrative conjugative elements (ICEs) belonging to the Tn916 family of ICE, Tn916 and Tn5801, both conferring tetracycline resistance. This study confirms the presence of enterococci in the bovine GI tract possessing ARGs on MGEs, but the predominant species in cattle, E. hirae is not commonly associated with infections in humans. Analysis using additional complete genomes of E. faecium from the NCBI database demonstrated differential clustering of commensal and clinical isolates, suggesting that these strains may be specifically adapted to their respective environments.

Synthesis and Evaluation of 1,2,4-Triazolo[1,5-a]pyrimidines as Antibacterial Agents Against Enterococcus faecium

PubMed Central

Wang, Huan; Lee, Mijoon; Peng, Zhihong; Blázquez, Blas; Lastochkin, Elena; Kumarasiri, Malika; Bouley, Renee; Chang, Mayland; Mobashery, Shahriar

2015-01-01

Rapid emergence of antibiotic resistance is one of the most challenging global public health concerns. In particular, vancomycin-resistant Enterococcus faecium infections have been increasing in frequency, representing 25% of enterococci infections in intensive care units. A novel class of 1,2,4-triazolo[1,5-a]pyrimidines active against E. faecium is reported herein. We used a three-component Biginelli-like heterocyclization reaction for the synthesis of a series of these derivatives based on reactions of aldehydes, β-dicarbonyl compounds, and 3-alkylthio-5-amino-1,2,4-tria-zoles. The resulting compounds were assayed for antimicrobial activity against the ESKAPE panel of bacteria, followed by investigation of their in vitro activities. These analyses identified a subset of 1,2,4-triazolo[1,5-a]pyrimidines that had good narrow-spectrum antibacterial activity against E. faecium and exhibited metabolic stability with low intrinsic clearance. Macromolecular synthesis assays revealed cell-wall biosynthesis as the target of these antibiotics. PMID:25923368

Dispersion of Multidrug-Resistant Enterococcus faecium Isolates Belonging to Major Clonal Complexes in Different Portuguese Settings▿

PubMed Central

Freitas, Ana R.; Novais, Carla; Ruiz-Garbajosa, Patricia; Coque, Teresa M.; Peixe, Luísa

2009-01-01

The population structure of 56 Enterococcus faecium isolates selected from a collection of enterococci from humans, animals, and the environment in Portugal (1997 to 2007) was analyzed by multilocus sequence typing. We identified 41 sequence types clustering into CC17, CC5, CC9, CC22 and CC94, all clonal lineages comprising isolates from different hosts. Our findings highlight the role of community-associated hosts as reservoirs of enterococci able to cause human infections. PMID:19447948

Differential Penicillin-Binding Protein 5 (PBP5) Levels in the Enterococcus faecium Clades with Different Levels of Ampicillin Resistance.

PubMed

Montealegre, Maria Camila; Roh, Jung Hyeob; Rae, Meredith; Davlieva, Milya G; Singh, Kavindra V; Shamoo, Yousif; Murray, Barbara E

2017-01-01

Ampicillin resistance in Enterococcus faecium is a serious concern worldwide, complicating the treatment of E. faecium infections. Penicillin-binding protein 5 (PBP5) is considered the main ampicillin resistance determinant in E. faecium The three known E. faecium clades showed sequence variations in the pbp5 gene that are associated with their ampicillin resistance phenotype; however, these changes alone do not explain the array of resistance levels observed among E. faecium clinical strains. We aimed to determine if the levels of PBP5 are differentially regulated between the E. faecium clades, with the hypothesis that variations in PBP5 levels could help account for the spectrum of ampicillin MICs seen in E. faecium We studied pbp5 mRNA levels and PBP5 protein levels as well as the genetic environment upstream of pbp5 in 16 E. faecium strains that belong to the different E. faecium clades and for which the ampicillin MICs covered a wide range. Our results found that pbp5 and PBP5 levels are increased in subclade A1 and A2 ampicillin-resistant strains compared to those in clade B and subclade A2 ampicillin-susceptible strains. Furthermore, we found evidence of major clade-associated rearrangements in the region upstream of pbp5, including large DNA fragment insertions, deletions, and single nucleotide polymorphisms, that may be associated with the differential regulation of PBP5 levels between the E. faecium clades. Overall, these findings highlight the contribution of the clade background to the regulation of PBP5 abundance and point to differences in the region upstream of pbp5 as likely contributors to the differential expression of ampicillin resistance. Copyright © 2016 American Society for Microbiology.

Complete sequence of Enterococcus faecium pVEF3 and the detection of an omega-epsilon-zeta toxin-antitoxin module and an ABC transporter.

PubMed

Sletvold, H; Johnsen, P J; Hamre, I; Simonsen, G S; Sundsfjord, A; Nielsen, K M

2008-07-01

Glycopeptide resistant Enterococcus faecium (GREF) persists on Norwegian poultry farms despite the ban on the growth promoter avoparcin. The biological basis for long-term persistence of avoparcin resistance is not fully understood. This study presents the complete DNA sequence of the E. faecium R-plasmid pVEF3 and functional studies of some plasmid-encoded traits (a toxin-antitoxin (TA) system and an ABC transporter) that may be of importance for plasmid persistence. The pVEF3 (63.1 kbp), isolated from an E. faecium strain of poultry origin sampled in Norway in 1999, has 71 coding sequences including the vanA avoparcin/vancomycin resistance encoding gene cluster. pVEF3 encodes the TA system omega-epsilon-zeta, and plasmid stability tests and transcription analysis show that omega-epsilon-zeta is functional in Enterococcus faecalis OGIX, although with decreasing effect over time. The predicted ABC transporter was not found to confer reduced susceptibility to any of the 28 substances tested. The TA system identified in the pVEF-type plasmids may contribute to vanA plasmid persistence on Norwegian poultry farms. However, size and compositional heterogeneity among E. faecium vanA plasmids suggest that additional plasmid maintenance systems in combination with host specific factors and frequent horizontal gene transfer and rearrangement causes the observed plasmid composition and distribution patterns.

Enterocin X, a Novel Two-Peptide Bacteriocin from Enterococcus faecium KU-B5, Has an Antibacterial Spectrum Entirely Different from Those of Its Component Peptides▿

PubMed Central

Hu, Chih-Bo; Malaphan, Wanna; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

2010-01-01

Enterocin X, composed of two antibacterial peptides (Xα and Xβ), is a novel class IIb bacteriocin from Enterococcus faecium KU-B5. When combined, Xα and Xβ display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known. PMID:20418437

The Fibronectin-Binding Protein Fnm Contributes to Adherence to Extracellular Matrix Components and Virulence of Enterococcus faecium

PubMed Central

Somarajan, Sudha R.; La Rosa, Sabina Leanti; Singh, Kavindra V.; Roh, Jung H.; Höök, Magnus

2015-01-01

The interaction between bacteria and fibronectin is believed to play an important role in the pathogenicity of clinically important Gram-positive cocci. In the present study, we identified a gene encoding a predicted fibronectin-binding protein of Enterococcus faecium (fnm), a homologue of Streptococcus pneumoniae pavA, in the genomes of E. faecium strain TX82 and all other sequenced E. faecium isolates. Full-length recombinant Fnm from strain TX82 bound to immobilized fibronectin in a concentration-dependent manner and also appeared to bind collagen type V and laminin, but not other proteins, such as transferrin, heparin, bovine serum albumin, mucin, or collagen IV. We demonstrated that the N-terminal fragment of Fnm is required for full fibronectin binding, since truncation of this region caused a 2.4-fold decrease (P < 0.05) in the adhesion of E. faecium TX82 to fibronectin. Deletion of fnm resulted in a significant reduction (P < 0.001) in the ability of the mutant, TX6128, to bind fibronectin relative to that of the wild-type strain; in situ reconstitution of fnm in the deletion mutant strain restored adherence. In addition, the Δfnm mutant was highly attenuated relative to TX82 (P ≤ 0.0001) in a mixed-inoculum rat endocarditis model. Taken together, these results demonstrate that Fnm affects the adherence of E. faecium to fibronectin and is important in the pathogenesis of experimental endocarditis. PMID:26371130

Transfer of antibiotic resistance from Enterococcus faecium of fermented meat origin to Listeria monocytogenes and Listeria innocua.

PubMed

Jahan, M; Holley, R A

2016-04-01

Listeria monocytogenes is an important foodborne pathogen that can cause infection in children, pregnant women, the immunocompromised and the elderly. Antibiotic resistance in this species would represent a significant public health problem since the organism has a high fatality/case ratio and resistance may contribute to failure of therapeutic treatment. This study was designed to explore whether the in vitro transferability of antibiotic resistance from enterococci to Listeria spp. could occur. It was found that 2/8 Listeria strains were able to acquire tetracycline resistance from Enterococcus faecium. Listeria monocytogenes GLM-2 acquired the resistance determinant tet(M) and additional streptomycin resistance through in vitro mating with Ent. faecium S27 isolated from commercial fermented dry sausage. Similarly, Listeria innocua became more resistant to tetracycline, but the genetic basis for this change was not confirmed. It has been suggested that enterococci may transfer antibiotic resistance genes via transposons to Listeria spp., and this may explain, in part, the origin of their antibiotic resistance. Thus, the presence of enterococci in food should not be ignored since they may actively contribute to enhanced antibiotic resistance of L. monocytogenes and other pathogens. Acquisition of antibiotic resistance by pathogenic bacteria in the absence of antibiotic pressure represents an unquantified threat to human health. In the present work resistance to tetracycline and streptomycin were transferred by nonplasmid-based conjugation from Enterococcus faecium isolated from fermented sausage to Listeria monocytogenes and Listeria innocua. Thus, natural transfer of antibiotic resistance to Listeria strains may occur in the future which reinforces the concern about the safety of enterococcal strains present in foods. © 2016 The Society for Applied Microbiology.

Genomic Analysis of Reduced Susceptibility to Tigecycline in Enterococcus faecium

PubMed Central

Isnard, Christophe; Cosquer, Thibaud; Odhiambo, Arlène; Bucquet, Fiona; Guérin, François; Giard, Jean-Christophe

2014-01-01

Tigecycline (TIG) is approved for use for the treatment of complicated intra-abdominal infections, skin and skin structure infections, as well as pneumonia. Acquired resistance or reduced susceptibility to TIG has been observed in Gram-negative rods, has seldom been reported in Gram-positive organisms, and has not yet been reported in Enterococcus faecium. Using the serial passage method, in vitro mutant AusTig and in vitro mutants HMtig1 and HMtig2 with decreased TIG susceptibility (MICs, 0.25 μg/ml) were obtained from strains E. faecium Aus0004 and HM1070 (MICs, 0.03 μg/ml), respectively. In addition, two vancomycin-resistant E. faecium clinical isolates (EF16 and EF22) with reduced susceptibility to TIG (MICs, 0.5 and 0.25 μg/ml, respectively) were studied. Compared to the wild-type strains, the in vitro mutants also showed an increase in the MICs of other tetracyclines. An efflux mechanism did not seem to be involved in the reduced TIG susceptibility, since the presence of efflux pump inhibitors (reserpine or pantoprazole) did not affect the MICs of TIG. Whole-genome sequencing of AusTig was carried out, and genomic comparison with the Aus0004 genome was performed. Four modifications leading to an amino acid substitution were found. These mutations affected the rpsJ gene (efau004_00094, coding for the S10 protein of the 30S ribosomal subunit), efau004_01228 (encoding a cation transporter), efau004_01636 (coding for a hypothetical protein), and efau004_02455 (encoding the l-lactate oxidase). The four other strains exhibiting reduced TIG susceptibility were screened for the candidate mutations. This analysis revealed that three of them showed an amino acid substitution in the same region of the RpsJ protein. In this study, we characterized for the first time genetic determinants linked to reduced TIG susceptibility in enterococci. PMID:25348531

Genomic analysis of reduced susceptibility to tigecycline in Enterococcus faecium.

PubMed

Cattoir, Vincent; Isnard, Christophe; Cosquer, Thibaud; Odhiambo, Arlène; Bucquet, Fiona; Guérin, François; Giard, Jean-Christophe

2015-01-01

Tigecycline (TIG) is approved for use for the treatment of complicated intra-abdominal infections, skin and skin structure infections, as well as pneumonia. Acquired resistance or reduced susceptibility to TIG has been observed in Gram-negative rods, has seldom been reported in Gram-positive organisms, and has not yet been reported in Enterococcus faecium. Using the serial passage method, in vitro mutant AusTig and in vitro mutants HMtig1 and HMtig2 with decreased TIG susceptibility (MICs, 0.25 μg/ml) were obtained from strains E. faecium Aus0004 and HM1070 (MICs, 0.03 μg/ml), respectively. In addition, two vancomycin-resistant E. faecium clinical isolates (EF16 and EF22) with reduced susceptibility to TIG (MICs, 0.5 and 0.25 μg/ml, respectively) were studied. Compared to the wild-type strains, the in vitro mutants also showed an increase in the MICs of other tetracyclines. An efflux mechanism did not seem to be involved in the reduced TIG susceptibility, since the presence of efflux pump inhibitors (reserpine or pantoprazole) did not affect the MICs of TIG. Whole-genome sequencing of AusTig was carried out, and genomic comparison with the Aus0004 genome was performed. Four modifications leading to an amino acid substitution were found. These mutations affected the rpsJ gene (efau004_00094, coding for the S10 protein of the 30S ribosomal subunit), efau004_01228 (encoding a cation transporter), efau004_01636 (coding for a hypothetical protein), and efau004_02455 (encoding the l-lactate oxidase). The four other strains exhibiting reduced TIG susceptibility were screened for the candidate mutations. This analysis revealed that three of them showed an amino acid substitution in the same region of the RpsJ protein. In this study, we characterized for the first time genetic determinants linked to reduced TIG susceptibility in enterococci. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Enterococcus faecium WEFA23 from infant lessens high-fat-diet-induced hyperlipidemia via cholesterol 7-alpha-hydroxylase gene by altering the composition of gut microbiota in rats.

PubMed

Huang, Fuqing; Zhang, Fen; Xu, Di; Zhang, Zhihong; Xu, Feng; Tao, Xueying; Qiu, Liang; Wei, Hua

2018-06-20

Enterococcus faecium WEFA23 is a potential probiotic strain from Chinese infants with the ability to decrease cholesterol levels. Aiming to explore the mechanism of E. faecium WEFA23 in lowering cholesterol in vivo, we examined the gene transcriptions related to cholesterol metabolism, the composition of bile acids in feces, the synthesis of trimethylamine N-oxide (TMAO) in liver, and the composition of the gut microbiota of rats. We found that E. faecium WEFA23 enhanced the synthesis of bile acids by promoting cholesterol excretion, upregulating the genes transcript level relevant to cholesterol decomposition and transportation, and downregulating the genes involved in cholesterol synthesis. In addition, E. faecium WEFA23 not only downregulated the transcript levels of farnesoid X receptor and fibroblast growth factor 15 as well as flavin-containing monooxygenase 3, but also decreased the TMAO production followed by increasing the CYP7A1 transcript level. Furthermore, when orally administered to rats for 35 d, E. faecium WEFA23 improved the gut microbiota diversity of rats fed a high-fat diet. Therein, the ratio of Bacteroidetes to Firmicutes and the abundance of Rikenellaceae increased, whereas the number of Veillonellaceae decreased. These results suggest that reduction of cholesterol level by E. faecium WEFA23 might be related to the changes in the gut microbiota. Our finding provides important information on lowering cholesterol by E. faecium and reveals that Enterococcus spp. might have the potential to decrease the TMAO level. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Antimicrobial resistance of Enterococcus faecium strains isolated from commercial probiotic products used in cattle and swine.

PubMed

Amachawadi, Raghavendra G; Giok, Felicia; Shi, Xiaorong; Soto, Jose; Narayanan, Sanjeev K; Tokach, Mike D; Apley, Mike D; Nagaraja, T G

2018-04-03

Probiotics, an antibiotic alternative, are widely used as feed additives for performance benefits in cattle and swine production systems. Among bacterial species contained in probiotics, Enterococcus faecium is common. Antimicrobial resistance (AMR), particularly multidrug resistance, is a common trait among enterococci because of their propensity to acquire resistance and horizontally transfer AMR genes. Also, E. faecium is an opportunistic pathogen, and in the United States, it is the second most common nosocomial pathogen. There has been no published study on AMR and virulence potential in E. faecium contained in probiotic products used in cattle and swine in the United States. Therefore, our objectives were to determine phenotypic susceptibilities or resistance to antimicrobials, virulence genes (asa1, gelE, cylA, esp, and hyl) and assess genetic diversity of E. faecium isolated from commercial products. Twenty-two commercially available E. faecium-based probiotic products used in cattle (n = 13) and swine (n = 9) were procured and E. faecium was isolated and species confirmed. Antimicrobial susceptibility testing to determine minimum inhibitory concentrations was done by micro-broth dilution method using National Antimicrobial Resistance Monitoring Systems Gram-positive Sensititre panel plate (CMV3AGPF), and categorization of strains as susceptible or resistant was as per Clinical Laboratory and Standards Institute's guidelines. E. faecium strains from 7 products (3 for swine and 4 for cattle) were pan-susceptible to the 16 antimicrobials tested. Strains from 15 products (6 for swine and 9 for cattle) exhibited resistance to at least one antimicrobial and a high proportion of strains was resistant to lincomycin (10/22), followed by tetracycline (4/22), daptomycin (4/22), ciprofloxacin (4/22), kanamycin (3/22), and penicillin (2/22). Four strains were multidrug resistant, with resistant phenotypes ranging from 3 to 6 antimicrobials or class. None of the E

Antimicrobial activity and safety evaluation of Enterococcus faecium KQ 2.6 isolated from peacock feces.

PubMed

Zheng, Wei; Zhang, Yu; Lu, Hui-Min; Li, Dan-Ting; Zhang, Zhi-Liang; Tang, Zhen-Xing; Shi, Lu-E

2015-05-12

The objective of this paper was to study antimicrobial activity and safety of Enterococcus faecium KQ 2.6 (E. faecium KQ 2.6) isolated from peacock feces. Agar well diffusion method was adopted in antimicrobial activity assay. Disk diffusion test was used to determine the antibiotic resistance. The identification and virulence potential of E. faecium KQ 2.6 were investigated using PCR amplification. The results indicated that cell free supernatant (CFS) of the strain had the good antimicrobial activity against selected gram-positive and gram-negative bacteria. The biochemical characteristics of antimicrobial substances were investigated. The results indicated that the antimicrobial substances were still active after treatment with catalase and proteinase, respectively. Moreover, the stability of antimicrobial substances did not change after heat treatment at 40, 50, 60, 70 and 80°C for 30 min, respectively. The activity of antimicrobial substances remained stable at 4 and -20°C after long time storage. The antimicrobial activity of CFS was compared with that of the buffer with similar strength and pH. The inhibitory zone of the buffer was apparently smaller than that of CFS, which meant that the acid in CFS was not the only factor that was contributed to antibacterial activity of CFS. The antibiotic resistance and virulence potential were evaluated using disk diffusion test and PCR amplification. The results showed that E. faecium KQ 2.6 did not harbor any tested virulence genes such as gelE, esp, asa1, cylA, efaA and hyl. It was susceptible to most of tested antibiotics except for vancomycin and polymyxin B. E. faecium KQ 2.6 may be used as bio-preservative cultures for the production of fermented foods.

High-resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms.

PubMed

Tong, Steven Y C; Xie, Shirley; Richardson, Leisha J; Ballard, Susan A; Dakh, Farshid; Grabsch, Elizabeth A; Grayson, M Lindsay; Howden, Benjamin P; Johnson, Paul D R; Giffard, Philip M

2011-01-01

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.

Effects of two novel amino acid substitutions on the penicillin binding properties of the PBP5 C‑terminal from Enterococcus faecium.

PubMed

Zhou, Chengjiang; Niu, Haiying; Yu, Hui; Zhou, Lishe; Wang, Zhanli

2015-10-01

The low‑affinity penicillin‑binding protein (PBP)5 is responsible for resistance to β‑lactam antibiotics in Enterococcus faecium. (E. faecium). In order to evaluate more fully the potential of this species for the development of resistance to β-lactam antibiotics, the present study aimed to examine the extent of penicillin-binding protein (PBP) variations in a collection of clinical E. faecium isolates. In the present study, the C‑terminal domain of PBP5 (PBP5‑CD) of 13 penicillin‑resistant clinical isolates of E. faecium were sequenced and the correlation between penicillin resistance and particular amino acid changes were analyzed. The present study identified for the first time, to the best of our knowledge, two novel substitutions (Tyr460Phe and Ala462Thr or Val462Thr) of E. faecium PBP5‑CD. The covalent interaction between penicillin and PBP5‑CD was also investigated using homology modeling and molecular docking methods. The theoretical calculation revealed that Phe460 and Thr462 were involved in penicillin binding, suggesting that substitutions at these positions exert effects on the affinity for penicillin, and this increased affinity translates into lower resistance in vitro.

A Vaccine Approach for the Prevention of Infections by Multidrug-resistant Enterococcus faecium*

PubMed Central

Kodali, Srinivas; Vinogradov, Evgeny; Lin, Fiona; Khoury, Nancy; Hao, Li; Pavliak, Vilo; Jones, C. Hal; Laverde, Diana; Huebner, Johannes; Jansen, Kathrin U.; Anderson, Annaliesa S.; Donald, Robert G. K.

2015-01-01

The incidence of multidrug-resistant Enterococcus faecium hospital infections has been steadily increasing. With the goal of discovering new vaccine antigens, we systematically fractionated and purified four distinct surface carbohydrates from E. faecium endocarditis isolate Tx16, shown previously to be resistant to phagocytosis in the presence of human serum. The two most abundant polysaccharides consist of novel branched heteroglycan repeating units that include signature sugars altruronic acid and legionaminic acid, respectively. A minor high molecular weight polysaccharide component was recognized as the fructose homopolymer levan, and a glucosylated lipoteichoic acid (LTA) was identified in a micellar fraction. The polysaccharides were conjugated to the CRM197 carrier protein, and the resulting glycoconjugates were used to immunize rabbits. Rabbit immune sera were evaluated for their ability to kill Tx16 in opsonophagocytic assays and in a mouse passive protection infection model. Although antibodies raised against levan failed to mediate opsonophagocytic killing, the other glycoconjugates induced effective opsonic antibodies, with the altruronic acid-containing polysaccharide antisera showing the greatest opsonophagocytic assay activity. Antibodies directed against either novel heteroglycan or the LTA reduced bacterial load in mouse liver or kidney tissue. To assess antigen prevalence, we screened a diverse collection of blood isolates (n = 101) with antibodies to the polysaccharides. LTA was detected on the surface of 80% of the strains, and antigens recognized by antibodies to the two major heteroglycans were co-expressed on 63% of these clinical isolates. Collectively, these results represent the first steps toward identifying components of a glycoconjugate vaccine to prevent E. faecium infection. PMID:26109072

Modification of IgE binding to αS1-casein by proteolytic activity of Enterococcus faecium isolated from Iranian camel milk samples.

PubMed

Kordesedehi, Reihane; Taheri-Kafrani, Asghar; Rabbani-Khorasgani, Mohammad; Kazemi, Rezvan; Mutangadura, Daniel; Haertle, Thomas

2018-06-20

Milk is a perfect source of nutrients for neonates. When breast feeding cannot be done, an infant's alimentation is usually initiated to cow's milk, among the primary foods. It has been reported that about 2.5% of juveniles under the age of 3 years manifest allergic reactions to cow's milk proteins. Among the cow's milk proteins, casein fractions are considered as the strongest allergenic proteins. The proteolytic enzymes of lactic acid bacteria (LAB), during fermentation of dairy products, can break down milk proteins especially caseins and subsequently reduce the immune reactivity of allergenic proteins. In this research, raw bovine and camel milk samples were screened for cocci LAB strains and after isolation, their proteolytic activity against bovine milk caseins were evaluated by SDS-PAGE and RP-HPLC. The potential of cocci LAB strains on α S1 -casein degradation and their potential to break down the principle allergenic epitopes of this protein was detected using indirect competitive ELISA. Molecular identification of the best proteolytic strain was fulfilled by 16S rDNA fragment sequencing with universal primers. The obtained results demonstrated that Enterococcus faecium isolated from raw camel milk samples was the most efficient isolate in hydrolyzing Na-caseinate and α S1 -casein. Hydrolysated α S1 -casein by Enterococcus faecium was also less recognized by IgE of bovine milk allergic patients' sera in comparison with native α S1 -casein. It has been proposed that Enterococcus faecium could be an efficient strain in allergenicity reduction of cow's milk proteins. So it could be an excellent candidate to be potentially used in dairy industries. Copyright © 2018 Elsevier B.V. All rights reserved.

Five Genes Encoding Surface-Exposed LPXTG Proteins Are Enriched in Hospital-Adapted Enterococcus faecium Clonal Complex 17 Isolates▿

PubMed Central

Hendrickx, Antoni P. A.; van Wamel, Willem J. B.; Posthuma, George; Bonten, Marc J. M.; Willems, Rob J. L.

2007-01-01

Most Enterococcus faecium isolates associated with hospital outbreaks and invasive infections belong to a distinct genetic subpopulation called clonal complex 17 (CC17). It has been postulated that the genetic evolution of CC17 involves the acquisition of various genes involved in antibiotic resistance, metabolic pathways, and virulence. To gain insight into additional genes that may have favored the rapid emergence of this nosocomial pathogen, we aimed to identify surface-exposed LPXTG cell wall-anchored proteins (CWAPs) specifically enriched in CC17 E. faecium. Using PCR and Southern and dot blot hybridizations, 131 E. faecium isolates (40 CC17 and 91 non-CC17) were screened for the presence of 22 putative CWAP genes identified from the E. faecium TX0016 genome. Five genes encoding LPXTG surface proteins were specifically enriched in E. faecium CC17 isolates. These five LPXTG surface protein genes were found in 28 to 40 (70 to 100%) of CC17 and in only 7 to 24 (8 to 26%) of non-CC17 isolates (P < 0.05). Three of these CWAP genes clustered together on the E. faecium TX0016 genome, which may comprise a novel enterococcal pathogenicity island covering E. faecium contig 609. Expression at the mRNA level was demonstrated, and immunotransmission electron microscopy revealed an association of the five LPXTG surface proteins with the cell wall. Minimal spanning tree analysis based on the presence and absence of 22 CWAP genes revealed grouping of all 40 CC17 strains together with 18 hospital-derived but evolutionary unrelated non-CC17 isolates in a distinct CWAP-enriched cluster, suggesting horizontal transfer of CWAP genes and a role of these CWAPs in hospital adaptation. PMID:17873043

Enterococcus faecium Biofilm Formation: Identification of Major Autolysin AtlAEfm, Associated Acm Surface Localization, and AtlAEfm-Independent Extracellular DNA Release

PubMed Central

Paganelli, Fernanda L.; Willems, Rob J. L.; Jansen, Pamela; Hendrickx, Antoni; Zhang, Xinglin; Bonten, Marc J. M.; Leavis, Helen L.

2013-01-01

ABSTRACT Enterococcus faecium is an important multidrug-resistant nosocomial pathogen causing biofilm-mediated infections in patients with medical devices. Insight into E. faecium biofilm pathogenesis is pivotal for the development of new strategies to prevent and treat these infections. In several bacteria, a major autolysin is essential for extracellular DNA (eDNA) release in the biofilm matrix, contributing to biofilm attachment and stability. In this study, we identified and functionally characterized the major autolysin of E. faecium E1162 by a bioinformatic genome screen followed by insertional gene disruption of six putative autolysin genes. Insertional inactivation of locus tag EfmE1162_2692 resulted in resistance to lysis, reduced eDNA release, deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and significant chaining compared to that of the wild type. Therefore, locus tag EfmE1162_2692 was considered the major autolysin in E. faecium and renamed atlAEfm. In addition, AtlAEfm was implicated in cell surface exposure of Acm, a virulence factor in E. faecium, and thereby facilitates binding to collagen types I and IV. This is a novel feature of enterococcal autolysins not described previously. Furthermore, we identified (and localized) autolysin-independent DNA release in E. faecium that contributes to cell-cell interactions in the atlAEfm mutant and is important for cell separation. In conclusion, AtlAEfm is the major autolysin in E. faecium and contributes to biofilm stability and Acm localization, making AtlAEfm a promising target for treatment of E. faecium biofilm-mediated infections. PMID:23592262

Pyrosequencing-based comparative genome analysis of the nosocomial pathogen Enterococcus faecium and identification of a large transferable pathogenicity island

PubMed Central

2010-01-01

Background The Gram-positive bacterium Enterococcus faecium is an important cause of nosocomial infections in immunocompromized patients. Results We present a pyrosequencing-based comparative genome analysis of seven E. faecium strains that were isolated from various sources. In the genomes of clinical isolates several antibiotic resistance genes were identified, including the vanA transposon that confers resistance to vancomycin in two strains. A functional comparison between E. faecium and the related opportunistic pathogen E. faecalis based on differences in the presence of protein families, revealed divergence in plant carbohydrate metabolic pathways and oxidative stress defense mechanisms. The E. faecium pan-genome was estimated to be essentially unlimited in size, indicating that E. faecium can efficiently acquire and incorporate exogenous DNA in its gene pool. One of the most prominent sources of genomic diversity consists of bacteriophages that have integrated in the genome. The CRISPR-Cas system, which contributes to immunity against bacteriophage infection in prokaryotes, is not present in the sequenced strains. Three sequenced isolates carry the esp gene, which is involved in urinary tract infections and biofilm formation. The esp gene is located on a large pathogenicity island (PAI), which is between 64 and 104 kb in size. Conjugation experiments showed that the entire esp PAI can be transferred horizontally and inserts in a site-specific manner. Conclusions Genes involved in environmental persistence, colonization and virulence can easily be aquired by E. faecium. This will make the development of successful treatment strategies targeted against this organism a challenge for years to come. PMID:20398277

Growth inhibition of heat-injured Enterococcus faecium by oligophosphates in a cured meat model.

PubMed

Houben, J H; Tjeerdsma-van Bokhoven, J L M

2004-12-01

Cells of two heat-resistant strains of Enterococcus faecium were heated and incubated in meat suspensions containing curing ingredients. The concentrations of the curing ingredients were those frequently used for pasteurized ham-type products, except that the concentrations of the oligophosphates (triphosphate and diphosphate) varied. Heating tests at 69 degrees C were performed with inoculated meat suspensions in heat-sealed plastic pouches. Numbers of bacteria were counted immediately after heating and in parallel series of heated pouches incubated at 37 degrees C. Plating was performed in Tryptone Dextrose Yeast Meat Peptonised Milk Agar (TDYMP); in TDYMP Agar to which the curing ingredients were added; and in TDYMP Agar to which the curing ingredients except oligophosphates were added. The inclusion of oligophosphates in the heating medium increased the heat-injury sustained by the E. faecium cells, and in combination with rather severe heat treatment even completely blocked the growth of surviving organisms in the meat suspension incubated at 37 degrees C. The presence of oligophosphates in the culture medium TDYMP Agar severely reduced the counts of freshly heated cells; however, this effect disappeared after repair and growth of the surviving organisms in the meat suspension.

Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50▿

PubMed Central

Basanta, Antonio; Herranz, Carmen; Gutiérrez, Jorge; Criado, Raquel; Hernández, Pablo E.; Cintas, Luis M.

2009-01-01

A segregationally stable expression and secretion vector for Saccharomyces cerevisiae, named pYABD01, was constructed by cloning the yeast gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s) into the S. cerevisiae high-copy-number expression vector pYES2. The structural genes of the two leaderless peptides of enterocin L50 (EntL50A and EntL50B) from Enterococcus faecium L50 were cloned, separately (entL50A or entL50B) and together (entL50AB), into pYABD01 under the control of the galactose-inducible promoter PGAL1. The generation of recombinant S. cerevisiae strains heterologously expressing and secreting biologically active EntL50A and EntL50B demonstrates the suitability of the MFα1s-containing vector pYABD01 to direct processing and secretion of these antimicrobial peptides through the S. cerevisiae Sec system. PMID:19218405

Enterococcus faecium LKE12 Cell-Free Extract Accelerates Host Plant Growth via Gibberellin and Indole-3-Acetic Acid Secretion.

PubMed

Lee, Ko-Eun; Radhakrishnan, Ramalingam; Kang, Sang-Mo; You, Young-Hyun; Joo, Gil-Jae; Lee, In-Jung; Ko, Jae-Hwan; Kim, Jin-Ho

2015-09-01

The use of microbial extracts containing plant hormones is a promising technique to improve crop growth. Little is known about the effect of bacterial cell-free extracts on plant growth promotion. This study, based on phytohormonal analyses, aimed at exploring the potential mechanisms by which Enterococcus faecium LKE12 enhances plant growth in oriental melon. A bacterial strain, LKE12, was isolated from soil, and further identified as E. faecium by 16S rDNA sequencing and phylogenetic analysis. The plant growth-promoting ability of an LKE12 bacterial culture was tested in a gibberellin (GA)-deficient rice dwarf mutant (waito-C) and a normal GA biosynthesis rice cultivar (Hwayongbyeo). E. faecium LKE12 significantly improved the length and biomass of rice shoots in both normal and dwarf cultivars through the secretion of an array of gibberellins (GA1, GA3, GA7, GA8, GA9, GA12, GA19, GA20, GA24, and GA53), as well as indole-3-acetic acid (IAA). To the best of our knowledge, this is the first study indicating that E. faecium can produce GAs. Increases in shoot and root lengths, plant fresh weight, and chlorophyll content promoted by E. faecium LKE12 and its cell-free extract inoculated in oriental melon plants revealed a favorable interaction of E. faecium LKE12 with plants. Higher plant growth rates and nutrient contents of magnesium, calcium, sodium, iron, manganese, silicon, zinc, and nitrogen were found in cell-free extract-treated plants than in control plants. The results of the current study suggest that E. faecium LKE12 promotes plant growth by producing GAs and IAA; interestingly, the exogenous application of its cell-free culture extract can be a potential strategy to accelerate plant growth.

Survey of Virulence Determinants among Vancomycin Resistant Enterococcus faecalis and Enterococcus faecium Isolated from Clinical Specimens of Hospitalized Patients of North west of Iran

PubMed Central

Sharifi, Yaeghob; Hasani, Alka; Ghotaslou, Reza; Varshochi, Mojtaba; Hasani, Akbar; Aghazadeh, Mohammad; Milani, Morteza

2012-01-01

Recent data indicates an increasing rate of vancomycin resistance in clinical enterococcal isolates worldwide. The nosocomial enterococci are likely to harbor virulence elements that increase their ability to colonize hospitalized patients. The aim of this study was to characterize virulence determinants in vancomycin-resistant enterococci (VRE) obtained from various clinical sources. During the years 2008 to 2010, a total of 48 VRE isolates were obtained from three University teaching hospitals in Northwest, Iran. Initially, phenotypic speciation was done and minimum inhibitory concentrations (MICs) of vancomycin were determined by agar dilution method and E-test. Then, species identification and resistance genotypes along with detection of virulence genes (asa1, esp, gelE, ace and cpd) of the isolates were performed by multiplex PCR. Thirty eight isolates were identified as vancomycin-resistant Enterococcus faecium (VREfm) and ten as E. faecalis (VREfs). Irrespective of the species, vanA gene (89.58%) was dominant and three phenotypically vancomycin susceptible E. faecium isolates carried the vanB gene. Among virulence genes investigated, the esp was found in 27(71%) VREfm strains, but did not in any VREfs. Other virulence determinants were highly detected in VREfs strains. Our data indicate a high prevalence of E. faecium harboring vancomycin resistance with vanA genotype and the two VRE species displayed different virulence genes. PMID:22582098

Multi-antibiotic resistant and putative virulence gene signatures in Enterococcus species isolated from pig farms environment.

PubMed

Beshiru, Abeni; Igbinosa, Isoken H; Omeje, Faith I; Ogofure, Abraham G; Eyong, Martin M; Igbinosa, Etinosa O

2017-03-01

The continuous misuse of antimicrobials in food animals both orally and subcutaneously as therapeutic and prophylactic agents to bacterial infections could be detrimental and contribute to the dissemination of resistant clones in livestock production. The present study was carried out to determine the antibiogram and virulence gene characteristics of Enterococcus species from pig farms. A total of 300 faecal samples were obtained from two pig farms in Benin City between February and July 2016. Standard culture-based and polymerase chain reaction (PCR) assay were adopted in the detection and characterization of the Enterococcus species. Antimicrobial susceptibility profile was determined using disc diffusion method. A total of 268 enterococci isolates were recovered from both farms investigated. In Farm A, 94/95 (99%) of E. faecalis isolates were resistant to clindamycin; while 23/25 (92%) of E. faecium isolates were resistant to clindamycin. In farm B, all E. faecalis isolates 119/119 (100%) were resistant to clindamycin; while 26/29 (90%) of E. faecium isolates were resistant to clindamycin. Virulence gene detected in the enterococci isolates includes aggregation (asa1) [Farm A (E. faecalis 66%, E. faecium 76%), Farm B (E. faecalis 71%, E. faecium 13%)] and others. Multidrug resistant profile of the isolates revealed that 17/95 (18%) of E. faecalis and 3/25 (12%) of E. faecium isolates from Farm A as well as, 16/119 (14%) of E. faecalis and 5/29 (17%) of E. faecium isolates from Farm B were resistant to CLI R , PEN R , ERY R , GEN R , TET R , MEM R , KAN R , and PTZ R . The high level of resistance observed in the study and their virulence gene signatures, calls for effective environmental monitoring to circumvent the environmental dissemination of resistant pathogenic clones. Thus environmental hygiene should be provided to food animals to prevent the proliferation and spread of resistant bacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

Polyclonal emergence of vanA vancomycin-resistant Enterococcus faecium in Australia.

PubMed

van Hal, Sebastiaan J; Espedido, Björn A; Coombs, Geoffrey W; Howden, Benjamin P; Korman, Tony M; Nimmo, Graeme R; Gosbell, Iain B; Jensen, Slade O

2017-04-01

To investigate the genetic context associated with the emergence of vanA VRE in Australia. The whole genomes of 18 randomly selected vanA -positive Enterococcus faecium patient isolates, collected between 2011 and 2013 from hospitals in four Australian capitals, were sequenced and analysed. In silico typing and transposon/plasmid assembly revealed that the sequenced isolates represented (in most cases) different hospital-adapted STs and were associated with a variety of different Tn 1546 variants and plasmid backbone structures. The recent emergence of vanA VRE in Australia was polyclonal and not associated with the dissemination of a single 'dominant' ST or vanA -encoding plasmid. Interestingly, the factors contributing to this epidemiological change are not known and future studies may need to consider investigation of potential community sources. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Analysis of Clonality and Antibiotic Resistance among Early Clinical Isolates of Enterococcus faecium in the United States

PubMed Central

Galloway-Peña, Jessica R.; Nallapareddy, Sreedhar R.; Arias, Cesar A.; Eliopoulos, George M.; Murray, Barbara E.

2009-01-01

Background The Enterococcus faecium genogroup, referred to as clonal complex 17 (CC17), seems to possess multiple determinants that increase its ability to survive and cause disease in nosocomial environments. Methods Using 53 clinical and geographically diverse US E. faecium isolates dating from 1971 to 1994 we determined the multi-locus sequence type, the presence of 16 putative virulence genes (hylEfm, espEfm and fms genes), resistance to ampicillin (AMPR), vancomycin (VANR) and high-levels of gentamicin and streptomycin. Results Overall, 16 different sequence types (STs), mostly CC17 isolates, were identified in 9 different regions of the US. The earliest CC17 isolates were part of an outbreak in 1982 in Richmond, VA. Characteristics of CC17 isolates included increases in AMPR, the presence of hylEfm and espEfm, emergence of VANR and the presence of at least 13/14 fms genes. Eight out of forty-one of the early AMPR isolates, however, were not within CC17. Conclusions While not all early US AMPR isolates were clonally related, E. faecium CC17 isolates have been circulating in the US since at least 1982 and appear to have progressively acquired additional virulence and antibiotic resistance determinants, perhaps explaining the recent success of this species in the hospital environment. PMID:19821720

Co-colonization of vanA and vanB Enterococcus faecium of clonal complex 17 in a patient with bacteremia due to vanA E. faecium.

PubMed

Seol, Chang Ahn; Park, Jeong Su; Sung, Heungsup; Kim, Mi-Na

2014-06-01

A 53-year-old Vietnamese man with liver cirrhosis was transferred from a Vietnamese hospital to our tertiary care hospital in Korea in order to undergo a liver transplantation. Bacteremia due to vanA Enterococcus faecium was diagnosed, and stool surveillance cultures for vancomycin-resistant enterococci (VRE) were positive for both vanA and vanB E. faecium. Pulsed-field gel electrophoresis analysis revealed that the 2 vanA VRE isolates from the blood and stool were clonal, but the vanB VRE was unrelated to the vanA VRE. vanA and vanB VRE were ST64 and ST18, single-allele variations of clonal complex 17, respectively. This is the first case report of vanA VRE bacteremia in a Vietnamese patient and demonstrates the reemergence of vanB VRE since a single outbreak occurred 15years ago in Korea. The reemergence of vanB VRE emphasizes the importance of VRE genotyping to prevent the spread of new VRE strains. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification of VanN-type vancomycin resistance in an Enterococcus faecium isolate from chicken meat in Japan.

PubMed

Nomura, Takahiro; Tanimoto, Koichi; Shibayama, Keigo; Arakawa, Yoshichika; Fujimoto, Shuhei; Ike, Yasuyoshi; Tomita, Haruyoshi

2012-12-01

Five VanN-type vancomycin-resistant Enterococcus faecium strains were isolated from a sample of domestic chicken meat in Japan. All isolates showed low-level resistance to vancomycin (MIC, 12 mg/liter) and had the same pulsed-field gel electrophoresis profile. The vancomycin resistance was encoded on a large plasmid (160 kbp) and was expressed constitutively. The VanN-type resistance operon was identical to the first resistance operon to be reported, with the exception of a 1-bp deletion in vanT(N) and a 1-bp substitution in vanS(N).

Intestinal microbiota and oral administration of Enterococcus faecium associated with the growth performance of new-born piglets.

PubMed

Wang, Y B; Du, W; Fu, A K; Zhang, X P; Huang, Y; Lee, K H; Yu, K; Li, W F; Li, Y L

2016-09-01

The oral administration of Enterococcus faecium EF1 to new-born suckling and weaning piglets along with their growth performances and intestinal microbiota was investigated in this study. Twenty-four new-born piglets were initially divided into 2 groups. The probiotics group received 2 ml of 10% sterilised skimmed milk by oral gavage supplemented with 6×10(8) cfu/ml viable E. faecium EF1 at the first, the third and the fifth day after birth, while the control group received 2 ml of 10% sterilised skimmed milk without probiotics at the same time. Results showed that oral administration of E. faecium EF1 was associated with a remarkable increase on the body weight of piglets for both suckling and weaning periods, by 30.73% (P<0.01) and 320.84% (P<0.01), and also decreased the diarrhoea rate, by 43.21% (P<0.05) and 71.42% (P<0.05), respectively. In addition, 454-pyrosequencing analysis revealed that there was no significant difference in the intestinal microbial diversity of the suckling piglets between the two groups; nevertheless, when compared to the control group, the relative abundance of Firmicutes in the probiotics group was substantially augmented, while the relative abundance of Proteobacteria, Bacteroidetes and Fusobacteria diminished. However, results indicated that oral administration of E. faecium EF1 did not have any influence on the relative abundance of Firmicutes in weaning piglets rather than increasing the relative abundance of Bacteroidetes and decreasing the relative abundance of Proteobacteria. Furthermore, at the level of the Firmicutes phylum, the relative abundance of Lactobacillales in the probiotic group increased significantly. These findings suggest that oral administration of E. faecium EF1 to new-born piglets could improve the growth performance and intestinal microbiota of piglets for both suckling and weaning periods.

Use of bacteriocin-producing, probiotic strain Enterococcus faecium AL41 to control intestinal microbiota in farm ostriches.

PubMed

Lauková, A; Kandričáková, A; Ščerbová, J

2015-06-01

Probiotic enterococci can produce bacteriocins. Enterococcus faecium AL41 is an Enterocin M-producing, probiotic strain which has previously shown beneficial effect in broiler chickens. In this study, it was used to control intestinal microbiota in farm ostriches in a 42-day experiment with an experimental group (EG, 40 ostriches) and a control group (CG, 46). In addition to feed mixture, the ostriches in EG received Ent. faecium AL41 (10(9) CFU ml(-1); by rifampicin-marked variant) 400 μl per animal per day in their drinking water for 21 days. Sampling was carried out at the start of the experiment (at day 0/1), at day 21 (after 21 days of AL41 application) and at day 42 (21 days after AL41 cessation). Faeces (mixture, n = 6) were treated using the standard microbiological dilution method and cultivated on selective media (ISO). The highest count of AL41 was found at day 42. Its identity was confirmed with PCR and Maldi-Tof. The ostriches were free of Salmonella and Campylobacter cells. At day 21, antimicrobial effect was demonstrated by significant reduction in coagulase-positive and negative staphylococci in EG compared to CG (P < 0·001) and coliforms, Enterobacteria and Pseudomonas-like bacteria (P < 0·001). We conclude that AL41 can be used to control intestinal microbiota in farm ostriches. Significance and impact of the study: Ostriches are excellent for high intensity farming in a wide range of climates, requiring only limited space and giving high yields per hectare. They are reared mainly for their meat. Although adult birds possess quite good immunity, young birds can be threatened by spoilage bacteria, especially when they are transferred from the nests to the farm area. Based on our previous results related to the beneficial effect of bacteriocin-producing, probiotic strain Enterococcus faecium AL41 in poultry or rabbits, we decided to test its ability to control intestinal microbiota in farming ostriches which has never been tested previously. �

A functional collagen adhesin gene, acm, in clinical isolates of Enterococcus faecium correlates with the recent success of this emerging nosocomial pathogen.

PubMed

Nallapareddy, Sreedhar R; Singh, Kavindra V; Okhuysen, Pablo C; Murray, Barbara E

2008-09-01

Enterococcus faecium recently evolved from a generally avirulent commensal into a multidrug-resistant health care-associated pathogen causing difficult-to-treat infections, but little is known about the factors responsible for this change. We previously showed that some E. faecium strains express a cell wall-anchored collagen adhesin, Acm. Here we analyzed 90 E. faecium isolates (99% acm(+)) and found that the Acm protein was detected predominantly in clinically derived isolates, while the acm gene was present as a transposon-interrupted pseudogene in 12 of 47 isolates of nonclinical origin. A highly significant association between clinical (versus fecal or food) origin and collagen adherence (P faecium infections showed reactivity with recombinant Acm, while only 4 of 30 community and hospitalized patient control group sera reacted (P faecium endocarditis patient sera. Although pulsed-field gel electrophoresis indicated that multiple strains expressed collagen adherence, multilocus sequence typing demonstrated that the majority of collagen-adhering isolates, as well as 16 of 17 endocarditis isolates, are part of the hospital-associated E. faecium genogroup referred to as clonal complex 17 (CC17), which has emerged globally. Taken together, our findings support the hypothesis that Acm has contributed to the emergence of E. faecium and CC17 in nosocomial infections.

Probiotic attributes, antioxidant, anti-inflammatory and neuromodulatory effects of Enterococcus faecium CFR 3003: in vitro and in vivo evidence.

PubMed

Divyashri, G; Krishna, G; Muralidhara; Prapulla, S G

2015-12-01

Accumulating evidence suggests that probiotic bacteria play a vital role in modulating various aspects integral to the health and well-being of humans. In the present study, probiotic attributes and the antioxidant, anti-inflammatory and neuromodulatory potential of Enterococcus faecium CFR 3003 were investigated by employing suitable model systems. E. faecium exhibited robust resistance to gastrointestinal stress conditions as it could withstand acid stress at pH 1.5, 2 and 3. The bacterium also survived at a bile salt concentration of 0.45 %, and better tolerance was observed towards pepsin and trypsin. E. faecium produced lactic acid as a major metabolic product, followed by butyric acid. Lyophilized cell-free supernatant (LCS) of E. faecium exhibited significant antioxidant capacity evaluated against 1,1-diphenyl-2-picryl-hydrazyl, ascorbate auto-oxidation, oxygen radical absorbance and reducing power. Interestingly, E. faecium, Lactobacillus rhamnosus GG MTCC 1408 and LCS showed a significant anti-inflammatory effect by negatively modulating TNF-α production and upregulating IL-10 levels in LPS-stimulated macrophage cell lines. In an in vivo mice model, the propensity of probiotic supplements to modulate endogenous oxidative markers and redox status in brain regions was assessed. Young mice provided with oral supplements (daily for 28 days) of E. faecium and L. rhamnosus exhibited diminished oxidative markers in the brain and enhanced activities of antioxidant enzymes with a concomitant increase in γ-aminobutyric acid and dopamine levels. Collectively, our findings clearly suggest the propensity of these bacteria to protect against tissue damage mediated through free radicals and inflammatory cytokines. Although the underlying molecular mechanisms need further studies, it is tempting to speculate that probiotics confer a neuroprotective advantage in vivo against oxidative damage-mediated neurodegenerative conditions.

Isolation and purification of two bacteriocins 3D produced by Enterococcus faecium with inhibitory activity against Listeria monocytogenes.

PubMed

Bayoub, Kaoutar; Mardad, Ilham; Ammar, Emna; Serrano, Aurelio; Soukri, Abdelaziz

2011-02-01

Strain 3D, isolated from fermented traditional Moroccan dairy product, and identified as Enterococcus faecium, was studied for its capability to produce two bacteriocins acting against Listeria monocytogenes. Bacteriocins 3 Da and 3Db were heat stable inactivated by proteinase K, pepsin, and trypsin but not when treated with catalase. The evidenced bacteriocins were stable in a wide pH range from 2 to 11 and bactericidal activity was kept during storage at 4°C. However, the combination of temperature and pH exhibited a stability of the bacteriocins. RP-HPLC purification of the anti-microbial compounds shows two active fractions eluted at 16 and 30.5 min, respectively. Mass spectrometry analysis showed that E. faecium 3D produce two bacteriocins Enterocin 3 Da (3893.080 Da) and Enterocin 3Db (4203.350 Da). This strain is food-grade organism and its bacteriocins were heat-stable peptides at basic, neutral, and acid pH: such bacteriocins may be of interest as food preservatives.

Behavior of Listeria monocytogenes in a multi-species biofilm with Enterococcus faecalis and Enterococcus faecium and control through sanitation procedures.

PubMed

da Silva Fernandes, Meg; Kabuki, Dirce Yorika; Kuaye, Arnaldo Yoshiteru

2015-05-04

The formation of mono-species biofilm (Listeria monocytogenes) and multi-species biofilms (Enterococcus faecium, Enterococcus faecalis, and L. monocytogenes) was evaluated. In addition, the effectiveness of sanitation procedures for the control of the multi-species biofilm also was evaluated. The biofilms were grown on stainless steel coupons at various incubation temperatures (7, 25 and 39°C) and contact times (0, 1, 2, 4, 6 and 8 days). In all tests, at 7°C, the microbial counts were below 0.4 log CFU/cm(2) and not characteristic of biofilms. In mono-species biofilm, the counts of L. monocytogenes after 8 days of contact were 4.1 and 2.8 log CFU/cm(2) at 25 and 39°C, respectively. In the multi-species biofilms, Enterococcus spp. were present at counts of 8 log CFU/cm(2) at 25 and 39°C after 8 days of contact. However, the L. monocytogenes in multi-species biofilms was significantly affected by the presence of Enterococcus spp. and by temperature. At 25°C, the growth of L. monocytogenes biofilms was favored in multi-species cultures, with counts above 6 log CFU/cm(2) after 8 days of contact. In contrast, at 39°C, a negative effect was observed for L. monocytogenes biofilm growth in mixed cultures, with a significant reduction in counts over time and values below 0.4 log CFU/cm(2) starting at day 4. Anionic tensioactive cleaning complemented with another procedure (acid cleaning, disinfection or acid cleaning+disinfection) eliminated the multi-species biofilms under all conditions tested (counts of all micro-organisms<0.4 log CFU/cm(2)). Peracetic acid was the most effective disinfectant, eliminating the multi-species biofilms under all tested conditions (counts of the all microorganisms <0.4 log CFU/cm(2)). In contrast, biguanide was the least effective disinfectant, failing to eliminate biofilms under all the test conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Microbiological quality assessment and validation of antimicrobials against unstressed or cold-stress adapted Salmonella and surrogate Enterococcus faecium on broiler carcasses and wings.

PubMed

Lemonakis, Lacey; Li, KaWang; Adler, Jeremy M; Shen, Cangliang

2017-09-01

This study aims to evaluate the microbiological quality and efficacy of antimicrobials to inactivate unstressed or cold-stress adapted Salmonella and Enterococcus on broiler carcasses and wings processed at a small USDA-inspected slaughter facility in West Virginia. The first part of the study included 42 carcasses that were pre- and secondarily-enriched in bacterial media followed by streak-plating onto XLT-4 and HardyCHROM™-agar Salmonella and confirmation using an API20E-kit. The aerobic plate counts (APC), Escherichia coli (ECC), total coliforms (TCC), and yeast/molds were analyzed on petri-films. The second part of the study included fresh broiler carcasses and wings that were inoculated with unstressed and cold-stress-adapted (4 °C, 7-day) Salmonella Typhimurium and Tennessee, and Enterococcus faecium ATCC 8459 (5.5 to 6.0 log10CFU/mL) and later dipped into peroxyacetic acid (PAA; 1,000 ppm), lactic acid (LA; 5%), lactic and citric acid blend (LCA; 2.5%), and sodium hypochlorite (SH; 70 ppm) for 30 s without (carcasses) or with 2-min drainage (wings). The surviving bacteria were recovered onto non-selective and selective agar to analyze the total microbial population, Salmonella and Enterococcus. APC, TCC, and Yeast/Molds were 2.62, 1.08, and 2.37 log10CFU/mL on broiler carcasses, respectively. A total of 30 and 40% of the carcasses tested positive for Salmonella spp. and E. coli (0.48 to 1.70 log10CFU/mL), respectively. For carcasses, antimicrobial reductions of cold-stress-adapted cells of Salmonella and Enterococcus were greater (P < 0.05) than the unstressed cells. For wings, cold-stress-adapted Salmonella were more (P < 0.05) sensitive to antimicrobials than unstressed cells; however, unstressed and cold-stress-adapted Enterococcus behaved similarly (P > 0.05). The reduction of Salmonella and Enterococcus on carcasses and wings increased in the order of SH ≤ LCA < LA < PAA and irrespective of unstressed or cold-stress-adapted cells. Applying

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets

PubMed Central

Guzmán Prieto, Ana M.; Urbanus, Rolf T.; Zhang, Xinglin; Bierschenk, Damien; Koekman, C. Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P.; Pape, Marieke; Paganelli, Fernanda L.; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P. A.; Bonten, Marc J. M.; Willems, Rob J. L.; van Schaik, Willem

2015-01-01

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets. PMID:26675410

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets.

PubMed

Guzmán Prieto, Ana M; Urbanus, Rolf T; Zhang, Xinglin; Bierschenk, Damien; Koekman, C Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P; Pape, Marieke; Paganelli, Fernanda L; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P A; Bonten, Marc J M; Willems, Rob J L; van Schaik, Willem

2015-12-17

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.

Detection of a New cfr-Like Gene, cfr(B), in Enterococcus faecium Isolates Recovered from Human Specimens in the United States as Part of the SENTRY Antimicrobial Surveillance Program.

PubMed

Deshpande, Lalitagauri M; Ashcraft, Deborah S; Kahn, Heather P; Pankey, George; Jones, Ronald N; Farrell, David J; Mendes, Rodrigo E

2015-10-01

Two linezolid-resistant Enterococcus faecium isolates (MICs, 8 μg/ml) from unique patients of a medical center in New Orleans were included in this study. Isolates were initially investigated for the presence of mutations in the V domain of 23S rRNA genes and L3, L4, and L22 ribosomal proteins, as well as cfr. Isolates were subjected to pulsed-field gel electrophoresis (just one band difference), and one representative strain was submitted to whole-genome sequencing. Gene location was also determined by hybridization, and cfr genes were cloned and expressed in a Staphylococcus aureus background. The two isolates had one out of six 23S rRNA alleles mutated (G2576T), had wild-type L3, L4, and L22 sequences, and were positive for a cfr-like gene. The sequence of the protein encoded by the cfr-like gene was most similar (99.7%) to that found in Peptoclostridium difficile, which shared only 74.9% amino acid identity with the proteins encoded by genes previously identified in staphylococci and non-faecium enterococci and was, therefore, denominated Cfr(B). When expressed in S. aureus, the protein conferred a resistance profile similar to that of Cfr. Two copies of cfr(B) were chromosomally located and embedded in a Tn6218 similar to the cfr-carrying transposon described in P. difficile. This study reports the first detection of cfr genes in E. faecium clinical isolates in the United States and characterization of a new cfr variant, cfr(B). cfr(B) has been observed in mobile genetic elements in E. faecium and P. difficile, suggesting potential for dissemination. However, further analysis is necessary to access the resistance levels conferred by cfr(B) when expressed in enterococci. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Efficacy of tigecycline alone and in combination with gentamicin in the treatment of experimental endocarditis due to linezolid-resistant Enterococcus faecium.

PubMed

Pontikis, Konstantinos; Pefanis, Angelos; Tsaganos, Thomas; Tzepi, Ira-Maria; Carrer, Dionyssia-Pinelopi; Giamarellou, Helen

2013-07-01

We evaluated the efficacy of tigecycline in a rabbit model of experimental endocarditis caused by a linezolid-resistant clinical strain of Enterococcus faecium. Tigecycline-treated animals had a 2.8-log10-CFU/g reduction in microbial counts in excised vegetations compared with controls. Addition of gentamicin caused a further arithmetical reduction in colony counts. The therapeutic effect was sustained 5 days after completion of treatment, as shown by relapse studies performed in treatment groups.

Antimicrobial resistance pattern and genetic correlation in Enterococcus faecium isolated from healthy volunteers.

PubMed

Asadian, M; Sadeghi, J; Rastegar Lari, A; Razavi, Sh; Hasannejad Bibalan, M; Talebi, M

2016-03-01

Enterococci are known as a cause of nosocomial infections and this aptitude is intensified by the growth of antibiotic resistance. In the present study, Enterococcus faecium isolates from healthy volunteers were considered to determine the antibiotic resistance profiles and genetic correlation. A total 91 normal flora isolates of enterococci were included in this study. Identification of Enterococcus genus and species were done by biochemical and PCR methods, respectively. Sensitivity for 10 antibiotics was determined and genetic relatedness of all isolates was assessed using Repetitive Element Palindromic PCR (REP-PCR) followed by Pulse Field Gel Electrophoresis (PFGE) on the representative patterns. None of the isolates were resistant to teicoplanin, vancomycin, quinupristin-dalfopristin, linezolid, chloramphenicol, ampicillin and high-level gentamicin. On the other hand, the resistance rate was detected in 30.7%, 23%, and 3.29% of isolates for erythromycin, tetracycline and ciprofloxacin, respectively. The results of PFGE showed 19 (61.5% of our isolates) common types (CT) and 35 (38.5%) single types (ST) amongst the isolates. This is the first study to describe antibiotic resistance pattern and genetic relationship among normal flora enterococci in Iran. This study showed no prevalence of Vancomycin Resistant Enterococci (VRE) and high degrees of diversity among normal flora isolates by genotyping using PFGE. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isolation and Biochemical Fingerprinting of Vancomycin-Resistant Enterococcus faecium From Meat, Chicken and Cheese.

PubMed

Talebi, Malihe; Sadeghi, Javad; Rahimi, Fateh; Pourshafie, Mohammad Reza

2015-04-01

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens and food chain has been considered as an assumed source for dissemination of VRE to human. The presence of VRE isolates from food samples and typing of these isolates with Phene plate, a biochemical fingerprinting method, were investigated. Thirty samples of meat, chicken and cheese were analyzed for VRE during 2010. Antibiotic susceptibility tests and minimum inhibitory concentration (MIC) were also examined. VRE isolates were typed with the Phene plate system (PhPlate), a biochemical fingerprinting method. A total of 70 VRE isolates were obtained and identified as Enterococcus faecium by species-specific PCR. All the isolates carried vanA, while none of them harbored vanB. The VRE isolates included 35, 27, and 8 isolates from meat, chicken and cheese, respectively. Typing with the PhPlate revealed a diversity index of 0.78 for E. faecium, containing 10 common and four single types. The results of antibiotic susceptibility and MIC tests showed an increased resistance to ciprofloxacin, erythromycin, ampicillin and gentamicin, to which, 100%, 100%, 100%, and 95% of VRE isolates were resistant, respectively. Only 5% of the isolates were resistant to chloramphenicol and the MIC of the isolates for vancomycin and teicoplanin was ≥ 256 µg/mL and for gentamicin-resistant isolates it was 1024 µg/mL. Conventional and molecular identification tests exhibited that all the isolates were E. faecium carrying vanA. None of the isolates harbored vanB. The results showed that enterococci are common contaminants in food. Indeed, this study indicates a high prevalence of multidrug-resistant enterococci in food of animal origin in Iran. Isolating some persisting enterococcal isolates revealed that continuous surveillance of antimicrobial resistance in enterococci from food is essential.

Effects of organic acids on thermal inactivation of acid and cold stressed Enterococcus faecium.

PubMed

Fernández, Ana; Alvarez-Ordóñez, Avelino; López, Mercedes; Bernardo, Ana

2009-08-01

In this study the adaptative response to heat (70 degrees C) of Enterococcus faecium using fresh and refrigerated (at 4 degrees C for up to 1 month) stationary phase cells grown in Brain Heart Infusion (BHI) buffered at pH 7.4 (non-acid-adapted cells) and acidified BHI at pH values of 6.4 and 5.4 with acetic, ascorbic, citric, lactic, malic and hydrochloric acids (acid-adapted cells) was evaluated. In all cases, the survival curves obtained were concave upward. A mathematical model based on the Weibull distribution accurately described the inactivation kinetic. The results indicate that previous adaptation to a low pH increased the bacterial heat resistance, whereas the subsequent cold storage of cells reduced E. faecium thermal tolerance. Fresh acid-adapted cells showed t(2.5)-values (time needed to obtain an inactivation level of 2.5 log10 cycles) ranging from 2.57 to 9.51 min, while non-acid-adapted cells showed t(2.5)-values of 1.92 min. The extent of increased heat tolerance varied with the acid examined, resulting in the following order: citric > or = acetic > malic > or = lactic > hydrochloric > or = ascorbic. In contrast, cold storage progressively decreased E. faecium thermal resistance. The t(2.5) values found at the end of the period studied were about 2-3-fold lower than those corresponding to non-refrigerated cells, although this decrease was more marked (about 5-fold) when cells were grown in buffered BHI and BHI acidified at pH 5.4 with hydrochloric acid. These findings highlight the need for a better understanding of microbial response to various preservation stresses in order to increase the efficiency of thermal processes and to indicate the convenience of counterbalancing the benefits of the hurdle concept.

A Novel High-Molecular-Mass Bacteriocin Produced by Enterococcus faecium: Biochemical Features and Mode of Action.

PubMed

Vasilchenko, A S; Vasilchenko, A V; Valyshev, A V; Rogozhin, E A

2018-02-08

Discovery of a novel bacteriocin is always an event in sciences, since cultivation of most bacterial species is a general problem in microbiology. This statement is reflected by the fact that number of bacteriocins is smaller for tenfold comparing to known antimicrobial peptides. We cultivated Enterococcus faecium on simplified medium to reduce amount of purification steps. This approach allows to purify the novel heavy weight bacteriocin produced by E. faecium ICIS 7. The novelty of this bacteriocin, named enterocin-7, was confirmed by N-terminal sequencing and by comparing the structural-functional properties with available data. Purified enterocin-7 is characterized by a sequence of amino acid residues having no homology in UniProt/SwissProt/TrEMBL databases: NH2 - Asp - Ala - His - Leu - Ser - Glu - Val - Ala - Glu - Arg - Phe - Glu - Asp - Leu - Gly. Isolated thermostable protein has a molecular mass of 65 kDa, which allows it to be classified into class III in bacteriocin classification schemes. Enterocin-7 displayed a broad spectrum of activity against some Gram-positive and Gram-negative microorganisms. Fluorescent microscopy and spectroscopy showed the permeabilizing mechanism of the action of enterocin-7, which is realized within a few minutes.

RelA Mutant Enterococcus faecium with Multiantibiotic Tolerance Arising in an Immunocompromised Host.

PubMed

Honsa, Erin S; Cooper, Vaughn S; Mhaissen, Mohammed N; Frank, Matthew; Shaker, Jessica; Iverson, Amy; Rubnitz, Jeffrey; Hayden, Randall T; Lee, Richard E; Rock, Charles O; Tuomanen, Elaine I; Wolf, Joshua; Rosch, Jason W

2017-01-03

Serious bacterial infections in immunocompromised patients require highly effective antibacterial therapy for cure, and thus, this setting may reveal novel mechanisms by which bacteria circumvent antibiotics in the absence of immune pressure. Here, an infant with leukemia developed vancomycin-resistant Enterococcus faecium (VRE) bacteremia that persisted for 26 days despite appropriate antibiotic therapy. Sequencing of 22 consecutive VRE isolates identified the emergence of a single missense mutation (L152F) in relA, which constitutively activated the stringent response, resulting in elevated baseline levels of the alarmone guanosine tetraphosphate (ppGpp). Although the mutant remained susceptible to both linezolid and daptomycin in clinical MIC testing and during planktonic growth, it demonstrated tolerance to high doses of both antibiotics when growing in a biofilm. This biofilm-specific gain in resistance was reflected in the broad shift in transcript levels caused by the mutation. Only an experimental biofilm-targeting ClpP-activating antibiotic was able to kill the mutant strain in an established biofilm. The relA mutation was associated with a fitness trade-off, forming smaller and less-well-populated biofilms on biological surfaces. We conclude that clinically relevant relA mutations can emerge during prolonged VRE infection, causing baseline activation of the stringent response, subsequent antibiotic tolerance, and delayed eradication in an immunocompromised state. The increasing prevalence of antibiotic-resistant bacterial pathogens is a major challenge currently facing the medical community. Such pathogens are of particular importance in immunocompromised patients as these individuals may favor emergence of novel resistance determinants due to lack of innate immune defenses and intensive antibiotic exposure. During the course of chemotherapy, a patient developed prolonged bacteremia with vancomycin-resistant Enterococcus faecium that failed to clear

Genome-wide Screening Identifies Phosphotransferase System Permease BepA to Be Involved in Enterococcus faecium Endocarditis and Biofilm Formation.

PubMed

Paganelli, Fernanda L; Huebner, Johannes; Singh, Kavindra V; Zhang, Xinglin; van Schaik, Willem; Wobser, Dominique; Braat, Johanna C; Murray, Barbara E; Bonten, Marc J M; Willems, Rob J L; Leavis, Helen L

2016-07-15

Enterococcus faecium is a common cause of nosocomial infections, of which infective endocarditis is associated with substantial mortality. In this study, we used a microarray-based transposon mapping (M-TraM) approach to evaluate a rat endocarditis model and identified a gene, originally annotated as "fruA" and renamed "bepA," putatively encoding a carbohydrate phosphotransferase system (PTS) permease (biofilm and endocarditis-associated permease A [BepA]), as important in infective endocarditis. This gene is highly enriched in E. faecium clinical isolates and absent in commensal isolates that are not associated with infection. Confirmation of the phenotype was established in a competition experiment of wild-type and a markerless bepA mutant in a rat endocarditis model. In addition, deletion of bepA impaired biofilm formation in vitro in the presence of 100% human serum and metabolism of β-methyl-D-glucoside. β-glucoside metabolism has been linked to the metabolism of glycosaminoglycans that are exposed on injured heart valves, where bacteria attach and form vegetations. Therefore, we propose that the PTS permease BepA is directly implicated in E. faecium pathogenesis. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Optimization of conditions for probiotic curd formulation by Enterococcus faecium MTCC 5695 with probiotic properties using response surface methodology.

PubMed

Ramakrishnan, Vrinda; Goveas, Louella Concepta; Prakash, Maya; Halami, Prakash M; Narayan, Bhaskar

2014-11-01

Enterococcus faecium MTCC 5695 possessing potential probiotic properties as well as enterocin producing ability was used as starter culture. Effect of time (12-24 h) and inoculum level (3-7 % v/v) on cell growth, bacteriocin production, antioxidant property, titrable acidity and pH of curd was studied by response surface methodology (RSM). The optimized conditions were 26.48 h and 2.17%v/v inoculum and the second order model validated. Co cultivation studies revealed that the formulated product had the ability to prevent growth of foodborne pathogens that affect keeping quality of the product during storage. The results indicated that application of E. faecium MTCC 5695 along with usage of optimized conditions attributed to the formation of highly consistent well set curd with bioactive and bioprotective properties. Formulated curd with potential probiotic attributes can be used as therapeutic agent for the treatment of foodborne diseases like Traveler's diarrhea and gastroenteritis which thereby help in improvement of bowel health.

Approaches for enhancing in situ detection of enterocin genes in thermized milk, and selective isolation of enterocin-producing Enterococcus faecium from Baird-Parker agar.

PubMed

Vandera, Elpiniki; Tsirka, Georgia; Kakouri, Athanasia; Koukkou, Anna-Irini; Samelis, John

2018-05-21

Enterococci are naturally selected for growth in thermized ewes'/goats' milk mixtures used for traditional cooked hard cheese processing in Greece. A culture-independent PCR-based approach was applied to detect the presence of enterocin-encoding genes in naturally culture-enriched thermized milk (TM). Portions of TM (63 °C, 30 s) collected from a commercial cheese plant before addition of starters were fermented at 37 °C for 48 h to facilitate growth of indigenous enterococci. The multiple enterocin-producing (m-Ent+) Enterococcus faecium KE82 and the nisin A-producing Lactococcus lactis subsp. cremoris M104 served as bacteriocin-positive inocula in separate TM treatments. The PCR results revealed a constant presence of the enterocin A, B and P genes in TM fermented naturally at 37 °C. Eleven out of 42 (26.2%) lactic isolates from the enriched TM cultures without inoculation were Ent+ E. faecium assigned to three biotypes. Biotype I (4 isolates) included single entA possessors, whereas biotype II (5 isolates) and biotype III (2 isolates) were m-Ent+ variants profiling entA-entB-entP and entA-entB genes, respectively. Biotype II displayed the strongest antilisterial activity in vitro. Surprisingly, 85.7% (6/7) of the m-Ent+ E. faecium were selectively isolated from Baird-Parker agar, reflecting their natural resistance to 0.01% tellurite contained in the egg yolk supplement. No cytolysin-positive E. faecalis or other Ent+ Enterococcus spp. were isolated. In conclusion, commercially thermized Greek milk is a natural pool or 'reservoir' of antagonistic Ent+ or m-Ent+ E. faecium strains that can be easily detected and recovered by applying this PCR-based approach to naturally fermented milks or cheese products. Copyright © 2018 Elsevier B.V. All rights reserved.

Efficacy of Tigecycline Alone and in Combination with Gentamicin in the Treatment of Experimental Endocarditis Due to Linezolid-Resistant Enterococcus faecium

PubMed Central

Pefanis, Angelos; Tsaganos, Thomas; Tzepi, Ira-Maria; Carrer, Dionyssia-Pinelopi; Giamarellou, Helen

2013-01-01

We evaluated the efficacy of tigecycline in a rabbit model of experimental endocarditis caused by a linezolid-resistant clinical strain of Enterococcus faecium. Tigecycline-treated animals had a 2.8-log10-CFU/g reduction in microbial counts in excised vegetations compared with controls. Addition of gentamicin caused a further arithmetical reduction in colony counts. The therapeutic effect was sustained 5 days after completion of treatment, as shown by relapse studies performed in treatment groups. PMID:23587961

Purification of a Novel Bacteriocin-Like Inhibitory Substance Produced by Enterococcus faecium ICIS 8 and Characterization of Its Mode of Action.

PubMed

Vasilchenko, Alexey S; Rogozhin, Eugene A; Valyshev, Alexander V

2017-06-01

The aim of this work was to purify and characterize a bacteriocin-like antimicrobial substance produced by an antagonistic active strain of Enterococcus faecium. A novel bacteriocin-like inhibitory substance (BLIS) produced by the E. faecium ICIS 8 strain was purified and characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and N-terminal amino acid sequencing revealed the following partial sequence: NH 2 -APKEKCFPKYCV. The proteinaceous nature of purified BLIS was assessed by treatment with proteolytic enzyme. Studies of the action of BLIS using bacteriological and bioluminescence assays revealed a dose-dependent inhibition of Listeria monocytogenes 88BK and Escherichia coli K12 TG1 lac::lux viability. The interaction of the BLIS with the bacterial surface led to the compensation of a negative charge value, as shown by zeta-potential measurements. Assessments of membrane integrity using fluorescent probes and atomic force microscopy revealed the permeabilization of the cellular barrier structures in both L. monocytogenes and E. coli. The novel BLIS from E. faecium ICIS 8 was characterized by a unique primary peptide sequence and exerted bactericidal activity against L. monocytogenes and E. coli by disrupting membrane integrity.

Diversity of Tn1546 in vanA-positive Enterococcus faecium clinical isolates with VanA, VanB, and VanD phenotypes and susceptibility to vancomycin.

PubMed

Cha, J O; Yoo, J I; Kim, H K; Kim, H S; Yoo, J S; Lee, Y S; Jung, Y H

2013-10-01

To investigate diversity in the vanA cluster in Enterococcus faecium isolates from nontertiary hospitals. We identified 43 vanA-positive Ent. faecium isolates, including two vancomycin-susceptible isolates, from hospitals between 2003 and 2006. Of these isolates, >85% were resistant to ampicillin, erythromycin and ciprofloxacin. The vanA cluster was classified into six types using overlapping PCR, but the prototype transposon Tn1546 was not found. Most vanA-positive vancomycin-resistant Enterococcus (VRE) carried IS1216V and belonged to Type III (58·1%) or Type II (20·9%). vanY, vanZ and IS1216V were observed in the left and right ends of Type III with long-range PCR. IS1216V was also observed within vanS and vanX in the two vancomycin-susceptible isolates and in two vancomycin-resistant isolates. No VRE isolates with VanB and VanD phenotypes contained point mutations in vanS, unlike in previous reports. Sequence types (STs) of all isolates belonged to clonal complex 17, and ST78 was predominant. Insertion sequences, especially IS1216V, cause structural variation in the vanA cluster. We report the first observation of vanY and vanZ at the left end of Tn1546 in clinical isolates. This is the first report of the frequency of vancomycin resistance and diversity of Tn1546 in vanA-positive Ent. faecium isolates from nontertiary hospitals. © 2013 The Society for Applied Microbiology.

Raising the Alarmone: Within-Host Evolution of Antibiotic-Tolerant Enterococcus faecium

PubMed Central

2017-01-01

ABSTRACT Enterococci are ancient commensal bacteria that recently emerged as leading causes of antibiotic-resistant, hospital-acquired infection. Vancomycin-resistant enterococci (VRE) epitomize why drug-resistant enterococcal infections are a problem: VRE readily colonize the antibiotic-perturbed gastrointestinal (GI) tract where they amplify to large numbers, and from there, they infect other body sites, including the bloodstream, urinary tract, and surgical wounds. VRE are resistant to many antimicrobials and host defenses, which facilitates establishment at the site of infection and confounds therapeutic clearance. Having evolved to colonize the GI tract, VRE are comparatively ill adapted to the human bloodstream. A recent study by Honsa and colleagues (E. S. Honsa et al., mBio 8:e02124-16, 2017, https://doi.org/10.1128/mBio.02124-16) found that a strain of vancomycin-resistant Enterococcus faecium evolved antibiotic tolerance within the bloodstream of an immunocompromised host by activating the stringent response through mutation of relA. Precisely how VRE colonize and infect and the selective pressures that led to the outgrowth of relA mutants are the subjects of ongoing research. PMID:28223450

Benefits of combinative application of probiotic, enterocin M-producing strain Enterococcus faecium AL41 and Eleutherococcus senticosus in rabbits.

PubMed

Lauková, Andrea; Simonová, Monika Pogány; Chrastinová, Ľubica; Plachá, Iveta; Čobanová, Klaudia; Formelová, Zuzana; Chrenková, Mária; Ondruška, Ľubomír; Strompfová, Viola

2016-03-01

This study presents the effects of the probiotic and enterocin M-producing strain Enterococcus faecium AL41 on microbiota, phagocytic activity (PA), oxidative stress, performance and biochemical parameters when applied individually or in combination with Eleutherococcus senticosus in rabbits. The novelty of the study lies in the use of our non-rabbit-derived strain (AL41 = CCM8558) which produces new enterocin M. Ninety-six post-weaned rabbits (Hyplus breed) aged 5 weeks were divided into three experimental groups, 24 in each: E. senticosus (ES, 30 g/100 kg) in feed, E. faecium AL41 (10(9) CFU/mL marked by rifampicin to differentiate it from other enterococci) in water, and ES + AL. AL41 colonized sufficiently in rabbits to reduce coliforms, staphylococci, pseudomonads and clostridia. Slight decrease in bacteria was also found in the caecum and appendix. Phagocytic activity was significantly increased in the experimental groups compared to the control group (CG) (p < 0.001; p < 0.05). Applications did not evoke oxidative stress. Biochemical parameters in blood and caecal organic acids were slightly influenced. Average daily weight gain was slightly higher in ES and AL + ES. Combinative application of E. faecium with E. senticosus can be beneficial in rabbits. AL41 strain alone and in combination with ES produced reduction in spoilage bacteria; the highest stimulation of PA was in the AL41 + ES group.

Influence of low-level resistance to vancomycin on efficacy of teicoplanin and vancomycin for treatment of experimental endocarditis due to Enterococcus faecium.

PubMed Central

Fantin, B; Leclercq, R; Arthur, M; Duval, J; Carbon, C

1991-01-01

Emergence of vancomycin-resistant strains among enterococci raises a new clinical challenge. Rabbits with aortic endocarditis were infected with Enterococcus faecium BM4172, a clinical strain resistant to low levels of vancomycin (MIC, 16 micrograms/ml) and susceptible to teicoplanin (MIC, 1 micrograms/ml), and against its susceptible variant E. faecium BM4172S obtained in vitro by insertional mutagenesis (MICs, 2 and 0.5 micrograms/ml, respectively). Control animals retained 8 to 10.5 log10 CFU/g of vegetation. We evaluated in this model the efficacy of vancomycin (30 mg/kg of body weight; mean peak and trough serum levels, 27 and 5 micrograms/ml, respectively), teicoplanin (standard dose, 10 mg/kg; mean peak and trough levels, 23 and 9 micrograms/ml, respectively; and high dose, 20 mg/kg; mean peak and trough levels, 63 and 25 micrograms/ml, respectively), gentamicin (6 mg/kg; mean peak and trough levels, 8.6 and less than 0.1 micrograms/ml, respectively), alone or in combination, given every 12 h intramuscularly for 5 days. Teicoplanin standard dose was as active as vancomycin against both strains. Vancomycin was not effective against E. faecium BM4172 but was highly effective against E. faecium BM4172S (7.5 +/- 1.1 log10 CFU/g of vegetation versus 4.9 +/- 1.0 log10 CFU/g of vegetation for vancomycin against E. faecium BM4172 and E. faecium BM4172S, respectively; P = 0.0012). A high dose of teicoplanin was more effective than vancomycin against E. faecium BM4172 (4.4 +/- 1.8 log10 CFU/g of vegetation versus 7.5 +/- 1.1 log10 CFU/g of vegetation for teicoplanin high dose and vancomycin, respectively; P less than 0.05). Against E. faecium BM4172 glycopeptide-gentamicin combinations were the most effective regimens in vitro and in vivo (2.8 +/- 0.7 and 3.5 +/- 1.3 log10 CFU/g of vegetation for vancomycin plus gentamicin and teicoplanin standard dose plus gentamicin, respectively; P < 0.05 versus single-drug regimens). We concluded that high-dose teicoplanin or the

Inhibition of Bacillus cereus Strains by Antimicrobial Metabolites from Lactobacillus johnsonii CRL1647 and Enterococcus faecium SM21.

PubMed

Soria, M Cecilia; Audisio, M Carina

2014-12-01

Bacillus cereus is an endospore-forming, Gram-positive bacterium able to cause foodborne diseases. Lactic acid bacteria (LAB) are known for their ability to synthesize organic acids and bacteriocins, but the potential of these compounds against B. cereus has been scarcely documented in food models. The present study has examined the effect of the metabolites produced by Lactobacillus johnsonii CRL1647 and Enterococcus faecium SM21 on the viability of select B. cereus strains. Furthermore, the effect of E. faecium SM21 metabolites against B. cereus strains has also been investigated on a rice food model. L. johnsonii CRL1647 produced 128 mmol/L of lactic acid, 38 mmol/L of acetic acid and 0.3 mmol/L of phenyl-lactic acid. These organic acids reduced the number of vegetative cells and spores of the B. cereus strains tested. However, the antagonistic effect disappeared at pH 6.5. On the other hand, E. faecium SM21 produced only lactic and acetic acid (24.5 and 12.2 mmol/L, respectively) and was able to inhibit both vegetative cells and spores of the B. cereus strains, at a final fermentation pH of 5.0 and at pH 6.5. This would indicate the action of other metabolites, different from organic acids, present in the cell-free supernatant. On cooked rice grains, the E. faecium SM21 bacteriocin(s) were tested against two B. cereus strains. Both of them were significantly affected within the first 4 h of contact; whereas B. cereus BAC1 cells recovered after 24 h, the effect on B. cereus 1 remained up to the end of the assay. The LAB studied may thus be considered to define future strategies for biological control of B. cereus.

Antimicrobial resistance profile of Enterococcus spp isolated from food in Southern Brazil

PubMed Central

Riboldi, Gustavo Pelicioli; Frazzon, Jeverson; d’Azevedo, Pedro Alves; Frazzon, Ana Paula Guedes

2009-01-01

Fifty-six Enterococcus spp. strains were isolated from foods in Southern Brazil, confirmed by PCR and classified as Enterococcus faecalis (27), Enterococcus faecium (23) and Enterococcus spp (6). Antimicrobial susceptibility tests showed resistance phenotypes to a range of antibiotics widely administrated in humans such as gentamycin, streptomycin, ampicillin and vancomycin. PMID:24031330

Bacteriocinogenic potential and virulence traits of Enterococcus faecium and E. faecalis isolated from human milk

PubMed Central

Khalkhali, Soodabeh; Mojgani, Naheed

2017-01-01

Background and Objectives: Human milk is a continuous supply of Lactic Acid bacteria (LAB), including enterococci with probiotic potentials. The aim of this study was to analyze two Enterococcus species, isolated from human milk for their probiotic potential, bacteriocin producing ability and virulence traits. Materials and Methods: Enterococcus faecium TA0033 and E. faecalis TA102 were tested for acid and bile tolerance, survival in simulated gastric and intestinal conditions. The antibacterial spectrum of the isolates was tested by agar well diffusion assay. The antagonistic agent was characterized by physico-chemical methods. The enterocin structural genes, virulence determinants, vancomycin resistance and biogenic amine genes, such as hdc1, hdc2, tdc, ldc and odc were also determined. Results: The tested isolates survived acidic conditions, high bile salt (1%), simulated gastric and intestinal conditions. The culture supernatant fluids of the two isolates inhibited the growth of Escherichia coli, Listeria monocytogenes, Salmonella typhi, Staphylococcus aureus, Shigella dysenteriae and Streptococcus agalactiae. The antagonistic activity was lost in the presence of proteolytic enzymes but tolerated the action of catalase, lysozyme and lipase. In contrast to enterocin TA102, enterocin TA0033 possessed bactericidal mode of action. Bacteriocin structural genes, entA and entB were present in the genome of the two isolates, while E. faecalis TA102 additionally harboured entP and bac31 genes. The phenotypic and genotypic virulence assessment studies indicated hyaluronidase (hyl) production and vancomycin resistance in E. faecalis TA102 while, none of the isolates harboured the biogenic amine genes. Conclusion: The presence of virulence genes in E. faecalis TA102 calls for careful monitoring of Enterococcus isolates for their safety parameters. PMID:29238458

Aging Eye Microbiota in Dry Eye Syndrome in Patients Treated with Enterococcus faecium and Saccharomyces boulardii.

PubMed

Chisari, Giuseppe; Chisari, Eleonora M; Borzi, Antonio M; Chisari, Clara G

2017-01-01

Aging and oxidative stress seem to play a key role in the onset and progression of ocular surface diseases. Dry Eye Syndrome (DES) is a multifactorial disease of the tears and ocular surface in which symptoms may interfere with the ability to work and carry out daily functions. This clinical trial was a pilot study to evaluate the effects of supplementation with mixture (Saccharomyces boulardii MUCL 53837 and Enterococcus faecium LMG S-28935) on the tear film. Following the run-in period subjects were randomized in two groups: group A (n.30 subjects) and group B (n.30 subjects). Group A (control) treated only with substitute tear and group B treated with substitute tear + mixture (probiotic). The data obtained in the two study groups A and B were, respectively the following: Schirmer I: 9.2±0.2 vs. 12.8±0.4 (p< 0.001); Schirmer II: 3.6±0.1 vs. 4.6±0.2 (p<0.001); BUT 3.8±0.3 vs. 6.2±0.2 (p<0.001). Culture test showed initial bacterial growth in group "A" (placebo) 27 out of 60 samples tested, corresponding to 45.0% and "B" after treatment (probiotic) was found positive culture whit growth of bacteria in 18 tests equal to 30.0%. The total numbers of isolations of aerobic and anaerobic bacteria found group A and B after treatment. A reduction of 23 to 16 strains of aerobic and anaerobic isolates from 13 to 7 has been found. The administration of probiotics strains was effective in reducing DES. In light of these results, we have identified our probiotic (Saccharomyces boulardii MUCL 53837 and Enterococcus faecium LMG S-28935) activity integration with the action of tear substitutes, along with standardization of clinical parameters of the tear film and microbiological activity in restoring of the microbiota ocular surface subject with DES. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Identification of Structural and Immunity Genes of a Class IIb Bacteriocin Encoded in the Enterocin A Operon of Enterococcus faecium Strain MXVK29.

PubMed

Escamilla-Martínez, E E; Cisneros, Y M Álvarez; Fernández, F J; Quirasco-Baruch, M; Ponce-Alquicira, E

2017-10-09

The Enterococcus faecium strain MXVK29, isolated from fermented sausages, produces a bacteriocin with a molecular mass of 3.5 kDa that belongs to the class of enterocins II.1, according to the terminal amino acid sequence, and has been identified as enterocin A. This bacteriocin is active against selected strains of Listeria, Staphylococcus, Pediococcus, and Enterococcus. In this study, we identified the genes adjacent to the structural gene for this bacteriocin, such as the immunity gene (entI) and the inducer gene (entF). Accessory genes for this bacteriocin, such as entK, entR, and entT, were identified as well, in addition to the orf2 and orf3, showing a high identity with class IIb peptides bacteriocins. The orf2 shows the consensus motif GxxxG, similar to those shown by bacteriocins such as PlnNC8α, EntCα, and Ent1071A, whereas orf3 shows a consensus motif SxxxS similar to that present in PlnNC8β (AxxxA). PlnNC8 is expressed only in bacterial cocultures, so there is the possibility that the expression of this two-peptide bacteriocin can be induced by a similar mechanism. So far, only the expression of enterocin A has been found in this strain; however, the presence of the genes ent29α and ent29β opens the possibility for further research on its induction, functionality, and origin. Although there are reports on this type of bacteriocin (EntX, EntC, and Ent1071) in other strains of E. faecium, no report exists yet on an Enterococcus strain producing two different classes of bacteriocin.

High prevalence of diverse vancomycin resistance Enterococcus faecium isolates in clinical and environmental sources in ICU wards in southwest of Iran.

PubMed

Arshadi, Maniya; Douraghi, Masoumeh; Shokoohizadeh, Leili; Moosavian, Seyed Mojtaba; Pourmand, Mohammad Reza

2017-10-01

This study aimed at determining the prevalence, antibiotic resistance patterns, and genetic linkage of Vancomycin Resistant Enterococcus faecium (VREfm) from different sources in the southwest of Iran. A total of 51 VREfm isolates were obtained and subjected to antibiotic susceptibility testing, carriage of virulence genes, and pulsed-field gel electrophoresis (PFGE) method. All the VRE isolates exhibited a high level of resistance to teicoplanin, ampicillin, erythromycin, ciprofloxacin, and gentamicin, also carried the vanA gene. A total of 59% and 34% of the VREfm strains harbored esp and hyl genes, respectively. The results from PFGE showed 31 PFGE patterns including 10 common types (CT) and 21 single types (ST) among the VRE isolates. Furthermore, isolates from different sources in each common type revealed cross transmission between clinical and environmental sources. Overall, the study showed a high prevalence of diverse VRE faecium strains with threatening resistance phenotypes in the environment and clinical sections among different ICU wards of Ahvaz hospitals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enhanced Control of Listeria monocytogenes by Enterococcus faecium KE82, a Multiple Enterocin-Producing Strain, in Different Milk Environments.

PubMed

Vandera, Elpiniki; Lianou, Alexandra; Kakouri, Athanasia; Feng, Jinbo; Koukkou, Anna-Irini; Samelis, John

2017-01-01

Enterococcus faecium KE82, isolated from traditional Greek Graviera cheese, was identified in pure broth cultures in vitro as a multiple enterocin-producing bacterial strain possessing the structural entA, entB, and entP enterocin genes. E. faecium KE82 was further assessed for in situ antilisterial activity in raw milk (RM) and commercially thermized milk (TM; 63°C for 30 s) in the presence of the indigenous microbiota and in sterile raw milk (SRM; 121°C for 5 min) with or without the addition of two commercial starter culture (CSC) strains Streptococcus thermophilus and Lactococcus lactis . Growth of Listeria monocytogenes was completely inhibited in RM incubated at 37°C for 6 h, whereas the pathogen was significantly inactivated in RM+KE82 samples during further incubation at 18°C for 66 h. In contrast, L. monocytogenes levels increased by approximately 2 log CFU/ml in TM, but in TM+KE82 samples, pathogen growth was retarded during the first 6 h at 37°C followed by growth cessation and partial inactivation at 18°C. After 48 to 72 h, growth of L. monocytogenes in SRM+CSC samples decreased by 4 to 5 log CFU/ml compared with the SRM control, whereas additional 10-fold decreases in the pathogen were observed in SRM+CSC+KE82 samples. Reverse transcription PCR analysis of SRM+KE82 and SRM+CSC+KE82 samples confirmed that the entA and entB genes were transcribed, but entP gene transcription was not detected. All RM and SRM samples inoculated with E. faecium KE82 displayed strong in situ inhibitory activity against L. monocytogenes in well diffusion bioassays, whereas activity was weaker to undetectable in comparable or additional TM+KE82 samples; no milk sample without E. faecium KE82 had activity against L. monocytogenes . The findings of this study indicate that E. faecium KE82 is an antilisterial agent that could be used in traditional dairy foods because it concomitantly produces enterocins A and B in situ in milk.

Molecular characterization of resistance, virulence and clonality in vancomycin-resistant Enterococcus faecium and Enterococcus faecalis: A hospital-based study in Beijing, China.

PubMed

Yang, Jing-xian; Li, Tong; Ning, Yong-zhong; Shao, Dong-hua; Liu, Jing; Wang, Shu-qin; Liang, Guo-wei

2015-07-01

The incidence of vancomycin-resistant enterococcus (VRE) in China is increasing, the molecular epidemiology of VRE in China is only partly known. This study was conducted to assess the molecular characterization of resistance, virulence and clonality of 69 vancomycin-resistant Enterococcus faecium (VREfm) and seven vancomycin-resistant Enterococcus faecalis (VREfs) isolates obtained from a Chinese hospital between July 2011 and July 2013. The glycopeptide resistance genes (VanA and VanB) were screened by multiplex PCR. The presence of five putative virulence genes (esp, gelE, asa1, hyl and cylA) were evaluated by another multiplex PCR. Multilocus sequence typing (MLST) scheme was used to assess the clonality. All 76 VRE isolates exhibited VanA phenotype and harbored VanA gene. Esp was the only gene detected both in VREfm and VREfs strains, accounting for 89.9% and 42.9%, respectively. The hyl gene was merely positive in 27.5% of VREfm strains. MLST analysis demonstrated three STs (ST6, ST4 and ST470) in VREfs and twelve STs (ST78, ST571, ST17, ST564, ST389, ST18, ST547, ST341, ST414, ST343, ST262 and ST203) in VREfm, which were all designated as CC17 by eBURST algorithm. An outbreak of VREfm belonging to ST571 was found to happen within the neurology ward in this hospital. To our knowledge, this is the first report of ST6 (CC2) VREfs strains in China and the first outbreak report of VREfm strains belonging to ST571 around the world. Our data could offer important information for understanding the molecular features of VRE in China. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative Genomics of Enterococci: Variation in Enterococcus faecalis, Clade Structure in E. faecium, and Defining Characteristics of E. gallinarum and E. casseliflavus

PubMed Central

Palmer, Kelli L.; Godfrey, Paul; Griggs, Allison; Kos, Veronica N.; Zucker, Jeremy; Desjardins, Christopher; Cerqueira, Gustavo; Gevers, Dirk; Walker, Suzanne; Wortman, Jennifer; Feldgarden, Michael; Haas, Brian; Birren, Bruce; Gilmore, Michael S.

2012-01-01

ABSTRACT The enterococci are Gram-positive lactic acid bacteria that inhabit the gastrointestinal tracts of diverse hosts. However, Enterococcus faecium and E. faecalis have emerged as leading causes of multidrug-resistant hospital-acquired infections. The mechanism by which a well-adapted commensal evolved into a hospital pathogen is poorly understood. In this study, we examined high-quality draft genome data for evidence of key events in the evolution of the leading causes of enterococcal infections, including E. faecalis, E. faecium, E. casseliflavus, and E. gallinarum. We characterized two clades within what is currently classified as E. faecium and identified traits characteristic of each, including variation in operons for cell wall carbohydrate and putative capsule biosynthesis. We examined the extent of recombination between the two E. faecium clades and identified two strains with mosaic genomes. We determined the underlying genetics for the defining characteristics of the motile enterococci E. casseliflavus and E. gallinarum. Further, we identified species-specific traits that could be used to advance the detection of medically relevant enterococci and their identification to the species level. PMID:22354958

Enterococcus faecium F58, a bacteriocinogenic strain naturally occurring in Jben, a soft, farmhouse goat's cheese made in Morocco.

PubMed

Achemchem, F; Martínez-Bueno, M; Abrini, J; Valdivia, E; Maqueda, M

2005-01-01

Characterization of Ent F-58 produced by Enterococcus faecium strain F58 isolated from Jben, a soft, farmhouse goat's cheese manufactured without starter cultures. E. faecium strain F58 was isolated because of its broad inhibitory spectrum, including activity against food-borne pathogenic and spoilage bacteria. The antimicrobial substance was produced during the growth phase, with maximum production after 16-20 h of incubation at 30 degrees C, and was stable over a wide pH range (4-8) and at high temperatures (5 min at 100 degrees C). The enterocin was purified to homogeneity using cation exchange and hydrophobic interaction on C-18 and reverse-phase high-performance liquid chromatography. The activity was eluted as two individual active fractions (F-58A and F-58B) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed masses of 5210.5 and 5234.3 Da respectively. Both peptides were partially sequenced by Edman degradation, and amino-acid sequencing revealed high similarity with enterocin L50 (I). PCR-amplified fragments containing the structural genes for F-58 A and B were located in a 22-kb plasmid harboured by this strain. We verified that it also holds the structural gene for P-like enterocin. E. faecium strain F58 from Jben cheese, a producer of enterocin L50, exerts an inhibitory effect against strains of genera such as Listeria, Staphylococcus, Clostridium, Brochothrix and Bacillus. Enterocin was characterized according to its functional and biological properties, purification to homogeneity and an analysis of its amino acid and genetic sequences. E. faecium strain F58 is a newly discovered producer of enterocin L50, the biotechnological characteristics of which indicate its potential for application as a protective agent against pathogens and spoilage bacteria in foods.

Dietary Enterococcus faecium NCIMB 10415 and Zinc Oxide Stimulate Immune Reactions to Trivalent Influenza Vaccination in Pigs but Do Not Affect Virological Response upon Challenge Infection

PubMed Central

Wang, Zhenya; Burwinkel, Michael; Chai, Weidong; Lange, Elke; Blohm, Ulrike; Breithaupt, Angele; Hoffmann, Bernd; Twardziok, Sven; Rieger, Juliane; Janczyk, Pawel; Pieper, Robert; Osterrieder, Nikolaus

2014-01-01

Swine influenza viruses (SIV) regularly cause significant disease in pigs worldwide. Since there is no causative treatment of SIV, we tested if probiotic Enterococcus (E.) faecium NCIMB 10415 or zinc (Zn) oxide as feed supplements provide beneficial effects upon SIV infection in piglets. Seventy-two weaned piglets were fed three different diets containing either E. faecium or different levels of Zn (2500 ppm, Znhigh; 50 ppm, Znlow). Half of the piglets were vaccinated intramuscularly (VAC) twice with an inactivated trivalent SIV vaccine, while all piglets were then infected intranasally with H3N2 SIV. Significantly higher weekly weight gains were observed in the E. faecium group before virus infection, and piglets in Znhigh and E. faecium groups gained weight after infection while those in the control group (Znlow) lost weight. Using ELISA, we found significantly higher H3N2-specific antibody levels in the E. faecium+VAC group 2 days before and at the day of challenge infection as well as at 4 and 6 days after challenge infection. Higher hemagglutination inhibition (HI) titers were also observed in the Znhigh+VAC and E. faecium+VAC groups at 0, 1 and 4 days after infection. However, there were no significant differences in virus shedding and lung lesions between the dietary groups. Using flow cytometry analysis significantly higher activated T helper cells and cytotoxic T lymphocyte percentages in the PBMCs were detected in the Znhigh and E. faecium groups at single time points after infection compared to the Znlow control group, but no prolonged effect was found. In the BAL cells no influence of dietary supplementation on immune cell percentages could be detected. Our results suggest that feeding high doses of zinc oxide and particularly E. faecium could beneficially influence humoral immune responses after vaccination and recovery from SIV infection, but not affect virus shedding and lung pathology. PMID:24489827

Enterococcus faecium as a Salmonella surrogate in the thermal processing of wheat flour: Influence of water activity at high temperatures.

PubMed

Liu, Shuxiang; Rojas, Rossana V; Gray, Peter; Zhu, Mei-Jun; Tang, Juming

2018-09-01

This study investigated the influence of temperature-dependent water activity (a w ) on thermal resistances of Enterococcus faecium NRRL B-2354 (E. faecium) and Salmonella Enteritidis PT 30 (S. Enteritidis) in wheat flour. The a w for wheat flour samples at 20, 40, and 60 °C was determined by a vapor sorption analyzer and at 75, 80 and 85 °C using custom-built thermal cells with high temperature humidity sensors. Full-factorial isothermal inactivation studies of both strains in sealed aluminum-test-cells included three temperatures (75, 80, and 85 °C) and three a w,25°C levels (0.30, 0.45 and 0.60 within ±0.02 range, prior to the thermal treatments). Isotherm results of wheat flour demonstrate a significant increase (P < 0.05) of a w as temperature rises (e.g. a w,25°C  = 0.45 ± 0.02 became a w,80°C  = 0.71 ± 0.02 in a closed system). Inactivation kinetics of both microorganisms fitted a log-linear model, the yielded D-values varied from 2.7 ± 0.2 min (D 85°C of S. Enteritidis at a w,25°C 0.60 ± 0.02) to 65.8 ± 2.5 min (D 75°C of E. faecium at a w,25°C 0.30 ± 0.02). The z T of E. faecium and S. Enteritidis decreased from 16.4 and 16.9 °C, respectively, to 10.2 °C with increased moisture content (dry basis) from 10 to 14%. Under all tested conditions, E. faecium exhibited equal or higher (1.0-3.1 times) D- and z T -values than those of Salmonella. Overall, E. faecium should be a conservative surrogate for Salmonella in thermal processing of wheat flour for control of Salmonella over a moisture content of 10-14% and treatment temperatures between 75 and 85 °C. Copyright © 2018 Elsevier Ltd. All rights reserved.

Inducer bacteria, unique signal peptides and low nutrient media stimulate in-vitro bacteriocin production by Lactobacillus spp. and Enterococcus spp. strains

USDA-ARS?s Scientific Manuscript database

Bacteriocins (BCN) provide promising potential to control bacterial infections in a variety of applications. We previously reported three Type IIa BCN produced by Lactobacillus salivarius B-30514 (OR-7), Enterococcus durans/faecium/hirae B-30745 (E 760) and Enterococcus faecium B-30746 (E 50-52). ...

Enterococcus Xinjiangensis sp. nov., Isolated from Yogurt of Xinjiang, China.

PubMed

Ren, Xiaopu; Li, Mingyang; Guo, Dongqi

2016-09-01

A Gram-strain-positive bacterial strain 48(T) was isolated from traditional yogurt in Xinjiang Province, China. The bacterium was characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, polymerase α subunit (rpoA) gene sequence analysis, determination of DNA G+C content, DNA-DNA hybridization with the type strain of Enterococcus ratti and analysis of phenotypic features. Strain 48(T) accounted for 96.1, 95.8, 95.8, and 95.7 % with Enterococcus faecium CGMCC 1.2136(T), Enterococcus hirae ATCC 9790(T), Enterococcus durans CECT 411(T), and E. ratti ATCC 700914(T) in the 16S rRNA gene sequence similarities, respectively. The sequence of rpoA gene showed similarities of 99.0, 96.0, 96.0, and 96 % with that of E. faecium ATCC 19434(T), Enterococcus villorum LMG12287, E. hirae ATCC 9790(T), and E. durans ATCC 19432(T), respectively. Based upon of polyphasic characterization data obtained in the study, a novel species, Enterococcus xinjiangensis sp. nov., was proposed and the type strain was 48(T)(=CCTCC AB 2014041(T) = JCM 30200(T)).

Tedizolid susceptibility in linezolid- and vancomycin-resistant Enterococcus faecium isolates.

PubMed

Klupp, E-M; Both, A; Belmar Campos, C; Büttner, H; König, C; Christopeit, M; Christner, M; Aepfelbacher, M; Rohde, H

2016-12-01

Vancomycin-resistant enterococci (VRE) are of ever-increasing importance, most notably in high-risk patient populations. Therapy options are often limited for these isolates, and apart from tigecycline and daptomycin, oxazolidinone linezolid is frequently administered. The broad usage of linezolid, however, has driven the emergence of linezolid-resistant VRE strains (LR-VRE), further shortening therapeutic options. Second-generation oxazolidinone tedizolid has the advantage of being active against a specific subset of LR-VRE, i.e. isolates expressing the plasmid-encoded chloramphenicol-florfenicol resistance (cfr) gene. Here we tested tedizolid activity in a collection of 30 LR Enterococcus faecium VRE (MIC range 32-256 mg/l) isolated between 2012 and 2015 from clinical and screening specimens. By pulsed field gel electrophoresis (PFGE) isolates were assigned to 16 clonal lineages. In three cases, linezolid-susceptible progenitor isolates of LR-VRE were isolated, thus demonstrating the de-novo emergence of the linezolid-resistant phenotype. PCR did not detect cfr, cfr(B) or novel oxazolidinone resistance gene optrA in LR-VRE. All isolates, however, carried mutations within the 23S rDNA. Compared to linezolid, tedizolid MICs were lower in all isolates (MIC range 2-32 mg/l), but remained above the FDA tedizolid breakpoint for E. faecalis at 0.5 mg/l. Thus, related to the predominant resistance mechanism, tedizolid is of limited value for treatment of most LR-VRE and represents a therapeutic option only for a limited subset of isolates.

Inactivation of Salmonella, Listeria monocytogenes and Enterococcus faecium NRRL B-2354 in a selection of low moisture foods.

PubMed

Rachon, Grzegorz; Peñaloza, Walter; Gibbs, Paul A

2016-08-16

The aims of this study were to obtain data on survival and heat resistance of cocktails of Salmonella, Listeria monocytogenes and the surrogate Enterococcus faecium (NRRL B-2354) in four low moisture foods (confectionery formulation, chicken meat powder, pet food and savoury seasoning) during storage before processing. Inoculated samples were stored at 16°C and cell viability examined at day 0, 3, 7 and 21. At each time point, the heat resistance at 80°C was determined. The purpose was to determine a suitable storage time of inoculated foods that can be applied in heat resistance studies or process validations with similar cell viability and heat resistance characteristics. The main inactivation study was carried out within 7days after inoculation, the heat resistance of each bacterial cocktail was evaluated in each low moisture food heated in thermal cells exposed to temperatures between 70 and 140°C. The Weibull model and the first order kinetics (D-value) were used to express inactivation data and calculate the heating time to achieve 5 log reduction at each temperature. Results showed that the pathogens Salmonella and L. monocytogenes and the surrogate E. faecium NRRL B-2354, can survive well (maximum reduction <0.8 log) in low moisture foods maintained at 16°C, as simulation of warehouse raw material storage in winter and before processing. The D80 value of the pathogens and surrogate did not significantly change during the 21day storage (p>0.05). The inactivation kinetics of the pathogens and surrogate at temperatures between 70 and 140°C, were different between each organism and product. E. faecium NRRL B-2354 was a suitable Salmonella surrogate for three of the low moisture foods studied, but not for the sugar-containing confectionery formulation. Heating low moisture food in moisture-tight environments (thermal cells) to 111.2, 105.3 or 111.8°C can inactivate 5 log of Salmonella, L. monocytogenes or E. faecium NRRL B-2354 respectively. Copyright �

Effects of Enterococcus faecium SLB 120 on growth performance, blood parameters, relative organ weight, breast muscle meat quality, excreta microbiota shedding, and noxious gas emission in broilers.

PubMed

Lan, R X; Lee, S I; Kim, I H

2017-09-01

This 5-week study was conducted to determine the effects of Enterococcus faecium (SLB 120) on growth performance, blood parameters, relative organ weight, breast muscle meat quality, excreta microbiota shedding, and noxious gas emission in broilers. A total of 816 one-day-old male broilers were allocated to 4 groups with 12 replications (17 broilers/pen) according to body weight (43.2 ± 0.32 g). Dietary treatment groups were: (1) CON, basal diet, (2) T1, CON + 0.05% E. faecium, (3) T2, CON + 0.10% E. faecium, (4) T3, CON + 0.20% E. faecium. From day 1 to 21, dietary E. faecium supplementation showed linear increase (P < 0.05) in gain:feed ratio. From day 21 to 35 and the overall period, dietary E. faecium supplementation showed a linear increase (P < 0.05) in body weight gain and gain:feed ratio. On day 35, dietary E. faecium supplementation showed a linear increase (P < 0.05) in the apparent total tract digestibility of dry matter and nitrogen, and the relative weight of bursa of Fabricius; no differences were observed in white blood cells, red blood cells or lymphocyte counts. Dietary E. faecium supplementation showed a linear decrease (P < 0.05) in excreta E.coli counts on day 7 and 35, while excreta Lactobacillus counts were linearly increased (P < 0.05) on day 35. On day 35, dietary E. faecium supplementation linearly decreased (P < 0.05) excreta NH3, H2S, and total mercaptans emission, but only H2S emission was linearly decreased (P < 0.05) on day 7. In conclusion, the supplementation of E. faecium improved growth performance, the digestibility of dry matter and nitrogen, the relative weight of bursa of Fabricius, and shifted excreta microbiota by increasing Lactobacillus and decreasing E.coli counts, as well as decreased excreta NH3, H2S, and total mercaptans gas emission. © 2017 Poultry Science Association Inc.

Antimicrobial Resistance of Enterococcus Species Isolated from Chicken in Turkey

PubMed Central

Sanlibaba, Pınar; Tezel, Basar Uymaz; Senturk, Esra

2018-01-01

Abstract The aim of the present work was to provide information about Enterococcus strains isolated from pre-packaged chicken samples in Ankara (Turkey), focusing on their prevalence, phenotypic and genotypic characteristics, and antibiotic resistance. We report the first study on the occurrence of antibiotic resistant enterococci in pre-packaged chicken samples in Ankara. A total of 97 suspicious enterococcal isolates were identified from 122 chicken samples. All isolates were identified to species level by phenotypic and molecular methods. In the 16S rDNA sequence analysis, Enterococcus faecium (61.85%) and Enterococcus faecalis (38.15%) were found to be the most frequently detected Enterococcus spp. Of the 97 isolates tested for hemolytic activity, 12.37% enterococcal strains were β-hemolytic. β-Hemolysin was most prevalent among E. faecium (58.33%) compared to E. faecalis (41.66%). Disk diffusion method was used for determining of antibiotic resistance. The analysis of the antimicrobial resistance of the 97 Enterococcus isolates revealed that the resistance to kanamycin (98.96%), rifampicin (80.41%) and ampicillin (60.82%) was most frequent. Furthermore, resistance to erythromycin (38.14%) and ciprofloxacin (34.02%) was also observed. The frequencies of resistance to tetracycline (9.27%), penicillin G (8.24%), and chloramphenicol (3.09%), gentamicin (2.06%) and streptomycin (1.03%) were low. None of the isolates was resistant to vancomycin. Multi-drug resistance was found in 97.93% of Enterococcus strains. E. faecium strains showed a more resistant phenotype than E. faecalis strains according to the antibiotic resistance levels. The results of this study indicated that chicken meat is a potential reservoir for the transmission of antibiotic resistance from animals to humans. PMID:29805287

Purification and characterization of multiple bacteriocins and an inducing peptide produced by Enterococcus faecium NKR-5-3 from Thai fermented fish.

PubMed

Ishibashi, Naoki; Himeno, Kohei; Fujita, Koji; Masuda, Yoshimitsu; Perez, Rodney Honrada; Zendo, Takeshi; Wilaipun, Pongtep; Leelawatcharamas, Vichien; Nakayama, Jiro; Sonomoto, Kenji

2012-01-01

Enterocins NKR-5-3A, B, C, and D were purified from the culture supernatant of Enterococcus faecium NKR-5-3 and characterized. Among the four purified peptides, enterocin NKR-5-3A (5242.3 Da) was identical to brochocin A, produced by Brochothrix campestris ATCC 43754, in mature peptides, and its putative synergistic peptide, enterocin NKR-5-3Z, was found to be encoded in ent53Z downstream of ent53A, encoding enterocin NKR-5-3A. Enterocin NKR-5-3B (6316.4 Da) showed a broad antimicrobial spectrum, and enterocin NKR-5-3C (4512.8 Da) showed high activity against Listeria. Enterocin NKR-5-3D (2843.5 Da), showing high homology to an inducing peptide produced by Lactobacillus sakei 5, induced the production of the enterocins. The enterocins showed different antimicrobial spectra and intensities. E. faecium NKR-5-3 concomitantly produced enterocins NKR-5-3A, B, C, and D which probably belong to different classes of bacteriocins. Furthermore, NKR-5-3 production was induced by enterocin NKR-5-3D.

Fatal Enterococcus durans aortic valve endocarditis: a case report and review of the literature

PubMed Central

Vijayakrishnan, Rajakrishnan; Rapose, Alwyn

2012-01-01

Most enterococcal endocarditis is caused by Enterococcus faecalis and Enterococcus faecium. Enterococcus durans is a rare member of non-faecalis, non-faecium enterococcal species and is found in the intestines of animals. E durans endocarditis is a very rare infection—only two cases of endocarditis in humans have been reported in the literature—and usually associated with good outcomes when treated with appropriate antibiotics. We report the first case of fatal E durans endocarditis. This patient had end-stage liver disease with associated compromised immune status that likely contributed to the progression of disease in spite of appropriate antibiotic coverage and clearance of bacteraemia. PMID:22684831

Wild corvid birds colonized with vancomycin-resistant Enterococcus faecium of human origin harbor epidemic vanA plasmids.

PubMed

Oravcová, Veronika; Peixe, Luísa; Coque, Teresa M; Novais, Carla; Francia, Maria V; Literák, Ivan; Freitas, Ana R

2018-06-02

The most prevalent type of acquired vancomycin resistance in Enterococcus faecium (VREfm) is encoded by the vanA transposon Tn1546, mainly located on transferable plasmids. vanA plasmids have been characterized in VREfm from a variety of sources but not wild birds. The aim of this study was to analyse the genetic context of VREfm strains recovered from wild corvid birds and to compare their plasmid and strain characteristics with human strains. To achieve that, 75 VREfm isolates, including strains from wild birds recovered during wide surveillance studies performed in Europe, Canada and the United States (2010-2013), and clinical and wastewater strains from Czech Republic, a region lacking data about vanA plasmids, were analysed. Their population structure, presence of major putative virulence markers and characterization of vanA transposons and plasmids were established. VREfm from wild birds were mainly associated with major human lineages (ST18 and ST78) circulating in hospitals worldwide and were enriched in putative virulence markers that are highly associated with clinical E. faecium from human infections. They also carried plasmids of the same families usually found in the clinical setting [RCR, small theta plasmids, RepA_N (pRUM/pLG1) and Inc18]. The clinically widespread IS1251-carrying Tn1546 type "F" was predominant and Tn1546-vanA was mainly located on pRUM/Axe-Txe (USA) and Inc18- or pLG1-like (Europe) plasmids. VREfm from hospitals and wastewaters carried Tn1546-vanA in different plasmid types including mosaic pRUM-Inc18 plasmids, not identified in wild birds. This is the first characterization of vanA plasmids obtained from wild birds. A similar plasmid pool seems to exist in different clonal E. faecium backgrounds of humans and wild birds. The isolation of VREfm strains from wild birds that belong to human E. faecium adapted lineages and carry virulence genes, Tn1546 and plasmid variants widespread in the clinical setting is of concern and highlight

Cross-talk Between Host, Microbiome and Probiotics: A Systems Biology Approach for Analyzing the Effects of Probiotic Enterococcus faecium NCIMB 10415 in Piglets.

PubMed

Twardziok, S O; Pieper, R; Aschenbach, J R; Bednorz, C; Brockmann, G A; Fromm, M; Klingspor, S; Kreuzer, S; Lodemann, U; Martens, H; Martin, L; Richter, J F; Scharek-Tedin, L; Siepert, B F; Starke, I C; Tedin, K; Vahjen, W; Wieler, L H; Zakrzewski, S S; Zentek, J; Wrede, P

2014-03-01

A comprehensive data-set from a multidisciplinary feeding experiment with the probiotic Enterococcus faecium was analyzed to elucidate effects of the probiotic on growing piglets. Sixty-two piglets were randomly assigned to a control (no probiotic treatment) and a treatment group (E. faecium supplementation). Piglets were weaned at 26 d. Age-matched piglets were sacrificed for the collection of tissue samples at 12, 26, 34 and 54 d. In addition to zootechnical data, the composition and activity of intestinal microbiota, immune cell types, and intestinal responses were determined. Our systems analysis revealed clear effects on several measured variables in 26 and 34 days old animals, while response patterns varied between piglets from different age groups. Correlation analyses identified reduced associations between intestinal microbial communities and immune system reactions in the probiotic group. In conclusion, the developed model is useful for comparative analyses to unravel systems effects of dietary components and their time resolution. The model identified that effects of E. faecium supplementation most prominently affected the interplay between intestinal microbiota and the intestinal immune system. These effects, as well as effects in other subsystems, clustered around weaning, which is the age where piglets are most prone to diarrhea. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Shape-dependent antibacterial activity of silver nanoparticles on Escherichia coli and Enterococcus faecium bacterium

NASA Astrophysics Data System (ADS)

Alshareef, A.; Laird, K.; Cross, R. B. M.

2017-12-01

Silver nanoparticles (AgNPs) have been shown to exhibit strong antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria including antibiotic resistant strains. This study aims to compare the bactericidal effect of different shaped AgNPs (spherical and truncated octahedral) against Escherichia coli and Enterococcus faecium. The antimicrobial activity of a range of concentrations (50, 100, 1000 μg/ml) was determined over 24 h using both optical density and viable counts. Truncated octahedral AgNPs (AgNOct) were found to be more active when compared with spherical AgNPs (AgNS). The difference in shape resulted in differences in efficacy which may be due to the higher surface area of AgNOct compared to AgNS, and differences in active facets and surface energies, with AgNPs having a bacteriostatic effect and AgNOct being bactericidal after 4 h. The results suggest that AgNPs can be used as effective growth inhibitors in different microorganisms, rendering them applicable to various medical devices and antimicrobial control systems.

Dietary Enterococcus faecium NCIMB 10415 and zinc oxide stimulate immune reactions to trivalent influenza vaccination in pigs but do not affect virological response upon challenge infection.

PubMed

Wang, Zhenya; Burwinkel, Michael; Chai, Weidong; Lange, Elke; Blohm, Ulrike; Breithaupt, Angele; Hoffmann, Bernd; Twardziok, Sven; Rieger, Juliane; Janczyk, Pawel; Pieper, Robert; Osterrieder, Nikolaus

2014-01-01

Swine influenza viruses (SIV) regularly cause significant disease in pigs worldwide. Since there is no causative treatment of SIV, we tested if probiotic Enterococcus (E.) faecium NCIMB 10415 or zinc (Zn) oxide as feed supplements provide beneficial effects upon SIV infection in piglets. Seventy-two weaned piglets were fed three different diets containing either E. faecium or different levels of Zn (2500 ppm, Zn(high); 50 ppm, Zn(low)). Half of the piglets were vaccinated intramuscularly (VAC) twice with an inactivated trivalent SIV vaccine, while all piglets were then infected intranasally with H3N2 SIV. Significantly higher weekly weight gains were observed in the E. faecium group before virus infection, and piglets in Zn(high) and E. faecium groups gained weight after infection while those in the control group (Zn(low)) lost weight. Using ELISA, we found significantly higher H3N2-specific antibody levels in the E. faecium+VAC group 2 days before and at the day of challenge infection as well as at 4 and 6 days after challenge infection. Higher hemagglutination inhibition (HI) titers were also observed in the Zn(high)+VAC and E. faecium+VAC groups at 0, 1 and 4 days after infection. However, there were no significant differences in virus shedding and lung lesions between the dietary groups. Using flow cytometry analysis significantly higher activated T helper cells and cytotoxic T lymphocyte percentages in the PBMCs were detected in the Zn(high) and E. faecium groups at single time points after infection compared to the Zn(low) control group, but no prolonged effect was found. In the BAL cells no influence of dietary supplementation on immune cell percentages could be detected. Our results suggest that feeding high doses of zinc oxide and particularly E. faecium could beneficially influence humoral immune responses after vaccination and recovery from SIV infection, but not affect virus shedding and lung pathology.

Role of the Emp Pilus Subunits of Enterococcus faecium in Biofilm Formation, Adherence to Host Extracellular Matrix Components, and Experimental Infection.

PubMed

Montealegre, Maria Camila; Singh, Kavindra V; Somarajan, Sudha R; Yadav, Puja; Chang, Chungyu; Spencer, Robert; Sillanpää, Jouko; Ton-That, Hung; Murray, Barbara E

2016-05-01

Enterococcus faecium is an important cause of hospital-associated infections, including urinary tract infections (UTIs), bacteremia, and infective endocarditis. Pili have been shown to play a role in the pathogenesis of Gram-positive bacteria, including E. faecium We previously demonstrated that a nonpiliated ΔempABC::cat derivative of E. faecium TX82 was attenuated in biofilm formation and in a UTI model. Here, we studied the contributions of the individual pilus subunits EmpA, EmpB, and EmpC to pilus architecture, biofilm formation, adherence to extracellular matrix (ECM) proteins, and infection. We identified EmpA as the tip of the pili and found that deletion of empA reduced biofilm formation to the same level as deletion of the empABC operon, a phenotype that was restored by reconstituting in situ the empA gene. Deletion of empB also caused a reduction in biofilm, while EmpC was found to be dispensable. Significant reductions in adherence to fibrinogen and collagen type I were observed with deletion of empA and empB, while deletion of empC had no adherence defect. Furthermore, we showed that each deletion mutant was significantly attenuated in comparison to the isogenic parental strain, TX82, in a mixed-inoculum UTI model (P < 0.001 to 0.048), that reconstitution of empA restored virulence in the UTI model, and that deletion of empA also resulted in attenuation in an infective endocarditis model (P = 0.0088). Our results indicate that EmpA and EmpB, but not EmpC, contribute to biofilm and adherence to ECM proteins; however, all the Emp pilins are important for E. faecium to cause infection in the urinary tract. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Role of the Emp Pilus Subunits of Enterococcus faecium in Biofilm Formation, Adherence to Host Extracellular Matrix Components, and Experimental Infection

PubMed Central

Montealegre, Maria Camila; Singh, Kavindra V.; Somarajan, Sudha R.; Yadav, Puja; Chang, Chungyu; Spencer, Robert; Sillanpää, Jouko; Ton-That, Hung

2016-01-01

Enterococcus faecium is an important cause of hospital-associated infections, including urinary tract infections (UTIs), bacteremia, and infective endocarditis. Pili have been shown to play a role in the pathogenesis of Gram-positive bacteria, including E. faecium. We previously demonstrated that a nonpiliated ΔempABC::cat derivative of E. faecium TX82 was attenuated in biofilm formation and in a UTI model. Here, we studied the contributions of the individual pilus subunits EmpA, EmpB, and EmpC to pilus architecture, biofilm formation, adherence to extracellular matrix (ECM) proteins, and infection. We identified EmpA as the tip of the pili and found that deletion of empA reduced biofilm formation to the same level as deletion of the empABC operon, a phenotype that was restored by reconstituting in situ the empA gene. Deletion of empB also caused a reduction in biofilm, while EmpC was found to be dispensable. Significant reductions in adherence to fibrinogen and collagen type I were observed with deletion of empA and empB, while deletion of empC had no adherence defect. Furthermore, we showed that each deletion mutant was significantly attenuated in comparison to the isogenic parental strain, TX82, in a mixed-inoculum UTI model (P < 0.001 to 0.048), that reconstitution of empA restored virulence in the UTI model, and that deletion of empA also resulted in attenuation in an infective endocarditis model (P = 0.0088). Our results indicate that EmpA and EmpB, but not EmpC, contribute to biofilm and adherence to ECM proteins; however, all the Emp pilins are important for E. faecium to cause infection in the urinary tract. PMID:26930703

Role of EfrAB efflux pump in biocide tolerance and antibiotic resistance of Enterococcus faecalis and Enterococcus faecium isolated from traditional fermented foods and the effect of EDTA as EfrAB inhibitor.

PubMed

Lavilla Lerma, Leyre; Benomar, Nabil; Valenzuela, Antonio Sánchez; Casado Muñoz, María del Carmen; Gálvez, Antonio; Abriouel, Hikmate

2014-12-01

Enterococcus faecalis and Enterococcus faecium isolated from various traditional fermented foods of both animal and vegetable origins have shown multidrug resistance to several antibiotics and tolerance to biocides. Reduced susceptibility was intra and inter-species dependent and was due to specific and unspecific mechanisms such as efflux pumps. EfrAB, a heterodimeric ABC transporter efflux pump, was detected in 100% of multidrug resistant (MDR) E. faecalis strains and only in 12% of MDR E. faecium strains. EfrAB expression was induced by half of minimum inhibitory concentration (MIC) of gentamicin, streptomycin and chloramphenicol. However, expression of efrA and efrB genes was highly dependent on the strain tested and on the antimicrobial used. Our results indicated that 3 mM EDTA highly reduced the MICs of almost all drugs tested. Nevertheless, the higher reductions (>8 folds) were obtained with gentamicin, streptomycin, chlorhexidine and triclosan. Reductions of MICs were correlated with down-regulation of EfrAB expression (10-140 folds) in all three MDR enterococci strains. This is the first report describing the role of EfrAB in the efflux of antibiotics and biocides which reflect also the importance of EfrAB in multidrug resistance in enterococci. EDTA used at low concentration as food preservative could be one of the best choices to prevent spread of multidrug resistant enterococci throughout food chain by decreasing EfrAB expression. EfrAB could be an attractive target not only in enterococci present in food matrix but also those causing infections as well by using EDTA as therapeutic agent in combination with low doses of antibiotics. Copyright © 2014 Elsevier Ltd. All rights reserved.

Emergence of daptomycin non-susceptibility in colonizing vancomycin-resistant Enterococcus faecium isolates during daptomycin therapy.

PubMed

Lellek, Heinrich; Franke, Gefion C; Ruckert, Carolin; Wolters, Manuel; Wolschke, Christiane; Christner, Martin; Büttner, Henning; Alawi, Malik; Kröger, Nicolaus; Rohde, Holger

2015-12-01

Infections due to vancomycin-resistant enterococci (VRE) are of significant importance in high-risk populations, and daptomycin is a bactericidal antibiotic to treat multidrug-resistant VRE in these patients. The emergence of daptomycin non-susceptibility invasive VRE during daptomycin therapy is a major clinical issue. Here the hypothesis was tested that systemic daptomycin therapy also induces the emergence of daptomycin non-susceptible (DNS-) isolates in colonizing VRE populations. 11 vancomycin-resistant Enterococcus faecium strain pairs recovered from rectal swabs were available for analysis. All initial isolates exhibited daptomycin MICs within the wild type MIC distribution of E. faecium (MIC≤4 mg/L). In follow-up isolates from five patients a 4-16-fold daptomycin MIC increase was detected. All patients carrying DNS-VRE received daptomycin (14-28 days) at 4 mg/kg body weight, while two patients in whom no DNS-VRE emerged were only treated with daptomycin for 1 and 4 days, respectively. Comparative whole genome sequencing identified DNS-VRE-specific single nucleotide polymorphisms (SNP), including mutations in cardiolipin synthase (Cls), and additional SNPs in independent genes potentially relevant for the DNS phenotype. Mutations within cls were also identified in three additional, colonizing DNS-VRE. Of these, at least one strain was transmitted within the hospital. In none of the VRE isolates tested, pre-existing or de novo mutations in the liaFSR operon were detected. This is the first report documenting the emergence of DNS-VRE in colonizing strains during daptomycin treatment, putting the patient at risk for subsequent DNS-VRE infections and priming the spread of DNS-VRE within the hospital environment. Copyright © 2015 Elsevier GmbH. All rights reserved.

Population Biology of Intestinal Enterococcus Isolates from Hospitalized and Nonhospitalized Individuals in Different Age Groups

PubMed Central

Tedim, Ana P.; Ruiz-Garbajosa, Patricia; Corander, Jukka; Rodríguez, Concepción M.; Cantón, Rafael; Willems, Rob J.; Baquero, Fernando

2014-01-01

The diversity of enterococcal populations from fecal samples from hospitalized (n = 133) and nonhospitalized individuals (n = 173) of different age groups (group I, ages 0 to 19 years; group II, ages 20 to 59 years; group III, ages ≥60 years) was analyzed. Enterococci were recovered at similar rates from hospitalized and nonhospitalized persons (77.44% to 79.77%) of all age groups (75.0% to 82.61%). Enterococcus faecalis and Enterococcus faecium were predominant, although seven other Enterococcus species were identified. E. faecalis and E. faecium (including ampicillin-resistant E. faecium) colonization rates in nonhospitalized persons were age independent. For inpatients, E. faecalis colonization rates were age independent, but E. faecium colonization rates (particularly the rates of ampicillin-resistant E. faecium colonization) significantly increased with age. The population structure of E. faecium and E. faecalis was determined by superimposing goeBURST and Bayesian analysis of the population structure (BAPS). Most E. faecium sequence types (STs; 150 isolates belonging to 75 STs) were linked to BAPS groups 1 (22.0%), 2 (31.3%), and 3 (36.7%). A positive association between hospital isolates and BAPS subgroups 2.1a and 3.3a (which included major ampicillin-resistant E. faecium human lineages) and between community-based ampicillin-resistant E. faecium isolates and BAPS subgroups 1.2 and 3.3b was found. Most E. faecalis isolates (130 isolates belonging to 58 STs) were grouped into 3 BAPS groups, BAPS groups 1 (36.9%), 2 (40.0%), and 3 (23.1%), with each one comprising widespread lineages. No positive associations with age or hospitalization were established. The diversity and dynamics of enterococcal populations in the fecal microbiota of healthy humans are largely unexplored, with the available knowledge being fragmented and contradictory. The study offers a novel and comprehensive analysis of enterococcal population landscapes and suggests that E. faecium

Solution Structure of Enterocin HF, an Antilisterial Bacteriocin Produced by Enterococcus faecium M3K31.

PubMed

Arbulu, Sara; Lohans, Christopher T; van Belkum, Marco J; Cintas, Luis M; Herranz, Carmen; Vederas, John C; Hernández, Pablo E

2015-12-16

The solution structure of enterocin HF (EntHF), a class IIa bacteriocin of 43 amino acids produced by Enterococcus faecium M3K31, was evaluated by CD and NMR spectroscopy. Purified EntHF was unstructured in water, but CD analysis supports that EntHF adopts an α-helical conformation when exposed to increasing concentrations of trifluoroethanol. Furthermore, NMR spectroscopy indicates that this bacteriocin adopts an antiparallel β-sheet structure in the N-terminal region (residues 1-17), followed by a well-defined central α-helix (residues 19-30) and a more disordered C-terminal end (residues 31-43). EntHF could be structurally organized into three flexible regions that might act in a coordinated manner. This is in agreement with the absence of long-range nuclear Overhauser effect signals between the β-sheet domain and the C-terminal end of the bacteriocin. The 3D structure recorded for EntHF fits emerging facts regarding target recognition and mode of action of class IIa bacteriocins.

Characterization and Heterologous Expression of the Genes Encoding Enterocin A Production, Immunity, and Regulation in Enterococcus faecium DPC1146

PubMed Central

O’Keeffe, Triona; Hill, Colin; Ross, R. Paul

1999-01-01

Enterocin A is a small, heat-stable, antilisterial bacteriocin produced by Enterococcus faecium DPC1146. The sequence of a 10,879-bp chromosomal region containing at least 12 open reading frames (ORFs), 7 of which are predicted to play a role in enterocin biosynthesis, is presented. The genes entA, entI, and entF encode the enterocin A prepeptide, the putative immunity protein, and the induction factor prepeptide, respectively. The deduced proteins EntK and EntR resemble the histidine kinase and response regulator proteins of two-component signal transducing systems of the AgrC-AgrA type. The predicted proteins EntT and EntD are homologous to ABC (ATP-binding cassette) transporters and accessory factors, respectively, of several other bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia coli hemolysin A. Immediately downstream of the entT and entD genes are two ORFs, the product of one of which, ORF4, is very similar to the product of the yteI gene of Bacillus subtilis and to E. coli protease IV, a signal peptide peptidase known to be involved in outer membrane lipoprotein export. Another potential bacteriocin is encoded in the opposite direction to the other genes in the enterocin cluster. This putative bacteriocin-like peptide is similar to LafX, one of the components of the lactacin F complex. A deletion which included one of two direct repeats upstream of the entA gene abolished enterocin A activity, immunity, and ability to induce bacteriocin production. Transposon insertion upstream of the entF gene also had the same effect, but this mutant could be complemented by exogenously supplied induction factor. The putative EntI peptide was shown to be involved in the immunity to enterocin A. Cloning of a 10.5-kb amplicon comprising all predicted ORFs and regulatory regions resulted in heterologous production of enterocin A and induction factor in Enterococcus faecalis, while a four-gene construct (entAITD) under the

Characterization and susceptibility patterns of clinically important Enterococcus species in eastern Nepal.

PubMed

Acharya, A; Khanal, A; Kanungo, R; Mohapatra, T

2007-12-01

Life threatening infections caused by enterococcus species with multidrug resistance has emerged as a threat to medical care in the present era. This study was conducted to characterize enterococcus species isolated from different clinical samples and to detect the pattern of susceptibility to some of the commonly used antibiotics in B.P Koirala Institute of Health Sciences (BPKIHS), a tertiary care hospital in eastern Nepal. Clinical samples submitted to the microbiology unit of Central Laboratory Service (CLS) for culture and sensitivity during March 2002 - February 2003 was analyzed. Enterococcus species were identified by colony characteristics, gram staining and relevant biochemical tests. Antibiotic susceptibility test was done by the Kirby Bauer disc diffusion technique. Of 50 Enterococcus species isolated, E. faecalis was the predominant isolate (48.0%) followed by E. faecium (32.0%) and E. avium (20.0%). Eighty-eight percent of E. faecalis showed sensitivity to cephotaxime and 87.0% to vancomycin. Multiple drug resistance was observed most commonly in E. faecium. Seventeen percent of E. faecium were resistant to vancomycin and 63.0% to ciprofloxacin and 44.0% to ampicillin. On the contrary E. avium rarely showed resistance to the antimicrobials tested including vancomycin. Enterococcal infections are common nowadays specially in hospitalized patients. Inappropriate use of antibiotics in clinical practice and poultry should be discouraged to prevent the emergence of multidrug resistant species.

Multilevel population genetic analysis of vanA and vanB Enterococcus faecium causing nosocomial outbreaks in 27 countries (1986-2012).

PubMed

Freitas, Ana R; Tedim, Ana P; Francia, Maria V; Jensen, Lars B; Novais, Carla; Peixe, Luísa; Sánchez-Valenzuela, Antonio; Sundsfjord, Arnfinn; Hegstad, Kristin; Werner, Guido; Sadowy, Ewa; Hammerum, Anette M; Garcia-Migura, Lourdes; Willems, Rob J; Baquero, Fernando; Coque, Teresa M

2016-12-01

Vancomycin-resistant Enterococcus faecium (VREfm) have been increasingly reported since the 1980s. Despite the high number of published studies about VRE epidemiology, the dynamics and evolvability of these microorganisms are still not fully understood. A multilevel population genetic analysis of VREfm outbreak strains since 1986, representing the first comprehensive characterization of plasmid content in E. faecium, was performed to provide a detailed view of potential transmissible units. From a comprehensive MeSH search, we identified VREfm strains causing hospital outbreaks (1986-2012). In total, 53 VanA and 18 VanB isolates (27 countries, 5 continents) were analysed and 82 vancomycin-susceptible E. faecium (VSEfm) were included for comparison. Clonal relatedness was established by PFGE and MLST (goeBURST/Bayesian Analysis of Population Structure, BAPS). Characterization of van transposons (PCR mapping, RFLP, sequencing), plasmids (transfer, ClaI-RFLP, PCR typing of relaxases, replication-initiation proteins and toxin-antitoxin systems, hybridization, sequencing), bacteriocins and virulence determinants (PCR, hybridization, sequencing) was performed. VREfm were mainly associated with major human lineages ST17, ST18 and ST78. VREfm and VSEfm harboured plasmids of different families [RCR, small theta plasmids, RepA_N (pRUM/pLG1) and Inc18] able to yield mosaic elements. Tn1546-vanA was mainly located on pRUM/Axe-Txe (USA) and Inc18-pIP186 (Europe) plasmids. The VanB2 type (Tn5382/Tn1549) was predominant among VanB strains (chromosome and plasmids). Both strains and plasmids contributed to the spread and persistence of vancomycin resistance among E. faecium. Horizontal gene transfer events among genetic elements from different clonal lineages (same or different species) result in chimeras with different stability and host range, complicating the surveillance of epidemic plasmids. © The Author 2016. Published by Oxford University Press on behalf of the British

Analysis of the world epidemiological situation among vancomycin-resistant Enterococcus faecium infections and the current situation in Poland

PubMed

Talaga-Ćwiertnia, Katarzyna; Bulanda, Małgorzata

2018-01-01

Vancomycin-resistant Enterococcus faecium (VREfm) strains have become an important hospital pathogen due to their rapid spread, high mortality rate associated with infections and limited therapeutic options. Vancomycin resistance is predominantly mediated by VanA or VanB phenotypes, which differ as regards maintaining sensitivity to teicoplanin in the VanB phenotype. The majority of VREfm cases in the United States, Europe, Korea, South America and Africa are currently caused by the VanA phenotype. However, the epidemics in Australia and Singapore are chiefly brought about by the VanB phenotype. The rate of VREfm isolate spread varies greatly. The greatest percentage of VREfm is now recorded in the USA, Ireland and Australia. Supervision of VRE is implemented to varying degrees. Therefore, the epidemiological situation in some countries is difficult to assess due to limited data or lack thereof.

Effect of calcium and magnesium on the antimicrobial action of enterocin LR/6 produced by Enterococcus faecium LR/6.

PubMed

Kumar, Manoj; Srivastava, Sheela

2011-06-01

Enterococci are well-known producers of antimicrobial peptides (enterocins) that possess potential as biopreservatives in food. In this study, divalent cations and release of intracellular potassium were used to assess the mechanism of interaction and killing of enterocin LR/6 produced by Enterococcus faecium LR/6 on three target Gram-positive and Gram-negative bacteria, namely Micrococcus luteus, Enterococcus sp. strain LR/3 and Escherichia coli K-12. Whilst treatment with enterocin LR/6 in all cases led to a significant loss of viability, suggesting a bactericidal mode of action, E. coli K-12 showed better tolerance than the other two strains. Bacteriocins have generally been reported to create pores in the membrane of sensitive cells and this function is diminished by divalent cations. In this study it was shown that Ca(2+) and Mg(2+) markedly improved the viability of enterocin LR/6-treated cells in a concentration-dependent manner. K(+) release as a sign of membrane leakiness was higher in M. luteus compared with the other two test strains. In agreement with the viability response, pre-exposure to Ca(2+) and Mg(2+) substantially reduced the amount of K(+) leakage by M. luteus and Enterococcus sp.; in the case of E. coli K-12, no leakage of K(+) was recorded. These results suggest that enterocin LR/6, which possesses good antibacterial potential, may not be very effective as a preservative in foods containing high concentrations of calcium and magnesium. Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium.

PubMed

Desbonnet, Charlene; Tait-Kamradt, Amelia; Garcia-Solache, Monica; Dunman, Paul; Coleman, Jeffrey; Arthur, Michel; Rice, Louis B

2016-04-05

The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as "cephalosporins") is reliant on the presence of class A penicillin-binding proteins (Pbps) PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2) of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP). Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system. β-Lactam antibiotics remain our most effective therapies against susceptible Gram-positive bacteria. The intrinsic resistance of Enterococcus faecium to β-lactams, particularly to cephalosporins, therefore represents a major limitation of therapy. Although the primary mechanism of resistance to β-lactams in E. faecium is the presence of low-affinity monofunctional transpeptidase (class B) penicillin-binding protein Pbp5, the interaction of Pbp5 with other proteins is fundamental to maintain a

Identification, Characterization, and Three-Dimensional Structure of the Novel Circular Bacteriocin, Enterocin NKR-5-3B, from Enterococcus faecium.

PubMed

Himeno, Kohei; Rosengren, K Johan; Inoue, Tomoko; Perez, Rodney H; Colgrave, Michelle L; Lee, Han Siean; Chan, Lai Y; Henriques, Sónia Troeira; Fujita, Koji; Ishibashi, Naoki; Zendo, Takeshi; Wilaipun, Pongtep; Nakayama, Jiro; Leelawatcharamas, Vichien; Jikuya, Hiroyuki; Craik, David J; Sonomoto, Kenji

2015-08-11

Enterocin NKR-5-3B, one of the multiple bacteriocins produced by Enterococcus faecium NKR-5-3, is a 64-amino acid novel circular bacteriocin that displays broad-spectrum antimicrobial activity. Here we report the identification, characterization, and three-dimensional nuclear magnetic resonance solution structure determination of enterocin NKR-5-3B. Enterocin NKR-5-3B is characterized by four helical segments that enclose a compact hydrophobic core, which together with its circular backbone impart high stability and structural integrity. We also report the corresponding structural gene, enkB, that encodes an 87-amino acid precursor peptide that undergoes a yet to be described enzymatic processing that involves adjacent cleavage and ligation of Leu(24) and Trp(87) to yield the mature (circular) enterocin NKR-5-3B.

Culture methods impact recovery of antibiotic-resistant Enterococci including Enterococcus cecorum from pre- and postharvest chicken.

PubMed

Suyemoto, M M; Barnes, H J; Borst, L B

2017-03-01

Pathogenic strains of Enterococcus cecorum (EC) expressing multidrug resistance have emerged. In National Antimicrobial Resistance Monitoring System (NARMS) data, EC is rarely recovered from chickens. Two NARMS methodologies (FDA and USDA) were compared with standard culture (SC) techniques for recovery of EC. NARMS methods failed to detect EC in 58 caecal samples, 20 chicken breast or six whole broiler samples. EC was recovered from 1 of 38 (2·6%) and 2 of 38 (5·2%) preharvest spinal lesions (USDA and FDA method, respectively). In contrast, using the SC method, EC was recovered from 44 of 53 (83%) caecal samples, all 38 (100%) spinal lesions, 14 of 20 (70%) chicken breast samples, and all three spinal lesions identified in whole carcasses. Compared with other Enterococcus spp., EC isolates had a higher prevalence of resistance to macrolides. The NARMS methods significantly affected recovery of enterococcal species other than EC. When the postharvest FDA method was applied to preharvest caecal samples, isolates of Enterococcus faecium were preferentially recovered. All 11 E. faecium isolates were multidrug resistant, including resistance to penicillin, daptomycin and linezolid. These findings confirm that current methodologies may not accurately identify the amount and range of antimicrobial resistance of enterococci from chicken sources. Enterococci are an important reservoir for antimicrobial resistance. This study demonstrates how current culture methods underreport resistance to macrolides in enterococci by selecting against strains of Enterococcus cecorum in pre- and postharvest chicken. Further, the application of postharvest surveillance methods to preharvest samples resulted in selective recovery of Enterococcus faecium over Enterococcus faecalis. Isolates of E. faecium recovered exhibited multidrug resistance including penicillin, daptomycin and linezolid resistance. These findings suggest that culture methodology significantly impacts the range and

Bacteriocidal activity of sanitizers against Enterococcus faecium attached to stainless steel as determined by plate count and impedance methods.

PubMed

Andrade, N J; Bridgeman, T A; Zottola, E A

1998-07-01

Enterococcus faecium attached to stainless steel chips (100 mm2) was treated with the following sanitizers: sodium hypochlorite, peracetic acid (PA), peracetic acid plus an organic acid (PAS), quaternary ammonium, organic acid, and anionic acid. The effectiveness of sanitizer solutions on planktonic cells (not attached) was evaluated by the Association of Official Analytical Chemists (AOAC) suspension test. The number of attached cells was determined by impedance measurement and plate count method after vortexing. The decimal reduction (DR) in numbers of the E. faecium population was determined for the three methods and was analyzed by analysis of variance (P < 0.05) using Statview software. The adhered cells were more resistant (P < 0.05) than nonadherent cells. The DR averages for all of the sanitizers for 30 s of exposure were 6.4, 2.2, and 2.5 for the AOAC suspension test, plate count method after vortexing, and impedance measurement, respectively. Plate count and impedance methods showed a difference (P < 0.05) after 30 s of sanitizer exposure but not after 2 min. The impedance measurement was the best method to measure adherent cells. Impedance measurement required the development of a quadratic regression. The equation developed from 82 samples is as follows: log CFU/chip = 0.2385T2-0.96T + 9.35, r2 = 0.92, P < 0.05, T = impedance detection time in hours. This method showed that the sanitizers PAS and PA were more effective against E. faecium than the other sanitizers. At 30 s, the impedance method recovered about 25 times more cells than the plate count method after vortexing. These data suggest that impedance measurement is the method of choice when evaluating the number of bacterial cells adhered to a surface.

Validation of Baking To Control Salmonella Serovars in Hamburger Bun Manufacturing, and Evaluation of Enterococcus faecium ATCC 8459 and Saccharomyces cerevisiae as Nonpathogenic Surrogate Indicators.

PubMed

Channaiah, Lakshmikantha H; Holmgren, Elizabeth S; Michael, Minto; Sevart, Nicholas J; Milke, Donka; Schwan, Carla L; Krug, Matthew; Wilder, Amanda; Phebus, Randall K; Thippareddi, Harshavardhan; Milliken, George

2016-04-01

This study was conducted to validate a simulated commercial baking process for hamburger buns to destroy Salmonella serovars and to determine the appropriateness of using nonpathogenic surrogates (Enterococcus faecium ATCC 8459 or Saccharomyces cerevisiae) for in-plant process validation studies. Wheat flour was inoculated (∼6 log CFU/g) with three Salmonella serovars (Typhimurium, Newport, or Senftenberg 775W) or with E. faecium. Dough was formed, proofed, and baked to mimic commercial manufacturing conditions. Buns were baked for up to 13 min in a conventional oven (218.3°C), with internal crumb temperature increasing to ∼100°C during the first 8 min of baking and remaining at this temperature until removal from the oven. Salmonella and E. faecium populations were undetectable by enrichment (>6-log CFU/g reductions) after 9.0 and 11.5 min of baking, respectively, and ≥5-log-cycle reductions were achieved by 6.0 and 7.75 min, respectively. D-values of Salmonella (three-serovar cocktail) and E. faecium 8459 in dough were 28.64 and 133.33, 7.61 and 55.67, and 3.14 and 14.72 min at 55, 58, and 61°C, respectively, whereas D-values of S. cerevisiae were 18.73, 5.67, and 1.03 min at 52, 55, and 58°C, respectivly. The z-values of Salmonella, E. faecium, and S. cerevisiae were 6.58, 6.25, and 4.74°C, respectively. A high level of thermal lethality was observed for baking of typical hamburger bun dough, resulting in rapid elimination of high levels of the three-strain Salmonella cocktail; however, the lethality and microbial destruction kinetics should not be extrapolated to other bakery products without further research. E. faecium demonstrated greater thermal resistance compared with Salmonella during bun baking and could serve as a conservative surrogate to validate thermal process lethality in commercial bun baking operations. Low thermal tolerance of S. cerevisiae relative to Salmonella serovars limits its usefulness as a surrogate for process validations.

Molecular Analysis of VanA Outbreak of Enterococcus faecium in Two Warsaw Hospitals: The Importance of Mobile Genetic Elements

PubMed Central

Wardal, Ewa; Markowska, Katarzyna; Żabicka, Dorota; Wróblewska, Marta; Giemza, Małgorzata; Mik, Ewa; Połowniak-Pracka, Hanna; Woźniak, Agnieszka; Hryniewicz, Waleria; Sadowy, Ewa

2014-01-01

Vancomycin-resistant Enterococcus faecium represents a growing threat in hospital-acquired infections. Two outbreaks of this pathogen from neighboring Warsaw hospitals have been analyzed in this study. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA, multilocus VNTR analysis (MLVA), and multilocus sequence typing (MLST) revealed a clonal variability of isolates which belonged to three main lineages (17, 18, and 78) of nosocomial E. faecium. All isolates were multidrug resistant and carried several resistance, virulence, and plasmid-specific genes. Almost all isolates shared the same variant of Tn1546 transposon, characterized by the presence of insertion sequence ISEf1 and a point mutation in the vanA gene. In the majority of cases, this transposon was located on 50 kb or 100 kb pRUM-related plasmids, which lacked, however, the axe-txe toxin-antitoxin genes. 100 kb plasmid was easily transferred by conjugation and was found in various clonal backgrounds in both institutions, while 50 kb plasmid was not transferable and occurred solely in MT159/ST78 strains that disseminated clonally in one institution. Although molecular data indicated the spread of VRE between two institutions or a potential common source of this alert pathogen, epidemiological investigations did not reveal the possible route by which outbreak strains disseminated. PMID:25003118

Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages.

PubMed

Gröbner, Sabine; Fritz, Evelyn; Schoch, Friederike; Schaller, Martin; Berger, Alexander C; Bitzer, Michael; Autenrieth, Ingo B

2010-10-01

Enterococci are commensal organisms in the alimentary tract. However, they can cause a variety of life-threatening infections, especially in nosocomial settings. We hypothesized that induction of cell death might enable these facultative pathogenic bacteria to evade the innate immune response and to cause infections of their host. We demonstrate that E. faecium when exposed to lysozyme induces cell death in macrophages in vitro and in vivo. Flow cytometric analyses of J774A.1 macrophages infected with E. faecium revealed loss of cell membrane integrity indicated by uptake of propidium iodide and decrease of the inner mitochondrial transmembrane potential DeltaPsi(m). Inhibition of caspases, treatment of macrophages with cytochalasin D, or rifampicin did not prevent cells from dying, suggesting cell death mechanisms that are independent of caspase activation, bacterial uptake, and intracellular bacterial replication. Characteristics of necrotic cell death were demonstrated by both lack of procaspase 3 activation and cell shrinkage, electron microscopy, and release of lactate dehydrogenase. Pretreatment of E. faecium with lysozyme and subsequently with broad spectrum protease considerably reduced cell death, suggesting that a bacterial surface protein is causative for cell death induction. Moreover, in a mouse peritonitis model we demonstrated that E. faecium induces cell death of peritoneal macrophages in vivo. Altogether, our results show that enterococci, under specific conditions such as exposure to lysozyme, induce necrotic cell death in macrophages, which might contribute to disseminated infections by these facultative pathogenic bacteria.

Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria.

PubMed

Klocke, Michael; Mundt, Kerstin; Idler, Frank; Jung, Sabrina; Backhausen, Jan E

2005-06-01

The genes for the bacteriocins enterocin A and B were isolated from Enterococcus faecium ATB 197a. Using the pET37b(+) vector, the enterocin genes were fused to an Escherichia coli specific export signal sequence, a cellulose-binding domain (CBD(cenA)) and a S-tag under the control of a T7lac promotor. The constructs were subsequently cloned into E. coli host cells. The expression of the recombinant enterocins had different effects on both the host cells and other Gram-positive bacteria. The expression of entA in Esc. coli led to the synthesis and secretion of functional active enterocin A fusion proteins, which were active against some Gram-positive indicator bacteria, but did not influence the viability of the host cells. In contrast, the expression of enterocin B fusion proteins led to a reduced viability of the host cells, indicating a misfolding of the protein or interference with the cellular metabolism of Esc. coli. Indicator strains of Gram-positive bacteria were not inhibited by purified enterocin B fusion proteins. However, recombinant enterocin B displayed inhibitory activity after the proteolytic cleavage of the fused peptides.

Relentless spread and adaptation of non-typeable vanA vancomycin-resistant Enterococcus faecium: a genome-wide investigation.

PubMed

van Hal, Sebastiaan J; Beukers, Alicia G; Timms, Verlaine J; Ellem, Justin A; Taylor, Peter; Maley, Michael W; Newton, Peter J; Ferguson, John K; Lee, Andie; Chen, Sharon C-A; Sintchenko, Vitali

2018-03-16

VRE are prevalent among patients in ICUs. Non-typeable vanA VRE, due to loss of one of the genes used for MLST (pstS), have increased in Australia, suggestive of a new, hospital-acquired lineage. To understand the significance of this lineage and its transmission using WGS of strains isolated from patients in ICUs across New South Wales, Australia. A total of 240 Enterococcus faecium isolates collected between February and May 2016, and identified by conventional PCR as vanA positive, were sequenced. Isolates originated from 12 ICUs in New South Wales, grouped according to six local health districts, and represented both rectal screening swab (n = 229) and clinical (n = 11) isolates. ST analysis revealed the absence of the pstS gene in 84.2% (202 of 240) of vanA isolates. Two different non-typeable STs were present based on different allelic backbone patterns. Loss of the pstS gene appeared to be the result of multiple recombination events across this region. Evidence for pstS-negative lineage spread across all six local health districts was observed suggestive of inter-hospital transmission. In addition, multiple outbreaks were detected, some of which were protracted and lasted for the duration of the study. These findings confirmed the evolution, emergence and dissemination of non-typeable vanA E. faecium. This study has highlighted the utility of WGS when attempting to describe accurately the hospital-based pathogen epidemiology, which in turn will continue to inform optimal infection control measures necessary to halt the spread of this important nosocomial organism.

Isolation and characterization of a new bacteriocin, termed enterocin M, produced by environmental isolate Enterococcus faecium AL41.

PubMed

Mareková, Mária; Lauková, Andrea; Skaugen, Morten; Nes, Ingolf

2007-08-01

The new bacteriocin, termed enterocin M, produced by Enterococcus faecium AL 41 showed a wide spectrum of inhibitory activity against the indicator organisms from different sources. It was purified by (NH4)2SO4 precipitation, cation-exchange chromatography and reverse phase chromatography (FPLC). The purified peptide was sequenced by N-terminal amino acid Edman degradation and a mass spectrometry analysis was performed. By combining the data obtained from amino acid sequence (39 N-terminal amino acid residues was determined) and the molecular weight (determined to be 4628 Da) it was concluded that the purified enterocin M is a new bacteriocin, which is very similar to enterocin P. However, its molecular weight is different from enterocin P (4701.25). Of the first 39 N-terminal residues of enterocin M, valine was found in position 20 and a lysine in position 35, while enterocin P has tryptophane residues in these positions.

Genetic identification of the bacteriocins produced by Enterococcus faecium IT62 and evidence that bacteriocin 32 is identical to enterocin IT.

PubMed

Izquierdo, Esther; Cai, Yimin; Marchioni, Eric; Ennahar, Saïd

2009-05-01

Enterococcus faecium IT62, a strain isolated from ryegrass in Japan, produces three bacteriocins (enterocins L50A, L50B, and IT) that have been previously purified and the primary structures of which have been determined by amino acid sequencing (E. Izquierdo, A. Bednarczyk, C. Schaeffer, Y. Cai, E. Marchioni, A. Van Dorsselaer, and S. Ennahar, Antimicrob. Agents Chemother., 52:1917-1923, 2008). Genetic analysis showed that the bacteriocins of E. faecium IT62 are plasmid encoded, but with the structural genes specifying enterocin L50A and enterocin L50B being carried by a plasmid (pTAB1) that is separate from the one (pTIT1) carrying the structural gene of enterocin IT. Sequencing analysis of a 1,475-bp region from pTAB1 identified two consecutive open reading frames corresponding, with the exception of 2 bp, to the genes entL50A and entL50B, encoding EntL50A and EntL50B, respectively. Both bacteriocins are synthesized without N-terminal leader sequences. Genetic analysis of a sequenced 1,380-bp pTIT1 fragment showed that the genes entIT and entIM, encoding enterocin IT and its immunity protein, respectively, were both found in E. faecium VRE200 for bacteriocin 32. Enterocin IT, a 6,390-Da peptide made up of 54 amino acids, has been previously shown to be identical to the C-terminal part of bacteriocin 32, a 7,998-Da bacteriocin produced by E. faecium VRE200 whose structure was deduced from its structural gene (T. Inoue, H. Tomita, and Y. Ike, Antimicrob. Agents Chemother., 50:1202-1212, 2006). By combining the biochemical and genetic data on enterocin IT, it was concluded that bacteriocin 32 is in fact identical to enterocin IT, both being encoded by the same plasmid-borne gene, and that the N-terminal leader peptide for this bacteriocin is 35 amino acids long and not 19 amino acids long as previously reported.

Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium

PubMed Central

Sillanpää, Jouko; Nallapareddy, Sreedhar R.; Prakash, Vittal P.; Qin, Xiang; Hook, Magnus; Weinstock, George M.; Murray, Barbara E.

2009-01-01

SUMMARY Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about pathogenic determinants of this organism. We have previously identified a cell wall anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad spectrum binding to extracellular matrix proteins. Here, we analyzed the draft genome of strain TX0016 for potential MSCRAMMs (microbial surface component recognizing adhesive matrix molecules). Genome-based bioinformatics identified 22 predicted cell wall anchored E. faeciumsurface proteins (Fms) of which 15 (including Acm) have typical characteristics of MSCRAMMs including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one (Fms10, redesignated Scm for second collagen adhesin of E. faeciu m) revealed that recombinant Scm65 (A- and B-domains) and Scm36 (A-domain) bound efficiently to collagen type V in a concentration dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism of recombinant Scm36 and of Acm37 indicated that these proteins are rich in β-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; 9 of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated as EbpCfm), detected a “ladder” pattern of high-molecular weight protein bands in a Western blot

Comparison of Genomic Methods for Differentiating Strains of Enterococcus faecium: Assessment Using Clinical Epidemiologic Data

PubMed Central

Savor, Connie; Pfaller, Michael A.; Kruszynski, Julie A.; Hollis, Richard J.; Noskin, Gary A.; Peterson, Lance R.

1998-01-01

Genomic DNA extracted from 45 vancomycin-resistant Enterococcus faecium (VRE) isolates was cleaved with HindIII and HaeIII and subjected to agarose gel electrophoresis. The ability of this method (restriction endonuclease analysis [REA]) to distinguish strains at the subspecies level was compared with results previously determined by pulsed-field gel electrophoresis (PFGE). Chart reviews were performed to provide a clinical correlation of possible epidemiologic relatedness. A likely clinical association was found for 29 patients as part of two outbreaks. REA found 21 of 21 isolates were the same type in the first outbreak, with PFGE calling 19 strains the same type. In the second outbreak with eight patient isolates, HindIII found six were the same type and two were unique types. HaeIII found three strains were the same type, two strains were a separate type, and three more strains were unique types, while PFGE found three were the same type and five were unique types. No single “ideal” method can be used without clinical epidemiologic investigation, but any of these techniques is helpful in providing focus to infection control practitioners assessing possible outbreaks of nosocomial infection. PMID:9774587

RelA Mutant Enterococcus faecium with Multiantibiotic Tolerance Arising in an Immunocompromised Host

PubMed Central

Honsa, Erin S.; Mhaissen, Mohammed N.; Frank, Matthew; Shaker, Jessica; Iverson, Amy; Rubnitz, Jeffrey; Hayden, Randall T.; Lee, Richard E.; Rock, Charles O.; Tuomanen, Elaine I.

2017-01-01

ABSTRACT Serious bacterial infections in immunocompromised patients require highly effective antibacterial therapy for cure, and thus, this setting may reveal novel mechanisms by which bacteria circumvent antibiotics in the absence of immune pressure. Here, an infant with leukemia developed vancomycin-resistant Enterococcus faecium (VRE) bacteremia that persisted for 26 days despite appropriate antibiotic therapy. Sequencing of 22 consecutive VRE isolates identified the emergence of a single missense mutation (L152F) in relA, which constitutively activated the stringent response, resulting in elevated baseline levels of the alarmone guanosine tetraphosphate (ppGpp). Although the mutant remained susceptible to both linezolid and daptomycin in clinical MIC testing and during planktonic growth, it demonstrated tolerance to high doses of both antibiotics when growing in a biofilm. This biofilm-specific gain in resistance was reflected in the broad shift in transcript levels caused by the mutation. Only an experimental biofilm-targeting ClpP-activating antibiotic was able to kill the mutant strain in an established biofilm. The relA mutation was associated with a fitness trade-off, forming smaller and less-well-populated biofilms on biological surfaces. We conclude that clinically relevant relA mutations can emerge during prolonged VRE infection, causing baseline activation of the stringent response, subsequent antibiotic tolerance, and delayed eradication in an immunocompromised state. PMID:28049149

Molecular analysis and distribution of multidrug-resistant Enterococcus faecium isolates belonging to clonal complex 17 in a tertiary care center in Mexico City

PubMed Central

2013-01-01

Background Enterococcus faecium has recently emerged as a multidrug-resistant nosocomial pathogen involved in outbreaks worldwide. A high rate of resistance to different antibiotics has been associated with virulent clonal complex 17 isolates carrying the esp and hyl genes and the purK1 allele. Results Twelve clinical vancomycin-resistant Enterococcus faecium (VREF) isolates were obtained from pediatric patients at the Hospital Infantil de México Federico Gómez (HIMFG). Among these VREF isolates, 58.3% (7/12) were recovered from urine, while 41.7% (5/12) were recovered from the bloodstream. The VREF isolates showed a 100% rate of resistance to ampicillin, amoxicillin-clavulanate, ciprofloxacin, clindamycin, chloramphenicol, streptomycin, gentamicin, rifampicin, erythromycin and teicoplanin. In addition, 16.7% (2/12) of the isolates were resistant to linezolid, and 66.7% (8/12) were resistant to tetracycline and doxycycline. PCR analysis revealed the presence of the vanA gene in all 12 VREF isolates, esp in 83.3% (10/12) of the isolates and hyl in 50% (6/12) of the isolates. Phylogenetic analysis via molecular typing was performed using pulsed-field gel electrophoresis (PFGE) and demonstrated 44% similarity among the VREF isolates. MLST analysis identified four different sequence types (ST412, ST757, ST203 and ST612). Conclusion This study provides the first report of multidrug-resistant VREF isolates belonging to clonal complex 17 from a tertiary care center in Mexico City. Multidrug resistance and genetic determinants of virulence confer advantages among VREF in the colonization of their host. Therefore, the prevention and control of the spread of nosocomial infections caused by VREF is crucial for identifying new emergent subclones that could be challenging to treat in subsequent years. PMID:24330424

Efficacy of Daptomycin Monotherapy and In Combination with β-lactams for Daptomycin-Susceptible Enterococcus faecium Harboring LiaSR Substitutions: Influence of The Inoculum Effect.

PubMed

Kebriaei, Razieh; Rice, Seth A; Singh, Kavindra V; Stamper, Kyle C; Dinh, An Q; Rios, Rafael; Diaz, Lorena; Murray, Barbara E; Munita, Jose M; Tran, Truc T; Arias, Cesar A; Rybak, Michael J

2018-05-14

Enterococcus faecium that harbor LiaFSR substitutions but are phenotypically susceptible to daptomycin (DAP) by current breakpoints are problematic since predisposition to resistance may lead to therapeutic failure. Using a simulated endocardial vegetation (SEV) PK/PD model, we investigated DAP regimens (6, 8 and 10 mg/kg/day) as monotherapy and in combination with ampicillin (AMP), ceftaroline (CPT) or ertapenem (ERT) against E. faecium HOU503, a DAP-susceptible strain that harbors common LiaS and LiaR substitutions found in clinical isolates (T120S and W73C, respectively). Of interest, the efficacy of DAP monotherapy, at any dose regimen, was dependent on the size of the inoculum. At an inoculum of ∼10 9 CFU/g, DAP doses of 6-8 mg/kg/d were not effective and led to significant regrowth with emergence of resistant derivatives. In contrast, at an inoculum of ∼10 7 , marked reductions in bacterial counts were observed with DAP 6 mg/kg/d with no resistance. The inoculum effect was confirmed in a rat model using humanized DAP exposures. Combinations of DAP with AMP, CPT or ERT demonstrated enhanced eradication and reduced potential for resistance allowing for de-escalation of the DAP dose. Persistence of the LiaRS substitutions were identified in DAP-resistant isolates recovered from the SEV model and in DAP-resistant derivatives of an initially DAP-susceptible clinical isolate of E. faecium (HOU668) harboring LiaSR substitutions and recovered from a patient with a recurrent bloodstream infection. Our results provide novel data for the use of DAP monotherapy and combinations for recalcitrant E. faecium infections and paves the way for testing these approaches in humans. Copyright © 2018 American Society for Microbiology.

Control of Listeria monocytogenes in goat's milk and goat's jben by the bacteriocinogenic Enterococcus faecium F58 strain.

PubMed

Achemchem, Fouad; Abrini, Jamal; Martínez-Bueno, Manuel; Valdivia, Eva; Maqueda, Mercedes

2006-10-01

The bacteriocinogenic Enterococcus faecium F58 strain, a natural goat's jben cheese isolate, lacks decarboxylase activity involved in most biogenic amine formation. It was also sensitive to 13 antibiotics assayed and free of virulence and vancomycin resistance genes. The F58 strain reached the stationary phase after 12 h of growth in sterile goat's milk, and the production of enterocin F-58 (Ent L50) was first detected after 48 h (400 AU/ml), thereafter remaining stable up to 5 days. The effectiveness of the F58 strain in controlling Listeria monocytogenes serovar 4b in reduced fat and whole goat's milk, and in goat's jben has been examined. Coculture experiments of F58-L. monocytogenes in both types of milk demonstrated that listeriae were not eliminated, although reductions by 1 to 4 log units were found. Nevertheless, when the F58 strain was previously inoculated in whole milk and left to grow for 12 h before contamination, the pathogen was completely eliminated after 130 h of coculture. Production of jben cheese contaminated with L. monocytogenes prior to packaging, using preparations of F58-producer strain, caused a significant decrease in the number of viable listeriae, which were undetectable after 1 week of cheese storage at 22 degrees C. Altogether, results from this study suggest that E. faecium F58 strain may be used as an adjunct culture in cheese to control contamination and growth of L. monocytogenes by in situ enterocin production, thus providing an additional hurdle to enhance control of this pathogen.

Influence of oral application of Enterococcus faecium AL41 on TGF-β4 and IL-17 expression and immunocompetent cell distribution in chickens challenged with Campylobacter jejuni.

PubMed

Letnická, Alica; Karaffová, Viera; Levkut, Mikuláš; Revajová, Viera; Herich, Róbert

2017-09-01

Campylobacteriosis is mainly caused by infection with Campylobacter jejuni following consumption or handling of Campylobacter-contaminated poultry meat. The aim of this study was to investigate the effect of probiotic Enterococcus faecium AL41 on TGF-β4 and IL-17 expression and on immunocompetent cell distribution after C. jejuni infection in broiler chicken, as a second part of the previous study of Karaffová et al. (2017). Accordingly, day-old chicks were randomly divided into four experimental groups of 10 chicks each (n = 10): control (C), E. faecium AL41 (EFAL41), C. jejuni CCM6191 (CJ), and combined E. faecium AL41 + C. jejuni CCM6191 (EFAL41 + CJ). Samples from the caecum were collected on days 4 and 7 post Campylobacter infection (dpi), for the isolation of mRNA of TGF-β4, IL-17 and for immunohistochemistry. The relative mRNA expression of TGF-β4 was upregulated in the combined (EFAL41 + CJ) group compared to other groups during both samplings, but the expression of IL-17 was downregulated. Similarly, the highest density of CD3+ was detected in the combined group at 7 dpi, but the number of IgA+ cells was increased in both groups with EFAL41. It was concluded that the EFAL41 probiotic E. faecium strain can modulate the expression of selected cytokines (upregulation of TGF-β4 but downregulation of IL-17 relative expression), and activate IgA-producing cells in the caeca of chicks infected with C. jejuni CCM6191.

Molecular characterization and antibiotic resistance of Enterococcus species from gut microbiota of Chilean Altiplano camelids

PubMed Central

Guerrero-Olmos, Katheryne; Báez, John; Valenzuela, Nicomédes; Gahona, Joselyne; del Campo, Rosa; Silva, Juan

2014-01-01

Background Enterococcus is one of the major human pathogens able to acquire multiple antibiotic-resistant markers as well as virulence factors which also colonize remote ecosystems, including wild animals. In this work, we characterized the Enterococcus population colonizing the gut of Chilean Altiplano camelids without foreign human contact. Material and methods Rectal swabs from 40 llamas and 10 alpacas were seeded in M-Enterococcus agar, and we selected a total of 57 isolates. Species identification was performed by biochemical classical tests, semi-automated WIDER system, mass spectrometry analysis by MALDI-TOF (matrix-assisted laser desorption/ionization with a time-of-flight mass spectrometer), and, finally, nucleotide sequence of internal fragments of the 16S rRNA, rpoB, pheS, and aac(6)-I genes. Genetic diversity was measured by pulsed field gel electrophoresis (PFGE)-SmaI, whereas the antibiotic susceptibility was determined by the WIDER system. Carriage of virulence factors was explored by polymerase chain reaction (PCR). Results Our results demonstrated that the most prevalent specie was Enterococcus hirae (82%), followed by other non–Enterococcus faecalis and non–Enterococcus faecium species. Some discrepancies were detected among the identification methods used, and the most reliable were the rpoB, pheS, and aac(6)-I nucleotide sequencing. Selected isolates exhibited susceptibility to almost all studied antibiotics, and virulence factors were not detected by PCR. Finally, some predominant clones were characterized by PFGE into a diverse genetic background. Conclusion Enterococcus species from the Chilean camelids’ gut microbiota were different from those adapted to humans, and they remained free of antibiotic resistance mechanisms as well as virulence factors. PMID:25405007

A novel enterocin T1 with anti-Pseudomonas activity produced by Enterococcus faecium T1 from Chinese Tibet cheese.

PubMed

Liu, Hui; Zhang, Lanwei; Yi, Huaxi; Han, Xue; Gao, Wei; Chi, Chunliang; Song, Wei; Li, Haiying; Liu, Chunguang

2016-02-01

An enterocin-producing Enterococcus faecium T1 was isolated from Chinese Tibet cheese. The enterocin was purified by SP-Sepharose and reversed phase HPLC. It was identified as unique from other reported bacteriocins based on molecular weight (4629 Da) and amino acid compositions; therefore it was subsequently named enterocin T1. Enterocin T1 was stable at 80-100 °C and over a wide pH range, pH 3.0-10.0. Protease sensitivity was observed to trypsin, pepsin, papain, proteinase K, and pronase E. Importantly, enterocin T1 was observed to inhibit the growth of numerous Gram-negative and Gram-positive bacteria including Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas fluorescens, Escherichia coli, Salmonella typhimurium, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Listeria monocytogenes. Take together, these results suggest that enterocin T1 is a novel bacteriocin with the potential to be used as a bio-preservative to control Pseudomonas spp. in food.

The Leaderless Bacteriocin Enterocin K1 Is Highly Potent against Enterococcus faecium: A Study on Structure, Target Spectrum and Receptor.

PubMed

Ovchinnikov, Kirill V; Kristiansen, Per Eugen; Straume, Daniel; Jensen, Marianne S; Aleksandrzak-Piekarczyk, Tamara; Nes, Ingolf F; Diep, Dzung B

2017-01-01

Enterocin K1 (EntK1), enterocin EJ97 (EntEJ97), and LsbB are three sequence related leaderless bacteriocins. Yet LsbB kills only lactococci while EntK1 and EntEJ97 target wider spectra with EntK1 being particularly active against Enterococcus faecium , including nosocomial multidrug resistant isolates. NMR study of EntK1 showed that it had a structure very similar to LsbB - both having an amphiphilic N-terminal α-helix and an unstructured C-terminus. The α-helix in EntK1 is, however, about 3-4 residues longer than that of LsbB. Enterococcal mutants highly resistant to EntEJ97 and EntK1 were found to have mutations within rseP , a gene encoding a stress response membrane-bound Zn-dependent protease. Heterologous expression of the enterococcal rseP rendered resistant cells of Streptococcus pneumoniae sensitive to EntK1 and EntEJ97, suggesting that RseP likely serves as the receptor for EntK1 and EntEJ97. It was also shown that the conserved proteolytic active site in E. faecalis RseP is partly required for EntK1 and EntEJ97 activity, since alanine substitutions of its conserved residues (HExxH) reduced the sensitivity of the clones to the bacteriocins. RseP is known to be involved in bacterial stress response. As expected, the growth of resistant mutants with mutations within rseP was severely affected when they were exposed to higher (stressing) growth temperatures, e.g., at 45°C, at which wild type cells still grew well. These findings allow us to design a hurdle strategy with a combination of the bacteriocin(s) and higher temperature that effectively kills bacteriocin sensitive bacteria and prevents the development of resistant cells.

The Identification and Functional Characterization of WxL Proteins from Enterococcus faecium Reveal Surface Proteins Involved in Extracellular Matrix Interactions

PubMed Central

Galloway-Peña, Jessica R.; Liang, Xiaowen; Singh, Kavindra V.; Yadav, Puja; Chang, Chungyu; La Rosa, Sabina Leanti; Shelburne, Samuel; Ton-That, Hung; Höök, Magnus

2014-01-01

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6 Enterococcus faecium proteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence in E. faecium and possibly other Gram-positive bacterial species. PMID:25512313

Cloning, purification, crystallization and preliminary crystallographic analysis of SecA from Enterococcus faecalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meining, Winfried, E-mail: wim@csb.ki.se; Scheuring, Johannes; Fischer, Markus

2006-06-01

SecA ATPase from E. faecalis has been cloned, overexpressed, purified and crystallized. Crystals belong to space group C2 and diffract to 2.4 Å resolution. The gene coding for SecA from Enterococcus faecalis was cloned and overexpressed in Escherichia coli. In this protein, the lysine at position 6 was replaced by an asparagine in order to reduce sensitivity towards proteases. The modified protein was purified and crystallized. Crystals diffracting to 2.4 Å resolution were obtained using the vapour-diffusion technique. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 203.4, b = 49.8, c = 100.8 Å,more » α = γ = 90.0, β = 119.1°. A selenomethionine derivative was prepared and is currently being tested in crystallization trials.« less

Evaluation of species-specific PCR, Bruker MS, VITEK MS and the VITEK 2 system for the identification of clinical Enterococcus isolates.

PubMed

Fang, H; Ohlsson, A-K; Ullberg, M; Ozenci, V

2012-11-01

The purpose of this investigation was to compare the performance of species-specific polymerase chain reaction (PCR), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and phenotypic identification systems for the identification of Enterococcus species. A total of 132 clinical isolates were investigated by the following: (1) a multiplex real-time PCR assay targeting ddl Enterococcus faecium, ddl Enterococcus faecalis, vanC1 and vanC2/C3 genes, and a high-resolution melting (HRM) analysis of the groESL gene for the differentiation of Enterococcus casseliflavus and Enterococcus gallinarum; (2) Bruker MS; (3) VITEK MS; and (4) the VITEK 2 system. 16S rRNA gene sequencing was used as a reference method in the study. The 132 isolates were identified as 32 E. faecalis, 63 E. faecium, 16 E. casseliflavus and 21 E. gallinarum. The multiplex PCR, Bruker MS and VITEK MS were able to identify all the isolates correctly at the species level. The VITEK 2 system could identify 131/132 (99.2 %) and 121/132 (91.7 %) of the isolates at the genus and species levels, respectively. The HRM-groESL assay identified all (21/21) E. gallinarum isolates and 81.3 % (13/16) of the E. casseliflavus isolates. The PCR methods described in the present study are effective in identifying the enterococcal species. MALDI-TOF MS is a rapid, reliable and cost-effective identification technique for enterococci. The VITEK 2 system is less efficient at detecting non-faecalis and non-faecium Enterococcus species.

Evaluation of High-Resolution Melting Curve Analysis of Ligation-Mediated Real-Time PCR, a Rapid Method for Epidemiological Typing of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Species) Pathogens

PubMed Central

Ryberg, Anna; Billström, Hanna; Hällgren, Anita; Nilsson, Lennart E.; Marklund, Britt-Inger; Olsson-Liljequist, Barbro; Schön, Thomas

2014-01-01

A single-tube method, ligation-mediated real-time PCR high-resolution melt analysis (LMqPCR HRMA), was modified for the rapid typing of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens. A 97% agreement (60/62 isolates) was achieved in comparison to pulsed-field gel electrophoresis (PFGE) results, which indicates that LMqPCR HRMA is a rapid and accurate screening tool for monitoring nosocomial outbreaks. PMID:25232168

Characterization of veterinary hospital-associated isolates of Enterococcus species in Korea.

PubMed

Chung, Yeon Soo; Kwon, Ka Hee; Shin, Sook; Kim, Jae Hong; Park, Yong Ho; Yoon, Jang Won

2014-03-28

Possible cross-transmission of hospital-associated enterococci between human patients, medical staff, and hospital environments has been extensively studied. However, limited information is available for veterinary hospital-associated Enterococcus isolates. This study investigated the possibility of cross-transmission of antibiotic-resistant enterococci between dog patients, their owners, veterinary staff, and hospital environments. Swab samples (n =46 5) were obtained from five veterinary hospitals in Seoul, Korea, during 2011. Forty-three Enterococcus strains were isolated, representing seven enterococcal species. E. faecalis and E. faecium were the most dominant species (16 isolates each, 37.2%). Although slight differences in the antibiotic resistance profiles were observed between the phenotypic and the genotypic data, our antibiogram analysis demonstrated high prevalence of the multiple drug-resistant (MDR) isolates of E. faecalis (10/16 isolates, 62.5%) and E. faecium (12/16 isolates, 75.0%). Pulsed-field gel electrophoretic comparison of the MDR isolates revealed three different clonal sets of E. faecalis and a single set of E. faecium, which were isolated from different sample groups or dog patients at the same or two separate veterinary hospitals. These results imply a strong possibility of cross-transmission of the antibiotic-resistant enterococcal species between animal patients, owners, veterinary staff, and hospital environments.

Association of Enterococcus spp. with Severe Combat Extremity Injury, Intensive Care, and Polymicrobial Wound Infection.

PubMed

Heitkamp, Rae A; Li, Ping; Mende, Katrin; Demons, Samandra T; Tribble, David R; Tyner, Stuart D

2018-01-01

Combat-related extremity wound infections can complicate the recovery of injured military personnel. The Enterococcus genus contains both commensal and pathogenic bacteria found in many combat wounds. We describe the patient population susceptible to Enterococcus infection, the characteristics of Enterococcus spp. isolated from combat-related wounds, and the microbiological profile of Enterococcus-positive wounds. Patient and culture data were obtained from the Trauma Infectious Disease Outcomes Study. Subjects were divided into a case group with enterococcal extremity wound infections and a comparator group with wound infections caused by other micro-organisms. Case and comparator subjects had similar patterns of injury and infection. Case subjects had higher Injury Severity Scores (33 vs. 30; p < 0.001), longer hospitalization at U.S. facilities (55 vs. 40 days; p = 0.004), and required more large-volume blood transfusions (>20 units) within 24 h post-injury (53% vs. 30%; p < 0.001). Approximately 60% of case subjects had three or more infections, and 91% had one or more polymicrobial infections, compared with 43% and 50%, respectively, in the comparator group. The thigh was the most common site of Enterococcus spp. isolation, contributing 50% of isolates. Enterococcus faecium was the predominant species isolated from case-group infections overall (66%), as well as in polymicrobial infections (74%). Frequent co-colonizing microbes in polymicrobial wound infections with Enterococcus were other ESKAPE pathogens (64%) (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae [and Escherichia coli], Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) and fungi (35%). The specific pathogenicity of Enterococcus relative to other pathogens in polymicrobial wounds is unknown. Identifying strain-specific outcomes and investigating the interactions of Enterococcus strains with other wound pathogens could provide additional tools

Determination of resistance and virulence genes in Enterococcus faecalis and E. faecium strains isolated from poultry and their genotypic characterization by ADSRRS-fingerprinting.

PubMed

Nowakiewicz, A; Ziólkowska, G; Troscianczyk, A; Zieba, P; Gnat, S

2017-04-01

The aim of this study was to determine the antimicrobial resistance of E. faecalis and E. faecium strains isolated from poultry and to carry out genotypic characterization thereof with the ADSRRS-fingerprinting method (amplification of DNA fragments surrounding rare restriction sites) and analysis of the genetic relatedness between the isolates with different resistance and virulence determinants. Samples were collected from 70 4-week-old chickens and tested for Enterococcus. Minimum inhibitory concentrations of 11 antimicrobials were determined using the broth microdilution method. Detection of antibiotic resistance and virulence genes was performed using PCR, and molecular analysis was carried out using the ADSRRS-fingerprinting method. The highest percentage of strains was resistant to tetracycline (60.5%) and erythromycin (54.4%), and a large number exhibited high-level resistance to both kanamycin (42.1%) and streptomycin (34.2%). Among 8 genes encoding AME, the tested strains showed mainly the presence of [aph(3΄)-IIIa], [ant(6)-Ia], [aac(6΄)-Ie-aph(2΄΄)-Ia], and [ant(9)-Ia] genes. Phenotypic resistance to erythromycin was encoded in 98.4% strains by the ermB gene. Genotypic resistance to tetracycline in E. faecium was associated with the presence of tetM and tetL (respectively, in 95.5 and 57.7% of the isolates); in contrast, E. faecalis strains were characterized mainly by the presence of tetO (83.3%). The virulence profile was homogenous for all E. faecium strains and included only efaAfm and ccf genes. All E. faecalis strains exhibited efaAfs, gelE, and genes encoding sex pheromones. The strains tested exhibited 34 genotypic profiles. Comparative analysis of phenotypic and genotypic resistance and virulence profiles and confrontation thereof with the genotypes of the strains tested showed that strains assigned to a particular genotype have an identical phenotypic resistance profile and a panel of resistance and virulence genes. The results of this

Prevalence of enterococcus species and their virulence genes in fresh water prior to and after storm events.

PubMed

Sidhu, J P S; Skelly, E; Hodgers, L; Ahmed, W; Li, Y; Toze, S

2014-01-01

Enterococcus spp. isolates (n = 286) collected from six surface water bodies in subtropical Brisbane, Australia, prior to and after storm events, were identified to species level and tested for the presence of seven clinically important virulence genes (VGs). Enterococcus faecalis (48%), Enterococcus faecium (14%), Enterococcus mundtii (13%), and Enterococcus casseliflavus (13%) were frequently detected at all sites. The frequency of E. faecium occurrence increased from 6% in the dry period to 18% after the wet period. The endocarditis antigen (efaA), gelatinase (gelE), collagen-binding protein (ace), and aggregation substance (asa1) were detected in 61%, 43%, 43%, and 23% of Enterococcus isolates, respectively. The chances of occurrence of ace, gelE, efaA, and asa1 genes in E. faecalis were found to be much higher compared to the other Enterococcus spp. The observed odds ratio of occurrence of ace and gelE genes in E. faecalis was much higher at 7.96 and 6.40 times, respectively. The hyl gene was 3.84 times more likely to be detected in E. casseliflavus. The presence of multiple VGs in most of the E. faecalis isolates underscores the importance of E. faecalis as a reservoir of VGs in the fresh water aquatic environment. Consequently, if contaminated surface water is to be used for production of potable and nonpotable water some degree of treatment depending upon intended use such as detention in basins prior to use or chlorination is required.

Novel Structure of Enterococcus faecium-Originated ermB-Positive Tn1546-Like Element in Staphylococcus aureus.

PubMed

Wan, Tsai-Wen; Hung, Wei-Chun; Tsai, Jui-Chang; Lin, Yu-Tzu; Lee, Hao; Hsueh, Po-Ren; Lee, Tai-Fen; Teng, Lee-Jene

2016-10-01

We determined the resistance determinants in 274 erythromycin-resistant methicillin-susceptible Staphylococcus aureus (MSSA) isolates during a 13-year period, 2000 to 2012. The resistance phenotypes, inducible macrolide-lincosamide-streptogramin (iMLS), constitutive MLS (cMLS), and macrolide-streptogramin (MS) resistance phenotypes, were examined by a double-disk diffusion D test. The ermB gene was more frequent (35%; 97/274) than ermC (27%; 75/274) or ermA (21%; 58/274). All 97 ermB-positive isolates harbored Tn551 and IS1216V The majority (89/97) of ermB-positive isolates displayed the cMLS phenotype and carried mobile element structure (MES)-like structures, which has been previously reported in sequence type 59 (ST59) methicillin-resistant S. aureus (MRSA). The remaining 8 ermB-carrying isolates, belonging to ST7 (n = 4), ST5 (n = 3), and ST59 (n = 1), were sasK intact and did not carry MES-like structures. Unlike a MES-like structure that was located on the chromosome, the ermB elements on sasK-intact isolates were located on plasmids by S1 nuclease pulsed-field gel electrophoresis (PFGE) analysis and conjugation tests. Sequence data for the ermB-containing region (14,566 bp) from ST59 NTUH_3874 revealed that the best match was a Tn1546-like element in plasmid pMCCL2 DNA (GenBank accession number AP009486) of Macrococcus caseolyticus Tn1546 is recognized as an enterococcal transposon and was known from the vancomycin resistance gene cluster in vancomycin-resistant Enterococcus (VRE). So far, acquisitions of Tn1546 in S. aureus have occurred in clonal complex 5 (CC5) MRSA, but not in MSSA. This is the first report that MSSA harbors an Enterococcus faecium-originated ermB-positive Tn1546-like element located on a plasmid. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

AsrR Is an Oxidative Stress Sensing Regulator Modulating Enterococcus faecium Opportunistic Traits, Antimicrobial Resistance, and Pathogenicity

PubMed Central

Lebreton, François; van Schaik, Willem; Sanguinetti, Maurizio; Posteraro, Brunella; Torelli, Riccardo; Le Bras, Florian; Verneuil, Nicolas; Zhang, Xinglin; Giard, Jean-Christophe; Dhalluin, Anne; Willems, Rob J. L.; Leclercq, Roland; Cattoir, Vincent

2012-01-01

Oxidative stress serves as an important host/environmental signal that triggers a wide range of responses in microorganisms. Here, we identified an oxidative stress sensor and response regulator in the important multidrug-resistant nosocomial pathogen Enterococcus faecium belonging to the MarR family and called AsrR (antibiotic and stress response regulator). The AsrR regulator used cysteine oxidation to sense the hydrogen peroxide which results in its dissociation to promoter DNA. Transcriptome analysis showed that the AsrR regulon was composed of 181 genes, including representing functionally diverse groups involved in pathogenesis, antibiotic and antimicrobial peptide resistance, oxidative stress, and adaptive responses. Consistent with the upregulated expression of the pbp5 gene, encoding a low-affinity penicillin-binding protein, the asrR null mutant was found to be more resistant to β-lactam antibiotics. Deletion of asrR markedly decreased the bactericidal activity of ampicillin and vancomycin, which are both commonly used to treat infections due to enterococci, and also led to over-expression of two major adhesins, acm and ecbA, which resulted in enhanced in vitro adhesion to human intestinal cells. Additional pathogenic traits were also reinforced in the asrR null mutant including greater capacity than the parental strain to form biofilm in vitro and greater persistance in Galleria mellonella colonization and mouse systemic infection models. Despite overexpression of oxidative stress-response genes, deletion of asrR was associated with a decreased oxidative stress resistance in vitro, which correlated with a reduced resistance to phagocytic killing by murine macrophages. Interestingly, both strains showed similar amounts of intracellular reactive oxygen species. Finally, we observed a mutator phenotype and enhanced DNA transfer frequencies in the asrR deleted strain. These data indicate that AsrR plays a major role in antimicrobial resistance and

Utilization of tannery fleshings: Optimization of conditions for fermenting delimed tannery fleshings using Enterococcus faecium HAB01 by response surface methodology.

PubMed

Kumar Rai, Amit; General, Thiyam; Bhaskar, N; Suresh, P V; Sakhare, P Z; Halami, P M; Gowda, Lalitha R; Mahendrakar, N S

2010-03-01

Conditions for fermentation of delimed tannery fleshings--to obtain higher degree of protein hydrolysis and reasonably better antioxidant activity--using Enterococcus faecium HAB01 (GenBank #FJ418568) were optimized. Three independent variables--viz., inoculum level (X1), glucose level (X2) and fermentation time (X3)--were optimized using response surface method considering degree of hydrolysis (DH; %) and total titrable acidity (TTA) as response variables. The optimized conditions were found to be 12.5% (v/w) inoculum, 17.5% (w/w) glucose and 96h of fermentation at 37+/-1 degrees C to obtain a maximum DH%. The usefulness of the predicted model was further validated by considering random combinations of the independent factors. The chemical score of the hydrolysate revealed an excess amount of essential amino acids, viz., arginine and leucine compared to reference protein. The liquor portion had relatively high antioxidant activities, indicating its potential for use as a high value feed ingredient. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

High-level vancomycin resistant Enterococcus faecium related to humans and pigs found in dust from pig breeding facilities.

PubMed

Braga, Teresa M; Pomba, Constança; Lopes, M Fátima Silva

2013-01-25

Environmental dust from animal breeding facilities was never screened for the presence of enterococci, nor of vancomycin-resistant enterococci (VRE), despite the possibility of being a vehicle of transmission of strains and antibiotic resistance genes between food-producing animals and man. Bio-security measures in pig facilities include disinfection with biocides to avoid the dissemination of opportunistic pathogenic bacteria, namely enterococci and in particular VRE. We thus undertook collection of enterococci and VRE in a representative number of breeding pig facilities in Portugal (n=171) and analyzed their susceptibility to benzalkonium chloride (BC) and chlorhexidine (CHX). A prevalence of 15% of VRE was found, with 6% high-level resistance found, and MIC values for CHX and BC were similar to those commonly found among enterococcal isolates from related environments, 8 μg/ml and 4 μg/ml, respectively. Among the isolated high-level vancomycin resistant Enterococcus faecium carrying the vanA genotype, we found multilocus sequence types closely related to pig and human isolates from European countries and Brazil. These results strongly advise constant surveillance of this environment and its inclusion in future epidemiologic studies on VRE. Copyright © 2012 Elsevier B.V. All rights reserved.

A novel method for simultaneous Enterococcus species identification/typing and van genotyping by high resolution melt analysis.

PubMed

Gurtler, Volker; Grando, Danilla; Mayall, Barrie C; Wang, Jenny; Ghaly-Derias, Shahbano

2012-09-01

In order to develop a typing and identification method for van gene containing Enterococcus faecium, two multiplex PCR reactions were developed for use in HRM-PCR (High Resolution Melt-PCR): (i) vanA, vanB, vanC, vanC23 to detect van genes from different Enterococcus species; (ii) ISR (intergenic spacer region between the 16S and 23S rRNA genes) to detect all Enterococcus species and obtain species and isolate specific HRM curves. To test and validate the method three groups of isolates were tested: (i) 1672 Enterococcus species isolates from January 2009 to December 2009; (ii) 71 isolates previously identified and typed by PFGE (pulsed-field gel electrophoresis) and MLST (multi-locus sequence typing); and (iii) 18 of the isolates from (i) for which ISR sequencing was done. As well as successfully identifying 2 common genotypes by HRM from the Austin Hospital clinical isolates, this study analysed the sequences of all the vanB genes deposited in GenBank and developed a numerical classification scheme for the standardised naming of these vanB genotypes. The identification of Enterococcus faecalis from E. faecium was reliable and stable using ISR PCR. The typing of E. faecium by ISR PCR: (i) detected two variable peaks corresponding to different copy numbers of insertion sequences I and II corresponding to peak I and II respectively; (ii) produced 7 melt profiles for E. faecium with variable copy numbers of sequences I and II; (iii) demonstrated stability and instability of peak heights with equal frequency within the patient sample (36.4±4.5 days and 38.6±5.8 days respectively for 192 patients); (iv) detected ISR-HRM types with as much discrimination as PFGE and more than MLST; and (v) detected ISR-HRM types that differentiated some isolates that were identical by PFGE and MLST. In conjunction with the rapid and accurate van genotyping method described here, this ISR-HRM typing and identification method can be used as a stable identification and typing method with

Evaluation of high-resolution melting curve analysis of ligation-mediated real-time PCR, a rapid method for epidemiological typing of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Species) pathogens.

PubMed

Woksepp, Hanna; Ryberg, Anna; Billström, Hanna; Hällgren, Anita; Nilsson, Lennart E; Marklund, Britt-Inger; Olsson-Liljequist, Barbro; Schön, Thomas

2014-12-01

A single-tube method, ligation-mediated real-time PCR high-resolution melt analysis (LMqPCR HRMA), was modified for the rapid typing of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens. A 97% agreement (60/62 isolates) was achieved in comparison to pulsed-field gel electrophoresis (PFGE) results, which indicates that LMqPCR HRMA is a rapid and accurate screening tool for monitoring nosocomial outbreaks. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Use of the Yeast Pichia pastoris as an Expression Host for Secretion of Enterocin L50, a Leaderless Two-Peptide (L50A and L50B) Bacteriocin from Enterococcus faecium L50▿

PubMed Central

Basanta, Antonio; Gómez-Sala, Beatriz; Sánchez, Jorge; Diep, Dzung B.; Herranz, Carmen; Hernández, Pablo E.; Cintas, Luis M.

2010-01-01

In this work, we report the expression and secretion of the leaderless two-peptide (EntL50A and EntL50B) bacteriocin enterocin L50 from Enterococcus faecium L50 by the methylotrophic yeast Pichia pastoris X-33. The bacteriocin structural genes entL50A and entL50B were fused to the Saccharomyces cerevisiae gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s) and cloned, separately and together (entL50AB), into the P. pastoris expression and secretion vector pPICZαA, which contains the methanol-inducible alcohol oxidase promoter (PAOX1) to express the fusion genes. After transfer into the yeast, the recombinant plasmids were integrated into the genome, resulting in three bacteriocinogenic yeast strains able to produce and secrete the individual bacteriocin peptides EntL50A and EntL50B separately and together. The secretion was efficiently directed by MFα1s through the Sec system, and the precursor peptides were found to be correctly processed to form mature and active bacteriocin peptides. The present work describes for the first time the heterologous expression and secretion of a two-peptide non-pediocin-like bacteriocin by a yeast. PMID:20348300

Clinical and molecular epidemiology of hospital Enterococcus faecium isolates in eastern France. Members of Réseau Franc-Comtois de Lutte contr les Infections Nosocomiales.

PubMed

Bertrand, X; Thouverez, M; Bailly, P; Cornette, C; Talon, D

2000-06-01

We carried out a surveillance study of Enterococcus faecium isolates in the Franche-Comtéregion of France over three years. Clinical and epidemiological strains were characterized by antibiotype and genotype (pulsed field gel electrophoresis, PFGE). Three case-control studies were performed to identify risk factors for colonization/infection with three defined resistant phenotypes (amoxycillin, high-level gentamicin and high-level kanamycin). The crude incidence of colonization/infection was 0.156%, and 68.8% of cases were classified as hospital-acquired. Incidence did not differ according to the type of hospitalization (middle term or acute care). The urinary tract was the major site of infection. Resistance rates were: 45.8% (amoxycillin), 18.7% (high-level gentamicin), 61.4% (high-level kanamycin) and 3.1% (vancomycin). No isolate produced b-lactamase and one isolate carried the vanA gene. PFGE revealed two major epidemic patterns each including resistant strains isolated in different hospitals and during different periods in the study. Previous antimicrobial treatment was not identified as a risk factor for colonization/infection with any resistant phenotype. Despite the low frequency of vancomycin-resistant isolates in this study, resistant strains were widely disseminated and had characteristics enabling them to persist and spread. If these strains acquired the vanA gene, the risk of an outbreak would be large. So, the prevalence of vancomycin-resistant E. faecium in hospitals should be carefully monitored in the future. Copyright 2000 The Hospital Infection Society.

Comparison of two methods for purification of enterocin B, a bacteriocin produced by Enterococcus faecium W3.

PubMed

Dündar, Halil; Atakay, Mehmet; Çelikbıçak, Ömür; Salih, Bekir; Bozoğlu, Faruk

2015-01-01

This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography, and sequential reverse-phase high-performance liquid chromatography. The latter approach exploited a pH-mediated cell adsorption-desorption method to extract cell-bound bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide were confirmed again by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) analysis. Although both approaches were satisfactory to obtain a sufficient amount of enterocin B for use in MS and amino acid sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B.

[Influence of staphylococcin T on Enterococcus sp. growth].

PubMed

Białucha, Agata; Kozuszko, Sylwia; Gospodarek, Eugenia; Bugalski, Roman Marian; Gierlotka, Krzysztof

2007-01-01

Bacteriocins are ribosomally synthesised, extracellular bacterial products. Generally, spectrum of inhibition is limited to the same or closely related species to bacteriocin producer. Staphylococcin T is produced by Staphylococcus cohnii strain. The present study concerns influence of StT to 267 Enterococcus sp. strains growth isolated between 2003 and 2006 in Department of Microbiology University Hospital of dr. A. Jurasz in Bydgoszcz. S. cohnii T antagonistic ability evaluated towards bacteries on Mueller-Hinton Agar (bio Mérieux) in aerobic conditions. After 24 and 48 hours tested enterococci suspensions were plated perpendiculary. Susceptibility to antibiotics was assessed by disc diffusion method according to the guideless of Clinical and Laboratory Standards Institute and National Reference Centre for Antimicrobial Susceptibility. Among Enterococcus sp. strains tested 7.1% were sensitive to StT. The highest percentage of sensitive enterococci isolated from wound swabs, urine, blood and pus. Enterococcus faecium strains dominated (63.2%) among enterococci sensitive to StT. Moderate inhibition degree on S. cohnii T bacteriocin action was observed in majority sensitive enterococci strains. Enterococcus sp. sensitive to StT strains were frequently multidrug resistant (68.4%). According to the study results and increasing resistance to antibiotics, StT could be an alternative agent used to treat infections caused by Enterococcus sp.

Safety Evaluation of Enterocin Producer Enterococcus sp. Strains Isolated from Traditional Turkish Cheeses.

PubMed

Avcı, Mine; Özden Tuncer, Banu

2017-07-06

The purpose of this study was to determine the antimicrobial activity and occurrence of bacteriocin structural genes in Enterococcus spp. isolated from different cheeses and also investigate some of their virulence factors. Enterococcus strains were isolated from 33 different cheeses. Enterococcus faecium (6 strains) and Enterococcus faecalis (5 strains) enterocin-producing strains were identified by 16S rDNA analyses. Structural genes entA, entB, entP and entX were detected in some isolates. Multiple enterocin structural genes were found in 7 strains. None of the tested enterococci demonstrated anyβ-haemolytic activity and only one strain had gelatinase activity. Six strains showed multiple antibiotic resistance patterns and in addition, vanA and several virulence genes were detected in many strains. Only E. faecalis MBE1-9 showed tyrosine decarboxylase activity and tdc gene was detected only in this strain.

Reduced susceptibility of Enterococcus spp. isolates from Cairo University Hospital to tigecycline: Highlight on the influence of proton pump inhibitors.

PubMed

Hassan, Reem Mostafa; Ghaith, Doaa Mohammad; Ismail, Dalia Kadry; Zafer, Mai Mahmoud

2018-03-01

The incidence of reduced susceptibility to tigecycline (TIG) is increasing. This study aimed to analyse the in vitro activity of TIG against Enterococcus spp. isolates recovered from hospitalised patients and to evaluate the effect of omeprazole on the in vitro antimicrobial activity of TIG against several enterococcal species. A total of 67 Enterococcus clinical isolates were identified by MALDI-TOF/MS and multiplex PCR. Minimum inhibitory concentrations (MICs) of TIG alone and in combination with omeprazole (10, 30 and 60mg/L) were determined by broth microdilution. Antibiotic susceptibility to other antibiotics was determined by disk diffusion. The presence of van, tet(X) and tet(X1) genes was tested by multiplex PCR. Of the 67 Enterococcus isolates, 2 (3.0%) were resistant to TIG and 13 (19.4%) were intermediate-resistant according to EUCAST. The frequencies of resistance to norfloxacin (80.6%), doxycycline (80.6%), levofloxacin (74.6%) and ciprofloxacin (71.6%) were highest, whilst that of vancomycin (25.4%) was lowest. The vanA gene was detected in 11 Enterococcus isolates (8 Enterococcus faecalis, 3 Enterococcus faecium), vanB in 3 Enterococcus isolates (2 E. faecium, 1 E. faecalis) and vanC-2/3 in 3 Enterococcus casseliflavus. Nine isolates (13.4%) were positive for tet(X1). TIG resistance occurred both in patients receiving or not TIG and/or omeprazole. Omeprazole increased TIG MICs by 4-128-fold. The possibility of selection of TIG-non-susceptible Enterococcus in the gut may occur with long-term use of omeprazole. Omeprazole influenced TIG activity in a concentration-dependent manner. To our knowledge; this is the first report of TIG-non-susceptible Enterococcus spp. in Egypt. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Effect of a probiotic beverage consumption (Enterococcus faecium CRL 183 and Bifidobacterium longum ATCC 15707) in rats with chemically induced colitis.

PubMed

Celiberto, Larissa Sbaglia; Bedani, Raquel; Dejani, Naiara Naiana; Ivo de Medeiros, Alexandra; Sampaio Zuanon, José Antonio; Spolidorio, Luis Carlos; Tallarico Adorno, Maria Angela; Amâncio Varesche, Maria Bernadete; Carrilho Galvão, Fábio; Valentini, Sandro Roberto; Font de Valdez, Graciela; Rossi, Elizeu Antonio; Cavallini, Daniela Cardoso Umbelino

2017-01-01

Some probiotic strains have the potential to assist in relieving the symptoms of inflammatory bowel disease. The impact of daily ingestion of a soy-based product fermented by Enterococcus faecium CRL 183 and Lactobacillus helveticus 416 with the addition of Bifidobacterium longum ATCC 15707 on chemically induced colitis has been investigated thereof within a period of 30 days. Colitis was induced by dextran sulfate sodium. The animals were randomly assigned into five groups: Group C: negative control; Group CL: positive control; Group CLF: DSS with the fermented product; Group CLP: DSS with the non-fermented product (placebo); Group CLS: DSS with sulfasalazine. The following parameters were monitored: disease activity index, fecal microbial analyses, gastrointestinal survival of probiotic microorganisms and short-chain fatty acids concentration in the feces. At the end of the protocol the animals' colons were removed so as to conduct a macroscopical and histopathological analysis, cytokines and nitrite quantification. Animals belonging to the CLF group showed fewer symptoms of colitis during the induction period and a lower degree of inflammation and ulceration in their colon compared to the CL, CLS and CLP groups (p<0.05). The colon of the animals in groups CL and CLS presented severe crypt damage, which was absent in CLF and CLP groups. A significant increase in the population of Lactobacillus spp. and Bifidobacterium spp. at the end of the protocol was verified only in the CLF animals (p<0.05). This group also showed an increase in short-chain fatty acids (propionate and acetate). Furthermore, the intestinal survival of E. faecium CRL 183 and B. longum ATCC 15707 in the CLF group has been confirmed by biochemical and molecular analyzes. The obtained results suggest that a regular intake of the probiotic product, and placebo to a lesser extent, can reduce the severity of DSS-induced colitis on rats.

Eight-year Surveillance of Antimicrobial Resistance among Enterococcus Spp. Isolated in the First Bethune Hospital

NASA Astrophysics Data System (ADS)

Xu, Jiancheng; Wang, Liqiang; Wang, Kai; Zhou, Qi

This study was to investigate the antimicrobial resistance of Enterococcus spp. isolated in 8 consecutive years in the First Bethune Hospital. Disk diffusion test was used to study the antimicrobial resistance. The data were analyzed by WHONET 5 software according to Clinical and Laboratory Standards Institute (CLSI). Most of 1446 strains of Enterococcus spp. were collected from urine 640 (44.3%), sputum 315 (21.8%), secretions and pus 265 (18.3%) during the past 8 years. The rates of high-level aminoglycoside resistance in Enterococcus faecalis and Enterococcus faecium were 57.4%∼75.9% and 69.0%∼93.8% during the past 8 years, respectively. No Enterococcus spp. was resistant to vancomycin. The antimicrobial resistance of Enterococcus spp. had increased in recent 8 years. The change of the antimicrobial resistance should be investigated in order to direct rational drug usage in the clinic and prevent bacterial strain of drug resistance from being transmitted.

Prevalence and antimicrobial resistance of Enterococcus spp and Staphylococcus spp isolated from surfaces in a veterinary teaching hospital.

PubMed

Hamilton, Elizabeth; Kaneene, John B; May, Katherine J; Kruger, John M; Schall, William; Beal, Matthew W; Hauptman, Joe G; DeCamp, Charles E

2012-06-15

To determine the prevalence and antimicrobial resistance of enterococci and staphylococci collected from environmental surfaces at a veterinary teaching hospital (VTH). Longitudinal study. Samples collected from surfaces in 5 areas (emergency and critical care, soft tissue and internal medicine, and orthopedic wards; surgery preparation and recovery rooms; and surgery office and operating rooms) of a VTH. Selected surfaces were swabbed every 3 months during the 3-year study period (2007 to 2009). Isolates of enterococci and staphylococci were identified via biochemical tests, and antimicrobial susceptibility was evaluated with a microbroth dilution technique. A subset of isolates was analyzed to assess clonality by use of pulsed-field gel electrophoresis. 430 samples were collected, and isolates of enterococci (n = 75) and staphylococci (110) were identified. Surfaces significantly associated with isolation of Enterococcus spp and Staphylococcus spp included cages and a weight scale. Fourteen Enterococcus spp isolates and 17 Staphylococcus spp isolates were resistant to ≥ 5 antimicrobials. Samples collected from the scale throughout the study suggested an overall increase in antimicrobial resistance of Enterococcus faecium over time. Clonality was detected for E faecium isolates collected from 2 different surfaces on the same day. Although not surprising, the apparent increase in antimicrobial resistance of E faecium was of concern because of the organism's ability to transmit antimicrobial resistance genes to other pathogens. Results reported here may aid in identification of critical control points to help prevent the spread of pathogens in VTHs.

Production of enterocins L50A, L50B, and IT, a new enterocin, by Enterococcus faecium IT62, a strain isolated from Italian ryegrass in Japan.

PubMed

Izquierdo, Esther; Bednarczyk, Audrey; Schaeffer, Christine; Cai, Yimin; Marchioni, Eric; Van Dorsselaer, Alain; Ennahar, Saïd

2008-06-01

Enterococcus faecium IT62, isolated from ryegrass in Japan, was shown to produce three different bacteriocins, two of which had molecular masses and amino acid sequences that corresponded to those of enterocin L50A and enterocin L50B. These peptides existed, however, as chemically modified forms that were either N formylated or N formylated and oxidized at Met(24). The third bacteriocin, named enterocin IT, had a molecular mass of 6,390 Da, was made up of 54 amino acids, and did not correspond to any known bacteriocin. However, enterocin IT was identical to the C-terminal part of the 16-amino-acid-longer bacteriocin 32 (T. Inoue, H. Tomita, and Y. Ike, Antimicrob. Agents Chemother., 50:1202-1212, 2006). For the first time, the antimicrobial activity spectra for enterocins L50A and L50B were determined separately and included a wide range of gram-positive bacteria but also a few gram-negative strains that were weakly sensitive. Slight differences in the activities of enterocins L50A and L50B were observed, as gram-positive bacteria showed an overall higher level of sensitivity to L50A than to L50B, as opposed to gram-negative ones. Conversely, enterocin IT showed a very narrow antimicrobial spectrum that was limited to E. faecium strains, one strain of Bacillus subtilis, and one strain of Lactococcus lactis. This study showed that E. faecium IT62, a grass-borne strain, produces bacteriocins with very different activity features and structures that may be found in strains associated with food or those of clinical origin, which demonstrates that a particular enterocin structure may be widespread and not related to the producer's origin.

Production of Enterocins L50A, L50B, and IT, a New Enterocin, by Enterococcus faecium IT62, a Strain Isolated from Italian Ryegrass in Japan▿

PubMed Central

Izquierdo, Esther; Bednarczyk, Audrey; Schaeffer, Christine; Cai, Yimin; Marchioni, Eric; Van Dorsselaer, Alain; Ennahar, Saïd

2008-01-01

Enterococcus faecium IT62, isolated from ryegrass in Japan, was shown to produce three different bacteriocins, two of which had molecular masses and amino acid sequences that corresponded to those of enterocin L50A and enterocin L50B. These peptides existed, however, as chemically modified forms that were either N formylated or N formylated and oxidized at Met24. The third bacteriocin, named enterocin IT, had a molecular mass of 6,390 Da, was made up of 54 amino acids, and did not correspond to any known bacteriocin. However, enterocin IT was identical to the C-terminal part of the 16-amino-acid-longer bacteriocin 32 (T. Inoue, H. Tomita, and Y. Ike, Antimicrob. Agents Chemother., 50:1202-1212, 2006). For the first time, the antimicrobial activity spectra for enterocins L50A and L50B were determined separately and included a wide range of gram-positive bacteria but also a few gram-negative strains that were weakly sensitive. Slight differences in the activities of enterocins L50A and L50B were observed, as gram-positive bacteria showed an overall higher level of sensitivity to L50A than to L50B, as opposed to gram-negative ones. Conversely, enterocin IT showed a very narrow antimicrobial spectrum that was limited to E. faecium strains, one strain of Bacillus subtilis, and one strain of Lactococcus lactis. This study showed that E. faecium IT62, a grass-borne strain, produces bacteriocins with very different activity features and structures that may be found in strains associated with food or those of clinical origin, which demonstrates that a particular enterocin structure may be widespread and not related to the producer's origin. PMID:18391036

Antibacterial potential and genetic profile of Enterococcus faecium strains isolated from human normal flora.

PubMed

Karimaei, Samira; Sadeghi, Javad; Asadian, Mahla; Esghaei, Maryam; Pourshafie, Mohammad Reza; Talebi, Malihe

2016-07-01

Enterococci have a widespread attendance in the circumference and belongs to the enteric commensal microbiota. Most of them produce the antimicrobial compounds and have an inhibition effect on pathogenic microorganisms. The objective of this study was to characterize the enterococcal strains isolated from human normal flora and assess their antibacterial activity. Enterococcal isolates were obtained from the feces of eighteen healthy humans. All enterococcal species were identified by biochemical and species-specific polymerase chain reaction (PCR). These isolates were investigated further to examine their ability to inhibit growth of Salmonella typhi, Shigella flexneri and Escherichia coli by well diffusion assay. Furthermore, antibiotic susceptibility test was performed and genetic relatedness of all isolates was evaluated by Pulse Field Gel Electrophoresis (PFGE). In all, 432 isolates were obtained from fecal samples. All of the isolates identified as Enterococcus faecium by biochemical and molecular (PCR) methods. Using repetitive element palindromic (REP)-PCR method 54 patterns have been obtained and were selected for further evaluation. The results indicated that 66%, 38% and 24% of our isolates had antimicrobial effect against S. typhi, S flexneri and enteroaggregative Escherichia coli (EAEC), respectively. On the other hand, there was no significant inhibition effect against enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). All isolates were sensitive to vancomycin, teicoplanin, linezolid, ampicillin, chloramphenicol and gentamicin. On the other hand, the resistance rates for erythromycin, tetracycline and ciprofloxacin were 20%, 22%, and 1.8% respectively. In addition, the analysis of PFGE showed forty patterns with eight (40.7%) common types (CT) and thirty two (59.2%) single types (ST). Among eight common types, only one common type (CT5) had similar antimicrobial effect. These results suggested that enterococcal isolates obtained from

Mosaic Structure of a Multiple-Drug-Resistant, Conjugative Plasmid from Campylobacter jejuni

DTIC Science & Technology

2005-01-30

allele of each gene in the respective clones. There were three genes predicted to encode alleles of strep- tomycin-inactivating enzymes from Enterococcus ...aminoglycoside 6-adenyltransferase/E. faecium /NP_863159 3 cpp50 2599–2811 26.3 473 70 100/100 (70) Unknown of pTet plasmid/C. jejuni strain 81-176/YP_063493 4... faecium /NP_863159 24 sat4 17692–18222 37.7 180 176 94/94 (176) Streptothricin acetyltransferase/E. faecium /AAM77897 25 aphA-3 18315–19109 44.9 264

Prevalence and antibiotic resistance of Enterococcus spp. isolated from retail cheese, ready-to-eat salads, ham, and raw meat.

PubMed

Pesavento, G; Calonico, C; Ducci, B; Magnanini, A; Lo Nostro, A

2014-08-01

Food specimens were analyzed in order to research Enterococcus spp.: 636 samples of raw meat (227 beef, 238 poultry, and 171 pork), 278 samples of cheese (110 fresh soft cheese and 168 mozzarella cheese), 214 samples of ready-to-eat salads, and 187 samples of ham. 312 strains of Enterococcus spp samples were isolated, then identified and submitted to susceptibility tests against 11 antimicrobial agents. The predominant species were Enterococcus faecalis in raw meat and Enterococcus faecium in retail products. Low percentages of microorganisms were resistant to vancomycin (3.53%), teicoplanin (2.24%), linezolid (0.32%), and amoxicillin in combination with clavulanic acid (0.32%). A high percentage of resistance was noted in E. faecalis at high level gentamicin (21.9%) and tetracycline (60.6%). In general, strains of E. faecalis were more resistant than E. faecium. Enterococci should be considered not only potential pathogens, but also a reservoir of genes encoding antibiotic resistance which can be transferred to other microorganisms. Continuous monitoring of their incidence and emerging resistance is important in order to identify foods which potentially represent a real risk to the population, and to ensure effective treatment of human enterococcal infections. Copyright © 2014 Elsevier Ltd. All rights reserved.

Combined effect of enterocin and lipase from Enterococcus faecium NCIM5363 against food borne pathogens: mode of action studies.

PubMed

Ramakrishnan, Vrinda; Narayan, Bhaskar; Halami, Prakash M

2012-08-01

Food borne diseases have a major impact on public health whose epidemiology is rapidly changing. The whole cells of pathogens involved or their toxins/metabolites affect the human health apart from spoiling sensory properties of the food products finally affecting the food industry as well as consumer health. With pathogens developing mechanisms of antibiotic resistance, there has been an increased need to replace antibiotics as well as chemical additives with naturally occurring bacteriocins. Bacteriocins are known to act mainly against Gram-positive pathogens and with little or no effect towards Gram-negative enteric bacteria. In the present study, combination effect of lipase and bacteriocin produced by Enterococcus faecium NCIM5363, a highly lipolytic lactic acid bacterium against various food pathogens was assessed. The lipase in combination with enterocin exhibited a lethal effect against Gram-negative pathogens. Scanning electron microscopy studies carried out to ascertain the constitutive mode of action of lipase and enterocin revealed that the lipase degrades the cell wall of Gram-negative bacteria and creates a pore through which enterocin enters thereby resulting in cell death. The novelty of this work is the fact that this is the first report revealing the synergistic effect of lipase with enterocin against Gram-negative bacteria.

Prevalence and antibiotic resistance of Enterococcus strains isolated from poultry.

PubMed

Stępień-Pyśniak, Dagmara; Marek, Agnieszka; Banach, Tomasz; Adaszek, Łukasz; Pyzik, Ewelina; Wilczyński, Jarosław; Winiarczyk, Stanisław

2016-06-01

The aim of this study was to evaluate the frequency of occurrence of bacteria of the genus Enterococcus in poultry, to identify them by means of matrixassisted laser desorption/ionisation time-of-flight mass spectrometry (MALDITOF MS), and to analyse the antimicrobial susceptibility of the isolated strains to the drugs most frequently used in poultry. The material for the bacteriological tests was obtained mainly from the heart (97%) of the birds investigated. Of a total of 2,970 samples tested, 911 (30.7%) tested positive for Enterococcus spp. Enterococci were detected in broilers (88.1%), laying hens (5.3%), turkeys (3.9%), breeding hens (2.2%), and geese (0.4%). The most commonly identified species were Enterococcus (E.) faecalis (74.7%), E. faecium (10.1%), E. gallinarum (5.5%), E. hirae (4.6%), and E. cecorum (4.1%). The most frequent resistance properties were resistance to sulphamethoxazole/trimethoprim (88%), tylosin (71.4%), enrofloxacin (69.4%), doxycycline (67.3%), and lincomycin/spectinomycin (56.1%). Only one vancomycin-resistant Enterococcus, E. cecorum from a broiler, was found.

Development of a Dry Inoculation Method for Thermal Challenge Studies in Low-Moisture Foods by Using Talc as a Carrier for Salmonella and a Surrogate (Enterococcus faecium).

PubMed

Enache, Elena; Kataoka, Ai; Black, D Glenn; Napier, Carla D; Podolak, Richard; Hayman, Melinda M

2015-06-01

The objective of this study was to obtain dry inocula of Salmonella Tennessee and Enterococcus faecium, a surrogate for thermal inactivation of Salmonella in low-moisture foods, and to compare their thermal resistance and stability over time in terms of survival. Two methods of cell growth were compared: cells harvested from a lawn on tryptic soy agar (TSA-cells) and from tryptic soy broth (TSB-cells). Concentrated cultures of each organism were inoculated onto talc powder, incubated at 35 °C for 24 h, and dried for additional 24 h at room temperature (23 ± 2 °C) to achieve a final water activity of ≤ 0.55 before sieving. Cell reductions of Salmonella and E. faecium during the drying process were between 0.14 and 0.96 log CFU/g, depending on growth method used. There was no difference between microbial counts at days 1 and 30. Heat resistance of the dry inoculum on talc inoculated into a model peanut paste (50 % fat and 0.6 water activity) was determined after 1 and 30 days of preparation, using thermal death time tests conducted at 85 °C. For Salmonella, there was no significant difference between the thermal resistance (D(85 °C)) for the TSB-cells and TSA-cells (e.g. day 1 cells D(85 °C) = 1.05 and 1.07 min, respectively), and there was no significant difference in D(85 °C) between dry inocula on talc used either 1 or 30 days after preparation (P > 0.05). However, the use the dry inocula of E. faecium yielded different results: the TSB-grown cells had a significantly (P < 0.05) greater heat resistance than TSA-grown cells (e.g. D(85 °C) for TSB-cells = 3.42 min versus 2.60 min for TSA-cells). E. faecium had significantly (P < 0.05) greater heat resistance than Salmonella Tennessee regardless what cell type was used for dry inoculum preparation; therefore, it proved to be a conservative but appropriate surrogate for thermal inactivation of Salmonella in low-moisture food matrices under the tested conditions.

Efficacy of the direct-fed microbial Enterococcus faecium alone or in combination with Saccharomyces cerevisiae or Lactococcus lactis during induced subacute ruminal acidosis.

PubMed

Chiquette, J; Lagrost, J; Girard, C L; Talbot, G; Li, S; Plaizier, J C; Hindrichsen, I K

2015-01-01

This study aimed at investigating Enterococcus faecium alone or E. faecium in combination with Saccharomyces cerevisiae or Lactococcus lactis during a subacute ruminal acidosis (SARA) challenge. Four ruminally fistulated Holstein dairy cows were assigned to the following treatments in a 4×4 Latin square design: (1) control (CON); (2) E. faecium (EF); (3) EF + S. cerevisiae (EFSC); (4) EF + L. lactis DSM 11037 (EFLL). Each experimental period consisted of 18 d of adaptation to the respective direct-fed microbial, 3 d of SARA challenge, and 7d of rest. Rumen pH was recorded every 10 min over 24 h on d 17 of adaptation, d 2 of SARA, and d 6 of rest. On the last day of adaptation, SARA, and rest, samples of rumen content (0 and 3 h after feeding) were taken for volatile fatty acids, lactate, vitamin B12, rumen microbes, and lipopolysaccharides determination. Blood samples (0 and 6 h after feeding) were taken for the measurement of acute-phase proteins. Dry matter intake and milk yield were recorded daily. During SARA, mean rumen pH with EFSC (5.94) was not different from that of EFLL (5.95) and tended to be higher than with CON (5.82) or EF (5.82). Postfeeding vitamin B12 concentrations in the rumen were greater with EFSC (134.5ng/g) than with EF (99.6ng/g) and tended to be greater when compared with CON (101.2ng/g) or EFLL (104.9ng/g). During rest, prefeed vitamin B12 was greater with EFSC (166.5ng/g) compared with CON (132.3ng/g). The EFSC treatment did better than EF alone on pH characteristics during adaptation and SARA and on maintenance of ruminal vitamin B12 status during SARA. Milk yield drop from d 1 to 3 of SARA was smaller with EFSC (-0.8kg/d), EF (-0.9kg/d), or EFLL (-0.9kg/d) compared with CON (-7.5kg/d). Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Persistence of Animal and Human Glycopeptide-Resistant Enterococci on Two Norwegian Poultry Farms Formerly Exposed to Avoparcin Is Associated with a Widespread Plasmid-Mediated vanA Element within a Polyclonal Enterococcus faecium Population

PubMed Central

Johnsen, P. J.; Østerhus, J. I.; Sletvold, H.; Sørum, M.; Kruse, H.; Nielsen, K.; Simonsen, G. S.; Sundsfjord, A.

2005-01-01

The evolutionary processes responsible for the long-term persistence of glycopeptide-resistant Enterococcus faecium (GREF) in nonselective environments were addressed by genetic analyses of E. faecium populations in animals and humans on two Norwegian poultry farms that were previously exposed to avoparcin. A total of 222 fecal GREF (n = 136) and glycopeptide-susceptible (n = 86) E. faecium (GSEF) isolates were obtained from farmers and poultry on three separate occasions in 1998 and 1999. Pulsed-field gel electrophoresis (PFGE) and plasmid DNA analyses discerned 22 GREF and 32 GSEF PFGE types within shifting polyclonal animal and human E. faecium populations and indicated the presence of transferable plasmid-mediated vanA resistance, respectively. Examples of dominant, persistent GREF PFGE types supported the notion that environmentally well-adapted GREF types may counteract the reversal of resistance. PFGE analyses, sequencing of the purK housekeeping gene, and partial typing of vanA-containing Tn1546 suggested a common animal and human reservoir of glycopeptide resistance. Inverse PCR amplification and sequence analyses targeting the right end of the Tn1546-plasmid junction fragment strongly indicated the presence of a common single Tn1546-plasmid-mediated element in 20 of 22 GREF PFGE types. This observation was further strengthened by vanY-vanZ hybridization analyses of plasmid DNAs as well as the finding of a physical linkage between Tn1546 and a putative postsegregation killing system for seven GREF PFGE types. In conclusion, our observations suggest that the molecular unit of persistence of glycopeptide resistance is a common mobile plasmid-mediated vanA-containing element within a polyclonal GREF population that changes over time. In addition, we propose that “plasmid addiction systems” may contribute to the persistence of GREF in nonselective environments. PMID:15640183

Rapid disc diffusion antibiotic susceptibility testing for Pseudomonas aeruginosa, Acinetobacter baumannii and Enterococcus spp.

PubMed Central

Hombach, Michael; Jetter, Marion; Blöchliger, Nicolas; Kolesnik-Goldmann, Natalia; Keller, Peter M; Böttger, Erik C

2018-01-01

Abstract Background We investigated the feasibility of rapid disc diffusion antibiotic susceptibility testing (rAST) with reading of inhibition zones after 6 and/or 8 h of incubation for Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa and Acinetobacter baumannii. In addition, we evaluated discrimination of resistant populations from the WT populations at early timepoints and the requirement for clinical breakpoint adaptations for proper interpretation of rAST data. Methods In total, 815 clinical strains [E. faecalis (n = 135), E. faecium (n = 227), P. aeruginosa (n = 295) and A. baumannii (n = 158)] were included in this study. Disc diffusion plates were streaked, incubated and imaged using the WASPLabTM automation system. WT populations and non-WT populations were defined using epidemiological cut-offs. Results and conclusions rAST at 6 and 8 h was possible for A. baumannii and enterococci with readability of inhibition zones >90%. Overall categorical agreement of rAST at 6 h with AST at 18 h was 97.2%, 97.4% and 95.3% for E. faecalis, E. faecium and A. baumannii, respectively. With few exceptions, major categorization error rates were <1% for A. baumannii, and vancomycin-resistant E. faecium were clearly separated from the WT at 6 h. For P. aeruginosa the average readability of inhibition zones was 68.9% at 8 h and we found an overall categorical agreement of 94.8%. Adaptations of clinical breakpoints and/or introduction of technical buffer zones, preferably based on aggregated population data from various epidemiological settings, are required for proper interpretation of rAST. PMID:29186434

Identification and tracing of Enterococcus spp. by RAPD-PCR in traditional fermented sausages and meat environment.

PubMed

Martín, B; Corominas, L; Garriga, M; Aymerich, T

2009-01-01

Four local small-scale factories were studied to determine the sources of enterococci in traditional fermented sausages. Different points during the production of a traditional fermented sausage type (fuet) were evaluated. Randomly amplified polymorphic DNA (RAPD)-PCR was used to type 596 Enterococcus isolates from the final products, the initial meat batter, the casing, the workers' hands and the equipment. Species-specific PCR-multiplex and the partial sequencing of atpA gene and 16S rRNA gene sequencing allowed the identification of the isolates: Enterococcus faecalis (31.4%), Enterococcus faecium (30.7%), Enterococcus sanguinicola (14.9%), Enterococcus devriesei (9.7%), Enterococcus malodoratus (7.2%), Enterococcus gilvus (1.0%), Enterococcus gallinarum (1.3%), Enterococcus casseliflavus (3.4%), Enterococcus hermanniensis (0.2%), and Enterococcus durans (0.2%). A total of 92 different RAPD-PCR profiles were distributed among the different factories and samples evaluated. Most of the genotypes found in fuet samples were traced back to their source. The major sources of enterococci in the traditional fermented sausages studied were mainly the equipment followed by the raw ingredients, although a low proportion was traced back to human origin. This work contributes to determine the source of enterococcal contamination in fermented sausages and also to the knowledge of the meat environment.

Purification and characterization of enterocin 62-6, a two-peptide bacteriocin produced by a vaginal strain of Enterococcus faecium: Potential significance in bacterial vaginosis

PubMed Central

Dezwaan, Diane C.; Mequio, Michael J.; Littell, Julia S.; Allen, Jonathan P.; Rossbach, Silvia; Pybus, Vivien

2009-01-01

A bacteriocin produced by a vaginal isolate of Enterococcus faecium strain 62-6, designated enterocin 62-6, was characterized following purification and DNA sequence analysis and compared to previously described bacteriocins. Enterocin 62-6 was isolated from brain heart infusion (BHI) culture supernatants using ammonium sulfate precipitation followed by elution from a Sepharose cation exchange column using a continuous salt gradient (0.1–0.7 M NaCl). SDS-PAGE of an active column fraction resulted in an electrophoretically pure protein, which corresponded to the growth inhibition of the sensitive Lactobacillus indicator strain in the gel overlay assay. Purified enterocin 62-6 was shown to be heat- and pH-stable, and sensitive to the proteolytic enzymes α-chymotrypsin and pepsin. Results from mass spectrometry suggested that it comprised two peptides of 5206 and 5219±1 Da, which was confirmed by DNA sequence analysis. The characteristics of enterocin 62-6 as a small, heat- and pH-stable, cationic, hydrophobic, two-peptide, plasmid-borne bacteriocin, with an inhibitory spectrum against a broad range of Gram-positive but not Gram-negative bacteria, were consistent with its classification as a class IIc bacteriocin. Furthermore, its wide spectrum of growth inhibitory activity against Gram-positive bacteria of vaginal origin including lactobacilli, and stability under the acidic conditions of the vagina, are consistent with our hypothesis that it could have potential significance in disrupting the ecology of the vaginal tract and pave the way for the establishment of the abnormal microbiota associated with the vaginal syndrome bacterial vaginosis. This is the first class IIc bacteriocin produced by a strain of E. faecium of vaginal origin to be characterized. PMID:19578555

Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from Bermuda

PubMed Central

Akpaka, Patrick Eberechi; Kissoon, Shivnarine; Wilson, Clyde; Jayaratne, Padman; Smith, Ashley; Golding, George R.

2017-01-01

Molecular characteristics of vancomycin resistant enterococci isolates from Bermuda Island is currently unknown. This study was conducted to investigate phenotypic and genotypic characteristics of VRE isolates from Bermuda Island using the chromogenic agar, E-tests, polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Eighteen E. faecium isolates were completely analyzed and were all resistant to vancomycin, susceptible to linezolid and quinupristin/dalfopristin, positive for vanA and esp genes. The MLST analysis confirmed most isolates were of the sequence types linked to clonal complex 17 (CC17) that is widely associated with outbreaks in hospitals. Infection control measures, antibiotic stewardship, and surveillance activities will continue to be a priority in hospital on the Island. PMID:28267763

Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from Bermuda.

PubMed

Akpaka, Patrick Eberechi; Kissoon, Shivnarine; Wilson, Clyde; Jayaratne, Padman; Smith, Ashley; Golding, George R

2017-01-01

Molecular characteristics of vancomycin resistant enterococci isolates from Bermuda Island is currently unknown. This study was conducted to investigate phenotypic and genotypic characteristics of VRE isolates from Bermuda Island using the chromogenic agar, E-tests, polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Eighteen E. faecium isolates were completely analyzed and were all resistant to vancomycin, susceptible to linezolid and quinupristin/dalfopristin, positive for vanA and esp genes. The MLST analysis confirmed most isolates were of the sequence types linked to clonal complex 17 (CC17) that is widely associated with outbreaks in hospitals. Infection control measures, antibiotic stewardship, and surveillance activities will continue to be a priority in hospital on the Island.

Effect of a probiotic beverage consumption (Enterococcus faecium CRL 183 and Bifidobacterium longum ATCC 15707) in rats with chemically induced colitis

PubMed Central

Celiberto, Larissa Sbaglia; Bedani, Raquel; Dejani, Naiara Naiana; Ivo de Medeiros, Alexandra; Sampaio Zuanon, José Antonio; Spolidorio, Luis Carlos; Tallarico Adorno, Maria Angela; Amâncio Varesche, Maria Bernadete; Carrilho Galvão, Fábio; Valentini, Sandro Roberto; Font de Valdez, Graciela; Rossi, Elizeu Antonio

2017-01-01

Background Some probiotic strains have the potential to assist in relieving the symptoms of inflammatory bowel disease. The impact of daily ingestion of a soy-based product fermented by Enterococcus faecium CRL 183 and Lactobacillus helveticus 416 with the addition of Bifidobacterium longum ATCC 15707 on chemically induced colitis has been investigated thereof within a period of 30 days. Methods Colitis was induced by dextran sulfate sodium. The animals were randomly assigned into five groups: Group C: negative control; Group CL: positive control; Group CLF: DSS with the fermented product; Group CLP: DSS with the non-fermented product (placebo); Group CLS: DSS with sulfasalazine. The following parameters were monitored: disease activity index, fecal microbial analyses, gastrointestinal survival of probiotic microorganisms and short-chain fatty acids concentration in the feces. At the end of the protocol the animals’ colons were removed so as to conduct a macroscopical and histopathological analysis, cytokines and nitrite quantification. Results Animals belonging to the CLF group showed fewer symptoms of colitis during the induction period and a lower degree of inflammation and ulceration in their colon compared to the CL, CLS and CLP groups (p<0.05). The colon of the animals in groups CL and CLS presented severe crypt damage, which was absent in CLF and CLP groups. A significant increase in the population of Lactobacillus spp. and Bifidobacterium spp. at the end of the protocol was verified only in the CLF animals (p<0.05). This group also showed an increase in short-chain fatty acids (propionate and acetate). Furthermore, the intestinal survival of E. faecium CRL 183 and B. longum ATCC 15707 in the CLF group has been confirmed by biochemical and molecular analyzes. Conclusions The obtained results suggest that a regular intake of the probiotic product, and placebo to a lesser extent, can reduce the severity of DSS-induced colitis on rats. PMID:28437455

Antimicrobial resistance of Enterococcus isolates in Turkey: A meta-analysis of current studies.

PubMed

Kilbas, Imdat; Ciftci, Ihsan Hakki

2018-03-01

In this study, a meta-analysis of Enterococcus isolates collected in 2000-2015 in Turkey and their susceptibility/resistance to antibiotics, clinical indications for initial drug treatment, and identification of alternative treatments was conducted. The meta-analysis examined antibiotic susceptibility/resistance in Enterococcus spp. isolates. The study was planned and conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Statements on antimicrobial resistance were grouped according to the antimicrobial stewardship programme (ASP). The mean resistance rates of Enterococcus faecalis to vancomycin (VAN) and linezolid (LNZ) were 1.0±2.2% and 1.9±2.6%, respectively, whereas the mean resistance rates of Enterococcus faecium to VAN and LNZ were 10.3±11.3% and 2.4±0%, respectively. This study is the first meta-analysis of the resistance of clinical Enterococcus isolates in Turkey to antimicrobial agents, which is a major problem stemming from the excessive usage of antibiotics. The development of antibiotic resistance in Turkey has changed over time. To support the practice of evidence-based medicine, more notifications about Enterococcus resistance status are needed, especially notifications following ASP rules. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Proteomic characterization of vanA-containing Enterococcus recovered from Seagulls at the Berlengas Natural Reserve, W Portugal.

PubMed

Radhouani, Hajer; Poeta, Patrícia; Pinto, Luís; Miranda, Júlio; Coelho, Céline; Carvalho, Carlos; Rodrigues, Jorge; López, María; Torres, Carmen; Vitorino, Rui; Domingues, Pedro; Igrejas, Gilberto

2010-09-21

Enterococci have emerged as the third most common cause of nosocomial infections, requiring bactericidal antimicrobial therapy. Although vancomycin resistance is a major problem in clinics and has emerged in an important extend in farm animals, few studies have examined it in wild animals. To determine the prevalence of vanA-containing Enterococcus strains among faecal samples of Seagulls (Larus cachinnans) of Berlengas Natural Reserve of Portugal, we developed a proteomic approach integrated with genomic data. The purpose was to detect the maximum number of proteins that vary in different enterococci species which are thought to be connected in some, as yet unknown, way to antibiotic resistance. From the 57 seagull samples, 54 faecal samples showed the presence of Enterococcus isolates (94.7%). For the enterococci, E. faecium was the most prevalent species in seagulls (50%), followed by E. faecalis and E. durans (10.4%), and E. hirae (6.3%). VanA-containing enterococcal strains were detected in 10.5% of the 57 seagull faecal samples studied. Four of the vanA-containing enterococci were identified as E. faecium and two as E. durans. The tet(M) gene was found in all five tetracycline-resistant vanA strains. The erm(B) gene was demonstrated in all six erythromycin-resistant vanA strains. The hyl virulence gene was detected in all four vanA-containing E. faecium isolates in this study, and two of them harboured the purK1 allele. In addition these strains also showed ampicillin and ciprofoxacin resistance. The whole-cell proteomic profile of vanA-containing Enterococcus strains was applied to evaluate the discriminatory power of this technique for their identification. The major differences among species-specific profiles were found in the positions corresponding to 97-45 kDa. Sixty individualized protein spots for each vanA isolate was identified and suitable for peptide mass fingerprinting measures by spectrometry measuring (MALDI/TOF MS) and their identification

Proteomic characterization of vanA-containing Enterococcus recovered from Seagulls at the Berlengas Natural Reserve, W Portugal

PubMed Central

2010-01-01

Background Enterococci have emerged as the third most common cause of nosocomial infections, requiring bactericidal antimicrobial therapy. Although vancomycin resistance is a major problem in clinics and has emerged in an important extend in farm animals, few studies have examined it in wild animals. To determine the prevalence of vanA-containing Enterococcus strains among faecal samples of Seagulls (Larus cachinnans) of Berlengas Natural Reserve of Portugal, we developed a proteomic approach integrated with genomic data. The purpose was to detect the maximum number of proteins that vary in different enterococci species which are thought to be connected in some, as yet unknown, way to antibiotic resistance. Results From the 57 seagull samples, 54 faecal samples showed the presence of Enterococcus isolates (94.7%). For the enterococci, E. faecium was the most prevalent species in seagulls (50%), followed by E. faecalis and E. durans (10.4%), and E. hirae (6.3%). VanA-containing enterococcal strains were detected in 10.5% of the 57 seagull faecal samples studied. Four of the vanA-containing enterococci were identified as E. faecium and two as E. durans. The tet(M) gene was found in all five tetracycline-resistant vanA strains. The erm(B) gene was demonstrated in all six erythromycin-resistant vanA strains. The hyl virulence gene was detected in all four vanA-containing E. faecium isolates in this study, and two of them harboured the purK1 allele. In addition these strains also showed ampicillin and ciprofoxacin resistance. The whole-cell proteomic profile of vanA-containing Enterococcus strains was applied to evaluate the discriminatory power of this technique for their identification. The major differences among species-specific profiles were found in the positions corresponding to 97-45 kDa. Sixty individualized protein spots for each vanA isolate was identified and suitable for peptide mass fingerprinting measures by spectrometry measuring (MALDI/TOF MS) and their

Purification and Genetic Characterization of Enterocin I from Enterococcus faecium 6T1a, a Novel Antilisterial Plasmid-Encoded Bacteriocin Which Does Not Belong to the Pediocin Family of Bacteriocins

PubMed Central

Floriano, Belén; Ruiz-Barba, José L.; Jiménez-Díaz, Rufino

1998-01-01

Enterocin I (ENTI) is a novel bacteriocin produced by Enterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C2/C18 reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E. faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI. entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1. PMID:9835578

Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins.

PubMed Central

Aymerich, T; Holo, H; Håvarstein, L S; Hugas, M; Garriga, M; Nes, I F

1996-01-01

A new bacteriocin has been isolated from an Enterococcus faecium strain. The bacteriocin, termed enterocin A, was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and mass spectrometry analysis. By combining the data obtained from amino acid and DNA sequencing, the primary structure of enterocin A was determined. It consists of 47 amino acid residues, and the molecular weight was calculated to be 4,829, assuming that the four cysteine residues form intramolecular disulfide bridges. This molecular weight was confirmed by mass spectrometry analysis. The amino acid sequence of enterocin A shared significant homology with a group of bacteriocins (now termed pediocin-like bacteriocins) isolated from a variety of lactic acid-producing bacteria, which include members of the genera Lactobacillus, Pediococcus, Leuconostoc, and Carnobacterium. Sequencing of the structural gene of enterocin A, which is located on the bacterial chromosome, revealed an N-terminal leader sequence of 18 amino acid residues, which was removed during the maturation process. The enterocin A leader belongs to the double-glycine leaders which are found among most other small nonlantibiotic bacteriocins, some lantibiotics, and colicin V. Downstream of the enterocin A gene was located a second open reading frame, encoding a putative protein of 103 amino acid residues. This gene may encode the immunity factor of enterocin A, and it shares 40% identity with a similar open reading frame in the operon of leucocin AUL 187, another pediocin-like bacteriocin. PMID:8633865

In vitro activity of a polyhexanide-betaine solution against high-risk clones of multidrug-resistant nosocomial pathogens.

PubMed

López-Rojas, Rafael; Fernández-Cuenca, Felipe; Serrano-Rocha, Lara; Pascual, Álvaro

2017-01-01

To determine the in vitro activity of a polyhexanide-betaine solution against collection strains and multidrug-resistant (MDR) nosocomial isolates, including high-risk clones. We studied of 8 ATCC and 21 MDR clinical strains of Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, including the multiresistant high-risk clones. The MICs and MBCs of a 0.1% polyhexanide-0.1% betaine solution were determined by microdilution. For each species, strains with the highest MICs were selected for further experiments. The dilution-neutralization test (PrEN 12054) was performed by incubating bacterial inocula of 10 6 CFU/mL for 1min with undiluted 0.1% polyhexanide-betaine solution. The CFUs were counted after neutralization. Growth curves and time-kill curves at concentrations of 0.25, 1, 4, and 8×MIC, were performed. MICs of recovered strains were determined when regrowth was observed in time-kill studies after 24h of incubation. Strains with reduced susceptibility were selected by serial passage on plates with increasing concentrations of polyhexanide-betaine, and MICs were determined. Polyhexanide-betaine MIC range was 0.5-8mg/L. MBCs equalled or were 1 dilution higher than MICs. The dilution-neutralization method showed total inoculum clearance of all strains. In time-kill curves, no regrowth was observed at 4×MIC, except for S. aureus (8×MIC). Increased MICs were not observed in time-kill curves, or after serial passages after exposure to polyhexanide-betaine. Polyhexanide-betaine presented bactericidal activity against all MDR clinical isolates tested, including high-risk clones, at significantly lower concentrations and time of activity than those commercially used. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

d-Ala-d-Ser VanN-Type Transferable Vancomycin Resistance in Enterococcus faecium▿

PubMed Central

Lebreton, François; Depardieu, Florence; Bourdon, Nancy; Fines-Guyon, Marguerite; Berger, Pierre; Camiade, Sabine; Leclercq, Roland; Courvalin, Patrice; Cattoir, Vincent

2011-01-01

Enterococcus faecium UCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 μg/ml) but susceptible to teicoplanin (MIC, 0.5 μg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene called vanN was obtained using degenerate primers and was sequenced. The deduced VanN protein was related (65% identity) to the d-alanine:d-serine VanL ligase. The organization of the vanN gene cluster, determined using degenerate primers and by thermal asymmetric interlaced (TAIL)-PCR, was similar to that of the vanC operons. A single promoter upstream from the resistance operon was identified by rapid amplification of cDNA ends (RACE)-PCR. The presence of peptidoglycan precursors ending in d-serine and d,d-peptidase activities in the absence of vancomycin indicated constitutive expression of the resistance operon. VanN-type resistance was transferable by conjugation to E. faecium. This is the first report of transferable d-Ala-d-Ser-type resistance in E. faecium. PMID:21807981

Colonisation of poultry by Salmonella Enteritidis S1400 is reduced by combined administration of Lactobacillus salivarius 59 and Enterococcus faecium PXN-33.

PubMed

Carter, Alun; Adams, Martin; La Ragione, Roberto M; Woodward, Martin J

2017-02-01

Salmonella Enteritidis remains a significant issue within the poultry industry and one potential solution is to use probiotic bacteria to prevent Salmonella colonisation through competitive exclusion (CE). We demonstrate that combined administration of Lactobacillus salivarius 59 and Enterococcus faecium PXN33 were effective competitive excluders of Salmonella Enteritidis S1400 in poultry. Two models were developed to evaluate the efficacy of probiotic where birds received Salmonella Enteritidis S1400 by a) oral gavage and b) sentinel bird to bird transmission. A statistically significant (p<0.001) 2 log reduction of Salmonella Enteritidis S1400 colonisation was observed in the ileum, caecum and colon at day 43 using combined administration of the two probiotic bacteria. However, no Salmonella Enteritidis S1400 colonisation reduction was observed when either probiotic was administered individually. In the sentinel bird model the combined probiotic administered at days 12 and 20 was more effective than one-off or double administrations at age 1 and 12days. In vitro cell free culture supernatant studies suggest the mechanism of Salmonella Enteritidis S1400 inhibition was due to a reduction in pH by the probiotic bacteria. Our current study provides further evidence that probiotics can significantly reduce pathogenic bacterial colonisation in poultry and that mixed preparation of probiotics provide superior performance when compared to individual bacterial preparations. Copyright © 2016 Elsevier B.V. All rights reserved.

COMPARISON BETWEEN AUTOMATED SYSTEM AND PCR-BASED METHOD FOR IDENTIFICATION AND ANTIMICROBIAL SUSCEPTIBILITY PROFILE OF CLINICAL Enterococcus spp

PubMed Central

Furlaneto-Maia, Luciana; Rocha, Kátia Real; Siqueira, Vera Lúcia Dias; Furlaneto, Márcia Cristina

2014-01-01

Enterococci are increasingly responsible for nosocomial infections worldwide. This study was undertaken to compare the identification and susceptibility profile using an automated MicrosScan system, PCR-based assay and disk diffusion assay of Enterococcus spp. We evaluated 30 clinical isolates of Enterococcus spp. Isolates were identified by MicrosScan system and PCR-based assay. The detection of antibiotic resistance genes (vancomycin, gentamicin, tetracycline and erythromycin) was also determined by PCR. Antimicrobial susceptibilities to vancomycin (30 µg), gentamicin (120 µg), tetracycline (30 µg) and erythromycin (15 µg) were tested by the automated system and disk diffusion method, and were interpreted according to the criteria recommended in CLSI guidelines. Concerning Enterococcus identification the general agreement between data obtained by the PCR method and by the automatic system was 90.0% (27/30). For all isolates of E. faecium and E. faecalis we observed 100% agreement. Resistance frequencies were higher in E. faecium than E. faecalis. The resistance rates obtained were higher for erythromycin (86.7%), vancomycin (80.0%), tetracycline (43.35) and gentamicin (33.3%). The correlation between disk diffusion and automation revealed an agreement for the majority of the antibiotics with category agreement rates of > 80%. The PCR-based assay, the van(A) gene was detected in 100% of vancomycin resistant enterococci. This assay is simple to conduct and reliable in the identification of clinically relevant enterococci. The data obtained reinforced the need for an improvement of the automated system to identify some enterococci. PMID:24626409

Oven, microwave, and combination roasting of peanuts: comparison of inactivation of salmonella surrogate Enterococcus faecium, color, volatiles, flavor, and lipid oxidation.

PubMed

Smith, Alicia L; Perry, Jennifer J; Marshall, Julie A; Yousef, Ahmed E; Barringer, Sheryl A

2014-08-01

Peanut safety and quality were evaluated for different roasting technologies. Shelled raw peanuts were roasted using an oven at 163 to 204 °C, microwave, or oven and microwave combinations. The lethal effect of these treatments was investigated on peanuts inoculated with the Salmonella surrogate, Enterococcus faecium and stored at room temperature for 1 h, 24 h, or 7 d before roasting. Roasted peanut color, odor activity values (OAVs), descriptive sensory panel analysis, free fatty acid, and peroxide values were determined. Color and OAVs were also analyzed on 2 commercial peanut butters. OAVs were calculated using volatile levels quantified with selected ion flow tube mass spectrometry and known odor thresholds. All treatments resulted in a minimum of 3 log reduction of inoculated bacterial population. Resistance to the process was not influenced by storage of inoculated peanuts prior to treatment. Roasting by different methods produced equivalent, commercially ideal L* color. Based on the OAVs, treatments had similar volatiles important to flavor compared to the commercial samples. Descriptive sensory analysis showed no significant difference between the roasting treatments for most of the sensory attributes. Lipid oxidation was not significantly different between the roasting methods, displaying no evidence that roasting time or temperature affected lipid oxidation, when ideal color was produced. These results suggest that oven, microwave, or combination roasting should be sufficient to mitigate the threat of Salmonella contamination and produce similar color, OAVs, sensory attributes, and lipid oxidation results. © 2014 Institute of Food Technologists®

Biochemical and Genetic Evidence that Enterococcus faecium L50 Produces Enterocins L50A and L50B, the sec-Dependent Enterocin P, and a Novel Bacteriocin Secreted without an N-Terminal Extension Termed Enterocin Q

PubMed Central

Cintas, Luis M.; Casaus, Pilar; Herranz, Carmen; Håvarstein, Leiv Sigve; Holo, Helge; Hernández, Pablo E.; Nes, Ingolf F.

2000-01-01

Enterococcus faecium L50 grown at 16 to 32°C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47°C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Håvarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63:4321–4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of E. faecium L50 containing the enterocin P structural gene (entP) and the putative immunity protein gene (entiP) reveals a genetic organization identical to that previously found in E. faecium P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was located immediately upstream of and in opposite orientation to entQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E. faecium L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47°C and maximally detected at 47 and 37 to 47�

Molecular Epidemiology of Enterococcal Bacteremia in Australia

PubMed Central

Pearson, Julie C.; Daley, Denise A.; Le, Tam; Robinson, Owen J.; Gottlieb, Thomas; Howden, Benjamin P.; Johnson, Paul D. R.; Bennett, Catherine M.; Stinear, Timothy P.; Turnidge, John D.

2014-01-01

Enterococci are a major cause of health care-associated infections and account for approximately 10% of all bacteremias globally. The aim of this study was to determine the proportion of enterococcal bacteremia isolates in Australia that are antimicrobial resistant, with particular emphasis on susceptibility to ampicillin and the glycopeptides, and to characterize the molecular epidemiology of the Enterococcus faecalis and Enterococcus faecium isolates. From 1 January to 31 December 2011, 1,079 unique episodes of bacteremia were investigated, of which 95.8% were caused by either E. faecalis (61.0%) or E. faecium (34.8%). The majority of bacteremias were health care associated, and approximately one-third were polymicrobial. Ampicillin resistance was detected in 90.4% of E. faecium isolates but was not detected in E. faecalis isolates. Vancomycin nonsusceptibility was reported in 0.6% and 36.5% of E. faecalis and E. faecium isolates, respectively. Unlike Europe and the United States, where vancomycin resistance in E. faecium is predominately due to the acquisition of the vanA operon, 98.4% of E. faecium isolates harboring van genes carried the vanB operon, and 16.1% of the vanB E. faecium isolates had vancomycin MICs at or below the susceptible breakpoint of the CLSI. Although molecular typing identified 126 E. faecalis pulsed-field gel electrophoresis pulsotypes, >50% belonged to two pulsotypes that were isolated across Australia. E. faecium consisted of 73 pulsotypes from which 43 multilocus sequence types were identified. Almost 90% of the E. faecium isolates were identified as CC17 clones, of which approximately half were characterized as ST203, which was isolated Australia-wide. In conclusion, the Australian Enterococcal Sepsis Outcome Programme (AESOP) study has shown that although they are polyclonal, enterococcal bacteremias in Australia are frequently caused by ampicillin-resistant vanB E. faecium. PMID:24391201

Diversity of Antibiotic Resistance Genes in Enterococcus Strains Isolated from Ready-to-Eat Meat Products.

PubMed

Chajęcka-Wierzchowska, Wioleta; Zadernowska, Anna; Łaniewska-Trokenheim, Łucja

2016-10-25

The objective of the study was to answer the question of whether the ready-to-eat meat products can pose indirect hazard for consumer health serving as reservoir of Enterococcus strains harboring tetracyclines, aminoglycosides, and macrolides resistance genes. A total of 390 samples of ready-to-eat meat products were investigated. Enterococcus strains were found in 74.1% of the samples. A total of 302 strains were classified as: Enterococcus faecalis (48.7%), Enterococcus faecium (39.7%), Enterococcus casseliflavus (4.3%), Enterococcus durans (3.0%), Enterococcus hirae (2.6%), and other Enterococcus spp. (1.7%). A high percentage of isolates were resistant to streptomycin high level (45%) followed by erythromycin (42.7%), fosfomycin (27.2%), rifampicin (19.2%), tetracycline (36.4%), tigecycline (19.9%). The ant(6')-Ia gene was the most frequently found gene (79.6%). Among the other genes that encode aminoglycosides-modifying enzymes, the highest portion of the strains had the aac(6')-Ie-aph(2'')-Ia (18.5%) and aph(3'')-IIIa (16.6%), but resistance of isolates from food is also an effect of the presence of aph(2'')-Ib, aph(2'')-Ic, aph(2'')-Id genes. Resistance to tetracyclines was associated with the presence of tetM (43.7%), tetL (32.1%), tetK (14.6%), tetW (0.7%), and tetO (0.3%) genes. The ermB and ermA genes were found in 33.8% and 18.9% of isolates, respectively. Nearly half of the isolates contained a conjugative transposon of the Tn916/Tn1545 family. Enterococci are widely present in retail ready-to-eat meat products. Many isolated strains (including such species as E. casseliflavus, E. durans, E. hirae, and Enterococcus gallinarum) are antibiotic resistant and carry transferable resistance genes. © 2016 Institute of Food Technologists®.

Effect of the probiotic Enterococcus faecium SF68 on presence of diarrhea in cats and dogs housed in an animal shelter.

PubMed

Bybee, S N; Scorza, A V; Lappin, M R

2011-01-01

Beneficial effects of probiotics have never been analyzed in an animal shelter. Dogs and cats housed in an animal shelter and administered a probiotic are less likely to have diarrhea of ≥2 days duration than untreated controls. Two hundred and seventeen cats and 182 dogs. Double blinded and placebo controlled. Shelter dogs and cats were housed in 2 separate rooms for each species. For 4 weeks, animals in 1 room for each species was fed Enterococcus faecium SF68 while animals in the other room were fed a placebo. After a 1-week washout period, the treatments by room were switched and the study continued an additional 4 weeks. A standardized fecal score system was applied to feces from each animal every day by a blinded individual. Feces of animals with and without diarrhea were evaluated for enteric parasites. Data were analyzed by a generalized linear mixed model using a binomial distribution with treatment being a fixed effect and the room being a random effect. The percentage of cats with diarrhea ≥2 days was significantly lower (P = .0297) in the probiotic group (7.4%) when compared with the placebo group (20.7%). Statistical differences between groups of dogs were not detected but diarrhea was uncommon in both groups of dogs during the study. Cats fed SF68 had fewer episodes of diarrhea of ≥2 days when compared with controls suggests the probiotic may have beneficial effects on the gastrointestinal tract. Copyright © 2011 by the American College of Veterinary Internal Medicine.

MALDI-TOF Mass Spectrometry as a Useful Tool for Identification of Enterococcus spp. from Wild Birds and Differentiation of Closely Related Species.

PubMed

Stępień-Pyśniak, Dagmara; Hauschild, Tomasz; Różański, Paweł; Marek, Agnieszka

2017-06-28

The aim of this study was to explore the accuracy and feasibility of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying bacteria from environmental sources, as compared with rpoA gene sequencing, and to evaluate the occurrence of bacteria of the genus Enterococcus in wild birds. In addition, a phyloproteomic analysis of certain Enterococcus species with spectral relationships was performed. The enterococci were isolated from 25 species of wild birds in central Europe (Poland). Proteomic (MALDI-TOF MS) and genomic ( rpoA gene sequencing) methods were used to identify all the isolates. Using MALDI-TOF MS, all 54 (100%) isolates were identified as Enterococcus spp. Among these, 51 (94.4%) isolates were identified to the species level (log(score) > or =2.0), and three isolates (5.6%) were identified at a level of probable genus identification (log(score) 1.88-1.927). Phylogenetic analysis based on rpoA sequences confirmed that all enterococci had been correctly identified. Enterococcus faecalis was the most prevalent enterococcal species (50%) and Enterococcus faecium (33.3%) the second most frequent species, followed by Enterococcus hirae (9.3%), Enterococcus durans (3.7%), and Enterococcus casseliflavus (3.7%). The phyloproteomic analysis of the spectral profiles of the isolates showed that MALDI-TOF MS is able to differentiate among similar species of the genus Enterococcus .

Diversity, distribution and antibiotic resistance of Enterococcus spp. recovered from tomatoes, leaves, water and soil on U.S. Mid-Atlantic farms.

PubMed

Micallef, Shirley A; Goldstein, Rachel E Rosenberg; George, Ashish; Ewing, Laura; Tall, Ben D; Boyer, Marc S; Joseph, Sam W; Sapkota, Amy R

2013-12-01

Antibiotic-resistant enterococci are important opportunistic pathogens and have been recovered from retail tomatoes. However, it is unclear where and how tomatoes are contaminated along the farm-to-fork continuum. Specifically, the degree of pre-harvest contamination with enterococci is unknown. We evaluated the prevalence, diversity and antimicrobial susceptibilities of enterococci collected from tomato farms in the Mid-Atlantic United States. Tomatoes, leaves, groundwater, pond water, irrigation ditch water, and soil were sampled and tested for enterococci using standard methods. Antimicrobial susceptibility testing was performed using the Sensititre microbroth dilution system. Enterococcus faecalis isolates were characterized using amplified fragment length polymorphism to assess dispersal potential. Enterococci (n = 307) occurred in all habitats and colonization of tomatoes was common. Seven species were identified: Enterococcus casseliflavus, E. faecalis, Enterococcus gallinarum, Enterococcus faecium, Enterococcus avis, Enterococcus hirae and Enterococcus raffinosus. E. casseliflavus predominated in soil and on tomatoes and leaves, and E. faecalis predominated in pond water. On plants, distance from the ground influenced presence of enterococci. E. faecalis from samples within a farm were more closely related than those from samples between farms. Resistance to rifampicin, quinupristin/dalfopristin, ciprofloxacin and levofloxacin was prevalent. Consumption of raw tomatoes as a potential exposure risk for antibiotic-resistant Enterococcus spp. deserves further attention. Copyright © 2013 Elsevier Ltd. All rights reserved.

Vancomycin resistant Enterococcus spp. from crows and their environment in metropolitan Washington State, USA: Is there a correlation between VRE positive crows and the environment?

PubMed

Roberts, Marilyn C; No, David B; Marzluff, John M; Delap, Jack H; Turner, Robert

2016-10-15

Vancomycin-resistant enterococci [VRE] have been isolated from municipal, hospital and agricultural wastewater, recreational beaches, wild animals, birds and food animals around the world. In this study, American crows (Corvus brachyrhynchos) from sewage treatment plants (WWTP), dairy farms, and a large roost in a restored wetland with corresponding environmental samples were cultured for VRE. A total of 245 samples [156 crows, 89 environmental] were collected and screened for acquired vanA, vanB and/or intrinsic vanC1 genes. Samples were enriched overnight in BHI supplemented with 20μg/mL aztreonam, 4μg/mL vancomycin and plated on m-Enterococcus agar media supplemented with 6μg/mL vancomycin. Selected colonies were grown on BHI media supplemented with 18μg/mL vancomycin. Of these, 24.5% of the crow and 55% the environmental/cow samples were VRE positive as defined by Enterococcus spp. able to grow on media supplemented with 18μg/mL vancomycin. A total of 122 VRE isolates, 43 crow and 79 environmental isolates were screened, identified to species level using 16S sequencing and further characterized. Four vanA E. faecium and multiple vanC1 E. gallinarum were identified from crows isolated from three sites. E. faecium vanA and E. gallinarum vanC1 along with other Enterococcus spp. carrying vanA, vanB, vanC1 were isolated from three environments. All enterococci were multidrug resistant. Crows were more likely to carry vanA E. faecium than either the cow feces or wetland waters/soils. Comparing E. gallinarum vanC1 from crows and their environment would be useful in determining whether crows share VRE strains with their environment. Copyright © 2016 Elsevier B.V. All rights reserved.

Susceptibility to disinfectants in antimicrobial-resistant and -susceptible isolates of Escherichia coli, Enterococcus faecalis and Enterococcus faecium from poultry-ESBL/AmpC-phenotype of E. coli is not associated with resistance to a quaternary ammonium compound, DDAC.

PubMed

Wieland, N; Boss, J; Lettmann, S; Fritz, B; Schwaiger, K; Bauer, J; Hölzel, C S

2017-06-01

The spread of bacteria that are simultaneously resistant to disinfectants and antimicrobials would constitute an unsettling scenario. In order to explore an association between antimicrobial resistance and reduced susceptibility to biocides/microbicides (disinfectants) in agriculture, we investigated Escherichia coli (n = 438) and enterococci (n = 120) isolated from six different flocks of the same poultry farm with known history of antimicrobial treatment. Susceptibility to disinfectants (formic acid and a quaternary ammonium compound (QAC), didecyldimethylammoniumchloride-DDAC) was assessed by macrodilution according to guidelines of the German Veterinary Society. Escherichia coli, Enterococcus faecalis and Enterococcus faecium were screened (i) for reduced biocide susceptibility and (ii) for an association of biocide susceptibility and antimicrobial resistance including the production of extended-spectrum beta-lactamases (ESBL) and the hyperproduction of AmpC-type beta-lactamases. DDAC inhibited ESBL/AmpC(hyper)-producing E. coli (n = 53) from poultry at similar or slightly lower inhibitory concentrations, compared with non-ESBL/AmpC strains (median MIC = 0·36 vs 1·44 mg l -1 ). In contrast, DDAC-MICs were positively correlated with several other antibiotic MICs (e.g. piperacillin and sulphamethoxazole + trimethoprim in E. coli, chloramphenicol in E. faecalis) and increased DDAC-MICs were statistically linked to high-level aminoglycoside resistance in enterococci (streptomycin high level). DDAC-MICs did not correlate with the presence of the integron marker qacEDelta1. This study provides indication that residual disinfectant might be able to select antimicrobial-resistant enterococci, but not ESBL-/AmpC (hyper)producing E. coli from poultry. While ESBL-/AmpC-E. coli were inhibited at disinfectant concentrations comparable to or lower than wildtype values, low concentrations of QACs might be able to select other antimicrobial-resistant E

Purification, crystallization and preliminary X-ray analysis of Enterococcus faecium aminoglycoside-2′′-phosphotransferase-Ib [APH(2′′)-Ib

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walanj, Rupa; Young, Paul; Baker, Heather M.

2005-04-01

APH(2′′)-Ib is an enzyme responsible for high-level gentamicin resistance in E. faecium isolates. Native crystals of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, APH(2′′)-Ib, has been cloned and the protein (comprising 299 amino-acid residues) expressed in Escherichia coli, purified and crystallized in the presence of 16%(w/v) PEG 3350 and gentamicin. The crystals belong tomore » the monoclinic space group P2{sub 1}, with approximate unit-cell parameters a = 79.7, b = 58.8, c = 81.4 Å, β = 98.4°, and preliminary X-ray diffraction analysis is consistent with the presence of two molecules in the asymmetric unit. Synchrotron diffraction data to approximately 2.65 Å resolution were collected from a native APH(2′′)-Ib crystal at beamline BL9-2 at SSRL (Stanford, CA, USA). Selenium-substituted crystals have also been produced and structure determination is proceeding.« less

Comparison of risk factors and outcomes of daptomycin-susceptible and -nonsusceptible vancomycin-resistant Enterococcus faecium infections in liver transplant recipients.

PubMed

Lewis, J D; Barros, A J; Sifri, C D

2018-02-10

Vancomycin-resistant Enterococcus faecium (VRE) infections are common in liver transplant recipients (LTRs). Daptomycin (DAP) is an important treatment for such infections; however, DAP-nonsusceptible VRE (DNS-VRE) are increasingly frequent. The purpose of this study was to compare clinical characteristics and outcomes of LTRs with infections due to DNS-VRE and DAP-susceptible VRE (DS-VRE). A single center, retrospective review of patients who underwent liver transplantation between January 1, 2010 and December 31, 2015 and developed infections due to DS-VRE or DNS-VRE post transplant was performed. Patients with DNS-VRE and DS-VRE infections were compared using univariate and logistic regression analysis. Fourteen LTRs developed DNS-VRE and 20 LTRs developed DS-VRE infection post-transplantation. No significant differences were observed in demographics, model for end-stage liver disease (MELD) scores, causes of end-stage liver disease, or rate of pre-transplant perirectal VRE colonization between groups. Bleeding complications and renal replacement therapy were more common in the DNS-VRE group than in the DS-VRE group. The duration of transplant hospitalization and post-transplant intensive care unit (ICU) admission was longer in the DNS-VRE group than in the DS-VRE group. The 30-day and 6-month mortality rate associated with DNS-VRE infection was similar to that associated with DS-VRE infection. Liver transplant recipients who develop DNS-VRE infection have higher bleeding complications and longer, more complex hospitalizations compared to those who develop DS-VRE infection post transplantation; however, mortality at 30 days and 6 months is not significantly worse. Further study is needed to determine optimal strategies for the prevention and treatment of DNS-VRE infections in LTRs. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

[Vancomycin-resistant enterococcus--chronicle of a foretold problem].

PubMed

Bonten, Marc J M; Willems, Rob J

2012-01-01

There have recently been 12 outbreaks of infection caused by vancomycin-resistant enterococci (VRE) in Dutch hospitals. Although the first VRE outbreaks were reported almost 12 years ago, such outbreaks remained uncommon and the question is why they are occurring now. Based on molecular epidemiological studies we have learned that a subpopulation of Enterococcus faecium, resistant to amoxicillin but susceptible to vancomycin, has become highly endemic in Dutch hospitals in the past 12 years. Initial analyses suggest that several transposons containing vancomycin-resistance genes have been introduced into this population, followed by nosocomial spread. We recommend that hospitals without detected VRE outbreaks screen high-risk patients for the presence of VRE. If transmission has already occurred in many hospitals, it will be extremely difficult (and costly) to eradicate VRE.

Antibacterial efficacy of nisin, pediocin 34 and enterocin FH99 against L. monocytogenes, E. faecium and E. faecalis and bacteriocin cross resistance and antibiotic susceptibility of their bacteriocin resistant variants.

PubMed

Kaur, Gurpreet; Singh, Tejinder Pal; Malik, Ravinder Kumar; Bhardwaj, Arun; De, Sachinandan

2014-02-01

The bacteriocin susceptibility of Listeria monocytogenes MTCC 657, Enterococcus faecium DSMZ 20477, E. faecium VRE, and E. faecalis ATCC 29212 and their corresponding bacteriocin resistant variants was assessed. The single and combined effect of nisin and pediocin 34 and enterocin FH99 bacteriocins produced by Pediococcus pentosaceus 34, and E. faecium FH99, respectively, was determined. Pediocin34 proved to be more effective in inhibiting L. monocytogenes MTCC 657. A greater antibacterial effect was observed against E. faecium DSMZ 20477 and E. faecium (VRE) when the a combination of nisin, pediocin 34 and enterocin FH99 were used whereas in case of L. monocytogenes MTCC 657 a combination of pediocin 34 and enterocin FH99 was more effective in reducing the survival of pathogen. Bacteriocin cross-resistance and the antibiotic susceptibility of wild type and their corresponding resistant variants were assessed and results showed that resistance to a bacteriocin may extend to other bacteriocins within the same class and also the acquired resistance to bacteriocins can modify the antibiotic susceptibility/resistance profile of the bacterial species used in the study. According to the hydrophobicity nisin resistant variant of L. monocytogenes was more hydrophobic (p < 0.001), whereas the pediocin 34 and enterocin FH99 resistant variants were less hydrophobic than the wild type strain. Nisin, pediocin 34 and enterocin FH99 resistant variants of E. faecium DSMZ 20477 and E. faecium VRE were less hydrophobic than their wild type counterparts. Nisin resistant E. faecalis ATCC 29212 was less hydrophobic than its wild type counterpart.

Targeting Enterococcus faecalis Biofilms with Phage Therapy

PubMed Central

Khalifa, Leron; Brosh, Yair; Gelman, Daniel; Coppenhagen-Glazer, Shunit; Beyth, Shaul; Poradosu-Cohen, Ronit; Que, Yok-Ai; Beyth, Nurit

2015-01-01

Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment. PMID:25662974

Performance of Vitek 2 for Antimicrobial Susceptibility Testing of Staphylococcus spp. and Enterococcus spp.

PubMed Central

Bobenchik, April M.; Hindler, Janet A.; Giltner, Carmen L.; Saeki, Sandra

2014-01-01

Vitek 2 (bioMérieux, Inc., Durham, NC) is a widely used commercial antimicrobial susceptibility testing system. We compared MIC results obtained by Vitek 2 to those obtained by the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) reference method for 134 staphylococcal and 84 enterococcal clinical isolates. Nineteen agents were evaluated, including all those available on Vitek 2 for testing staphylococci and enterococci. The resistance phenotypes tested included methicillin-resistant Staphylococcus aureus (MRSA) (n = 58), S. aureus with inducible clindamycin resistance (ICR) (n = 30), trimethoprim-sulfamethoxazole-resistant MRSA (n = 10), vancomycin-resistant Enterococcus (n = 37), high-level gentamicin-resistant Enterococcus (n = 15), linezolid-resistant Enterococcus (n = 5), and daptomycin-nonsusceptible Enterococcus faecalis (n = 6). For the staphylococci, there was 98.9% categorical agreement (CA). There was one very major error (VME) for gentamicin in a Staphylococcus hominis isolate, six VMEs for inducible clindamycin in S. aureus isolates, and two major errors (ME) for daptomycin in an S. aureus and a Staphylococcus epidermidis isolate. For enterococci, there was 97.3% CA. Two VMEs were observed for daptomycin in isolates of E. faecalis and 2 ME, 1 for high-level gentamicin resistance and 1 for nitrofurantoin, in E. faecium isolates. Overall, there was 98.3% CA and 99% essential agreement for the testing of staphylococci and enterococci by the Vitek 2. With the exception of detecting ICR in S. aureus, Vitek 2 performed reliably for antimicrobial susceptibility testing of staphylococci and enterococci. PMID:24478467

Presence of the resistance genes vanC1 and pbp5 in phenotypically vancomycin and ampicillin susceptible Enterococcus faecalis.

PubMed

Schwaiger, Karin; Bauer, Johann; Hörmansdorfer, Stefan; Mölle, Gabriele; Preikschat, Petra; Kämpf, Peter; Bauer-Unkauf, Ilse; Bischoff, Meike; Hölzel, Christina

2012-08-01

Ampicillin and vancomycin are important antibiotics for the therapy of Enterococcus faecalis infections. The ampicillin resistance gene pbp5 is intrinsic in Enterococcus faecium. The vanC1 gene confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Both genes are chromosomally located. Resistance to ampicillin and vancomycin was determined in 484 E. faecalis of human and porcine origin by microdilution. Since E. faecalis are highly skilled to acquire resistance genes, all strains were investigated for the presence of pbp5 (and, in positive strains, for the penicillin-binding protein synthesis repressor gene psr) and vanC1 (and, in positive strains, for vanXYc and vanT) by using polymerase chain reaction (PCR). One porcine and one human isolate were phenotypically resistant to ampicillin; no strain was vancomycin resistant. Four E. faecalis (3/1 of porcine/human origin) carried pbp5 (MIC=1 mg/L), and four porcine strains were vanC1 positive (minimum inhibitory concentration [MIC]=1 mg/L). Real-time reverse transcriptase (RT)-PCR revealed that the genes were not expressed. The psr gene was absent in the four pbp5-positive strains; the vanXYc gene was absent in the four vanC1-positive strains. However, vanT of the vanC gene cluster was detected in two vanC1-positive strains. To our knowledge, this is the first report on the presence of pbp5, identical with the "E. faecium pbp5 gene," and of vanC1/vanT in E. faecalis. Even if resistance is not expressed in these strains, this study shows that E. faecalis have a strong ability to acquire resistance genes-and potentially to spread them to other bacteria. Therefore, close monitoring of this species should be continued.

Enterococcus phages as potential tool for identifying sewage inputs in the Great Lakes region

USGS Publications Warehouse

Vijayavel, K.; Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Ebdon, J.; Taylor, H.; Kashian, D.R.

2014-01-01

Bacteriophages are viruses living in bacteria that can be used as a tool to detect fecal contamination in surface waters around the world. However, the lack of a universal host strain makes them unsuitable for tracking fecal sources. We evaluated the suitability of two newly isolated Enterococcus host strains (ENT-49 and ENT-55) capable for identifying sewage contamination in impacted waters by targeting phages specific to these hosts. Both host strains were isolated from wastewater samples and identified as E. faecium by 16S rRNA gene sequencing. Occurrence of Enterococcus phages was evaluated in sewage samples (n = 15) from five wastewater treatment plants and in fecal samples from twenty-two species of wild and domesticated animals (individual samples; n = 22). Levels of Enterococcus phages, F + coliphages, Escherichia coli and enterococci were examined from four rivers, four beaches, and three harbors. Enterococcus phages enumeration was at similar levels (Mean = 6.72 Log PFU/100 mL) to F + coliphages in all wastewater samples, but were absent from all non-human fecal sources tested. The phages infecting Enterococcus spp. and F + coliphages were not detected in the river samples (detection threshold < 10 PFU/100 mL), but were present in the beach and harbor samples (range = 1.83 to 2.86 Log PFU/100 mL). Slightly higher concentrations (range = 3.22 to 3.69 Log MPN/100 mL) of E. coli and enterococci when compared to F + coliphages and Enterococcus phages, were observed in the river, beach and harbor samples. Our findings suggest that the bacteriophages associated with these particular Enterococcus host strains offer potentially sensitive and human-source specific indicators of enteric pathogen risk.

Systemic and local bactericidal potentiality in late lactation Holstein-Friesian cows following a combined antibiotics and Enterococcus faecium SF68 dry-cow treatment.

PubMed

Tiantong, Attapol; Piamya, Piya; Chen, Shuen-Ei; Liu, Wen-Bor; Chang, Fang-Yu; Lin, Pei-Chi; Nagahata, Hajime; Chang, Chai-Ju

2015-08-01

Antibiotic dry-cow treatment contributes a major part to the total use of antibiotics in dairy herds. Enterococcus faecium strain SF68 (SF68) was of human origin but has been authorized in EU as probiotic feed additive. In the present study, one of the front and rear quarters of twelve late lactation Holstein-Friesian cows were infused once with a commercial antibiotic dry-cow formula (antibiotics quarter) on the first milk-stasis day (d 1), when the contrallateral quarters were infused with 5 x 10(8)-CFU SF68 plus half-dose antibiotic dry-cow formula (SF68/antibiotics quarter) meanwhile. Gelatinase level and cellular reactive oxygen species (ROS) production capacity were measured for blood and quarter secretion. The results showed that the count of blood total leukocytes minorly decreased on d 3 only but the microscopic somatic cell count (MSCC) continuously increased up to d 7, especially in SF68/antibiotics quarters. Plasma level of gelatinase A remained similar up to d 7 but gelatinase B was not detectable in plasma throughout the study. The level of gelatinase A in quarter secretion increased up to d 7 but gelatinase B increased even more drastically, especially in SF68/antibiotics quarters. The ROS production capacity of blood leukocytes increased temporarily only on d 3, but that of milk cells continuously increased up to d 7, especially in SF68/antitiotics quarters. Overall, late lactation Holstein-Friesian cows were systemically adaptable to the combined antibiotics and SF68 dry-cow treatment, while the local bactericidal potentiality in mammary gland was actively responsive to additional SF68 intramammary treatment.

Antimicrobial resistance status of Enterococcus from Australian cattle populations at slaughter.

PubMed

Barlow, Robert S; McMillan, Kate E; Duffy, Lesley L; Fegan, Narelle; Jordan, David; Mellor, Glen E

2017-01-01

Antimicrobial agents are used in cattle production systems for the prevention and control of bacterial associated diseases. A consequence of their use is the potential development of antimicrobial resistance (AMR). Enterococcus faecium and Enterococcus faecalis that are resistant to antimicrobials are of increased concern to public health officials throughout the world as they may compromise the ability of various treatment regimens to control disease and infection in human medicine. Australia is a major exporter of beef; however it does not have an ongoing surveillance system for AMR in cattle or foods derived from these animals. This study examined 910 beef cattle, 290 dairy cattle and 300 veal calf faecal samples collected at slaughter for the presence of enterococci. Enterococcus were isolated from 805 (88.5%) beef cattle faeces, 244 (84.1%) dairy cattle faeces and 247 (82.3%) veal calf faeces with a total of 800 enterococci subsequently selected for AMR testing. The results of AMR testing identified high levels of resistance to antimicrobials that are not critically or highly important to human medicine with resistance to flavomycin (80.2%) and lincomycin (85.4-94.2%) routinely observed. Conversely, resistance to antibiotics considered critically or highly important to human medicine such as tigecycline, daptomycin, vancomycin and linezolid was not present in this study. There is minimal evidence that Australian cattle production practices are responsible for disproportionate contributions to AMR development and in general resistance to antimicrobials of critical and high importance in human medicine was low regardless of the isolate source. The low level of antimicrobial resistance in Enterococcus from Australian cattle is likely to result from comprehensive controls around the use of antimicrobials in food-production animals in Australia. Nevertheless, continued monitoring of the effects of all antimicrobial use is required to support Australia's reputation

Antimicrobial resistance status of Enterococcus from Australian cattle populations at slaughter

PubMed Central

McMillan, Kate E.; Duffy, Lesley L.; Fegan, Narelle; Jordan, David; Mellor, Glen E.

2017-01-01

Antimicrobial agents are used in cattle production systems for the prevention and control of bacterial associated diseases. A consequence of their use is the potential development of antimicrobial resistance (AMR). Enterococcus faecium and Enterococcus faecalis that are resistant to antimicrobials are of increased concern to public health officials throughout the world as they may compromise the ability of various treatment regimens to control disease and infection in human medicine. Australia is a major exporter of beef; however it does not have an ongoing surveillance system for AMR in cattle or foods derived from these animals. This study examined 910 beef cattle, 290 dairy cattle and 300 veal calf faecal samples collected at slaughter for the presence of enterococci. Enterococcus were isolated from 805 (88.5%) beef cattle faeces, 244 (84.1%) dairy cattle faeces and 247 (82.3%) veal calf faeces with a total of 800 enterococci subsequently selected for AMR testing. The results of AMR testing identified high levels of resistance to antimicrobials that are not critically or highly important to human medicine with resistance to flavomycin (80.2%) and lincomycin (85.4–94.2%) routinely observed. Conversely, resistance to antibiotics considered critically or highly important to human medicine such as tigecycline, daptomycin, vancomycin and linezolid was not present in this study. There is minimal evidence that Australian cattle production practices are responsible for disproportionate contributions to AMR development and in general resistance to antimicrobials of critical and high importance in human medicine was low regardless of the isolate source. The low level of antimicrobial resistance in Enterococcus from Australian cattle is likely to result from comprehensive controls around the use of antimicrobials in food-production animals in Australia. Nevertheless, continued monitoring of the effects of all antimicrobial use is required to support Australia�

Resistance of enterococci to heat and chemical agents.

PubMed

Renner, P; Peters, J

1999-06-01

Within the framework of the standardisation efforts on disinfectant testing on the European level the test germ Enterococcus hirae ATCC 10541 has been included for some time in the test requirements whereas the test strain Enterococcus faecium, which has frequently been used up to now, has been largely ignored. We compared the thermal and the chemical resistance of both test germ species. In the quantitative suspension test with active ingredients from the group of aldehydes, phenols, quaternaries and oxidizing agents with the exception of peracetic acid, no significant differences were determined between the two strains. In the case of the studies on thermal resistance at 65 degrees C and 68 degrees C, Enterococcus faecium ATCC 6057, by contrast, proved to be far more resistant than Enterococcus hirae ATCC 10541. According to these results, priority should be given to Enterococcus faecium over Enterococcus hirae as the test germ for chemical and also chemothermal disinfection.

Characterization of Vancomycin-Resistant Enterococcus faecalis and Enterococcus faecium Isolated from Fresh Produces and Human Fecal Samples.

PubMed

Kim, Min-Chan; Cha, Min-Hyeok; Ryu, Jae-Gee; Woo, Gun-Jo

2017-04-01

Increased enterococcal infections in hospitals and multidrug-resistant and vancomycin-resistant enterococci (VRE) isolated from humans, animals, and food sources raised public health concern on the presence of VRE in multiple sources. We performed a comparative analysis of the antimicrobial resistance and genetics of VRE isolates derived from fresh produce and human fecal samples. Of 389 Enterococcus isolates, 8 fecal and 3 produce isolates were resistant to vancomycin and teicoplanin; all harbored vanA gene. The VRE isolates showed multidrug-resistant properties. The isolates from fresh produce in this study showed to have the common shared characteristics with the isolates from humans by the results of antimicrobial resistance, multilocus sequence typing, and Tn 1546 transposon analysis. Therefore, VRE isolates from fresh produce are likely related to VRE derived from humans. The results suggested that VRE may contaminate vegetables through the environment, and the contaminated vegetables could then act as a vehicle for human infections. Ongoing nationwide surveillance of antibiotic resistance and the promotion of the proper use of antibiotics are necessary.

Clinico-Microbiological Investigation of Catheter Associated Urinary Tract Infection by Enterococcus faecalis: vanA Genotype

PubMed Central

Padmavathy, Kesavaram; Madhavan, Radha; Krithika, Nagarajan; Kiruthiga, Alexander

2015-01-01

Prolonged hospitalization and exposure to third generation cephalosporins are reported to facilitate the acquisition and colonization of Vancomycin Resistant Enterococci (VRE). Though VRE is not uncommon in India, urinary tract infection with a vanA genotype is a cause of serious concern as VRE co-exhibit resistance to aminoglycosides. In India, majority of the VRE isolates recovered from hospitalized patients include Enterococcus faecium. We report a case of catheter associated urinary tract infection by an endogenous, multidrug resistant E. faecalis of vanA genotype following prolonged hospitalization, ICU stay, catheterisation and exposure to 3G cephalosporin and metronidazole. The patient responded to linezolid therapy. PMID:26435949

Comparison of Enterococcus Species Diversity in Marine Water and Wastewater Using Enterolert and EPA Method 1600

PubMed Central

Ferguson, Donna M.; Griffith, John F.; McGee, Charles D.; Weisberg, Stephen B.; Hagedorn, Charles

2013-01-01

EPA Method 1600 and Enterolert are used interchangeably to measure Enterococcus for fecal contamination of public beaches, but the methods occasionally produce different results. Here we assess whether these differences are attributable to the selectivity for certain species within the Enterococcus group. Both methods were used to obtain 1279 isolates from 17 environmental samples, including influent and effluent of four wastewater treatment plants, ambient marine water from seven different beaches, and freshwater urban runoff from two stream systems. The isolates were identified to species level. Detection of non-Enterococcus species was slightly higher using Enterolert (8.4%) than for EPA Method 1600 (5.1%). E. faecalis and E. faecium, commonly associated with human fecal waste, were predominant in wastewater; however, Enterolert had greater selectivity for E. faecalis, which was also shown using a laboratory-created sample. The same species selectivity was not observed for most beach water and urban runoff samples. These samples had relatively higher proportions of plant associated species, E. casseliflavus (18.5%) and E. mundtii (5.7%), compared to wastewater, suggesting environmental inputs to beaches and runoff. The potential for species selectivity among water testing methods should be considered when assessing the sanitary quality of beaches so that public health warnings are based on indicators representative of fecal sources. PMID:23840233

Amylolytic Enzymes Acquired from L-Lactic Acid Producing Enterococcus faecium K-1 and Improvement of Direct Lactic Acid Production from Cassava Starch.

PubMed

Unban, Kridsada; Kanpiengjai, Apinun; Takata, Goro; Uechi, Keiko; Lee, Wen-Chien; Khanongnuch, Chartchai

2017-09-01

An amylolytic lactic acid bacterium isolate K-1 was isolated from the wastewater of a cassava starch manufacturing factory and identified as Entercoccus faecium based on 16S rRNA gene sequence analysis. An extracellular α-amylase was purified to homogeneity and the molecular weight of the purified enzyme was approximately 112 kDa with optimal pH value and temperature measured of 7.0 and 40 °C, respectively. It was stable at a pH range of 6.0-7.0, but was markedly sensitive to high temperatures and low pH conditions, even at a pH value of 5. Ba 2+ , Al 3+ , and Co 2+ activated enzyme activity. This bacterium was capable of producing 99.2% high optically pure L-lactic acid of 4.3 and 8.2 g/L under uncontrolled and controlled pH at 6.5 conditions, respectively, in the MRS broth containing 10 g/L cassava starch as the sole carbon source when cultivated at 37 °C for 48 h. A control pH condition of 6.5 improved and stabilized the yield of L-lactic acid production directly from starch even at a high concentration of starch at up to 150 g/L. This paper is the first report describing the properties of purified α-amylase from E. faecium. Additionally, pullulanase and cyclodextrinase activities were also firstly recorded from E. faecium K-1.

Genomic and functional characterisation of two Enterococcus strains isolated from Cotija cheese and their potential role in ripening.

PubMed

Olvera-García, Myrna; Sanchez-Flores, Alejandro; Quirasco Baruch, Maricarmen

2018-03-01

Enterococcus spp. are present in the native microbiota of many traditional fermented foods. Their ability to produce antibacterial compounds, mainly against Listeria monocytogenes, has raised interest recently. However, there is scarce information about their proteolytic and lipolytic potential, and their biotechnological application is currently limited because enterococcal strains have been related to nosocomial infections. In this work, next-generation sequencing and optimised bioinformatic pipelines were used to annotate the genomes of two Enterococcus strains-one E. faecium and one E. faecalis-isolated from the Mexican artisanal ripened Cotija cheese. A battery of genes involved in their proteolytic system was annotated. Genes coding for lipases, esterases and other enzymes whose final products contribute to cheese aroma and flavour were identified as well. As for the production of antibacterial compounds, several peptidoglycan hydrolase- and bacteriocin-coding genes were identified in both genomes experimentally and by bioinformatic analyses. E. faecalis showed resistance to aminoglycosides and E. faecium to aminoglycosides and macrolides, as predicted by the genome functional annotation. No pathogenicity islands were found in any of the strains, although traits such as the ability of biofilm formation and cell aggregation were observed. Finally, a comparative genomic analysis was able to discriminate between the food strains isolated and nosocomial strains. In summary, pathogenic strains are resistant to a wide range of antibiotics and contain virulence factors that cause host damage; in contrast, food strains display less antibiotic resistance, include genes that encode class II bacteriocins and express virulence factors associated with host colonisation rather than invasion.

Whole genome characterization of a naturally occurring vancomycin-dependent Enterococcus faecium from a patient with bacteremia.

PubMed

Mitchell, Stephanie L; Mattei, Lisa M; Alby, Kevin

2017-08-01

Vancomycin-dependent enterococci are a relatively uncommon phenotype recovered in the clinical laboratory. Recognition and recovery of these isolates are important, to provide accurate identification and susceptibility information to treating physicians. Herein, we describe the recovery of a vancomycin-dependent and revertant E. faecium isolates harboring vanB operon from a patient with bacteremia. Using whole genome sequencing, we found a unique single nucleotide polymorphism (S186N) in the D-Ala-D-Ala ligase (ddl) conferring vancomycin-dependency. Additionally, we found that a majority of in vitro revertants mutated outside ddl, with some strains harboring mutations in vanS, while others likely containing novel mechanisms of reversion. Copyright © 2017 Elsevier B.V. All rights reserved.

Use of synthetic genes for cloning, production and functional expression of the bacteriocins enterocin A and bacteriocin E 50-52 by Pichia pastoris and Kluyveromyces lactis.

PubMed

Jiménez, Juan J; Borrero, Juan; Gútiez, Loreto; Arbulu, Sara; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2014-06-01

The use of synthetic genes may constitute a successful approach for the heterologous production and functional expression of bacterial antimicrobial peptides (bacteriocins) by recombinant yeasts. In this work, synthetic genes with adapted codon usage designed from the mature amino acid sequence of the bacteriocin enterocin A (EntA), produced by Enterococcus faecium T136, and the mature bacteriocin E 50-52 (BacE50-52), produced by E. faecium NRRL B-32746, were synthesized. The synthetic entA and bacE50-52 were cloned into the protein expression vectors pPICZαA and pKLAC2 for transformation of derived vectors into Pichia pastoris X-33 and Kluyveromyces lactis GG799, respectively. The recombinant vectors were linearized and transformed into competent cells selecting for P. pastoris X-33EAS (entA), P. pastoris X-33BE50-52S (bacE50-52), K. lactis GG799EAS (entA), and K. lactis GG799BE50-52S (bacE50-52). P. pastoris X-33EAS and K. lactis GG799EAS, but not P. pastoris X-33BE50-52S and K. lactis GG799BE50-52S, showed antimicrobial activity in their supernatants. However, purification of the supernatants of the producer yeasts permitted recovery of the bacteriocins EntA and BacE50-52. Both purified bacteriocins were active against Gram-positive bacteria such as Listeria monocytogenes but not against Gram-negative bacteria, including Campylobacter jejuni.

Microbiology and Injury Characteristics in Severe Open Tibia Fractures from Combat

DTIC Science & Technology

2012-01-01

cloacae 17 18.3 Klebsiella pneumoniae 13 14 Enterococcus faecium 13 14 Pseudomonas aeruginosa 13 14 Staphylococcus epidermidis 6 6.5 Infection cultures 57...baumannii 10 19.6 Staphylococcus epidermidis 10 19.6 Klebsiella pneumoniae 6 11.8 Enterococcus faecium 4 7.8 Burns et al. J Trauma Volume 72, Number 4 © 2012

Cloning, overexpression, purification of bacteriocin enterocin-B and structural analysis, interaction determination of enterocin-A, B against pathogenic bacteria and human cancer cells.

PubMed

Ankaiah, Dasari; Palanichamy, Esakkiraj; Antonyraj, Christian Bharathi; Ayyanna, Repally; Perumal, Venkatesh; Ahamed, Syed Ibrahim Basheer; Arul, Venkatesan

2018-05-02

In this present study, a gene (ent-B) encoding the bacteriocin enterocin-B was cloned, overexpressed and purified from Enterococcus faecium por1. The molecular weight of the bacteriocin enterocin-B was observed around 7.2 kDa and exhibited antimicrobial activity against several human pathogenic bacteria. The antimicrobial activity of cloned enterocin-B was increased effectively by combining with another bacteriocin enterocin-A from the same microorganism. Protein-protein docking and molecular dynamics simulation studies revealed that the bacteriocin enterocin-B is interacting with enterocin-A and formation of a heterodimer (enterocin A + B). The heterodimer of bacteriocin enterocin-A + B exhibited potential anti-bacterial, anti-biofilm activity against Staphylococcus aureus, Acinetobacter baumannii, Listeria monocytogenes and Escherichia coli. The bacteriocin enterocin-B, A and heterodimer of bacteriocin enterocin A + B showed no haemolysis on human RBC cells. This is the first report that the cell growth inhibitory activity of the bacteriocin enterocin B against HeLa, HT-29 and AGS human cancer cells and this cell growth inhibitory activity was significantly increased when cancer cells treated with the heterodimer of bacteriocins enterocin-A + B. The cell growth inhibitory activity of the bacteriocin enterocin-B and the heterodimer of bacteriocin enterocin-A + B were not observed in non-cancerous INT-407 cells (intestinal epithelial cells). Copyright © 2018. Published by Elsevier B.V.

Identification, antimicrobial resistance and genotypic characterization of Enterococcus spp. isolated in Porto Alegre, Brazil

PubMed Central

Bender, Eduardo André; de Freitas, Ana Lúcia Peixoto; Reiter, Keli Cristine; Lutz, Larissa; Barth, Afonso Luís

2009-01-01

In the past two decades the members of the genus Enterococcus have emerged as important nosocomial pathogens worldwide. In the present study, we evaluated the antimicrobial resistance and genotypic characteristics of 203 Enterococcus spp. recovered from different clinical sources from two hospitals in Porto Alegre, Rio Grande do Sul, Brazil. The species were identified by conventional biochemical tests and by an automated system. The genetic diversity of E. faecalis presenting high-level aminoglycoside resistance (HLAR) was assessed by pulsed-field gel electrophoresis of chromosomal DNA after SmaI digestion. The E. faecalis was the most frequent specie (93.6%), followed by E. faecium (4.4%). The antimicrobial resistance profile was: 2.5% to ampicillin, 0.5% to vancomycin, 0.5% teicoplanin, 33% to chloramphenicol, 2% to nitrofurantoin, 66.1% to erythromycin, 66.5% to tetracycline, 24.6% to rifampicin, 30% to ciprofloxacin and 87.2% to quinupristin-dalfopristin. A total of 10.3% of the isolates proved to be HLAR to both gentamicin and streptomycin (HLR-ST/GE), with 23.6% resistant only to gentamicin (HLR-GE) and 37.4% only to streptomycin (HLR-ST). One predominant clonal group was found among E. faecalis HLR-GE/ST. The prevalence of resistance among beta-lactam antibiotics and glycopeptides was very low. However, in this study there was an increased number of HLR Enterococcus which may be spreading intra and inter-hospital. PMID:24031416

Effect of vancomycin, tylosin, and chlortetracycline on vancomycin-resistant Enterococcus faecium colonization of broiler chickens during grow-out

USDA-ARS?s Scientific Manuscript database

Broiler chickens may serve as reservoirs for human colonization by vancomycin-resistant Enterococcus (VRE). We examined the effects of vancomycin and two commonly-used antimicrobial feed additives on VRE colonization in broiler chickens during grow-out. Chicks received unsupplemented feed or feed ...

23S rRNA gene-based enterococci community signatures in Lake Pontchartrain, Louisiana, USA, following urban runoff inputs after Hurricane Katrina.

PubMed

Bae, Hee-Sung; Hou, Aixin

2013-02-01

Little is known about the impacts of fecal polluted urban runoff inputs on the structure of enterococci communities in estuarine waters. This study employed a 23S rRNA gene-based polymerase chain reaction (PCR) assay with newly designed genus-specific primers, Ent127F-Ent907R, to determine the possible impacts of Hurricane Katrina floodwaters via the 17th Street Canal discharge on the community structure of enterococci in Lake Pontchartrain. A total of 94 phylotypes were identified through the restriction fragment length polymorphism (RFLP) screening of 494 clones while only 8 phylotypes occurred among 88 cultivated isolates. Sequence analyses of representative phylotypes and their temporal and spatial distribution in the lake and the canal indicated the Katrina floodwater input introduced a large portion of Enterococcus flavescens, Enterococcus casseliflavus, and Enterococcus dispar into the lake; typical fecal groups Enterococcus faecium, Enterococcus durans, Enterococcus hirae, and Enterococcus mundtii were detected primarily in the floodwater-impacted waters. This study provides a global picture of enterococci in estuarine waters impacted by Hurricane Katrina-derived urban runoff. It also demonstrates the culture-independent PCR approach using 23S rRNA gene as a molecular marker could be a good alternative in ecological studies of enterococci in natural environments to overcome the limitation of conventional cultivation methods.

Immunochemical characterization of temperature-regulated production of enterocin L50 (EntL50A and EntL50B), enterocin P, and enterocin Q by Enterococcus faecium L50.

PubMed

Criado, Raquel; Gutiérrez, Jorge; Martín, María; Herranz, Carmen; Hernández, Pablo E; Cintas, Luis M

2006-12-01

Polyclonal antibodies with specificity for enterocin L50A (EntL50A), enterocin L50B (EntL50B), and enterocin Q (EntQ) produced by Enterococcus faecium L50 have been generated by immunization of rabbits with chemically synthesized peptides derived from the C terminus of EntL50A (LR1) and EntL50B (LR2) and from the complete enterocin Q (EntQ) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of these antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect ELISA (CI-ELISA). The NCI-ELISA was valuable for detecting anti-EntL50A-, anti-EntL50B-, and anti-EntQ-specific antibodies in the sera of the LR1-KLH-, LR2-KLH-, and EntQ-KLH-immunized animals, respectively. Moreover, these antibodies and those specific for enterocin P (EntP) obtained in a previous work (J. Gutiérrez, R. Criado, R. Citti, M. Martín, C. Herranz, M. F. Fernández, L. M. Cintas, and P. E. Hernández, J. Agric. Food Chem. 52:2247-2255, 2004) were used in an NCI-ELISA to detect and quantify the production of EntL50A, EntL50B, EntP, and EntQ by the multiple-bacteriocin producer E. faecium L50 grown at different temperatures (16 to 47 degrees C). Our results show that temperature has a strong influence on bacteriocin production by this strain. EntL50A and EntL50B are synthesized at 16 to 32 degrees C, but production becomes negligible when the growth temperature is above 37 degrees C, whereas EntP and EntQ are synthesized at temperatures ranging from 16 to 47 degrees C. Maximum EntL50A and EntL50B production was detected at 25 degrees C, while EntP and EntQ are maximally produced at 37 and 47 degrees C, respectively. The loss of plasmid pCIZ1 (50 kb) and/or pCIZ2 (7.4 kb), encoding EntL50A and EntL50B as well as EntQ, respectively, resulted in a significant increase in production and stability of the chromosomally encoded EntP.

Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

PubMed Central

Ryu, Hodon; Henson, Michael; Elk, Michael; Toledo-Hernandez, Carlos; Griffith, John; Blackwood, Denene; Noble, Rachel; Gourmelon, Michèle; Glassmeyer, Susan

2013-01-01

The detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium, although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water. PMID:23087032

Pilot Study of Antimicrobial Resistance in Northern Bobwhites (Colinus virginianus).

PubMed

Zhang, Michael; Shen, Zhenyu; Rollins, Dale; Fales, William; Zhang, Shuping

2017-09-01

Antimicrobial resistance (AMR) is an important issue for both wildlife conservation and public health. The purpose of this study was to screen for AMR in fecal bacteria isolated from northern bobwhite (Colinus virginianus), a species that is an ecologically and economically important natural resource in the southern United States. The antimicrobial susceptibility profiles of 45 Escherichia coli isolates, 20 Enterococcus faecalis isolates, and 10 Enterococcus faecium isolates were determined using the Sensititer TM microbroth dilution minimum inhibitory concentration (MIC) plate, AVIAN1F. Overall, E. coli isolates had high MIC values for the following classes of antimicrobials: aminocoumarins, beta-lactams, lincosamides, macrolides, florfenicol, and sulfonamides. Enterococcus faecalis and E. faecium isolates had high MICs for aminocyclitols, aminoglycosides, beta-lactams, lincosamides, and sulfonamides. Enterococcus faecalis isolates also showed high MICs for aminocoumarins, while E. faecium isolates had high MICs for trimethoprim/sulfamethoxazole and tetracycline. Based on available veterinary interpretive criteria, 15% and 33% of E. coli isolates were resistant to sulphathiazole and sulphadimethoxine, respectively. Intermediate susceptibility to florfenicol was seen with 17.8% of E. coli isolates. Twenty percent of E. faecalis and 80% of E. faecium isolates were resistant to high-concentration streptomycin. One third of E. faecalis and 70% of E. faecium isolates were intermediately susceptible to erythromycin. Ten percent of E. faecium isolates were resistant to tetracycline and oxytetracycline. A comparison of available MIC suggests that AMR in wild bobwhite is less severe than in domestic poultry. Further investigation is needed to determine the source of AMR in wild bobwhite.

Synthesis and In Vitro Activity of Polyhalogenated 2-phenylbenzimidazoles as a New Class of anti-MRSA and Anti-VRE Agents.

PubMed

Göker, Hakan; Karaaslan, Cigdem; Püsküllü, Mustafa Orhan; Yildiz, Sulhiye; Duydu, Yalcin; Üstündağ, Aylin; Yalcin, Can Özgür

2016-01-01

A series of novel polyhalogenated 2-phenylbenzimidazoles have been synthesized and evaluated for in vitro antistaphylococcal activity against drug-resistant bacterial strains (methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium. Certain compounds inhibit bacterial growth perfectly. 11 was active than vancomycin (0.78 μg/mL) with the lowest MIC values with 0.19 μg/mL against methicillin-resistant Staphylococcus aureus, 8 and 35 exhibited best inhibitory activity against vancomycin-resistant Enterococcus faecium (1.56 μg/mL). The mechanism of action for this class of compounds appears to be different than clinically used antibiotics. These polyhalogenated benzimidazoles have potential for further investigation as a new class of potent anti-methicillin-resistant Staphylococcus aureus and anti-vancomycin-resistant Enterococcus faecium agents. © 2015 John Wiley & Sons A/S.

Increasing Prevalence of Aminoglycoside-Resistant Enterococcus faecalis Isolates Due to the aac(6')-aph(2") Gene: A Therapeutic Problem in Kermanshah, Iran.

PubMed

Khani, Mitra; Fatollahzade, Mahdie; Pajavand, Hamid; Bakhtiari, Somaye; Abiri, Ramin

2016-03-01

Enterococci are important pathogens in nosocomial infections. Various types of antibiotics, such as aminoglycosides, are used for treatment of these infections. Enterococci can acquire resistant traits, which can lead to therapeutic problems with aminoglycosides. This study was designed to identify the prevalence of, and to compare, the aac(6')-aph(2") and aph(3)-IIIa genes and their antimicrobial resistance patterns among Enterococcus faecalis and E. faecium isolates from patients at Imam Reza hospital in Kermanshah in 2011 - 2012. One hundred thirty-eight clinical specimens collected from different wards of Imam Reza hospital were identified to the species level by biochemical tests. Antimicrobial susceptibility tests against kanamycin, teicoplanin, streptomycin, imipenem, ciprofloxacin, and ampicillin were performed by the disk diffusion method. The minimum inhibitory concentrations of gentamicin, streptomycin, kanamycin, and amikacin were evaluated with the microbroth dilution method. The aminoglycoside resistance genes aac(6')-aph(2") and aph(3")-IIIa were analyzed with multiplex PCR. The prevalence of isolates was 33 (24.1%) for E. faecium and 63 (46%) for E. faecalis. Eighty-nine percent of the isolates were high-level gentamicin resistant (HLGR), and 32.8% of E. faecium isolates and 67.2% of E. faecalis isolates carried aac(6')-aph(2"). The prevalence of aph(3")-IIIa among the E. faecalis and E. faecium isolates was 22.7% and 77.3%, respectively. Remarkably increased incidence of aac(6')-aph(2") among HLGR isolates explains the relationship between this gene and the high level of resistance to aminoglycosides. As the resistant gene among enterococci can be transferred, the use of new-generation antibiotics is necessary.

Immunochemical Characterization of Temperature-Regulated Production of Enterocin L50 (EntL50A and EntL50B), Enterocin P, and Enterocin Q by Enterococcus faecium L50▿

PubMed Central

Criado, Raquel; Gutiérrez, Jorge; Martín, María; Herranz, Carmen; Hernández, Pablo E.; Cintas, Luis M.

2006-01-01

Polyclonal antibodies with specificity for enterocin L50A (EntL50A), enterocin L50B (EntL50B), and enterocin Q (EntQ) produced by Enterococcus faecium L50 have been generated by immunization of rabbits with chemically synthesized peptides derived from the C terminus of EntL50A (LR1) and EntL50B (LR2) and from the complete enterocin Q (EntQ) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of these antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect ELISA (CI-ELISA). The NCI-ELISA was valuable for detecting anti-EntL50A-, anti-EntL50B-, and anti-EntQ-specific antibodies in the sera of the LR1-KLH-, LR2-KLH-, and EntQ-KLH-immunized animals, respectively. Moreover, these antibodies and those specific for enterocin P (EntP) obtained in a previous work (J. Gutiérrez, R. Criado, R. Citti, M. Martín, C. Herranz, M. F. Fernández, L. M. Cintas, and P. E. Hernández, J. Agric. Food Chem. 52:2247-2255, 2004) were used in an NCI-ELISA to detect and quantify the production of EntL50A, EntL50B, EntP, and EntQ by the multiple-bacteriocin producer E. faecium L50 grown at different temperatures (16 to 47°C). Our results show that temperature has a strong influence on bacteriocin production by this strain. EntL50A and EntL50B are synthesized at 16 to 32°C, but production becomes negligible when the growth temperature is above 37°C, whereas EntP and EntQ are synthesized at temperatures ranging from 16 to 47°C. Maximum EntL50A and EntL50B production was detected at 25°C, while EntP and EntQ are maximally produced at 37 and 47°C, respectively. The loss of plasmid pCIZ1 (50 kb) and/or pCIZ2 (7.4 kb), encoding EntL50A and EntL50B as well as EntQ, respectively, resulted in a significant increase in production and stability of the chromosomally encoded EntP. PMID:17056686

vanA-positive multi-drug-resistant Enterococcus spp. isolated from surfaces of a US hospital laundry facility.

PubMed

Michael, K E; No, D; Roberts, M C

2017-02-01

Enterococcus spp. are a normal part of the gastrointestinal tract of humans and animals. They are also important pathogens, being responsible for 14% of US nosocomial infections from 2007 to 2010. To examine a laundry facility that processes clinical linens for the presence and seasonality of vancomycin-resistant Enterococcus spp. Surface samples were collected four times in 2015 from the dirty and clean areas of the laundry facility. Isolates were confirmed using biochemical assays, and antibiotic susceptibility testing was performed. Further investigations included molecular characterization by multi-locus sequence typing (MLST), detection of acquired vanA and vanB and/or intrinsic vanC1 genes by polymerase chain reaction, and eBURST analysis. Seventy-four vanA-positive multi-drug-resistant Enterococcus spp. were identified: 64/120 (53%) in the dirty area and 10/120 (8%) in the clean area. There were 14 ST types among the E. faecium isolates identified (ST16, 17, 18, 117, 186, 280, 324, 412, 584, 664, 665, 736, 750 and 1038). Both E. faecalis isolates were ST109. Isolation of vancomycin-resistant enterococci (VRE) isolates was significantly higher (53% vs 8%) in the dirty area of the facility compared with the clean area. This is the first study to examine an industrial laundry facility for the presence of VRE, and may be an unrecognized reservoir. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Using Phage Lytic Enzymes to Destroy Pathogenic and BW Bacteria

DTIC Science & Technology

2005-07-14

against antibiotic resistant Enterococcus faecalis and Enterococcus faecium . J Bacteriol. 186:4808-12. Cheng, Q., D. Nelson, S. Zhu, and V.A...Lysins from Enterococcus faecalis RU-654 3. Fischetti, Vincent A. Schuch, Raymond Lytic Enzymes and spore surface antigens for detection and

Dogs Leaving the ICU Carry a Very Large Multi-Drug Resistant Enterococcal Population with Capacity for Biofilm Formation and Horizontal Gene Transfer

PubMed Central

Ghosh, Anuradha; Dowd, Scot E.; Zurek, Ludek

2011-01-01

The enterococcal community from feces of seven dogs treated with antibiotics for 2–9 days in the veterinary intensive care unit (ICU) was characterized. Both, culture-based approach and culture-independent 16S rDNA amplicon 454 pyrosequencing, revealed an abnormally large enterococcal community: 1.4±0.8×108 CFU gram−1 of feces and 48.9±11.5% of the total 16,228 sequences, respectively. The diversity of the overall microbial community was very low which likely reflects a high selective antibiotic pressure. The enterococcal diversity based on 210 isolates was also low as represented by Enterococcus faecium (54.6%) and Enterococcus faecalis (45.4%). E. faecium was frequently resistant to enrofloxacin (97.3%), ampicillin (96.5%), tetracycline (84.1%), doxycycline (60.2%), erythromycin (53.1%), gentamicin (48.7%), streptomycin (42.5%), and nitrofurantoin (26.5%). In E. faecalis, resistance was common to tetracycline (59.6%), erythromycin (56.4%), doxycycline (53.2%), and enrofloxacin (31.9%). No resistance was detected to vancomycin, tigecycline, linezolid, and quinupristin/dalfopristin in either species. Many isolates carried virulence traits including gelatinase, aggregation substance, cytolysin, and enterococcal surface protein. All E. faecalis strains were biofilm formers in vitro and this phenotype correlated with the presence of gelE and/or esp. In vitro intra-species conjugation assays demonstrated that E. faecium were capable of transferring tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin resistance traits to human clinical strains. Multi-locus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of E. faecium strains showed very low genotypic diversity. Interestingly, three E. faecium clones were shared among four dogs suggesting their nosocomial origin. Furthermore, multi-locus sequence typing (MLST) of nine representative MLVA types revealed that six sequence types (STs) originating from five

[Spectrum and drug sensitivity of pathogenic bacteria in children with nephrotic syndrome complicated by urinary tract infection: an analysis of 97 cases].

PubMed

Song, Shao-Na; Zhang, Bi-Li; Wang, Wen-Hong; Zhang, Xuan

2012-09-01

To investigate the spectrum and drug sensitivity of pathogenic bacteria in children with nephrotic syndrome (NS) complicated by urinary tract infection (UTI). A retrospective analysis was performed on the spectrum and drug sensitivity of pathogenic bacteria in 97 children with NS complicated by UTI, who hospitalized from January to December, 2011. The incidence of UTI in children with NS was 36.5%. It was significantly more common in children with recurrent NS than in those with primary NS (44.0% vs 31.9%; P<0.05). These cases mainly presented with asymptomatic bacteriuria. Enterococcus was the most common pathogenic bacteria (50.5%), including Enterococcus faecium (29.4%) and Enterococcus faecalis (21.1%), followed by Gram-negative bacteria, such as Escherichia coli (15.6%) and Klebsiella pneumoniae (14.7%). Enterococcus was highly sensitive to nitrofurantoin, vacomycin and linezolid, but was highly resistant to tetracycline and moxifloxacin. More multi-resistant strains were detected in Enterococcus faecium than in Enterococcus faecalis (72% vs 17%; P<0.05). Escherichia coli and Klebsiella pneumoniae were highly sensitive to amikacin, imipenem and piperacillin/tazobactam. Of the Gram-negative bacteria, 25% produced extended spectrum β-lactamases (ESBLs). ESBLs-producing bacteria had 100% sensitivity to imipenem, amikacin and piperacillin/tazobactam but were highly resistant to ampicillin, cefazolin and ceftriaxone. Children with recurrent NS are more susceptible to UTI than those with primary NS. Enterococcus is becoming major pathogenic bacteria for UTI in children with NS and has relatively high drug resistance, and most strains of Enterococcus faecium are multi-resistant.

Increasing Prevalence of Aminoglycoside-Resistant Enterococcus faecalis Isolates Due to the aac(6’)-aph(2”) Gene: A Therapeutic Problem in Kermanshah, Iran

PubMed Central

Khani, Mitra; Fatollahzade, Mahdie; Pajavand, Hamid; Bakhtiari, Somaye; Abiri, Ramin

2016-01-01

Background: Enterococci are important pathogens in nosocomial infections. Various types of antibiotics, such as aminoglycosides, are used for treatment of these infections. Enterococci can acquire resistant traits, which can lead to therapeutic problems with aminoglycosides. Objectives: This study was designed to identify the prevalence of, and to compare, the aac(6’)-aph(2”) and aph(3)-IIIa genes and their antimicrobial resistance patterns among Enterococcus faecalis and E. faecium isolates from patients at Imam Reza hospital in Kermanshah in 2011 - 2012. Patients and Methods: One hundred thirty-eight clinical specimens collected from different wards of Imam Reza hospital were identified to the species level by biochemical tests. Antimicrobial susceptibility tests against kanamycin, teicoplanin, streptomycin, imipenem, ciprofloxacin, and ampicillin were performed by the disk diffusion method. The minimum inhibitory concentrations of gentamicin, streptomycin, kanamycin, and amikacin were evaluated with the microbroth dilution method. The aminoglycoside resistance genes aac(6’)-aph(2”) and aph(3”)-IIIa were analyzed with multiplex PCR. Results: The prevalence of isolates was 33 (24.1%) for E. faecium and 63 (46%) for E. faecalis. Eighty-nine percent of the isolates were high-level gentamicin resistant (HLGR), and 32.8% of E. faecium isolates and 67.2% of E. faecalis isolates carried aac(6’)-aph(2”). The prevalence of aph(3”)-IIIa among the E. faecalis and E. faecium isolates was 22.7% and 77.3%, respectively. Conclusions: Remarkably increased incidence of aac(6’)-aph(2”) among HLGR isolates explains the relationship between this gene and the high level of resistance to aminoglycosides. As the resistant gene among enterococci can be transferred, the use of new-generation antibiotics is necessary. PMID:27217920

Salmonella surrogate reduction using industrial peanut dry roasting parameters

USDA-ARS?s Scientific Manuscript database

Studies were conducted to evaluate the effectiveness of industrial peanut dry roasting parameters in Salmonella reduction using a Salmonella surrogate, Enterococcus faecium, which is slightly more heat tolerant than Salmonella. Runner-type peanuts were inoculated with E. faecium and roasted in a lab...

Vancomycin tolerance in enterococci.

PubMed

Saribas, Suat; Bagdatli, Yasar

2004-11-01

Tolerance can be defined as the ability of bacteria to grow in the presence of high concentrations of bactericide antimicrobics, so that the killing action of the drug is avoided but the minimal inhibitory concentration (MIC) remains the same. We investigated vancomycin tolerance in the Enterococcus faecium and Enterococcus faecalis strains isolated from different clinical specimens. Vancomycin was obtained from Sigma Chemical Co. We studied 100 enterococci strains. Fifty-six and 44 of Enterococcus strains were idendified as E. feacalis and E. faecium, respectively. To determine MICs and minimal bactericidal concentration (MBC), we inoculated strains from an overnight agar culture to Muller-Hinton broth and incubated them for 4-6 h at 37 degrees C with shaking to obtain a logarithmic phase culture. The inoculum was controlled by performing a colony count for each test. We determined MBC values and MBC/MIC ratios to study tolerance to vancomycin. Vancomycin tolerance was defined as a high MBC value and an MBC/MIC ratio > or =32. Fifty-six and 44 of the Enterococcus strains were identified as E. faecium and E. faecalis, respectively. Thirty-one E. faecium and 48 E. faecalis were found to be susceptible to vancomycin and these susceptible strains were included in this study. The MICs of susceptible strains ranged from < or =1 to 4 mg/l, the MBCs were > or =512 mg/l. Tolerance was detected in all E. faecalis and E. faecium strains. The standard E. faecalis 21913 strain also exhibited tolerance according to the high MBC value and the MBC/MIC ratio. We defined the tolerant strains as having no bactericidal effect and MBC/MIC > or =32. We found that a 100% tolerance was present in susceptible strains. One of the hypotheses for tolerance is that tolerant cells fail to mobilize or create the autolysins needed for enlargement and division. Our data suggests that tolerance may compromise glycopeptide therapy of serious enterococci infections. To add an aminoglycoside to the

Effect of ingestion of soy yogurt on intestinal parameters of rats fed on a beef-based animal diet

PubMed Central

Bedani, Raquel; Pauly-Silveira, Nadiége Dourado; Cano, Veridiana Soares Pereira; Valentini, Sandro Roberto; de Rossi, Graciela Font; Valdez, Elizeu Antonio

2011-01-01

The aim of this study was to investigate whether the ingestion of soy yogurt fermented with Enterococcus faecium CRL 183 would modify the intestinal count of enterococci, fecal pH and ammonia content in rats fed on a diet containing red meat. The rats were placed in 4 groups: for 60 days, group I was given a standard casein-based rodent feed and groups II-IV, the beef-based feed. From day 30, groups III-IV also received the following products: III) soy yogurt; IV) suspension of E. faecium CRL 183. At the start and on days 30 and 60, feces were collected for the determination of pH, ammonia content, count of enterococci and identification of their species. On day 60, rats were sacrificed and their colons also removed for count of enterococci and identification of their species. Rats that ingested soy yogurt showed no significant change (P<0.05) in fecal counts of Enterococcus spp., but, this rat group showed a higher count of E. faecium than rats that ingested suspension of E. faecium CRL 183. The ingestion of soy yogurt and E. faecium culture caused a significant rise (P < 0.05) in fecal pH and ammonia content. Our results suggest that consumption of soy yogurt fermented with E. faecium CRL 183 and L. helveticus subsp. jugurti could change the species of Enterococcus spp. present in the feces and colon of rats fed on a beef-based diet. However, the fermented soy product and the pure culture of E. faecium CRL 183 also induced undesirable effects such as the increase of fecal pH and ammonia content in the feces of rats fed on a beef-based diet. PMID:24031747

Efficacy of vacuum steam pasteurization for inactivation of Salmonella PT 30, Escherichia coli O157:H7 and Enterococcus faecium on low moisture foods.

PubMed

Shah, Manoj K; Asa, Gladys; Sherwood, Julie; Graber, Kari; Bergholz, Teresa M

2017-03-06

Low moisture foods such as nuts, spices, and seeds have been implicated in several outbreaks due to Salmonella or E. coli O157:H7 contamination. Such foods may be consumed raw, and can be used as ingredients in other food products. While numerous thermal inactivation studies have been conducted for Salmonella on nuts, studies on other seeds and grains are minimal. Product water activity can influence the thermal resistance of pathogens, where thermal resistance increases as water activity decreases, leading to a requirement for higher temperatures and longer exposure times to achieve significant reduction of pathogen numbers. Vacuum steam pasteurization uses steam under vacuum, which can be operated at temperatures above and below 100°C. The objective of this study was to determine the efficacy of vacuum steam pasteurization for inactivation of pathogens on whole flaxseed, quinoa, sunflower kernels, milled flaxseed and whole black peppercorns. The use of E. faecium as a potential surrogate for Salmonella and E. coli O157:H7 in vacuum steam pasteurization was also evaluated. Pasteurization for 1min at 75°C yielded average log reductions of 5.48±1.22, 5.71±0.40 and 5.23±0.61 on flaxseed, 4.29±0.92, 5.89±0.26 and 2.39±0.83 on quinoa, and 4.01±0.74, 5.40±0.83 and 2.99±0.92 on sunflower kernels for Salmonella PT 30, E. coli O157:H7 and E. faecium, respectively. Similarly, on milled flaxseed and black peppercorns average log reductions of 3.02±0.79 and 6.10±0.64CFU/g were observed for Salmonella PT 30 after 1min of treatment at 75°C but, on average, >6.0 log reductions were observed after pasteurization at 85°C. Our data demonstrate that vacuum steam pasteurization can be effectively used to reduce pathogens on these low moisture foods at temperature as low as 75 and 85°C, and that E. faecium may be used as a potential surrogate for Salmonella PT 30 and E. coli O157:H7. Copyright © 2017 Elsevier B.V. All rights reserved.

Growth Behavior of E. coli, Enterococcus and Staphylococcus Species in the Presence and Absence of Sub-inhibitory Antibiotic Concentrations: Consequences for Interpretation of Culture-Based Data.

PubMed

Heß, Stefanie; Gallert, Claudia

2016-11-01

Culture-based approaches are used to monitor, e.g., drinking water or bathing water quality and to investigate species diversity and antibiotic resistance levels in environmental samples. For health risk assessment, it is important to know whether the growing cultures display the actual abundance of, e.g., clinically relevant antibiotic resistance phenotypes such as vancomycin-resistant Enterococcus faecium/Enterococcus faecalis (VRE) or methicillin-resistant Staphylococcus aureus. In addition, it is important to know whether sub-inhibitory antibiotic concentrations, which are present in surface waters, favor the growth of antibiotic-resistant strains. Therefore, clinically relevant bacteria were isolated from different water sources and the growth behavior of 58 Escherichia coli, 71 Enterococcus, and 120 Staphylococcus isolates, belonging to different species and revealing different antibiotic resistance patterns, was studied with respect to "environmental" antibiotic concentrations. The finding that VRE could only be detected after specific enrichment can be explained by their slow growth compared to non-resistant strains. Interpreting their absence in standardized culture-based methods as nonexistent might be a fallacy. Sub-inhibitory antibiotic concentrations that were detected in sewage and receiving river water did not specifically promote antibiotic-resistant strains. Generally, those antibiotics that influenced cell metabolism directly led to slightly reduced growth rates and less than maximal optical densities after 48 h of incubation.

Detection of human and animal sources of pollution by microbial and chemical methods

USDA-ARS?s Scientific Manuscript database

A multi-indicator approach comprising Enterococcus, bacterial source tracking (BST), and sterol analysis was tested for pollution source identification. Fecal contamination was detected in 100% of surface water sites tested. Enterococcus faecium was the dominant species in aged litter samples from p...

Mutations associated with reduced surotomycin susceptibility in Clostridium difficile and Enterococcus species.

PubMed

Adams, Hannah M; Li, Xiang; Mascio, Carmela; Chesnel, Laurent; Palmer, Kelli L

2015-07-01

Clostridium difficile infection (CDI) is an urgent public health concern causing considerable clinical and economic burdens. CDI can be treated with antibiotics, but recurrence of the disease following successful treatment of the initial episode often occurs. Surotomycin is a rapidly bactericidal cyclic lipopeptide antibiotic that is in clinical trials for CDI treatment and that has demonstrated superiority over vancomycin in preventing CDI relapse. Surotomycin is a structural analogue of the membrane-active antibiotic daptomycin. Previously, we utilized in vitro serial passage experiments to derive C. difficile strains with reduced surotomycin susceptibilities. The parent strains used included ATCC 700057 and clinical isolates from the restriction endonuclease analysis (REA) groups BI and K. Serial passage experiments were also performed with vancomycin-resistant and vancomycin-susceptible Enterococcus faecium and Enterococcus faecalis. The goal of this study is to identify mutations associated with reduced surotomycin susceptibility in C. difficile and enterococci. Illumina sequence data generated for the parent strains and serial passage isolates were compared. We identified nonsynonymous mutations in genes coding for cardiolipin synthase in C. difficile ATCC 700057, enoyl-(acyl carrier protein) reductase II (FabK) and cell division protein FtsH2 in C. difficile REA type BI, and a PadR family transcriptional regulator in C. difficile REA type K. Among the 4 enterococcal strain pairs, 20 mutations were identified, and those mutations overlap those associated with daptomycin resistance. These data give insight into the mechanism of action of surotomycin against C. difficile, possible mechanisms for resistance emergence during clinical use, and the potential impacts of surotomycin therapy on intestinal enterococci. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Evidence for Horizontal Gene Transfer in Evolution of Elongation Factor Tu in Enterococci

PubMed Central

Ke, Danbing; Boissinot, Maurice; Huletsky, Ann; Picard, François J.; Frenette, Johanne; Ouellette, Marc; Roy, Paul H.; Bergeron, Michel G.

2000-01-01

The elongation factor Tu, encoded by tuf genes, is a GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only one tuf gene. We have designed degenerate PCR primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two different tuf genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, Enterococcus casseliflavus, Enterococcus dispar, Enterococcus durans, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, and Enterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum, Enterococcus columbae, Enterococcus faecalis, Enterococcus sulfureus, Enterococcus saccharolyticus, and Enterococcus solitarius), only the tufA gene was present. Based on 16S rRNA gene sequence analysis, the 11 species having two tuf genes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis of tuf sequences demonstrated that the enterococcal tufA gene branches with the Bacillus, Listeria, and Staphylococcus genera, while the enterococcal tufB gene clusters with the genera Streptococcus and Lactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcal tufB genes and the tuf genes of streptococci and Lactococcus lactis. The data suggest that an ancestral streptococcus

Antibiotic resistance, virulence determinants and production of biogenic amines among enterococci from ovine, feline, canine, porcine and human milk

PubMed Central

2013-01-01

Background Recent studies have shown that mammalian milk represents a continuous supply of commensal bacteria, including enterococci. The objectives of this study were to evaluate the presence of enterococci in milk of different species and to screen them for several genetic and phenotypic traits of clinical significance among enterococci. Results Samples were obtained from, at least, nine porcine, canine, ovine, feline and human healthy hosts. Enterococci could be isolated, at a concentration of 1.00 × 102 -1.16 × 103 CFU/ml, from all the porcine samples and, also from 85, 50, 25 and 25% of the human, canine, feline and ovine ones, respectively. They were identified as Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus casseliflavus and Enterococcus durans. Among the 120 initial enterococcal isolates, 36 were selected on the basis of their different PFGE profiles and further characterized. MLST analysis revealed a wide diversity of STs among the E. faecalis and E. faecium strains, including some frequently associated to hospital infections and novel STs. All the E. faecalis strains possessed some of the potential virulence determinants (cad, ccf, cob, cpd, efaAfs, agg2, gelE, cylA, espfs) assayed while the E. faecium ones only harboured the efaAfm gene. All the tested strains were susceptible to tigecycline, linezolid and vancomycin, and produced tyramine. Their susceptibility to the rest of the antimicrobials and their ability to produce other biogenic amines varied depending on the strain. Enterococci strains isolated from porcine samples showed the widest spectrum of antibiotic resistance. Conclusions Enterococci isolated from milk of different mammals showed a great genetic diversity. The wide distribution of virulence genes and/or antibiotic resistance among the E. faecalis and E. faecium isolates indicates that they can constitute a reservoir of such traits and a risk to animal and human health. PMID:24325647

Comparative analysis on antibiotic resistance characteristics of Listeria spp. and Enterococcus spp. isolated from laying hens and eggs in conventional and organic keeping systems in Bavaria, Germany.

PubMed

Schwaiger, K; Schmied, E-M V; Bauer, J

2010-05-01

By investigating the prevalence and antimicrobial resistance characteristics of Gram-positive bacteria from organic and conventional keeping systems of laying hens, it was to be determined to what extent these properties are influenced by the different systems. For this purpose, a total of 799 cloacal swabs and 800 egg samples were examined. Prevalences for all selected bacteria from cloacal swabs were much the same for both organic and caged birds: Listeria spp.1.3%[org] versus 1.6%[con]; Enterococcus spp. 95.5%[org] versus 97.5%[con]. Egg contents and eggshells were generally contaminated to a lesser extent, primarily with Enterococcus spp. Listeria isolates were susceptible to almost all tested antibiotics, only three Listeria innocua from conventional keepings were resistant to clindamycin; one isolate additionally to imipenem. High percentages of Enterococcus faecalis were resistant to doxycycline and macrolides. Enterococcus faecium proved to have high resistance rates to clindamycin, fosfomycin and erythromycin; 9.1% were even resistant to the reserve antibiotic synercid. Further, Enterococcus spp. showed higher resistance rates to doxycycline, erythromycin, fosfomycin and rifampicin. No glycopeptide resistant enterococci were detected. A correlation between keeping system and resistance/susceptibility rates could be demonstrated. In detail, E. faecalis from organic laying hen husbandries showed significant lower resistance prevalences to tylosin, streptomycin and doxycycline; susceptibility rates were higher for enrofloxacin and ciprofloxacin. Rifampicin and imipenem were more effective in isolates from conventional keepings (P < 0.05). The amounts of resistant isolates of the Enterococcus raffinosus from organic farms were significantly lower, the amounts of sensitive isolates were significantly higher than from conventional farms concerning eight antibiotics (P < 0.05). When comparing the susceptibility/resistance rates, as well as the mean minimum

Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis.

PubMed

Borrero, Juan; Jiménez, Juan J; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2011-01-01

Replacement of the signal peptide (SP) of the bacteriocins enterocin P (EntP) and hiracin JM79 (HirJM79), produced by Enterococcus faecium P13 and Enterococcus hirae DCH5, respectively, by the signal peptide of Usp45 (SP(usp45)), the major Sec-dependent protein secreted by Lactococcus lactis, permits the production, secretion, and functional expression of EntP and HirJM79 by L. lactis. Chimeric genes encoding the SP(usp45) fused to either mature EntP (entP), with or without the immunity gene (entiP) or to mature HirJM79 (hirJM79), with or without the immunity gene (hiriJM79), were cloned into the expression vector pMG36c, carrying the P(32) constitutive promoter, and into pNZ8048 under control of the inducible PnisA promoter. The production of EntP and HirJM79 by most of the L. lactis recombinant strains was 1.5- to 3.7-fold higher and up to 3.6-fold higher than by the E. faecium P13 and E. hirae DCH5 control strains, respectively. However, the specific antimicrobial activity of the recombinant EntP was 1.1- to 6.2-fold higher than that produced by E. faecium P13, while that of the HirJM79 was a 40% to an 89% of that produced by E. hirae DCH5. Chimeras of SP(usp45) fused to mature EntP or HirJM79 drive the production and secretion of these bacteriocins in L. lactis in the absence of specific immunity and secretion proteins. The supernatants of the recombinant L. lactis NZ9000 strains, producers of EntP, showed a much higher antimicrobial activity against Listeria spp. than that of the recombinant L. lactis NZ9000 derivatives, producers of HirJM79.

Lactobacillus rhamnosus GG (ATCC 53103) and platelet aggregation in vitro.

PubMed

Korpela, R; Moilanen, E; Saxelin, M; Vapaatalo, H

1997-06-17

Lactobacillus rhamnosus GG is an experimentally and clinically well documented probiotic used in different dairy products. The present study aimed to investigate the safety aspects of Lactobacillus rhamnosus GG, particularly with respect to platelet aggregation, the initiating event in thrombosis. Platelet rich plasma was separated from the blood of healthy volunteers, and the effects of Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus rhamnosus (ATCC 7469) and Enterococcus faecium T2L6 in different dilutions on spontaneous, ADP- and adrenaline-induced aggregation were tested. The bacteria did not influence spontaneous aggregation. Only Enterococcus faecium T2L6 enhanced the adrenaline-induced aggregation, with a less clear effect on ADP-induced aggregation.

Novel Compounds From Shark and Stingray Epidermal Mucus With Antimicrobial Activity Against Wound Infection Pathogens

DTIC Science & Technology

2014-03-01

Micrococcus sp., Enterococcus faecalis, Enterococcus faecium, Vancomycin resistant Enterococcus, and Escherichia coli). During the second Quarter of...mm) 803 D10 Rhinoptera bonasus MRSA (9.5) MSSA (4.5) Bacillus cereus (6) Listeria monocytogenes (6) MRSA (2) Micrococcus sp (7) VRE (2) 803...E6 Rhinoptera bonasus MRSA (7.5) MSSA (8.5) VRE (4.5) Bacillus subtilis (10) MRSA (10) MRSA (10) Micrococcus (16) Listeria monocytogenes (13

Genes Important for Catalase Activity in Enterococcus faecalis

PubMed Central

Baureder, Michael; Hederstedt, Lars

2012-01-01

Little in general is known about how heme proteins are assembled from their constituents in cells. The Gram-positive bacterium Enterococcus faecalis cannot synthesize heme and does not depend on it for growth. However, when supplied with heme in the growth medium the cells can synthesize two heme proteins; catalase (KatA) and cytochrome bd (CydAB). To identify novel factors important for catalase biogenesis libraries of E. faecalis gene insertion mutants were generated using two different types of transposons. The libraries of mutants were screened for clones deficient in catalase activity using a colony zymogram staining procedure. Analysis of obtained clones identified, in addition to katA (encoding the catalase enzyme protein), nine genes distributed over five different chromosomal loci. No factors with a dedicated essential role in catalase biogenesis or heme trafficking were revealed, but the results indicate the RNA degradosome (srmB, rnjA), an ABC-type oligopeptide transporter (oppBC), a two-component signal transducer (etaR), and NADH peroxidase (npr) as being important for expression of catalase activity in E. faecalis. It is demonstrated that catalase biogenesis in E. faecalis is independent of the CydABCD proteins and that a conserved proline residue in the N-terminal region of KatA is important for catalase assembly. PMID:22590595

Surveillance of bacterial contamination in small animal veterinary hospitals with special focus on antimicrobial resistance and virulence traits of enterococci.

PubMed

KuKanich, Kate S; Ghosh, Anuradha; Skarbek, Jennifer V; Lothamer, Kale M; Zurek, Ludek

2012-02-15

To determine the prevalence of bacterial contamination on 4 surfaces of 4 types of standard equipment in small animal veterinary hospitals. Surveillance study. 10 small animal veterinary hospitals. Each hospital was visited 3 times at 4-month intervals; at each visit, a cage door, stethoscope, rectal thermometer, and mouth gag were swabbed. Swab samples were each plated onto media for culture of enterococci and organisms in the family Enterobacteriaceae. Enterococci were identified via a species-specific PCR assay and sodA gene sequencing; species of Enterobacteriaceae were identified with a biochemical test kit. Antimicrobial susceptibility was assessed via the disk diffusion method. Enterococci were screened for virulence traits and genotyped to assess clonality. Among the 10 hospitals, enterococci were isolated from cage doors in 7, from stethoscopes in 7, from thermometers in 6, and from mouth gags in 1; contamination with species of Enterobacteriaceae was rare. Enterococci were mainly represented by Enterococcus faecium (35.4%), Enterococcus faecalis (33.2%), and Enterococcus hirae (28.3%). Antimicrobial resistance was common in E. faecium, whereas virulence traits were present in 99% of E. faecalis isolates but not in E. faecium isolates. Clonal multidrug-resistant E. faecium was isolated from several surfaces at 1 hospital over multiple visits, whereas sporadic nonclonal contamination was detected in other hospitals. Contamination of surfaces in small animal veterinary hospitals with multidrug-resistant enterococci is a potential concern for pets and humans contacting these surfaces. Implementing precautions to minimize enterococcal contamination on these surfaces is recommended.

Major globally disseminated clonal complexes of antimicrobial resistant enterococci associated with infections in cancer patients in Brazil.

PubMed

Santos, Barbara A; Oliveira, Jéssica S; Cardoso, Nayara T; Barbosa, André V; Superti, Silvana V; Teixeira, Lúcia M; Neves, Felipe P G

2017-11-01

Cancer and hematological malignancies constitute major comorbidities in enterococcal infections, but little is known about the characteristics of enterococci affecting cancer patients. The aim of this study was to characterize 132 enterococcal clinical isolates obtained from cancer patients attending a Cancer Reference Center in Brazil between April 2013 and March 2014. Susceptibility to 17 antimicrobial agents was assessed by disk diffusion method. Resistance and virulence genes were investigated by PCR. Multilocus sequence typing (MLST) was performed for selected Enterococcus faecalis and Enterococcus faecium isolates. The predominant species was E. faecalis (108 isolates), followed by E. faecium (18), Enterococcus gallinarum (3), Enterococcus avium (2) and Enterococcus durans (1). Multidrug-resistant (MDR) isolates made up 44.7%, but all isolates were susceptible to fosfomycin, linezolid and glycopeptides. The most prevalent genes associated with erythromycin- and tetracycline-non susceptible isolates were erm(B) (47/71; 66.2%) and tet(M) (24/68; 35.3%), respectively. High-level resistance (HLR) to gentamicin was found in 22 (16.7%) isolates and 13 (59.1%) of them carried the aac(6')-Ie-aph(2″)-Ia gene. HLR to streptomycin was detected in 34 (25.8%) isolates, of which 15 (44.1%) isolates had the ant(6')-Ia gene. The most common virulence genes were gelE (48.9%), esp (30.5%) and asa1 (29.8%). MLST performed for 26 E. faecalis isolates revealed 18 different sequence-types (STs), with seven corresponding to novel STs (625, 626, 627, 628, 629, 630, and 635). On the other hand, nine of 10 E. faecium isolates analyzed by MLST belonged to a single clonal complex, comprised of mostly ST412, which emerged worldwide after mid-2000s, but also two novel STs (963 and 964). We detected major globally disseminated E. faecalis and E. faecium clonal complexes along with novel closely related STs, indicating the fitness and continuous evolution of these hospital

Vancomycin-resistant enterococci with vanA gene in treated municipal wastewater and their association with human hospital strains.

PubMed

Oravcova, Veronika; Mihalcin, Matus; Zakova, Jana; Pospisilova, Lucie; Masarikova, Martina; Literak, Ivan

2017-12-31

Vancomycin-resistant enterococci (VRE) are pathogens of increasing medical importance. In Brno, Czech Republic, we collected 37 samples from the effluent of a wastewater treatment plant (WWTP), 21 surface swabs from hospital settings, and 59 fecal samples from hospitalized patients and staff. Moreover, we collected 284 gull cloacal swabs from the colony situated 35km downstream the WWTP. Samples were cultured selectively. Enterococci were identified using MALDI-TOF MS, phenotypically tested for susceptibility to antibiotics, and by PCR for occurrence of resistance and virulence genes. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were used to examine genotypic diversity. VRE carrying the vanA gene were found in 32 (86%, n=37) wastewater samples, from which we obtained 49 isolates: Enterococcus faecium (44) and Enterococcus gallinarum (2), Enterococcus casseliflavus (2), and Enterococcus raffinosus (1). From 33 (69%) of 48 inpatient stool samples, we obtained 39 vanA-carrying VRE, which belonged to E. faecium (33 isolates), Enterococcus faecalis (4), and Enterococcus raffinosus (2). Nearly one-third of the samples from hospital surfaces contained VRE with the vanA gene. VRE were not detected among gulls. Sixty-seven (84%, n=80) E. faecium isolates carried virulence genes hyl and/or esp. Virulence of E. faecalis was encoded by gelE, asa1, and cylA genes. A majority of the E. faecium isolates belonged to the clinically important sequence types ST17 (WWTP: 10 isolates; hospital: 4 isolates), ST18 (9;8), and ST78 (5;0). The remaining isolates belonged to ST555 (2;0), ST262 (1;6), ST273 (3;0), ST275 (1;0), ST549 (2;0), ST19 (0;1), ST323 (3;0), and ST884 (7;17). Clinically important enterococci carrying the vanA gene were almost continually detectable in the effluent of the WWTP, indicating insufficient removal of VRE during wastewater treatment and permanent shedding of these antibiotic resistant pathogens into the environment from this

Angiotensin-converting enzyme inhibitory activity in Mexican Fresco cheese.

PubMed

Torres-Llanez, M J; González-Córdova, A F; Hernandez-Mendoza, A; Garcia, H S; Vallejo-Cordoba, B

2011-08-01

The objective of this study was to evaluate if Mexican Fresco cheese manufactured with specific lactic acid bacteria (LAB) presented angiotensin I-converting enzyme inhibitory (ACEI) activity. Water-soluble extracts (3 kDa) obtained from Mexican Fresco cheese prepared with specific LAB (Lactococcus, Lactobacillus, Enterococcus, and mixtures: Lactococcus-Lactobacillus and Lactococcus-Enterococcus) were evaluated for ACEI activity. Specific peptide fractions with high ACEI were analyzed using reverse phase-HPLC coupled to mass spectrometry for determination of amino acid sequence. Cheese containing Enterococcus faecium or a Lactococcus lactis ssp. lactis-Enterococcus faecium mixture showed the largest number of fractions with ACEI activity and the lowest half-maximal inhibitory concentration (IC(50); <10 μg/mL). Various ACEI peptides derived from β-casein [(f(193-205), f(193-207), and f(193-209)] and α(S1)-casein [f(1-15), f(1-22), f(14-23), and f(24-34)] were found. The Mexican Fresco cheese manufactured with specific LAB strains produced peptides with potential antihypertensive activity. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Model systems for the study of Enterococcal colonization and infection

PubMed Central

Goh, H. M. Sharon; Yong, M. H. Adeline; Chong, Kelvin Kian Long

2017-01-01

ABSTRACT Enterococcus faecalis and Enterococcus faecium are common inhabitants of the human gastrointestinal tract, as well as frequent opportunistic pathogens. Enterococci cause a range of infections including, most frequently, infections of the urinary tract, catheterized urinary tract, bloodstream, wounds and surgical sites, and heart valves in endocarditis. Enterococcal infections are often biofilm-associated, polymicrobial in nature, and resistant to antibiotics of last resort. Understanding Enterococcal mechanisms of colonization and pathogenesis are important for identifying new ways to manage and intervene with these infections. We review vertebrate and invertebrate model systems applied to study the most common E. faecalis and E. faecium infections, with emphasis on recent findings examining Enterococcal-host interactions using these models. We discuss strengths and shortcomings of each model, propose future animal models not yet applied to study mono- and polymicrobial infections involving E. faecalis and E. faecium, and comment on the significance of anti-virulence strategies derived from a fundamental understanding of host-pathogen interactions in model systems. PMID:28102784

Biodiversity of lactic acid bacteria in Moroccan soft white cheese (Jben).

PubMed

Ouadghiri, Mouna; Amar, Mohamed; Vancanneyt, Marc; Swings, Jean

2005-10-15

The bacterial diversity occurring in traditional Moroccan soft white cheese, produced in eight different regions in Morocco, was studied. A total of 164 lactic acid bacteria were isolated, purified and identified by whole-cell protein fingerprinting and rep-PCR genomic fingerprinting. The majority of the strains belonged to the genera Lactobacillus, Lactococcus, Leuconostoc and Enterococcus. Sixteen species were identified: Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus buchneri, Lactococcus lactis, Lactococcus garvieae, Lactococcus raffinolactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Leuconostoc citreum, Eterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus saccharominimus and Streptococcus sp.

Prevalence, antimicrobial resistance and genetic lineages of Enterococcus spp. from vegetable food, soil and irrigation water in farm environments in Tunisia.

PubMed

Ben Said, Leila; Klibi, Naouel; Dziri, Raoudha; Borgo, Francesca; Boudabous, Abdellatif; Ben Slama, Karim; Torres, Carmen

2016-03-30

The objective of this study was to determine the species, clonal diversity, antibiotic resistance and virulence of enterococci in different environments. Seventy-one samples of farm origin (34 of food vegetables, 27 of soil and ten of irrigation water) and 19 samples of vegetables from five markets, were inoculated in Slanetz-Bartley agar plates supplemented or not with gentamicin (SB-Gen and SB plates, respectively) for enterococci recovery. Enterococci were obtained from 72.2% of tested samples in SB media (food vegetables from farms, 88.2%; soil and irrigation water, 51%; food vegetables from markets, 84.2%), and 65 enterococcal isolates were obtained. Enterococcus faecium was the most prevalent species (52.3%), followed by E. hirae (35.4%), E. faecalis (6.15%), and E. casseliflavus (6.15%). Antibiotic resistance detected among these enterococci was as follows (percentage/detected gene): ciprofloxacin (60%), erythromycin (18.4%/erm(B)), tetracycline (15.4%/tet(M)-tet(L)), kanamycin (15.4%/aph(3')-III), chloramphenicol (7.7%), streptomycin (3%/ant(6)), vancomycin (6.15%/vanC2)), teicoplanin (0%) and ampicillin (0%). High-level gentamicin-resistant (HLR-G) enterococci were detected in SB-Gen plates in 14 of 90 tested samples (15.5%), and 15 isolates were characterized: ten E. faecalis, four E. faecium and one E. hirae. All HLR-G enterococci carried the aac(6')-aph(2″), erm(B) and tet(M) genes, among other resistance genes. The HLR-G isolates showed high genetic diversity (ten different PFGE profiles), and were ascribed to the sequence types ST2, ST16, ST28 and new ST528 (in E. faecalis), and ST56, new ST885 and new ST886 (in E. faecium). Food vegetables in the farm or market settings are frequently contaminated by HLR-G enterococci, and these microorganisms could reach the human intestine through the food chain, if hygienic conditions are not followed. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Both live and dead Enterococci activate Caenorhabditis elegans host defense via immune and stress pathways.

PubMed

Yuen, Grace J; Ausubel, Frederick M

2018-12-31

The innate immune response of the nematode Caenorhabditis elegans has been extensively studied and a variety of Toll-independent immune response pathways have been identified. Surprisingly little, however, is known about how pathogens activate the C. elegans immune response. Enterococcus faecalis and Enterococcus faecium are closely related enterococcal species that exhibit significantly different levels of virulence in C. elegans infection models. Previous work has shown that activation of the C. elegans immune response by Pseudomonas aeruginosa involves P. aeruginosa-mediated host damage. Through ultrastructural imaging, we report that infection with either E. faecalis or E. faecium causes the worm intestine to become distended with proliferating bacteria in the absence of extensive morphological changes and apparent physical damage. Genetic analysis, whole-genome transcriptional profiling, and multiplexed gene expression analysis demonstrate that both enterococcal species, whether live or dead, induce a rapid and similar transcriptional defense response dependent upon previously described immune signaling pathways. The host response to E. faecium shows a stricter dependence upon stress response signaling pathways than the response to E. faecalis. Unexpectedly, we find that E. faecium is a C. elegans pathogen and that an active wild-type host defense response is required to keep an E. faecium infection at bay. These results provide new insights into the mechanisms underlying the C. elegans immune response to pathogen infection.

Both live and dead Enterococci activate Caenorhabditis elegans host defense via immune and stress pathways

PubMed Central

2018-01-01

ABSTRACT The innate immune response of the nematode Caenorhabditis elegans has been extensively studied and a variety of Toll-independent immune response pathways have been identified. Surprisingly little, however, is known about how pathogens activate the C. elegans immune response. Enterococcus faecalis and Enterococcus faecium are closely related enterococcal species that exhibit significantly different levels of virulence in C. elegans infection models. Previous work has shown that activation of the C. elegans immune response by Pseudomonas aeruginosa involves P. aeruginosa-mediated host damage. Through ultrastructural imaging, we report that infection with either E. faecalis or E. faecium causes the worm intestine to become distended with proliferating bacteria in the absence of extensive morphological changes and apparent physical damage. Genetic analysis, whole-genome transcriptional profiling, and multiplexed gene expression analysis demonstrate that both enterococcal species, whether live or dead, induce a rapid and similar transcriptional defense response dependent upon previously described immune signaling pathways. The host response to E. faecium shows a stricter dependence upon stress response signaling pathways than the response to E. faecalis. Unexpectedly, we find that E. faecium is a C. elegans pathogen and that an active wild-type host defense response is required to keep an E. faecium infection at bay. These results provide new insights into the mechanisms underlying the C. elegans immune response to pathogen infection. PMID:29436902

Characterization and risk factors of vancomycin-resistant Enterococci (VRE) among animal-affiliated workers in Malaysia.

PubMed

Getachew, Y; Hassan, L; Zakaria, Z; Zaid, C Z M; Yardi, A; Shukor, R A; Marawin, L T; Embong, F; Aziz, S A

2012-11-01

This study determined the risk factors and characteristics of vancomycin-resistant Enterococci (VRE) among individuals working with animals in Malaysia. Targeted cross-sectional studies accompanied with laboratory analysis for the identification and characterization of resistance and virulence genes and with genotype of VRE were performed. VRE were detected in 9·4% (95% CI: 6·46-13·12) of the sampled populations. Enterococcus faecium, Enterococcus faecalis and Enterococcus gallinarum were isolated, and vanA was detected in 70% of the isolates. Enterococcus faecalis with vanB was obtained from one foreign poultry worker. At least one virulence gene was detected in >50% of Ent. faecium and Ent. faecalis isolates. The esp and gelE genes were common among Ent. faecium (58·3%) and Ent. faecalis (78%), respectively. The VRE species showed diverse RAPD profiles with some clustering of strains based on the individual's background. However, the risk factors found to be significantly associated with the prevalence of VRE were age (OR: 5·39, 95% CI: 1·98-14·61) and previous hospitalization (OR: 4·06, 95% CI: 1·33-12·35). VRE species isolated from individuals in this study have high level of vancomycin resistance, were genetically diverse and possessed the virulence traits. Age of individuals and history of hospitalization rather than occupational background determined VRE colonization. This study provides comprehensive findings on the epidemiological and molecular features of VRE among healthy individuals working with animals. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Molecular epidemiology of vancomycin resistant enterococci in a tertiary care hospital in Saudi Arabia.

PubMed

Somily, Ali M; Al-Mohizea, Maha M; Absar, Muhammed M; Fatani, Amal J; Ridha, Afaaf M; Al-Ahdal, Mohammed N; Senok, Abiola C; Al-Qahtani, Ahmed A

2016-08-01

Vancomycin-resistant enterococci (VRE) are a major cause of nosocomial infections with high mortality and morbidity. There is limited data on the molecular characterization of VRE in Saudi Arabia. This study was carried out to investigate the premise that a shift in VRE epidemiology is occurring in our setting. Enterococcus species identification and susceptibility testing plus VRE phenotypic confirmation by vancomycin and teicoplanin E-test were carried out. Vancomycin resistance genes were detected by PCR. Strain typing was conducted using PFGE. Among the strains of Enterococcus spp. investigated in this study, 17 (4.5%) were VRE. With the exception of one isolate from rectal swab, all others were clinical specimens with blood being the commonest source (n = 11; 64.7%), followed by urine (n = 3; 17.6%). The 17 VRE isolates were Enterococcus faecium (n/N = 13/17) and Enterococcus gallinarum (n/N = 4/17). Among E. faecium isolates, vanA(+)/vanB(+) (n/N = 8/13; 62%) exhibiting VanB phenotype were predominant. One of the five vanA(+)E. faecium isolates exhibited a VanB phenotype indicative of vanA genotype-VanB phenotype incongruence. E. gallinarum isolates exhibited a Van C phenotype although two were vanA(+)/vanC1(+). PFGE revealed a polyclonal distribution with eight pulsotypes. These findings indicate an evolving VRE epidemiology with vanA(+)/vanB(+) isolates and vanA genotype-VanB phenotype incongruence isolates, which were previously described as colonizers, are now causing clinical infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comprehensive molecular, genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40.

PubMed

Zischka, Melanie; Künne, Carsten T; Blom, Jochen; Wobser, Dominique; Sakιnç, Türkân; Schmidt-Hohagen, Kerstin; Dabrowski, P Wojtek; Nitsche, Andreas; Hübner, Johannes; Hain, Torsten; Chakraborty, Trinad; Linke, Burkhard; Goesmann, Alexander; Voget, Sonja; Daniel, Rolf; Schomburg, Dietmar; Hauck, Rüdiger; Hafez, Hafez M; Tielen, Petra; Jahn, Dieter; Solheim, Margrete; Sadowy, Ewa; Larsen, Jesper; Jensen, Lars B; Ruiz-Garbajosa, Patricia; Quiñones Pérez, Dianelys; Mikalsen, Theresa; Bender, Jennifer; Steglich, Matthias; Nübel, Ulrich; Witte, Wolfgang; Werner, Guido

2015-03-12

Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type. We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32

High Prevalence of Ceftazidime-Resistant Klebsiella pneumoniae and Increase of Imipenem-Resistant Pseudomonas aeruginosa and Acinetobacter spp. in Korea: a KONSAR Program in 2004

PubMed Central

Lee, Kyungwon; Lim, Chang Hyun; Cho, Ji Hyun; Lee, Wee Gyo; Uh, Young; Kim, Hwi Jun; Yong, Dongeun

2006-01-01

A nationwide antimicrobial resistance surveillance has been conducted since 1997 in Korea. In this study, susceptibility test data generated in 2004 by KONSAR group hospitals were analyzed and compared to those at a commercial laboratory. In hospitals, the rank orders of organisms in 2004 were identical to those in 2003. The most prevalent species was Staphylococcus aureus (20.2%) in hospitals, but Escherichia coli (29.7%) in the commercial laboratory. The proportions of Enterococcus faecium to all isolates of Enterococcus faecalis plus E. faecium were 47.2% in hospitals and 24.9% in the commercial laboratory. The mean resistance rates of significant antimicrobial-organism combinations in hospitals were: oxacillin-resistant S. aureus (68%), oxacillin-resistant (penicillin-nonsusceptible) Streptococcus pneumoniae (68%), vancomycin-resistant E. faecium (25%), cefotaxime-resistant E. coli (14%), ceftazidime- and cefoxitin-resistant Klebsiella pneumoniae (34% and 32%, respectively), and imipenem-resistant Acinetobacter spp. and Pseudomonas aeruginosa (17% and 24%, respectively). In conclusion, oxacillin-resistant staphylococci, expanded-spectrum cephalosporin-resistant K. pneumoniae, and imipenem-resistant Acinetobacter spp. and P. aeruginosa were prevalent in 2004. Increasing trends were observed for vancomycin-resistant E. faecium, cefoxitin-resistant E. coli and K. pneumoniae, and imipenem-resistant Acinetobacter spp. and P. aeruginosa. Certain antimicrobial-organism combinations were also prevalent among the commercial laboratory-tested strains. PMID:17066507

Nucleotide sequence of the beta-lactamase gene from Enterococcus faecalis HH22 and its similarity to staphylococcal beta-lactamase genes.

PubMed Central

Zscheck, K K; Murray, B E

1991-01-01

The nucleotide sequence of the constitutively produced beta-lactamase (Bla) gene from Enterococcus faecalis HH22 was shown to be identical to the published sequences of three of four staphylococcal type A beta-lactamase genes; more differences were seen with the genes for staphylococcal type C and D enzymes. One hundred forty nucleotides upstream of the beta-lactamase start codon were determined for an inducible staphylococcal beta-lactamase and were identical to those of the constitutively expressed enterococcal gene, indicating that the changes resulting in constitutive expression are not due to changes in the promoter or operator region. Moreover, complementation studies indicated that production of the enterococcal enzyme could be repressed. The genes for the enterococcal Bla and an inducible staphylococcal Bla were each cloned into a shuttle vector and transformed into enterococcal and staphylococcal recipients. The major difference between the backgrounds of the two hosts was that more enzyme was produced by the staphylococcal host, regardless of the source of the gene. The location of the enzyme was found to be host dependent, since each cloned gene generated extracellular (free) enzyme in the staphylococcus and cell-bound enzyme in the enterococcus. On the basis of the identities of the enterococcal Bla and several staphylococcal Bla sequences, these data suggest the recent spread of beta-lactamase to enterococci and also suggest the loss of a functional repressor. PMID:1952840

Detection of Multiple Waterborne Pathogens Using Microsequencing Arrays

EPA Science Inventory

Aims: A microarray was developed to simultaneously detect Cryptosporidium parvum, Cryptosporidium hominis, Enterococcus faecium, Bacillus anthracis and Francisella tularensis in water. Methods and Results: A DNA microarray was designed to contain probes that specifically dete...

The in vitro activity of flomoxef compared to four other cephalosporins and imipenem.

PubMed

Shah, P M; Knothe, H

1991-01-01

The antibacterial activity of the oxacephalosporin flomoxef was evaluated in comparison to cefpirome, cefuzoname, cefotaxime, ceftazidime, and imipenem against fresh clinical isolates. Flomoxef is an antibiotic with strong antibacterial activity against staphylococci including methicillin-resistant strains and streptococci with the exception of Enterococcus faecalis and Enterococcus faecium. It is very active against gram-negative cocci and rods including gram-positive and gram-negative anaerobes. Against Pseudomonas sp. flomoxef has no activity.

A Current Perspective on Daptomycin for the Clinical Microbiologist

PubMed Central

Pollett, Simon; Sakoulas, George

2013-01-01

SUMMARY Daptomycin is a lipopeptide antimicrobial with in vitro bactericidal activity against Gram-positive bacteria that was first approved for clinical use in 2004 in the United States. Since this time, significant data have emerged regarding the use of daptomycin for the treatment of serious infections, such as bacteremia and endocarditis, caused by Gram-positive pathogens. However, there are also increasing reports of daptomycin nonsusceptibility, in Staphylococcus aureus and, in particular, Enterococcus faecium and Enterococcus faecalis. Such nonsusceptibility is largely in the context of prolonged treatment courses and infections with high bacterial burdens, but it may occur in the absence of prior daptomycin exposure. Nonsusceptibility in both S. aureus and Enterococcus is mediated by adaptations to cell wall homeostasis and membrane phospholipid metabolism. This review summarizes the data on daptomycin, including daptomycin's unique mode of action and spectrum of activity and mechanisms for nonsusceptibility in key pathogens, including S. aureus, E. faecium, and E. faecalis. The challenges faced by the clinical laboratory in obtaining accurate susceptibility results and reporting daptomycin MICs are also discussed. PMID:24092854

OCCURRENCE OF HIGH-LEVEL AMINOGLYCOSIDE RESISTANCE IN ENVIRONMENTAL ISOLATES OF ENTEROCOCCI

EPA Science Inventory

High-level resistance fo aminoglycosides was observed in environmental isolates of enterococci. Various aquatic habitats, including agricultural runoff, creeks, rivers, wastewater, and wells, were analyzed. Strains of Enterococcus faecalis, e.faecium, E. gallinarum, and other Ent...

Microbiological quality of ready-to-eat salads: an underestimated vehicle of bacteria and clinically relevant antibiotic resistance genes.

PubMed

Campos, Joana; Mourão, Joana; Pestana, Nazaré; Peixe, Luísa; Novais, Carla; Antunes, Patrícia

2013-09-16

The increase demand for fresh vegetables is causing an expansion of the market for minimally processed vegetables along with new recognized food safety problems. To gain further insight on this topic we analyzed the microbiological quality of Portuguese ready-to-eat salads (RTS) and their role in the spread of bacteria carrying acquired antibiotic resistance genes, food products scarcely considered in surveillance studies. A total of 50 RTS (7 brands; split or mixed leaves, carrot, corn) were collected in 5 national supermarket chains in Porto region (2010). They were tested for aerobic mesophilic counts, coliforms and Escherichia coli counts as well as for the presence of Salmonella and Listeria monocytogenes. Samples were also plated in different selective media with/without antibiotics before and after enrichment. The E. coli, other coliforms and Enterococcus recovered were characterized for antibiotic resistance profiles and clonality with phenotypic and genetic approaches. A high number of RTS presented poor microbiological quality (86%--aerobic mesophilic counts, 74%--coliforms, 4%--E. coli), despite the absence of screened pathogens. In addition, a high diversity of bacteria (species and clones) and antibiotic resistance backgrounds (phenotypes and genotypes) were observed, mostly with enrichment and antibiotic selective media. E. coli was detected in 13 samples (n=78; all types and 4 brands; phylogenetic groups A, B1 and D; none STEC) with resistance to tetracycline [72%; tet(A) and/or tet(B)], streptomycin (58%; aadA and/or strA-strB), sulfamethoxazole (50%; sul1 and/or sul2), trimethoprim (50%; dfrA1 or dfrA12), ampicillin (49%; blaTEM), nalidixic acid (36%), ciprofloxacin (5%) or chloramphenicol (3%; catA). E. coli clones, including the widespread group D/ST69, were detected in different samples from the same brand or different brands pointing out to a potential cross-contamination. Other clinically relevant resistance genes were detected in 2 Raoultella

Tyrosine decarboxylase activity of enterococci grown in media with different nutritional potential: tyramine and 2-phenylethylamine accumulation and tyrDC gene expression.

PubMed

Bargossi, Eleonora; Tabanelli, Giulia; Montanari, Chiara; Lanciotti, Rosalba; Gatto, Veronica; Gardini, Fausto; Torriani, Sandra

2015-01-01

The ability to accumulate tyramine and 2-phenylethylamine by two strains of Enterococcus faecalis and two strains Enterococcus faecium was evaluated in two cultural media added or not with tyrosine. All the enterococcal strains possessed a tyrosine decarboxylase (tyrDC) which determined tyramine accumulation in all the conditions tested, independently on the addition of high concentration of free tyrosine. Enterococci differed in rate and level of biogenic amines accumulation. E. faecalis EF37 and E. faecium FC12 produced tyramine in high amount since the exponential growth phase, while 2-phenylethylamine was accumulated when tyrosine was depleted. E. faecium FC12 and E. faecalis ATCC 29212 showed a slower tyraminogenic activity which took place mainly in the stationary phase up to 72 h of incubation. Moreover, E. faecalis ATCC 29212 produced 2-phenylethylamine only in the media without tyrosine added. In BHI added or not with tyrosine the tyrDC gene expression level differed considerably depending on the strains and the growth phase. In particular, the tyrDC gene expression was high during the exponential phase in rich medium for all the strains and subsequently decreased except for E. faecium FC12. Even if tyrDC presence is common among enterococci, this study underlines the extremely variable decarboxylating potential of strains belonging to the same species, suggesting strain-dependent implications in food safety.

Antibiotic resistance in faecal bacteria isolated from horses receiving virginiamycin for the prevention of pasture-associated laminitis.

PubMed

Menzies-Gow, N J; Young, N J

2011-09-28

Enterococcus faecium, a major cause of potentially life-threatening hospital-acquired human infections, can be resistant to several antimicrobials, such that streptogramin quinupristin-dalfopristin (Q/D) is one of the few antibiotics still effective. Consequently use of the streptogramin virginiamycin as an animal growth promoter was banned in the EU in 1999 as some believed this contributed to the emergence of Q/D resistant E. faecium. Virginiamycin is advocated for preventing equine pasture-associated laminitis, but its effect on equine faecal bacterial Q/D resistance has not been determined. Faecal samples were obtained from horses receiving virginiamycin, horses co-grazing and horses not exposed to virginiamycin. Streptogramin resistant E. faecium were cultured from 70% (21/30) of animals treated with virginiamycin, 75% (18/24) of co-grazing animals and 69% (11/16) of animals not exposed. ermB and vatD genes were detected using real time PCR in 63% and 66% of animals treated with virginiamycin, 75% and 71% of co-grazing animals and 63% and 69% of animals not exposed. Antimicrobial resistance genes were present only in samples which had cultured Q/D resistant E. faecium. There was no significant difference between groups with respect to antimicrobial resistance. The gene load of vatD was significantly (p=0.04) greater in unexposed animals compared to those treated with virginiamycin. The use of virginiamycin to prevent pasture-associated laminitis does not appear to be related to an increased Q/D resistance frequency. However, in view of the high frequency of resistance within all groups, the horse is a reservoir of Q/D resistant genes and clones that potentially could be transferred transiently to humans. Copyright © 2011 Elsevier B.V. All rights reserved.

Vancomycin-resistant Enterococcus faecium bacteraemia as a complication of Kayexalate (sodium polystyrene sulfonate, SPS) in sorbitol-induced ischaemic colitis.

PubMed

Cerrud-Rodriguez, Roberto Christian; Alcaraz-Alvarez, Diego; Chiong, Brian Bobby; Ahmed, Abdurhman

2017-11-09

We present the case report of an 80-year-old woman with chronic kidney disease stage G5 admitted to the hospital with fluid overload and hyperkalaemia. Sodium polystyrene sulfonate (SPS, Kayexalate) in sorbitol suspension was given orally to treat her hyperkalaemia, which precipitated an episode of SPS in sorbitol-induced ischaemic colitis with the subsequent complication of vancomycin-resistant Enterococcus (VRE) bacteraemia. SPS (Kayexalate) in sorbitol suspension has been implicated in the development of intestinal necrosis. Sorbitol, which is added as a cathartic agent to decrease the chance of faecal impaction, may be primarily responsible through several proposed mechanisms. The gold standard of diagnosis is the presence of SPS crystals in the colon biopsy. On a MEDLINE search, no previous reports of a VRE bacteraemia as a complication of biopsy-confirmed SPS in sorbitol ischaemic colitis were found. To the best of our knowledge, ours would be the first such case ever reported. © BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Enterococcus caccae sp. nov., isolated from human stools.

PubMed

Carvalho, Maria da Glória S; Shewmaker, P Lynn; Steigerwalt, Arnold G; Morey, Roger E; Sampson, A J; Joyce, Kevin; Barrett, Timothy J; Teixeira, Lucia M; Facklam, Richard R

2006-07-01

The National Antimicrobial Resistance Monitoring System Laboratory at the Centers for Disease Control and Prevention (CDC) isolated two enterococcus-like strains that were referred to the CDC Streptococcus Laboratory for further identification. The isolates were recovered from human stool samples collected on different occasions from the same individual in Portland (OR, USA) in July 2000. Conventional physiological tests distinguished these strains from all known species of enterococci. Analyses of whole-cell-protein electrophoretic profiles showed the same unique profile for the two isolates, being most similar those of Enterococcus moraviensis and Enterococcus haemoperoxidus albeit not close enough to allow conclusive inclusion in any enterococcal species. Both isolates gave positive results in tests using the AccuProbe Enterococcus genetic probe, and Lancefield extracts reacted with CDC group D antiserum. Comparative 16S rRNA gene sequencing studies also revealed that these strains were closely related to the species E. moraviensis (99.6 % identity). The results of DNA-DNA relatedness experiments confirmed that these strains represented a single novel taxon. The highest level of DNA-DNA relatedness found between the novel taxon and any of the currently recognized species of Enterococcus was 32 %, for both E. moraviensis and E. haemoperoxidus. On the basis of this evidence, it is proposed that these stool isolates constitute a novel species, for which the name Enterococcus caccae sp. nov. is proposed. The type strain is 2215-02(T) (=SS-1777(T)=ATCC BAA-1240(T)=CCUG 51564(T)).

Characterization of a Bacteriocin-Like Substance Produced by a Vaginal Lactobacillus salivarius Strain

PubMed Central

Ocaña, Virginia S.; Pesce de Ruiz Holgado, Aída A.; Nader-Macías, María Elena

1999-01-01

A novel bacteriocin-like substance produced by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328 with activity against Enterococcus faecalis, Enterococcus faecium, and Neisseria gonorrhoeae was characterized. The highest level of production of this heat-resistant peptide or protein occurred during the late exponential phase. Its mode of action was shown to be bactericidal. L. salivarius subsp. salivarius CRL 1328 could be used for the design of a probiotic to prevent urogenital infections. PMID:10584033

Enterococcal Endocarditis: Can We Win the War?

PubMed Central

Munita, Jose M.

2015-01-01

Treatment of enterococcal infections has long been recognized as an important clinical challenge, particularly in the setting of infective endocarditis (IE). Furthermore, the increase prevalence of isolates exhibiting multidrug resistance (MDR) to traditional anti-enterococcal antibiotics such as ampicillin, vancomycin and aminoglycosides (high-level resistance) poses immense therapeutic dilemmas in hospitals around the world. Unlike IE caused by most isolates of Enterococcus faecalis, which still retain susceptibility to ampicillin and vancomycin, the emergence and dissemination of a hospital-associated genetic clade of multidrug resistant Enterococcus faecium, markedly limits the therapeutic options. The best treatment of IE MDR enterococcal endocarditis is unknown and the paucity of antibiotics with bactericidal activity against these organisms is a cause of serious concern. Although it appears that we are winning the war against E. faecalis, the battle rages on against isolates of multidrug-resistant E. faecium. PMID:22661339

Double-Serine Fluoroquinolone Resistance Mutations Advance Major International Clones and Lineages of Various Multi-Drug Resistant Bacteria.

PubMed

Fuzi, Miklos; Szabo, Dora; Csercsik, Rita

2017-01-01

The major international sequence types/lineages of methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae and ESBL-producing E. coli were demonstrated to have been advanced by favorable fitness balance associated with high-level resistance to fluoroquinolones. The paper shows that favorable fitness in the major STs/lineages of these pathogens was principally attained by the capacity of evolving mutations in the fluoroquinolone-binding serine residues of both the DNA gyrase and topoisomerase IV enzymes. The available information on fitness balance incurred by individual and various combinations of mutations in the enzymes is reviewed in multiple species. Moreover, strong circumstantial evidence is presented that major STs/lineages of other multi-drug resistant bacteria, primarily vancomycin-resistant Enterococcus faecium (VRE), emerged by a similar mechanism. The reason(s) why the major ST/lineage strains of various pathogens proved more adept at evolving favorable mutations than most isolates of the same species remains to be elucidated.

Double-Serine Fluoroquinolone Resistance Mutations Advance Major International Clones and Lineages of Various Multi-Drug Resistant Bacteria

PubMed Central

Fuzi, Miklos; Szabo, Dora; Csercsik, Rita

2017-01-01

The major international sequence types/lineages of methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae and ESBL-producing E. coli were demonstrated to have been advanced by favorable fitness balance associated with high-level resistance to fluoroquinolones. The paper shows that favorable fitness in the major STs/lineages of these pathogens was principally attained by the capacity of evolving mutations in the fluoroquinolone-binding serine residues of both the DNA gyrase and topoisomerase IV enzymes. The available information on fitness balance incurred by individual and various combinations of mutations in the enzymes is reviewed in multiple species. Moreover, strong circumstantial evidence is presented that major STs/lineages of other multi-drug resistant bacteria, primarily vancomycin-resistant Enterococcus faecium (VRE), emerged by a similar mechanism. The reason(s) why the major ST/lineage strains of various pathogens proved more adept at evolving favorable mutations than most isolates of the same species remains to be elucidated. PMID:29250038

Prevalence and Antimicrobial Resistance of Enterococci Isolated from Retail Meats in the United States, 2002 to 2014.

PubMed

Tyson, Gregory H; Nyirabahizi, Epiphanie; Crarey, Emily; Kabera, Claudine; Lam, Claudia; Rice-Trujillo, Crystal; McDermott, Patrick F; Tate, Heather

2018-01-01

Bacteria of the genus Enterococcus are important human pathogens that are frequently resistant to a number of clinically important antibiotics. They are also used as markers of animal fecal contamination of human foods and are employed as sentinel organisms for tracking trends in resistance to antimicrobials with Gram-positive activity. As part of the National Antimicrobial Resistance Monitoring System (NARMS), we evaluated several retail meat commodities for the presence of enterococci from 2002 to 2014, and we found 92.0% to be contaminated. The majority of isolates were either Enterococcus faecalis (64.0%) or Enterococcus faecium (28.6%), and the antimicrobial resistance of each isolate was assessed by broth microdilution. The resistance prevalences for several drugs, including erythromycin and gentamicin, were significantly higher among poultry isolates, compared to retail beef or pork isolates. None of the isolates was resistant to the clinically important human drug vancomycin, only 1 isolate was resistant to linezolid, and resistance to tigecycline was below 1%. In contrast, a majority of both E. faecalis (67.5%) and E. faecium (53.7%) isolates were resistant to tetracycline. Overall, the robust NARMS testing system employed consistent sampling practices and methods throughout the testing period, with the only significant trend in resistance prevalence being decreased E. faecium resistance to penicillin. These data provide excellent baseline levels of resistance that can be used to measure future changes in resistance prevalence that may result from alterations in the use of antimicrobials in food animal production. IMPORTANCE Enterococci, including E. faecalis and E. faecium , are present in the guts of food-producing animals and are used as a measure of fecal contamination of meat. We used the large consistent sampling methods of NARMS to assess the prevalence of Enterococcus strains isolated from retail meats, and we found over 90% of meats to be

Application of DNA probes for rRNA and vanA genes to investigation of a nosocomial cluster of vancomycin-resistant enterococci.

PubMed Central

Woodford, N; Morrison, D; Johnson, A P; Briant, V; George, R C; Cookson, B

1993-01-01

DNA probes specific for genes encoding rRNA and the glycopeptide resistance gene vanA were used to investigate a cluster of vancomycin-resistant (MICs, > 512 mg/liter) Enterococcus faecalis and Enterococcus faecium isolated from separate patients in a renal unit in a London hospital. When digested with BamHI, 12 of 13 vancomycin-resistant E. faecalis isolates exhibited a common restriction fragment length polymorphism pattern of rRNA genes (ribotype). A vanA probe hybridized with chromosomal DNA in these 12 isolates. The other isolate of vancomycin-resistant E. faecalis had a different ribotype and the vanA gene was located on plasmid DNA. These data suggest that cross-infection with a single strain of vancomycin-resistant E. faecalis occurred in most instances. In contrast, 23 vancomycin-resistant E. faecium isolates showed greater heterogeneity, comprising 8 ribotypes, suggesting that multiple strains were present in the unit. Twenty-one of these 23 isolates harbored a 24-MDa plasmid which hybridized with the vanA probe, implying that interstrain dissemination of a vancomycin resistance plasmid may have occurred among E. faecium isolates in the renal unit. Images PMID:8096216

Antimicrobial Resistance in Enterococcus spp. Isolated from Environmental Samples in an Area of Intensive Poultry Production

PubMed Central

Furtula, Vesna; Jackson, Charlene R.; Farrell, Erin Gwenn; Barrett, John B.; Hiott, Lari M.; Chambers, Patricia A.

2013-01-01

Enterococcus spp. from two poultry farms and proximate surface and ground water sites in an area of intensive poultry production were tested for resistance to 16 clinical antibiotics. Resistance patterns were compared to assess trends and possible correlations for specific antimicrobials and levels of resistance. Enterococci were detected at all 12 surface water sites and three of 28 ground water sites. Resistance to lincomycin, tetracycline, penicillin and ciprofloxacin in poultry litter isolates was high (80.3%, 65.3%, 61.1% and 49.6%, respectively). Resistance in the surface water to the same antibiotics was 87.1%, 24.1%, 7.6% and 12.9%, respectively. Overall, 86% of litter isolates, 58% of surface water isolates and 100% of ground water isolates were resistant to more than one antibiotic. Fifty-four different resistance patterns were recognised in isolates obtained from litter and environmental samples and several E. faecium and E. faecalis isolates from litter and environment samples shared the same resistance pattern. Multiple antibiotic resistant (MAR) indices calculated to assess health risks due to the presence of resistant enterococci suggested an increased presence of antibiotics in surface water, likely from poultry sources as no other wastewater contributions in the area were documented. PMID:23481592

Fosfomycin synergy in vitro with amoxicillin, daptomycin, and linezolid against vancomycin-resistant Enterococcus faecium from renal transplant patients with infected urinary stents.

PubMed

Descourouez, Jillian L; Jorgenson, Margaret R; Wergin, Justine E; Rose, Warren E

2013-03-01

Fosfomycin is a potential option for vancomycin-resistant enterococcus (VRE) infections despite limited in vitro and clinical data. In this study, 32 VRE isolates from renal transplant patients with urinary stent infections were susceptible to fosfomycin, daptomycin, and linezolid and resistant to amoxicillin, minocycline, and nitrofurantoin based on their MIC(50)s and MIC(90)s. Fosfomycin was bacteriostatic at 0.5 to 16× the MIC (32 to 2,048 μg/ml); synergy occurred when fosfomycin was combined with daptomycin (2.8 to 3.9 log(10) CFU/ml kill; P < 0.001) or amoxicillin (2.6 to 3.4; P < 0.05). These combinations may be potent options to treat VRE urinary infections pending investigation of clinical efficacy.

Diversity of enterococcal species and characterization of high-level aminoglycoside resistant enterococci of samples of wastewater and surface water in Tunisia.

PubMed

Ben Said, Leila; Klibi, Naouel; Lozano, Carmen; Dziri, Raoudha; Ben Slama, Karim; Boudabous, Abdellatif; Torres, Carmen

2015-10-15

One hundred-fourteen samples of wastewater (n=64) and surface-water (n=50) were inoculated in Slanetz-Bartley agar plates supplemented or not with gentamicin (SB-Gen and SB plates, respectively) for enterococci recovery. Enterococci were obtained from 75% of tested samples in SB media (72% in wastewater; 78% in surface-water), and 85 enterococcal isolates (one/positive-sample) were obtained. Enterococcus faecium was the most prevalent species (63.5%), followed by Enterococcus faecalis (20%), Enterococcus hirae (9.4%), Enterococcus casseliflavus (4.7%), and Enterococcus gallinarum/Enterococcus durans (2.4%). Antibiotic resistance detected among these enterococci was as follows [percentage/detected gene (number isolates)]: kanamycin [29%/aph(3')-IIIa (n=22)], streptomycin [8%/ant(6)-Ia (n=4)], erythromycin [44%/erm(B) (n=34)], tetracycline [18%/tet(M) (n=6)/tet(M)-tet(L) (n=9)], chloramphenicol [2%/cat(A) (n=1)], ciprofloxacin [7%] and trimethoprim-sulfamethoxazole [94%]. High-level-gentamicin resistant (HLR-G) enterococci were recovered from 15 samples in SB-Gen or SB plates [12/64 samples of wastewater (19%) and 3/50 samples of surface-water (6%)]; HLR-G isolates were identified as E. faecium (n=7), E. faecalis (n=6), and E. casseliflavus (n=2). These HLR-G enterococci carried the aac(6')-Ie-aph(2")-Ia and erm(B) genes, in addition to aph(3')-IIIa (n=10), ant(6)-Ia (n=9), tet(M) (n=13), tet(L) (n=8) and cat(A) genes (n=2). Three HLR-G enterococci carried the esp virulence gene. Sequence-types detected among HLR-G enterococci were as follows: E. faecalis (ST480, ST314, ST202, ST55, and the new ones ST531 and ST532) and E. faecium (ST327, ST12, ST296, and the new ones ST985 and ST986). Thirty-two different PFGE patterns were detected among 36 high-level-aminoglycoside-resistant enterococci recovered in water samples. Diverse genetic lineages of HLR-G enterococci were detected in wastewater and surface-water in Tunisia. Water can represent an important source for the

Pyrosequencing vs. culture-dependent approaches to analyze lactic acid bacteria associated to chicha, a traditional maize-based fermented beverage from Northwestern Argentina.

PubMed

Elizaquível, Patricia; Pérez-Cataluña, Alba; Yépez, Alba; Aristimuño, Cecilia; Jiménez, Eugenia; Cocconcelli, Pier Sandro; Vignolo, Graciela; Aznar, Rosa

2015-04-02

The diversity of lactic acid bacteria (LAB) associated with chicha, a traditional maize-based fermented alcoholic beverage from Northwestern Argentina, was analyzed using culture-dependent and culture-independent approaches. Samples corresponding to 10 production steps were obtained from two local producers at Maimará (chicha M) and Tumbaya (chicha T). Whereas by culture-dependent approach a few number of species (Lactobacillus plantarum and Weissella viridescens in chicha M, and Enterococcus faecium and Leuconostoc mesenteroides in chicha T) were identified, a higher quantitative distribution of taxa was found in both beverages by pyrosequencing. The relative abundance of OTUs was higher in chicha M than in chicha T; six LAB genera were common for chicha M and T: Enterococcus, Lactococcus, Streptococcus, Weissella, Leuconostoc and Lactobacillus while Pediococcus only was detected in chicha M. Among the 46 identified LAB species, those of Lactobacillus were dominant in both chicha samples, exhibiting the highest diversity, whereas Enterococcus and Leuconostoc were recorded as the second dominant genera in chicha T and M, respectively. Identification at species level showed the predominance of Lb. plantarum, Lactobacillus rossiae, Leuconostoc lactis and W. viridescens in chicha M while Enterococcus hirae, E. faecium, Lc. mesenteroides and Weissella confusa predominated in chicha T samples. In parallel, when presumptive LAB isolates (chicha M: 146; chicha T: 246) recovered from the same samples were identified by ISR-PCR and RAPD-PCR profiles, species-specific PCR and 16S rRNA gene sequencing, most of them were assigned to the Leuconostoc genus (Lc. mesenteroides and Lc. lactis) in chicha M, Lactobacillus, Weissella and Enterococcus being also present. In contrast, chicha T exhibited the presence of Enterococcus and Leuconostoc, E. faecium being the most representative species. Massive sequencing approach was applied for the first time to study the diversity and

AN EFFICIENT IMMUNOMAGNETIC CAPTURE SYSTEM FOR ENTEROCOCCUS FAECALIS AND ENTEROCOCCUS FAECIUM

EPA Science Inventory

Enterococci detection is one of the two approved procedures by the US Environmental Protection Agency (EPA) used for the assessment of the microbiological quality of recreational waters. The action levels established by the EPA for enterococci are 35 pr 100 ml in marine recreati...

Cloning strategies for heterologous expression of the bacteriocin enterocin A by Lactobacillus sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475.

PubMed

Jiménez, Juan J; Diep, Dzung B; Borrero, Juan; Gútiez, Loreto; Arbulu, Sara; Nes, Ingolf F; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2015-10-15

Bacteriocins produced by lactic acid bacteria (LAB) attract considerable interest as natural and nontoxic food preservatives and as therapeutics whereas the bacteriocin-producing LAB are considered potential probiotics for food, human and veterinary applications, and in the animal production field. Within LAB the lactobacilli are increasingly used as starter cultures for food preservation and as probiotics. The lactobacilli are also natural inhabitants of the gastrointestinal (GI) tract and attractive vectors for delivery of therapeutic peptides and proteins, and for production of bioactive peptides. Research efforts for production of bacteriocins in heterologous hosts should be performed if the use of bacteriocins and the LAB bacteriocin-producers is ever to meet the high expectations deposited in these antimicrobial peptides. The recombinant production and functional expression of bacteriocins by lactobacilli would have an additive effect on their probiotic functionality. The heterologous production of the bacteriocin enterocin A (EntA) was evaluated in different Lactobacillus spp. after fusion of the versatile Sec-dependent signal peptide (SP usp45 ) to mature EntA plus the EntA immunity gene (entA + entiA) (fragment UAI), and their cloning into plasmid vectors that permitted their inducible (pSIP409 and pSIP411) or constitutive (pMG36c) production. The amount, antimicrobial activity (AA) and specific antimicrobial activity (SAA) of the EntA produced by Lactobacillus sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475 transformed with the recombinant plasmids pSIP409UAI, pSIP411UAI and pMGUAI varied depending of the expression vector and the host strain. The Lb. casei CECT475 recombinant strains produced the largest amounts of EntA, with the highest AA and SAA. Supernatants from Lb. casei CECT (pSIP411UAI) showed a 4.9-fold higher production of EntA with a 22.8-fold higher AA and 4.7-fold higher SAA than those from Enterococcus faecium T136, the natural

Lower Prevalence of Antibiotic-Resistant Enterococci on U.S. Conventional Poultry Farms that Transitioned to Organic Practices

PubMed Central

Hulet, R. Michael; Zhang, Guangyu; McDermott, Patrick; Kinney, Erinna L.; Schwab, Kellogg J.; Joseph, Sam W.

2011-01-01

Background: In U.S. conventional poultry production, antimicrobials are used for therapeutic, prophylactic, and nontherapeutic purposes. Researchers have shown that this can select for antibiotic-resistant commensal and pathogenic bacteria on poultry farms and in poultry-derived products. However, no U.S. studies have investigated on-farm changes in resistance as conventional poultry farms transition to organic practices and cease using antibiotics. Objective: We investigated the prevalence of antibiotic-resistant Enterococcus on U.S. conventional poultry farms that transitioned to organic practices. Methods: Poultry litter, feed, and water samples were collected from 10 conventional and 10 newly organic poultry houses in 2008 and tested for Enterococcus. Enterococcus (n = 259) was identified using the Vitek® 2 Compact System and tested for susceptibility to 17 antimicrobials using the Sensititre™ microbroth dilution system. Data were analyzed using SAS software (version 9.2), and statistical associations were derived based on generalized linear mixed models. Results: Litter, feed, and water samples were Enterococcus positive. The percentages of resistant Enterococcus faecalis and resistant Enterococcus faecium were significantly lower (p < 0.05) among isolates from newly organic versus conventional poultry houses for two (erythromycin and tylosin) and five (ciprofloxacin, gentamicin, nitrofurantoin, penicillin, and tetracycline) antimicrobials, respectively. Forty-two percent of E. faecalis isolates from conventional poultry houses were multidrug resistant (MDR; resistant to three or more antimicrobial classes), compared with 10% of isolates from newly organic poultry houses (p = 0.02); 84% of E. faecium isolates from conventional poultry houses were MDR, compared with 17% of isolates from newly organic poultry houses (p < 0.001). Conclusions: Our findings suggest that the voluntary removal of antibiotics from large-scale U.S. poultry farms that transition to

Sewage sludge and liquid pig manure as possible sources of antibiotic resistant bacteria.

PubMed

Hölzel, Christina S; Schwaiger, Karin; Harms, Katrin; Küchenhoff, Helmut; Kunz, Anne; Meyer, Karsten; Müller, Christa; Bauer, Johann

2010-05-01

Within the last decades, the environmental spread of antibiotic resistant bacteria has become a topic of concern. In this study, liquid pig manure (n=305) and sewage sludge (n=111) - used as agricultural fertilizers between 2002 and 2005 - were investigated for the presence of Escherichia coli, Enterococcus faecalis and Enterococcus faecium. Bacteria were tested for their resistance against 40 chemotherapeutics including several "reserve drugs". E. coli (n=613) from pig manure were at a significantly higher degree resistant to streptomycin, doxycycline, spectinomycin, cotrimoxazole, and chloramphenicol than E. coli (n=116) from sewage sludge. Enterococci (Ent. faecalis, n=387, and Ent. faecium, n=183) from pig manure were significantly more often resistant to high levels of doxycycline, rifampicin, erythromycin, and streptomycin than Ent. faecalis (n=44) and Ent. faecium (n=125) from sewage sludge. Significant differences in enterococcal resistance were also seen for tylosin, chloramphenicol, gentamicin high level, fosfomycin, clindamicin, enrofloxacin, moxifloxacin, nitrofurantoin, and quinupristin/dalfopristin. By contrast, aminopenicillins were more effective in enterococci from pig manure, and mean MIC-values of piperacillin+tazobactam and third generation cefalosporines were significantly lower in E. coli from pig manure than in E. coli from sewage sludge. 13.4% (E. coli) to 25.3% (Ent. faecium) of pig manure isolates were high-level multiresistant to substances from more than three different classes of antimicrobial agents. In sewage sludge, high-level-multiresistance reached from 0% (Ent. faecalis) to 16% (Ent. faecium). High rates of (multi-) resistant bacteria in pig manure emphasize the need for a prudent - cautious - use of antibiotics in farm animals. Copyright 2010 Elsevier Inc. All rights reserved.

Changes in Enterococcal Populations and Related Antibiotic Resistance along a Medical Center-Wastewater Treatment Plant-River Continuum

PubMed Central

Oberlé, Kenny; Galopin, Sébastien; Cattoir, Vincent; Budzinski, Hélène; Petit, Fabienne

2013-01-01

To determine if hospital effluent input has an ecological impact on downstream aquatic environment, antibiotic resistance in Enterococcus spp. along a medical center-retirement home-wastewater treatment plant-river continuum in France was determined using a culture-based method. Data on antibiotic consumption among hospitalized and general populations and levels of water contamination by antibiotics were collected. All isolated enterococci were genotypically identified to the species level, tested for in vitro antibiotic susceptibility, and typed by multilocus sequence typing. The erm(B) and mef(A) (macrolide resistance) and tet(M) (tetracycline resistance) genes were detected by PCR. Along the continuum, from 89 to 98% of enterococci, according to the sampled site, were identified as Enterococcus faecium. All E. faecium isolates from hospital and retirement home effluents were multiply resistant to antibiotics, contained erm(B) and mef(A) genes, and belonged to hospital-adapted clonal complex 17 (CC17). Even though this species remained dominant in the downstream continuum, the relative proportion of CC17 isolates progressively decreased in favor of other subpopulations of E. faecium that were more diverse, less resistant to antibiotics, and devoid of the classical macrolide resistance genes and that belonged to various sequence types. Antibiotic concentrations in waters were far below the MICs for susceptible isolates. CC17 E. faecium was probably selected in the gastrointestinal tract of patients under the pressure of administered antibiotics and then excreted together with the resistance genes in waters to progressively decrease along the continuum. PMID:23377946

Screening municipal wastewater effluent and surface water used for drinking water production for the presence of ampicillin and vancomycin resistant enterococci.

PubMed

Taučer-Kapteijn, Maja; Hoogenboezem, Wim; Heiliegers, Laura; de Bolster, Danny; Medema, Gertjan

2016-07-01

The emergence of clinical enterococcal isolates that are resistant to both ampicillin and vancomycin is a cause of great concern, as therapeutic alternatives for the treatment of infections caused by such organisms are becoming limited. Aquatic environments could play a role in the dissemination of antibiotic resistant enterococci. This study investigated the presence of ampicillin and vancomycin resistant enterococci in the treated effluent of six wastewater treatment plants (WWTPs) and in surface water used as a source for drinking water production in the Netherlands. Membrane filtration in combination with selective media with ampicillin or vancomycin was applied to determine the presence of ampicillin resistant Enterococcus (ARE) and vancomycin resistant Enterococcus (VRE) species. Ampicillin resistant Enterococcus faecium (minimal inhibitory concentration (MIC) >16μg/mL; n=1033) was observed in all studied WWTP effluents. In surface water used for drinking water production (intake locations), no ARE or VRE were observed. At both types of location, intrinsic vancomycin resistant Pediococcus spp., Leuconostoc spp. and Lactobacillus spp. were isolated with the vancomycin medium. The ampicillin resistant E. faecium (AREfm) isolates (n=113) did not contain the vanA or vanB gene, but MIC testing for vancomycin showed intermediate vancomycin resistance (2-8μgmL(-1)) to occur in these AREfm strains. This study documents the discharge of ampicillin resistant E. faecium strains with intermediate vancomycin resistance by the WWTPs into the surface water, but no presence of these strains downstream at intake locations for drinking water production. Copyright © 2016 Elsevier GmbH. All rights reserved.

Heterologous expression of glycopeptide resistance vanHAX gene clusters from soil bacteria in Enterococcus faecalis.

PubMed

Hasman, Henrik; Aarestrup, Frank M; Dalsgaard, Anders; Guardabassi, Luca

2006-04-01

The aim of the study was to determine whether glycopeptide resistance gene clusters from soil bacteria could be heterologously expressed in Enterococcus faecalis and adapt to the new host following exposure to vancomycin. The vanHAX clusters from Paenibacillus thiaminolyticus PT-2B1, Paenibacillus apiarius PA-B2B and Amycolatopsis coloradensis DSM 44225 were separately cloned in an appropriately constructed shuttle vector containing the two-component regulatory system (vanRS) of Tn1546. The complete vanA(PT) operon (vanRSHAXY) from P. thiaminolyticus PT-2B1 was cloned in the same shuttle vector lacking enterococcal vanRS. All plasmid constructs were electroporated into E. faecalis JH2-2 and the MICs of vancomycin and teicoplanin were determined for each recombinant strain before and following exposure to sublethal concentrations of vancomycin. The vanHAX clusters from P. thiaminolyticus and P. apiarius conferred high-level vancomycin resistance (MIC > or = 125 mg/L) in E. faecalis JH2-2. In contrast, cloning of the vanHAX cluster from A. coloradensis did not result in a significant increase of vancomycin resistance (MIC = 0.7 mg/L). Resistance to vancomycin was not observed after cloning the complete vanA(PT) operon from P. thiaminolyticus (MIC = 2 mg/L), but this recombinant rapidly adapted to high concentrations of vancomycin (MIC = 500 mg/L) following exposure to sub-lethal concentrations of this antibiotic. The results showed that vanA(PT) in P. thiaminolyticus is a possible ancestor of vanA-mediated glycopeptide resistance in enterococci. Experimental evidence supported the hypothesis that enterococci did not acquire glycopeptide resistance directly from glycopeptide-producing organisms such as A. coloradensis.

Complete genome sequence of Brachybacterium faecium type strain (Schefferle 6-10T)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lapidus, Alla; Pukall, Rudiger; LaButti, Kurt

2009-05-20

Brachybacterium faecium Collins et al. 1988 is the type species of the genus, and is of phylogenetic interest because of its location in the Dermabacteraceae, a rather isolated family within the actinobacterial suborder Micrococcineae. B. faecium is known for its rod-coccus growth cycle and the ability to degrade uric acid. It grows aerobically or weakly anaerobically. The strain described in this report is a free-living, nonmotile, Gram-positive bacterium, originally isolated from poultry deep litter. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a membermore » of the actinobacterial family Dermabacteraceae, and the 3,614,992 bp long single replicon genome with its 3129 protein-coding and 69 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.« less

Comparative in-vitro activities of quinupristin-dalfopristin against Gram-positive bloodstream isolates.

PubMed

Schouten, M A; Hoogkamp-Korstanje, J A

1997-08-01

The in-vitro activity of quinupristin-dalfopristin was compared with those of vancomycin, teicoplanin, erythromycin, clarithromycin, rifampicin, imipenem, meropenem, ciprofloxacin and sparfloxacin against 414 bloodstream isolates of Gram-positive cocci. Quinupristin-dalfopristin inhibited strains of Streptococcus pyogenes and Streptococcus agalactiae at 0.12 mg/L, methicillin- and/or erythromycin-resistant Staphylococcus aureus and Staphylococcus epidermidis at 0.5 mg/L, Staphylococcus haemolyticus, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus mitis, Streptococcus bovis, Streptococcus sanguis and Streptococcus anginosus at 1 mg/L and Enterococcus faecalis at 8 mg/L.

Controlling growth of Streptococcus faecium in a ham model with heat and ethylenediamine tetraacetic acid, tertiary butylhydroquinone or nisin.

PubMed

Houben, J H; Krol, B

1985-01-01

The study described in this paper was designed to test the effect of heat and EDTA, TBHQ or nisin on the stability of a ham model with regard to Streptococcus faecium. Comminuted ham tissue containing brine and test ingredients was inoculated with two strains of Str. faecium and heated for 0, 30, 60, 90 and 120 min at 68·9°C. Growth was followed during incubation at 7°C and 37°C. In combination with heating at 68·9°C for 30 min, a stable (180 days 7°C) model was obtained by adding (ppm): 400, EDTA; 1000, TBHQ or 20, nisin. EDTA and nisin seem promising compounds for enhancing the stability of pasteurized ham with regard to Str. faecium. Copyright © 1985. Published by Elsevier Ltd.

Antibiotic susceptibility of enterococci isolated from traditional fermented meat products.

PubMed

Barbosa, J; Ferreira, V; Teixeira, P

2009-08-01

Antibiotic susceptibility was evaluated for 182 Enterococcus spp. isolated from Alheira, Chouriça de Vinhais and Salpicão de Vinhais, fermented meat products produced in the North of Portugal. Previously, a choice was made from a group of 1060 isolates, using phenotypic and genotypic tests. From these, 76 were previously identified as Enterococcus faecalis, 44 as Enterococcus faecium, one as Enterococcus casseliflavus and 61 as Enteroccocus spp. In order to encompass several of the known chemical and functional classes of antibiotics, resistance to ampicillin, penicillin G, ciprofloxacin, chloramphenicol, erythromycin, nitrofurantoin, rifampicin, tetracycline and vancomycin was evaluated. All the isolates were sensitive to antibiotics of clinical importance, such as penicillins and vancomycin. Some differences in Minimal Inhibitory Concentrations (MICs) of antibiotics, could be associated with the enterococcal species.

Evaluation of a New System, VITEK 2, for Identification and Antimicrobial Susceptibility Testing of Enterococci

PubMed Central

Garcia-Garrote, Fernando; Cercenado, Emilia; Bouza, Emilio

2000-01-01

We evaluated the new automated VITEK 2 system (bioMérieux) for the identification and antimicrobial susceptibility testing of enterococci. The results obtained with the VITEK 2 system were compared to those obtained by reference methods: standard identification by the scheme of Facklam and Sahm [R. R. Facklam and D. F. Sahm, p. 308–314, in P. R. Murray et al., ed., Manual of Clinical Microbiology, 6th ed., 1995] and with the API 20 STREP system and, for antimicrobial susceptibility testing, broth microdilution and agar dilution methods by the procedures of the National Committee for Clinical Laboratory Standards. The presence of vanA and vanB genes was determined by PCR. A total of 150 clinical isolates were studied, corresponding to 60 Enterococcus faecalis, 55 Enterococcus faecium, 26 Enterococcus gallinarum, 5 Enterococcus avium, 2 Enterococcus durans, and 2 Enterococcus raffinosus isolates. Among those isolates, 131 (87%) were correctly identified to the species level with the VITEK 2 system. Approximately half of the misidentifications were for E. faecium with low-level resistance to vancomycin, identified as E. gallinarum or E. casseliflavus; however, a motility test solved the discrepancies and increased the agreement to 94%. Among the strains studied, 66% were vancomycin resistant (57 VanA, 16 VanB, and 26 VanC strains), 23% were ampicillin resistant (MICs, ≥16 μg/ml), 31% were high-level gentamicin resistant, and 45% were high-level streptomycin resistant. Percentages of agreement for susceptibility and resistance to ampicillin, vancomycin, and teicoplanin and for high-level gentamicin resistance and high-level streptomycin resistance were 93, 95, 97, 97, and 96%, respectively. The accuracy of identification and antimicrobial susceptibility testing of enterococci with the VITEK 2 system, together with the significant reduction in handling time, will have a positive impact on the work flow of the clinical microbiology laboratory. PMID:10834961

Enterococcus faecalis phosphomevalonate kinase

PubMed Central

Doun, Stephanie S.; Burgner, John W.; Briggs, Scott D.; Rodwell, Victor W.

2005-01-01

The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni++ affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37°C. The activation energy was ~5.6 kcal/mol. Activity with Mn++, the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). Km values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 μmol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed. PMID:15802646

Enterococcus faecalis urinary-tract infections: Do they have a zoonotic origin?

PubMed

Abat, Cédric; Huart, Michael; Garcia, Vincent; Dubourg, Grégory; Raoult, Didier

2016-10-01

Major human pathogens are frequently isolated from meat-producing animals, particularly poultry. Among them is Enterococcus faecalis, which is known to be one of the main cause of human urinary-tract infections worldwide. Early in 2015, we detected several, consecutive abnormal increases in the weekly number of human E. faecalis infections in various medical settings in the Provence-Alpes-Côte d'Azur region of France, especially including community-acquired urinary-tract infections. Speculating that this region-wide epidemiological event may have originated from animal-based food, we initiated this work to provide an overview of the epidemiology of E. faecalis, with a particular focus on the possible link between E. faecalis clones isolated from food-producing animals and those responsible for human urinary-tract infections. At that time, only one study had clearly identified strong epidemiological links between E. faecalis clones isolated from food-producing animals and human E. faecalis urinary-tract infections. This observation, coupled with our region-wide epidemiological experience, leads us to strongly believe that E. faecalis is a real zoonotic pathogen with potentially highly significant impact on human health. This is of particular concern because of its ability to acquire antibiotic-resistance genes and to infect animals and humans. Various strategies must be urgently implemented to address this public health threat, in particular through the development and implementation of large integrated automated surveillance systems based on animal and human health data to enable us to detect E. faecalis epidemiological events. Copyright © 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Heat injury and recovery of Streptococcus faecium associated with the souring of chub-packed luncheon meat.

PubMed

Bell, R G; De Lacy, K M

1984-10-01

The presence of NaCl in the heating medium provided some protection from lethal heat damage for cells of a Streptococcus faecium strain isolated from luncheon meat whereas the presence of NaNO2 either alone or in addition to NaCl, had no significant effect on cell survival. Subsequent recovery and growth of heat-damaged cells was retarded by the presence of NaCl. When NaNO2 was present in addition to NaCl the inhibitory effect of the latter was reduced. These principal components of the luncheon-meat-cure are apparently opposed in their activities on post-heating recovery and growth of Strep. faecium. Product stability, i.e. duration of the lag before growth occurs, is directly related to the severity of the heat treatment and to the concentration of NaCl in the product. Therefore the resistance of pasteurized chub-packed luncheon meat to streptococcal spoilage during storage at temperatures conducive to microbial growth results from a prolonged heat-induced salt-maintained pre-growth adjustment phase rather than to any inherent inhibitory property of the luncheon meat to the growth of non-heat-damaged Strep. faecium cells.

Significance and survival of Enterococci during the house fly development.

PubMed

Ghosh, Anuradha; Akhtar, Mastura; Holderman, Chris; Zurek, Ludek

2014-01-01

House flies are among the most important nonbiting insect pests of medical and veterinary importance. Larvae develop in decaying organic substrates and their survival strictly depends on an active microbial community. House flies have been implicated in the ecology and transmission of enterococci, including multi-antibiotic-resistant and virulent strains of Enterococcus faecalis. In this study, eight American Type Culture Collection type strains of enterococci including Enterococcus avium, Enterococcus casseliflavus, Enterococcus durans, Enterococcus hirae, Enterococcus mundtii, Enterococcus gallinarum, Enterococcusfaecalis, and Enterococcusfaecium were evaluated for their significance in the development of house flies from eggs to adults in bacterial feeding assays. Furthermore, the bacterial colonization of the gut of teneral flies as well as the importance of several virulence traits of E. faecalis in larval mortality was assessed. Overall survival of house flies (egg to adult) was significantly higher when grown with typically nonpathogenic enterococcal species such as E. hirae (76.0% survival), E. durans (64.0%), and E. avium (64.0%) compared with that with clinically important species E. faecalis (24.0%) and E. faecium (36.0%). However, no significant differences in survival of house fly larvae were detected when grown with E. faecalis strains carrying various virulence traits, including isogenic mutants of the human clinical isolate E. faecalis V583 with in-frame deletions of gelatinase, serine protease, and capsular polysaccharide serotype C. Enterococci were commonly detected in fly puparia (range: 75-100%; concentration: 103-105 CFU/puparium);however, the prevalence of enterococci in teneral flies varied greatly: from 25.0 (E. casseliflavus) to 89.5% (E. hirae). In conclusion, depending on the species, enterococci variably support house fly larval development and colonize the gut of teneral adults. The human pathogenic species, E. faecalis and E. faecium

9230 FECAL ENTEROCOCCUS/STREPTOCOCCUS GROUPS

EPA Science Inventory

In 1903 the genus name Enterococcus was proposed for gram-positive, catalase-negative, coccoid-shaped bacterial of intestinal origin. Several years later, it was suggested that the genus name be changed to Streptococcus because of the organisms' ability to form chains of coccoid...