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Sample records for cronobacter sakazakii baa-894

  1. Genome Sequence of Cronobacter sakazakii BAA-894 and Comparative Genomic Hybridization Analysis with Other Cronobacter Species

    PubMed Central

    Kucerova, Eva; Clifton, Sandra W.; Xia, Xiao-Qin; Long, Fred; Porwollik, Steffen; Fulton, Lucinda; Fronick, Catrina; Minx, Patrick; Kyung, Kim; Warren, Wesley; Fulton, Robert; Feng, Dongyan; Wollam, Aye; Shah, Neha; Bhonagiri, Veena; Nash, William E.; Hallsworth-Pepin, Kymberlie; Wilson, Richard K.

    2010-01-01

    Background The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified. Methodology/Principal Findings We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 kb (51% GC) and 131 kb (56% GC). The genome was used to construct a 387,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10–17% absence of genes. Conclusions/Significance CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from

  2. Immunoproteomic identification of immunogenic proteins in Cronobacter sakazakii strain BAA-894.

    PubMed

    Wang, Jian; Du, Xin-Jun; Lu, Xiao-Nan; Wang, Shuo

    2013-03-01

    Cronobacter spp. are emerging opportunistic pathogens. Cronobacter sakazakii is considered as the predominant species in all infections. So far, our understanding of the species' immunogens and potential virulence factors of Cronobacter spp. remains limited. In this study, an immunoproteomic approach was used to investigate soluble and insoluble proteins from the genome-sequenced strain C. sakazakii ATCC BAA-894. Proteins were separated using two-dimensional electrophoresis, detected by Western blotting with polyclonal antibodies of C. sakazakii BAA-894, and identified using tandem mass spectrometry (MALDI-MS and MALDI-MS/MS, MS/MSMS). A total of 11 immunoreactive proteins were initially identified in C. sakazakii BAA-894, including two outer membrane proteins, four periplasmic proteins, and five cytoplasmic proteins. In silico functional analysis of the 11 identified proteins indicated three proteins that were initially described as immunogens of pathogenic bacteria. For the remaining eight proteins, one protein was categorized as a potential virulence factor involved in protection against reactive oxygen species, and seven proteins were considered to play potential roles in adhesion, invasion, and biofilm formation. To our knowledge, this is the first time that immunogenic proteins of C. sakazakii BAA-894 have been identified as immunogens and potential virulence factors by an immunoproteomics approach. Future studies should investigate the roles of these proteins in bacterial pathogenesis and modulation of host immune responses during infection to identify their potential as molecular therapeutic targets.

  3. Reconstruction of the Carotenoid Biosynthetic Pathway of Cronobacter sakazakii BAA894 in Escherichia coli

    PubMed Central

    Zhang, Wei; Hu, Xiaoqing; Wang, Liqin; Wang, Xiaoyuan

    2014-01-01

    Cronobacter sakazakii could form yellow-pigmented colonies. However, the chemical structure and the biosynthetic pathway of the yellow pigments have not been identified. In this study, the yellow pigments of C. sakazakii BAA894 were purified and analyzed. The major components of the yellow pigments were confirmed as zeaxanthin-monoglycoside and zeaxanthin-diglycoside. A gene cluster containing seven genes responsible for the yellow pigmentation in C. sakazakii BAA894 was identified. The seven genes of C. sakazakii BAA894 or parts of them were reconstructed in a heterologous host Escherichia coli DH5α. The pigments formed in these E. coli strains were isolated and analyzed by thin layer chromatography, UV-visible spectroscopy, high performance liquid chromatography, and electron spray ionization-mass spectrometry. These redesigned E. coli strains could produce different carotenoids. E. coli strain expressing all the seven genes could produce zeaxanthin-monoglycoside and zeaxanthin-diglycoside; E. coli strains expressing parts of the seven genes could produce lycopene, β-carotene, cryptoxanthin or zeaxanthin. This study identified the gene cluster responsible for the yellow pigmentation in C. sakazakii BAA894. PMID:24466219

  4. Transcriptome analysis of Cronobacter sakazakii ATCC BAA-894 after interaction with human intestinal epithelial cell line HCT-8.

    PubMed

    Jing, Chun-e; Du, Xin-jun; Li, Ping; Wang, Shuo

    2016-01-01

    Cronobacter spp. are opportunistic pathogens that are responsible for infections including severe meningitis, septicemia, and necrotizing enterocolitis in neonates and infants. To date, questions still remain regarding the mechanisms of pathogenicity and virulence determinants for each bacterial strain. In this study, we established an in vitro model for Cronobacter sakazakii ATCC BAA-894 infection of HCT-8 human colorectal epithelial cells. The transcriptome profile of C. sakazakii ATCC BAA-894 after interaction with HCT-8 cells was determined using high-throughput whole-transcriptome sequencing (RNA sequencing (RNA-seq)). Gene expression profiles indicated that 139 genes were upregulated and 72 genes were downregulated in the adherent C. sakazakii ATCC BAA-894 strain on HCT-8 cells compared to the cultured bacteria in the cell-free medium. Expressions of some flagella genes and virulence factors involved in adherence were upregulated. High osmolarity and osmotic stress-associated genes were highly upregulated, as well as genes responsible for the synthesis of lipopolysaccharides and outer membrane proteins, iron acquisition systems, and glycerol and glycerophospholipid metabolism. In sum, our study provides further insight into the mechanisms underlying C. sakazakii pathogenesis in the human gastrointestinal tract.

  5. Computational identification and analysis of arsenate reductase protein in Cronobacter sakazakii ATCC BAA-894 suggests potential microorganism for reducing arsenate.

    PubMed

    Chaturvedi, Navaneet; Singh, Vinay Kumar; Pandey, Paras Nath

    2013-06-01

    This study focuses a bioinformatics-based prediction of arsC gene product arsenate reductase (ArsC) protein in Cronobacter sakazakii BAA-894 strain. A protein structure-based study encloses three-dimensional structural modeling of target ArsC protein, was carried out by homology modeling method. Ultimately, the detection of active binding regions was carried out for characterization of functional sites in protein. The ten probable ligand binding sites were predicted for target protein structure and highlighted the common binding residues between target and template protein. It has been first time identified that modeled ArsC protein structure in C. sakazakii was structurally and functionally similar to well-characterized ArsC protein of Escherichia coli because of having same structural motifs and fold with similar protein topology and function. Investigation revealed that ArsC from C. sakazakii can play significant role during arsenic resistance and potential microorganism for bioremediation of arsenic toxicity.

  6. Caenorhabditis elegans as a model for studying Cronobacter sakazakii ATCC BAA-894 pathogenesis.

    PubMed

    Sivamaruthi, Bhagavathi Sundaram; Ganguli, Abhijit; Kumar, Mukesh; Bhaviya, Sheker; Pandian, Shunmugiah Karutha; Balamurugan, Krishnaswamy

    2011-10-01

    Cronobacter sakazakii is occasionally associated with food-borne illness seen in neonates and infants with weakened immune system. It can cause meningitis, local necrotizing enterocolitis and systemic bacteremia leading to infant mortality rates upto 33-80%. With the aim of investigating whether C. sakazakii is also a pathogen of the model organism C. elegans, we have performed killing assays and monitored the mortality of host fed with pathogen. C. elegans fed with C. sakazakii die over the course of several days, as a consequence of an accumulation of bacteria in the host intestine. Further, the rate of C. sakazakii mediated infection in C. elegans depends on the accumulation of the bacterial load inside the host. C. sakazakii killed C. elegans with an LT(50) (time for half to die) of 134 ± 2.8 h in liquid assay conditions, whereas the mortality of C. elegans infected with C. sakazakii was less pronounced during solid assays. We found that 24 h of C. sakazakii infection is enough to cause gametogenesis defects and increased cell damage in intestinal tract of host. To monitor the immune regulations during C. sakazakii infection in C. elegans at molecular level, total RNA was isolated and few candidate genes (lys-7, clec-60 and clec-87) were kinetically analyzed by using the semi-quantitative RT-PCR. The level of expression of lys-7, clec-60 and clec-87 mRNAs isolated from C. elegans infected with C. sakazakii was significantly higher when compared to C. elegans exposed to E. coli OP50 control. This is the first report in which physiological changes and an induction of host immunity mediated antimicrobial genes by C. sakazakii are shown in C. elegans.

  7. Protein sequences insight into heavy metal tolerance in Cronobacter sakazakii BAA-894 encoded by plasmid pESA3.

    PubMed

    Chaturvedi, Navaneet; Kajsik, Michal; Forsythe, Stephen; Pandey, Paras Nath

    2015-12-01

    The recently annotated genome of the bacterium Cronobacter sakazakii BAA-894 suggests that the organism has the ability to bind heavy metals. This study demonstrates heavy metal tolerance in C. sakazakii, in which proteins with the heavy metal interaction were recognized by computational and experimental study. As the result, approximately one-fourth of proteins encoded on the plasmid pESA3 are proposed to have potential interaction with heavy metals. Interaction between heavy metals and predicted proteins was further corroborated using protein crystal structures from protein data bank database and comparison of metal-binding ligands. In addition, a phylogenetic study was undertaken for the toxic heavy metals, arsenic, cadmium, lead and mercury, which generated relatedness clustering for lead, cadmium and arsenic. Laboratory studies confirmed the organism's tolerance to tellurite, copper and silver. These experimental and computational study data extend our understanding of the genes encoding for proteins of this important neonatal pathogen and provide further insights into the genotypes associated with features that can contribute to its persistence in the environment. The information will be of value for future environmental protection from heavy toxic metals.

  8. Functional Screening of the Cronobacter sakazakii BAA-894 Genome reveals a role for ProP (ESA_02131) in carnitine uptake.

    PubMed

    Feeney, Audrey; Sleator, Roy D

    2015-01-01

    Cronobacter sakazakii is a neonatal pathogen responsible for up to 80% of fatalities in infected infants. Low birth weight infants and neonates infected with C. sakazakii suffer necrotizing enterocolitis, bacteraemia and meningitis. The mode of transmission most often associated with infection is powdered infant formula (PIF) which, with an aw of ∼0.2, is too low to allow most microorganisms to persist. Survival of C. sakazakii in environments subject to extreme hyperosmotic stress has previously been attributed to the uptake of compatible solutes including proline and betaine. Herein, we report the construction and screening of a C. sakazakii genome bank and the identification of ProP (ESA_02131) as a carnitine uptake system.

  9. Functional Screening of the Cronobacter sakazakii BAA-894 Genome reveals a role for ProP (ESA_02131) in carnitine uptake

    PubMed Central

    Feeney, Audrey; Sleator, Roy D

    2015-01-01

    Cronobacter sakazakii is a neonatal pathogen responsible for up to 80% of fatalities in infected infants. Low birth weight infants and neonates infected with C. sakazakii suffer necrotizing enterocolitis, bacteraemia and meningitis. The mode of transmission most often associated with infection is powdered infant formula (PIF) which, with an aw of ∼0.2, is too low to allow most microorganisms to persist. Survival of C. sakazakii in environments subject to extreme hyperosmotic stress has previously been attributed to the uptake of compatible solutes including proline and betaine. Herein, we report the construction and screening of a C. sakazakii genome bank and the identification of ProP (ESA_02131) as a carnitine uptake system. PMID:25915804

  10. Microarray-based Comparative Genomic Indexing of the Cronobacter genus (Enterobacter sakazakii)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cronobacter is a recently defined genus synonymous with Enterobacter sakazakii. This new genus currently comprises 6 genomospecies. To extend our understanding of the genetic relationship between Cronobacter sakazakii BAA-894 and the other species of this genus, microarray-based comparative genomi...

  11. Cronobacter (Enterobacter) sakazakii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cronobacter sakazakii has been identified as an infrequently isolated opportunistic pathogen. Over 120 cases of C. sakazakii-related illness have been reported and most reported cases are life-threatening infections. Many of these outbreaks have been linked the consumption of C. sakazakii-contamina...

  12. Analysis of the role of the Cronobacter sakazakii ProP homologues in osmotolerance

    PubMed Central

    2014-01-01

    Bacteria respond to elevated osmolality by the accumulation of a range of low molecular weight molecules, known as compatible solutes (owing to their compatibility with the cells' normal physiology at high internal concentrations). The neonatal pathogen Cronobacter sakazakii is uniquely osmotolerant, surviving in powdered infant formula (PIF) which typically has a water activity (aw) of 0.2 – inhospitable to most micro-organisms. Mortality rates of up to 80% in infected infants have been recorded making C. sakazakii a serious cause for concern. In silico analysis of the C. sakazakii BAA-894 genome revealed seven copies of the osmolyte uptake system ProP. Herein, we test the physiological role of each of these homologues following heterologous expression against an osmosensitive Escherichia coli host. PMID:24910715

  13. Effect of polymyxin resistance (pmr) on biofilm formation of Cronobacter sakazakii.

    PubMed

    Bao, Xuerui; Jia, Xiangyin; Chen, Lequn; Peters, Brian M; Lin, Chii-Wann; Chen, Dingqiang; Li, Lin; Li, Bing; Li, Yanyan; Xu, Zhenbo; Shirtliff, Mark E

    2016-12-22

    Cronobacter sakazakii (C.sakazakii) has been identified as a wide-spread conditioned pathogen associated with series of serious illnesses, such as neonatal meningitis, enterocolitis, bacteremia or sepsis. As food safety is concerned, microbial biofilm has been considered to be a potential source of food contamination. The current study aims to investigate the ability of biofilm formation of two C. sakazakii strains (wild type BAA 894 and pmrA mutant). Crystal violet (CV), XTT (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino carbonyl)-2H-(tetrazolium hydroxide)] assays, and scanning electron microscopy (SEM) are performed on different time points during biofilm formation of C. sakazakii strains. Furthermore, RNA-seq strategy is utilized and the transcriptome data is analyzed to study the expression of genes related to biofilm formation along with whole genome sequencing. For biomass, in the first 24 h, pmrA mutant produced approximately 5 times than wildtype. However, the wild type exhibited more biomass than pmrA mutant during the post maturation stage (7-14 d). In addition, the wildtype showed higher viability than pmrA mutant during the whole biofilm formation. This study represents the first evidence on the biofilm formation of C. sakazakii pmrA mutant, which may further aid in the prevention and control for the food contamination caused by C. sakazakii.

  14. Cronobacter sakazakii reduction by blueberry proanthocyanidins.

    PubMed

    Joshi, Snehal S; Howell, Amy B; D'Souza, Doris H

    2014-05-01

    Blueberry juice and blueberry polyphenols reportedly have antimicrobial properties against foodborne pathogens, without much currently known on their effects against Cronobacter sakazakii. This study evaluated the antimicrobial effects of blueberry proanthocyanidins (PAC) and commercial blueberry juice (BJ) against two strains of C. sakazakii, ATCC 29004 and 29544. BJ (pH 2.8), blueberry PAC (5 mg/ml) and controls (phosphate buffered saline (PBS), pH 7.2, and malic acid pH 3.0) were mixed with equal volumes of washed overnight cultures of C. sakazakii and incubated for 30 min, 1 h, 3 h and 6 h at 37°C. Reductions of ∼1 and 1.50 log CFU/ml were obtained for strains 29004 and 29544, respectively after 30 min with BJ or blueberry PAC. Both C. sakazakii strains 29004 and 29544 were reduced to undetectable levels from 8.25 ± 0.12 log CFU/ml and 8.48 ± 0.03 log CFU/ml, respectively with BJ (pH 2.8) or blueberry PAC after 1 h, while malic acid (pH 3.0) showed ∼1.3 log CFU/ml reduction for both strains. Scanning electron microscopy studies showed differences in cell membrane morphology with clumping and formation of blebs of the treated strains compared to untreated controls. These results warrant further in vivo studies with blueberry bioactives to determine potential for preventing and treating C. sakazakii infections.

  15. Supersize me: Cronobacter sakazakii phage GAP32

    SciTech Connect

    Abbasifar, Reza; Griffiths, Mansel W.; Sabour, Parviz M.; Ackermann, Hans-Wolfgang; Vandersteegen, Katrien; Lavigne, Rob; Noben, Jean-Paul; Alanis Villa, Argentina; Abbasifar, Arash; Nash, John H.E.; Kropinski, Andrew M.

    2014-07-15

    Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB{sub C}saM{sub G}AP32 (GAP32), which possesses the second largest sequenced phage genome (358,663 bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB{sub K}leM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily. - Highlights: • Cronobacter sakazakii phage vB{sub C}saM{sub G}AP32 has a genome of 358,663 bp. • It encodes 545 proteins which is more than Mycoplasma genitalium G37. • It is a member of the Myoviridae. • It is peripherally related to coliphage PBECO4 and Klebsiella phage vB{sub K}leM-RaK2. • GAP32 encodes a chromosome condensation protein.

  16. Duplex Real-Time PCR Method for the Differentiation of Cronobacter sakazakii and Cronobacter malonaticus.

    PubMed

    Li, Xiaofang; Cui, Jinghua; DU, Xiaoli; Cui, Zhigang; Huang, Yibing; Kan, Biao

    2017-01-01

    Cronobacter sakazakii and Cronobacter malonaticus are the most common species of Cronobacter , so it is necessary to detect the two species as soon as possible in surveillance programs. We developed a real-time PCR method for identifying C. sakazakii and C. malonaticus from the genus Cronobacter . In this study, the two pairs of primers and probes were designed, targeting 16S rRNA and fusA, respectively. The specificity of the real-time PCR assay was validated with 112 strains of Cronobacter , including 56 C. sakazakii , 32 C. malonaticus , 16 Cronobacter dublinensis , 6 Cronobacter turicensis , and 2 Cronobacter muytjensii . The results showed that C. sakazakii and C. malonaticus were all correctly identified, consistent with the results of another method by analyzing the clustering of the fusA sequence. The detection limit for pure culture was 10(2) CFU/ml and 10(3) CFU/g for artificially contaminated rehydrated powdered infant formula. Therefore, the developed real-time PCR was a rapid, sensitive, and reliable method for the identification of C. sakazakii and C. malonaticus .

  17. Cronobacter ('Enterobacter sakazakii'): current status and future prospects.

    PubMed

    Chenu, J W; Cox, J M

    2009-08-01

    The genus Cronobacter accommodates the 16 biogroups of the emerging opportunistic pathogen known formerly as Enterobacter sakazakii. Cronobacter spp. are occasional contaminants of milk powder and, consequently, powdered infant formula and represent a significant health risk to neonates. This review presents current knowledge of the food safety aspects of Cronobacter, particularly in infant formula milk powder. Sources of contamination, ecology, disease characteristics and risk management strategies are discussed. Future directions for research are indicated, with a particular focus on the management of this increasingly important bacterium in the production environment.

  18. Isolation of Cronobacter spp. (Enterobacter Sakazakii) from Artisanal Mozzarella

    PubMed Central

    Rippa, Paola; Battaglia, Luciana; Parisi, Nicola

    2014-01-01

    Cronobacter spp. (Enterobacter sakazakii) is an opportunistic bacterial pathogen capable of causing disease and even fatalities in newborn infants within the first weeks of life if consumed as part of the diet. Premature and immunocompromised newborn infants are at particular risk. The microorganism has been isolated from a variety of foods including contaminated infant milk formula powder and milk powder substitute. The study aimed to evaluate the level of microbiological contamination in 47 samples of mozzarella cheese made with cow’s milk collected from artisan cheese producers in Southern Italy. Samples were collected from commercial sales points and underwent qualitative and quantitative microbiological analyses to test for the bacterial contaminants most commonly found in milk and cheese products. The 47 samples underwent qualitative and quantitative microbiological tests according to ISO UNI EN standards. Analyses focused on Staphylococcus aures, Salmonella spp., Listeria monocytogenes, Pseudomonas spp., E. coli, Yersinia spp., total coliforms and Cronobacter sakazakii. The ISO/TS 22964:2006 method was used to investigate possible contamination by C. sakazakii. Biochemical identification was carried out using an automated system for identification and susceptibility tests. None of the samples examined resulted positive for Salmonella spp. or Listeria spp. Only one sample resulted positive for Staphylococcus aureus. Pseudomonas spp. was isolated in 10 (21%) of 47 samples. High levels of total coliforms were found in 10 of 47 samples. Cronobacter spp. (Enterobacter sakazakii) was isolated in one sample. This is the first study to confirm isolation of C. sakazakii in artisan mozzarella cheese made from cow’s milk. The presence of C. sakazakii could be related to external contamination during the phases of production or to the use of contaminated milk. Since mozzarella is recommended in the diet of children and adults of all ages, this present study helps

  19. Inhibition of Cronobacter sakazakii Virulence Factors by Citral

    PubMed Central

    Shi, Chao; Sun, Yi; Liu, Zhiyuan; Guo, Du; Sun, Huihui; Sun, Zheng; Chen, Shan; Zhang, Wenting; Wen, Qiwu; Peng, Xiaoli; Xia, Xiaodong

    2017-01-01

    Cronobacter sakazakii is a foodborne pathogen associated with fatal forms of necrotizing enterocolitis, meningitis and sepsis in neonates and infants. The aim of this study was to determine whether citral, a major component of lemongrass oil, could suppress putative virulence factors of C. sakazakii that contribute to infection. Sub-inhibitory concentrations of citral significantly decreased motility, quorum sensing, biofilm formation and endotoxin production. Citral substantially reduced the adhesion and invasion of C. sakazakii to Caco-2 cells and decreased bacterial survival and replication within the RAW 264.7 macrophage cells. Citral also repressed the expression of eighteen genes involved in the virulence. These findings suggest that citral has potential to be developed as an alternative or supplemental agent to mitigate the infections caused by C. sakazakii. PMID:28233814

  20. Genes Involved in Cronobacter sakazakii Biofilm Formation ▿

    PubMed Central

    Hartmann, Isabel; Carranza, Paula; Lehner, Angelika; Stephan, Roger; Eberl, Leo; Riedel, Kathrin

    2010-01-01

    Cronobacter spp. are opportunistic food-borne pathogens that can cause severe and sometimes lethal infections in neonates. In some outbreaks, the sources of infection were traced to contaminated powdered infant formula (PIF) or contaminated utensils used for PIF reconstitution. In this study, we investigated biofilm formation in Cronobacter sakazakii strain ES5. To investigate the genetic basis of biofilm formation in Cronobacter on abiotic surfaces, we screened a library of random transposon mutants of strain ES5 for reduced biofilm formation using a polystyrene microtiter assay. Genetic characterization of the mutants led to identification of genes that are associated with cellulose biosynthesis and flagellar structure and biosynthesis and genes involved in basic cellular processes and virulence, as well as several genes whose functions are currently unknown. In two of the mutants, hypothetical proteins ESA_00281 and ESA_00282 had a strong impact on flow cell biofilm architecture, and their contribution to biofilm formation was confirmed by genetic complementation. In addition, adhesion of selected biofilm formation mutants to Caco-2 intestinal epithelial cells was investigated. Our findings suggest that flagella and hypothetical proteins ESA_00281 and ESA_00282, but not cellulose, contribute to adhesion of Cronobacter to this biotic surface. PMID:20118366

  1. Antibacterial activities of plant-derived compounds and essential oils toward Cronobacter sakazakii and Cronobacter malonaticus.

    PubMed

    Fraňková, Adéla; Marounek, Milan; Mozrová, Věra; Weber, Jaroslav; Klouček, Pavel; Lukešová, Daniela

    2014-10-01

    Cronobacter sakazakii and C. malonaticus are opportunistic pathogens that cause infections in children and immunocompromised adults. In the present study, the antibacterial activity of 19 plant-derived compounds, 5 essential oils, and an extract of propolis were assessed against C. sakazakii and C. malonaticus. The effects of most of these antimicrobials have not been reported previously. Both strains were susceptible to thymol, carvacrol, thymoquinone, p-cymene, linalool, camphor, citral, eugenol, and trans-cinnamaldehyde as well as cinnamon, lemongrass, oregano, clove, and laurel essential oils; their minimum inhibitory concentrations varied between 0.1 and 2.0 mg/mL. As an alternative treatment method, vapors of the volatiles were tested as an indirect treatment. Vapors of trans-cinnamaldehyde, eugenol, oregano, and cinnamon essential oils inhibited both tested strains, while vapors of linalool were only active against C. sakazakii. To our knowledge, this study is the first time that the inhibitory activity of the vapors of these compounds and essential oils has been reported against Cronobacter spp.

  2. The phenotypic and genotypic characterization of Korean isolates of Cronobacter spp. (Enterobacter sakazakii).

    PubMed

    Kim, Jung-Beom; Kang, Suk-Ho; Park, Yong-Bae; Choi, Jae-Ho; Park, Sung-Jin; Cho, Seung-Hak; Park, Mi-Sun; Lee, Hae Kyung; Choi, Na-Jung; Kim, Ha-Na; Oh, Deog-Hwan

    2011-05-01

    This study was conducted to investigate the phenotypic and genotypic characteristics of Korean isolates of Cronobacter spp. (Enterobacter sakazakii). A total of 43 Cronobacter spp., including 5 clinical isolates, 34 food isolates, 2 environmental isolates, and 2 reference strains (C. sakazakii ATCC 29004 and C. muytjensii ATCC51329) were used in this study. Korean isolates of Cronobacter spp. were divided into 11 biogroups according to their biochemical profiles and 3 genomic groups based on the analysis of their 16S rRNA gene sequences. Biogroups 1 and 2 contained the majority of isolates (n=26), most of which were contained in 16S rRNA cluster 1 (n=34). Korean isolates of Cronobacter spp. showed diverse biochemical profiles. Biogroup 1 contained C. sakazakii GIHE (Gyeonggido Research Institute of Health and Environment) 1 and 2, which were isolated from babies that exhibited symptoms of Cronobacter spp. infection such as gastroenteritis, sepsis, and meningitis. Our finding revealed that Biogroup 1, C. sakazakii, is more prevalent and may be a more pathogenic biogroup than other biogroups, but the pathogenic biogroup was not represented clearly among the 11 biogroups tested in this study. Thus, all biogroups of Cronobacter spp. were recognized as pathogenic bacteria, and the absence of Cronobacter spp. in infant foods should be constantly regulated to prevent food poisoning and infection caused by Cronobacter spp.

  3. Reevaluation of a Suspected Cronobacter sakazakii Outbreak in Mexico.

    PubMed

    Jackson, Emily E; Flores, Julio Parra; Fernández-Escartín, Eduardo; Forsythe, Stephen J

    2015-06-01

    In 2010, two infants became ill at a hospital in Mexico. Subsequently, a range of clinical, environmental, and powdered and rehydrated infant formula isolates were identified by using a combination of phenotyping and PCR probes. The strains were clustered according to pulsed-field gel electrophoresis. The causative agent was reported as Cronobacter sakazakii, with powdered infant formula (PIF) identified as the likely source of the infections. This new study further characterized the isolates from this outbreak by using multilocus sequence typing and whole genome sequencing of selected strains. Though four PIF isolates and one hospital environmental isolate were identified as C. sakazakii sequence type 297 by multilocus sequence typing, they were isolated 6 months prior to the outbreak. Genotypic analyses of patient isolates identified them as Enterobacter hormaechei and Enterobacter spp. The pulsed-field gel electrophoresis profile of the Enterobacter spp. isolates matched those of isolates from previously unopened tins of PIF. E. hormaechei was only isolated from the two infants and not PIF. The reevaluation of this outbreak highlights the need for accurate detection and identification assays, particularly during outbreak investigations in which incorrect identifications may mislead the investigation and attribution of the source. Though the species responsible for the symptoms could not be determined, this outbreak demonstrated the possible transmission of Enterobacter spp. from PIF to infants. These are possibly the first reported cases of Enterobacter spp. infection of infants from bacterial-contaminated PIF.

  4. Nonthermal Inactivation of Cronobacter sakazakii in Infant Formula Milk: A Review.

    PubMed

    Pina-Pérez, M C; Rodrigo, D; Martínez, A

    2016-07-26

    Up-to-date, nonthermal technologies and combinations of them, in accordance with the "hurdle technology" concept, are being applied by different research groups in response to calls by the International Food and Human Health Organizations (ESPGHAN, 2004; FAO/WHO, 2006, 2008) for alternatives to thermal control of Cronobacter sakazakii in reconstituted powdered infant formula milk. This review highlights (i) current knowledge on the application of nonthermal technologies to control C. sakazakii in infant formula milk and (ii) the importance of the application of nonthermal technologies for the control of C. sakazakii as part of the development of strategies in the context of improving food safety and quality of this product.

  5. Growth kinetics and model comparison of cronobacter sakazakii in reconstituted powdered infant formula

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cronobacter sakazakii is a life-threatening bacterium, primarily implicated in illnesses associated with the consumption of powdered infant formula (PIF). It can cause rare but invasive infections, leading to sepsis, meningitis, or necrotizing enterocolitis in infants fed with contaminated PIF. Th...

  6. Cronobacter sakazakii clinical isolates overcome host barriers and evade the immune response.

    PubMed

    Almajed, Faisal S; Forsythe, Stephen J

    2016-01-01

    Cronobacter sakazakii is the most frequently clinically isolated species of the Cronobacter genus. However the virulence factors of C. sakazakii including their ability to overcome host barriers remains poorly studied. In this study, ten clinical isolates of C. sakazakii were assessed for their ability to invade and translocate through human colonic carcinoma epithelial cells (Caco-2) and human brain microvascular endothelial cells (HBMEC). Their ability to avoid phagocytosis in human macrophages U937 and human brain microglial cells was investigated. Additionally, they were tested for serum sensitivity and the presence of the Cronobacter plasminogen activation gene (cpa) gene, which is reported to confer serum resistance. Our data showed that the clinical C. sakazakii strains invaded and translocated through Caco-2 and HBMEC cell lines and some strains showed significantly higher levels of invasion and translocation. Moreover, C. sakazakii was able to persist and even multiply in phagocytic macrophage and microglial cells. All strains, except one, were able to withstand human serum exposure, the single serum sensitive strain was also the only one which did not encode for the cpa gene. These results demonstrate that C. sakazakii clinical isolates are able to overcome host barriers and evade the host immune response indicating their capacity to cause diseases such as necrotizing enterocolitis (NEC) and meningitis. Our data showed for the first time the ability of C. sakazakii clinical isolates to survive and multiply within human microglial cells. Additionally, it was shown that C. sakazakii clinical strains have the capacity to translocate through the Caco-2 and HBMEC cell lines paracellularly.

  7. Antimicrobial Activity and Possible Mechanism of Action of Citral against Cronobacter sakazakii

    PubMed Central

    Shi, Chao; Song, Kaikuo; Zhang, Xiaorong; Sun, Yi; Sui, Yue; Chen, Yifei; Jia, Zhenyu; Sun, Huihui; Sun, Zheng; Xia, Xiaodong

    2016-01-01

    Citral is a flavor component that is commonly used in food, beverage and fragrance industries. Cronobacter sakazakii is a food-borne pathogen associated with severe illness and high mortality in neonates and infants. The objective of the present study was to evaluate antimicrobial effect of citral against C. sakazakii strains. The minimum inhibitory concentration (MIC) of citral against C. sakazakii was determined via agar dilution method, then Gompertz models were used to quantitate the effect of citral on microbial growth kinetics. Changes in intracellular pH (pHin), membrane potential, intracellular ATP concentration, and membrane integrity were measured to elucidate the possible antimicrobial mechanism. Cell morphology changes were also examined using a field emission scanning electron microscope. The MICs of citral against C. sakazakii strains ranged from 0.27 to 0.54 mg/mL, and citral resulted in a longer lag phase and lower growth rate of C. sakazakii compared to the control. Citral affected the cell membrane of C. sakazakii, as evidenced by decreased intracellular ATP concentration, reduced pHin, and cell membrane hyperpolarization. Scanning electron microscopy analysis further confirmed that C. sakazakii cell membranes were damaged by citral. These findings suggest that citral exhibits antimicrobial effect against C. sakazakii strains and could be potentially used to control C. sakazakii in foods. However, how it works in food systems where many other components may interfere with its efficacy should be tested in future research before its real application. PMID:27415761

  8. Sub-Inhibitory Concentrations of Trans-Cinnamaldehyde Attenuate Virulence in Cronobacter sakazakii in Vitro

    PubMed Central

    Amalaradjou, Mary Anne Roshni; Kim, Kwang Sik; Venkitanarayanan, Kumar

    2014-01-01

    Cronobacter sakazakii is a foodborne pathogen, which causes a life-threatening form of meningitis, necrotizing colitis and meningoencephalitis in neonates and children. Epidemiological studies implicate dried infant formula as the principal source of C. sakazakii. In this study, we investigated the efficacy of sub-inhibitory concentrations (SIC) of trans-cinnamaldehyde (TC), an ingredient in cinnamon, for reducing C. sakazakii virulence in vitro using cell culture, microscopy and gene expression assays. TC significantly (p ≤ 0.05) suppressed C. sakazakii adhesion to and invasion of human and rat intestinal epithelial cells, and human brain microvascular endothelial cells. In addition, TC inhibited C. sakazakii survival and replication in human macrophages. We also observed that TC reduced the ability of C. sakazakii to cause cell death in rat intestinal cells, by inhibiting nitric oxide production. Results from gene expression studies revealed that TC significantly downregulated the virulence genes critical for motility, host tissue adhesion and invasion, macrophage survival, and LPS (Lipopolysaccharide) synthesis in C. sakazakii. The efficacy of TC in attenuating these major virulence factors in C. sakazakii underscores its potential use in the prevention and/or control of infection caused by this pathogen. PMID:24837831

  9. Effect of trans-cinnamaldehyde on reducing resistance to environmental stresses in Cronobacter sakazakii.

    PubMed

    Amalaradjou, Mary Anne Roshni; Venkitanarayanan, Kumar

    2011-03-01

    Cronobacter sakazakii is an emerging foodborne pathogen transmitted exclusively through contaminated infant formula (IFM), and associated with life-threatening infections in infants. C. sakazakii has the ability to tolerate a variety of environmental stress conditions, including heat stress, acidity, high osmotic pressure, and desiccation. In this study, we investigated the efficacy of a subinhibitory concentration (750 μM) of trans-cinnamaldehyde (TC), an ingredient in cinnamon, for reducing C. sakazakii's tolerance to these environmental stresses. Three strains of TC-treated C. sakazakii were separately subjected to high temperature (50°C, 55°C, and 60°C), acidic pH (3.3), high osmotic pressure (a(w) 0.81), and desiccation. TC (750 μM) substantially (p < 0.05) compromised stress tolerance of C. sakazakii compared to C. sakazakii cells not exposed to TC. Real-time quantitative polymerase chain reaction results revealed that TC significantly (p < 0.05) downregulated C. sakazakii genes critical for stress tolerance and survival, including rpoS, chaperonins, phoP/Q, outer membrane porins, and osmolyte transporter genes. The efficacy of TC in reducing C. sakazakii stress tolerance underscores its potential use for controlling the pathogen by increasing its susceptibility to commonly applied hurdles in food processing.

  10. Sub-inhibitory concentrations of trans-cinnamaldehyde attenuate virulence in Cronobacter sakazakii in vitro.

    PubMed

    Amalaradjou, Mary Anne Roshni; Kim, Kwang Sik; Venkitanarayanan, Kumar

    2014-05-15

    Cronobacter sakazakii is a foodborne pathogen, which causes a life-threatening form of meningitis, necrotizing colitis and meningoencephalitis in neonates and children. Epidemiological studies implicate dried infant formula as the principal source of C. sakazakii. In this study, we investigated the efficacy of sub-inhibitory concentrations (SIC) of trans-cinnamaldehyde (TC), an ingredient in cinnamon, for reducing C. sakazakii virulence in vitro using cell culture, microscopy and gene expression assays. TC significantly (p ≤ 0.05) suppressed C. sakazakii adhesion to and invasion of human and rat intestinal epithelial cells, and human brain microvascular endothelial cells. In addition, TC inhibited C. sakazakii survival and replication in human macrophages. We also observed that TC reduced the ability of C. sakazakii to cause cell death in rat intestinal cells, by inhibiting nitric oxide production. Results from gene expression studies revealed that TC significantly downregulated the virulence genes critical for motility, host tissue adhesion and invasion, macrophage survival, and LPS (Lipopolysaccharide) synthesis in C. sakazakii. The efficacy of TC in attenuating these major virulence factors in C. sakazakii underscores its potential use in the prevention and/or control of infection caused by this pathogen.

  11. International Life Science Institute North America Cronobacter (Formerly Enterobacter sakazakii) isolate set.

    PubMed

    Ivy, Reid A; Farber, Jeffrey M; Pagotto, Franco; Wiedmann, Martin

    2013-01-01

    Foodborne pathogen isolate collections are important for the development of detection methods, for validation of intervention strategies, and to develop an understanding of pathogenesis and virulence. We have assembled a publicly available Cronobacter (formerly Enterobacter sakazakii) isolate set that consists of (i) 25 Cronobacter sakazakii isolates, (ii) two Cronobacter malonaticus isolates, (iii) one Cronobacter muytjensii isolate, which displays some atypical phenotypic characteristics, biochemical profiles, and colony color on selected differential media, and (iv) two nonclinical Enterobacter asburiae isolates, which show some phenotypic characteristics similar to those of Cronobacter spp. The set consists of human (n = 10), food (n = 11), and environmental (n = 9) isolates. Analysis of partial 16S rDNA sequence and seven-gene multilocus sequence typing data allowed for reliable identification of these isolates to species and identification of 14 isolates as sequence type 4, which had previously been shown to be the most common C. sakazakii sequence type associated with neonatal meningitis. Phenotypic characterization was carried out with API 20E and API 32E test strips and streaking on two selective chromogenic agars; isolates were also assessed for sorbitol fermentation and growth at 45°C. Although these strategies typically produced the same classification as sequence-based strategies, based on a panel of four biochemical tests, one C. sakazakii isolate yielded inconclusive data and one was classified as C. malonaticus. EcoRI automated ribotyping and pulsed-field gel electrophoresis (PFGE) with XbaI separated the set into 23 unique ribotypes and 30 unique PFGE types, respectively, indicating subtype diversity within the set. Subtype and source data for the collection are publicly available in the PathogenTracker database (www. pathogentracker. net), which allows for continuous updating of information on the set, including links to publications that include

  12. Cronobacter sakazakii DNA Detection in Cerebrospinal Fluid of a Patient with Amyotrophic Lateral Sclerosis Mimic Syndrome

    PubMed Central

    Piombo, Marianna; Chiarello, Daniela; Corbetto, Marzia; Di Pino, Giovanni; Dicuonzo, Giordano; Angeletti, Silvia; Riva, Elisabetta; De Florio, Lucia; Capone, Fioravante; Di Lazzaro, Vincenzo

    2015-01-01

    A 45-year-old male noticed progressive weakness of the right lower limb with gait disturbance. Over the following months, motor deficits worsened, spreading to the right upper limb. Electromyography showed active denervation in the upper and lower limb muscles. A diagnosis of amyotrophic lateral sclerosis (ALS) was made. About 2 years after symptom onset, gradual improvement occurred. Cerebrospinal fluid analysis performed about 3 years after the beginning of symptoms identified Cronobacter sakazakii. Since no other possible causes were identified, we suggest that an almost completely reversible ALS-like syndrome had been triggered by Cronobacter infection in our immunocompetent patient. PMID:26955334

  13. Inhibition of Cronobacter sakazakii by Lactobacillus acidophilus n.v. Er2 317/402

    PubMed Central

    Lee, Jong Suk; Lee, Junsoo

    2016-01-01

    Lactobacillus acidophilus n.v. Er2 317/402 strain Narine is known as a health beneficial functional probiotic culture and supplementary source of nutrition for newborns. In this study, in vitro antimicrobial activities of Narine-lyophilized (Narine-L), Narine-heat treated (Narine-HT), and Narine crude cell-free extract (Narine-CCFE) were evaluated against pathogen Cronobacter sakazakii (C. sakazakii) in agar as well as in a reconstituted powdered infant formula (RPIF) model. Inhibition zones of 30 mg Narine-L and Narine-HT were both 150 U, whereas inhibition zone of 30 mg Narine-CCFE was 200 U. Narine-L (1 g) and Narine-HT (1 g) were added to 10 mL of artificially contaminated RPIF, respectively, containing 100 μL of C. sakazakii (1.62×108 colony forming unit (CFU)/mL). After treatment with Narine-L and Narine-HT for 3 h and 6 h at 37℃, less than ≤107 CFU/mL of C. sakazakii was detected in RPIF. Without Narine-L and Narine-HT treatment, the population of C. sakazakii increased up to 5.36×109 CFU/mL after 6 h. Examination by transmission electron microscopy confirmed C. sakazakii cells were damaged by Narine-CCFE. Thus, employing Narine culture as a natural and safe bio-preservative may protect infants from C. sakazakii. PMID:27857539

  14. The taxonomy of Enterobacter sakazakii: proposal of a new genus Cronobacter gen. nov. and descriptions of Cronobacter sakazakii comb. nov. Cronobacter sakazakii subsp. sakazakii, comb. nov., Cronobacter sakazakii subsp. malonaticus subsp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov. and Cronobacter genomospecies 1

    PubMed Central

    Iversen, Carol; Lehner, Angelika; Mullane, Niall; Bidlas, Eva; Cleenwerck, Ilse; Marugg, John; Fanning, Séamus; Stephan, Roger; Joosten, Han

    2007-01-01

    Background Enterobacter sakazakii is an opportunistic pathogen that can cause infections such as necrotizing enterocolitis, bacteraemia, meningitis and brain abscess/lesions. When the species was defined in 1980, 15 biogroups were described and it was suggested that these could represent multiple species. In this study the taxonomic relationship of strains described as E. sakazakii was further investigated. Results Strains identified as E. sakazakii were divided into separate groups on the basis of f-AFLP fingerprints, ribopatterns and full-length 16S rRNA gene sequences. DNA-DNA hybridizations revealed five genomospecies. The phenotypic profiles of the genomospecies were determined and biochemical markers identified. Conclusion This study clarifies the taxonomy of E. sakazakii and proposes a reclassification of these organisms. PMID:17439656

  15. Efficiency of bacteriophage therapy against Cronobacter sakazakii in Galleria mellonella (greater wax moth) larvae.

    PubMed

    Abbasifar, Reza; Kropinski, Andrew M; Sabour, Parviz M; Chambers, James R; MacKinnon, Joanne; Malig, Thomas; Griffiths, Mansel W

    2014-09-01

    Cronobacter sakazakii, an opportunistic pathogen found in milk-based powdered infant formulae, has been linked to meningitis in infants, with high fatality rates. A set of phages from various environments were purified and tested in vitro against strains of C. sakazakii. Based on host range and lytic activity, the T4-like phage vB_CsaM_GAP161, which belongs to the family Myoviridae, was selected for evaluation of its efficacy against C. sakazakii. Galleria mellonella larvae were used as a whole-animal model for pre-clinical testing of phage efficiency. Twenty-one Cronobacter strains were evaluated for lethality in G. mellonella larvae. Different strains of C. sakazakii caused 0 to 98% mortality. C. sakazakii 3253, with an LD50 dose of ~2.0×10(5) CFU/larva (24 h, 37 °C) was selected for this study. Larvae infected with a dose of 5×LD50 were treated with phage GAP161 (MOI=8) at various time intervals. The mortality rates were as high as 100% in the groups injected with bacteria only, compared to 16.6% in the group infected with bacteria and treated with phage. Phage GAP161 showed the best protective activity against C. sakazakii when the larvae were treated prior to or immediately after infection. The results obtained with heat-inactivated phage proved that the survival of the larvae is not due to host immune stimulation. These results suggest that phage GAP161 is potentially a useful control agent against C. sakazakii. In addition, G. mellonella may be a useful whole-animal model for pre-screening phages for efficacy and safety prior to clinical evaluation in mammalian models.

  16. Cronobacter sakazakii: stress survival and virulence potential in an opportunistic foodborne pathogen.

    PubMed

    Feeney, Audrey; Kropp, Kai A; O'Connor, Roxana; Sleator, Roy D

    2014-01-01

    A characteristic feature of the opportunistic foodborne pathogen Cronobacter sakazakii is its ability to survive in extremely arid environments, such as powdered infant formula, making it a dangerous opportunistic pathogen of individuals of all age groups, especially infants and neonates. Herein, we provide a brief overview of the pathogen; clinical manifestations, environmental reservoirs and our current understanding of stress response mechanisms and virulence factors which allow it to cause disease.

  17. Cronobacter sakazakii: stress survival and virulence potential in an opportunistic foodborne pathogen

    PubMed Central

    Feeney, Audrey; Kropp, Kai A; O’Connor, Roxana; Sleator, Roy D

    2014-01-01

    A characteristic feature of the opportunistic foodborne pathogen Cronobacter sakazakii is its ability to survive in extremely arid environments, such as powdered infant formula, making it a dangerous opportunistic pathogen of individuals of all age groups, especially infants and neonates. Herein, we provide a brief overview of the pathogen; clinical manifestations, environmental reservoirs and our current understanding of stress response mechanisms and virulence factors which allow it to cause disease. PMID:25562731

  18. Cronobacter sakazakii infection alters serotonin transporter and improved fear memory retention in the rat

    PubMed Central

    Sivamaruthi, Bhagavathi S.; Madhumita, Rajkumar; Balamurugan, Krishnaswamy; Rajan, Koilmani E.

    2015-01-01

    It is well established that Cronobacter sakazakii infection cause septicemia, necrotizing enterocolitis and meningitis. In the present study, we tested whether the C. sakazakii infection alter the learning and memory through serotonin transporter (SERT). To investigate the possible effect on SERT, on postnatal day-15 (PND-15), wistar rat pups were administered with single dose of C. sakazakii culture (infected group; 107 CFU) or 100 μL of Luria-Bertani broth (medium control) or without any treatment (naïve control). All the individuals were subjected to passive avoidance test on PND-30 to test their fear memory. We show that single dose of C. sakazakii infection improved fear memory retention. Subsequently, we show that C. sakazakii infection induced the activation of toll-like receptor-3 and heat-shock proteins-90 (Hsp-90). On the other hand, level of serotonin (5-hydroxytryptamine) and SERT protein was down-regulated. Furthermore, we show that C. sakazakii infection up-regulate microRNA-16 (miR-16) expression. The observed results highlight that C. sakazakii infections was responsible for improved fear memory retention and may have reduced the level of SERT protein, which is possibly associated with the interaction of up-regulated Hsp-90 with SERT protein or miR-16 with SERT mRNA. Taken together, observed results suggest that C. sakazakii infection alter the fear memory possibly through SERT. Hence, this model may be effective to test the C. sakazakii infection induced changes in synaptic plasticity through SERT and effect of other pharmacological agents against pathogen induced memory disorder. PMID:26388777

  19. Proteomic analysis of the mode of antibacterial action of trans-cinnamaldehyde against Cronobacter sakazakii 415.

    PubMed

    Amalaradjou, Mary Anne Roshni; Venkitanarayanan, Kumar

    2011-10-01

    Cronobacter sakazakii is an opportunistic foodborne pathogen that contaminates powdered infant formula, causing a rare but life-threatening infection in neonates and infants. Contaminated powdered infant formula represents the only known source of infection in infants. We previously reported that trans-cinnamaldehyde (TC), an ingredient in cinnamon, inactivated C. sakazakii in powdered infant formula. Although the antimicrobial properties of TC have been well established, only limited information is available on its antimicrobial mechanisms, especially at the molecular level. Therefore, we performed a proteomic analysis of the outer membrane and whole cell proteins from TC-treated C. sakazakii to investigate its potential antimicrobial mechanisms against C. sakazakii. The proteomic data revealed that TC exerts antimicrobial effects by several mechanisms, including disruption of carbohydrate, amino acid, and lipid metabolism. Additionally, TC compromises motility, attachment, and invasion ability and cellular defenses of C. sakazakii against oxidative stress, thereby reducing its virulence. The results of this study suggest that TC could be potentially used for controlling C. sakazakii.

  20. The driving force of prophages and CRISPR-Cas system in the evolution of Cronobacter sakazakii

    PubMed Central

    Zeng, Haiyan; Zhang, Jumei; Li, Chensi; Xie, Tengfei; Ling, Na; Wu, Qingping; Ye, Yingwang

    2017-01-01

    Cronobacter sakazakii is an important foodborne pathogens causing rare but life-threatening diseases in neonates and infants. CRISPR-Cas system is a new prokaryotic defense system that provides adaptive immunity against phages, latter play an vital role on the evolution and pathogenicity of host bacteria. In this study, we found that genome sizes of C. sakazakii strains had a significant positive correlation with total genome sizes of prophages. Prophages contributed to 16.57% of the genetic diversity (pan genome) of C. sakazakii, some of which maybe the potential virulence factors. Subtype I-E CRISPR-Cas system and five types of CRISPR arrays were found in the conserved site of C. sakazakii strains. CRISPR1 and CRISPR2 loci with high variable spacers were active and showed potential protection against phage attacks. The number of spacers from two active CRISPR loci in clinical strains was significant less than that of foodborne strains, it maybe a reason why clinical strains were found to have more prophages than foodborne strains. The frequently gain/loss of prophages and spacers in CRISPR loci is likely to drive the quick evolution of C. sakazakii. Our study provides a new insight into the co-evolution of phages and C. sakazakii. PMID:28057934

  1. First clinical isolates of Cronobacter spp. (Enterobacter sakazakii) in Argentina: characterization and subtyping by pulsed-field gel electrophoresis.

    PubMed

    Asato, Valeria C; Vilches, Viviana E; Pineda, María G; Casanueva, Enrique; Cane, Alejandro; Moroni, Mirian P; Brengi, Silvina P; Pichel, Mariana G

    2013-01-01

    Cronobacter species are opportunistic pathogens associated with severe infections in neonates and immunocompromised infants. From January 2009 through September 2010, two cases of neonatal infections associated with Cronobacter malonaticus and one case associated with Cronobacter sakazakii, two of them fatal, were reported in the same hospital. These are the first clinical isolates of Cronobacter spp. in Argentina. The objective of this work was to characterize and subtype clinical isolates of Cronobacter spp. in neonate patients, as well as to establish the genetic relationship between these isolates and the foodborne isolates previously identified in the country. Pulsed-field gel electrophoresis analysis showed a genetic relationship between the C. malonaticus isolates from two patients. Different results were found when the pulsed-field gel electrophoresis patterns of clinical isolates were compared with those deposited in the National Database of Cronobacter spp.

  2. Growth Inhibition of Cronobacter sakazakii in Experimentally Contaminated Powdered Infant Formula by Kefir Supernatant.

    PubMed

    Kim, Dong-Hyeon; Chon, Jung-Whan; Kang, Il-Byeong; Kim, Hyunsook; Kim, Hong-Seok; Song, Kwang-Young; Seo, Kun-Ho

    2015-09-01

    Kefir is a type of fermented milk containing lactic and acetic acid bacteria and yeast. In this study, we evaluated the antimicrobial activity of kefir supernatant against Cronobacter sakazakii in powdered infant formula (PIF). In a spot-on-lawn test, the growth of 20 C. sakazakii strains, including 10 clinical and 10 food isolates, was completely inhibited in the presence of kefir supernatant. Significant differences in the diameters of inhibition zones were observed upon treatment with kefir compared with the results for Lactobacillus kefiri and Candida kefyr culture supernatants or solutions of lactic and acetic acid and ethyl alcohol in the agar well diffusion test (P < 0.05). The addition of 100 μl of kefir supernatant to 1 ml of nutrient broth completely inhibited the growth of C. sakazakii, as evaluated by spectrophotometry. The antimicrobial activity of kefir supernatant in experimentally contaminated PIF was also tested; we found no viable C. sakazakii cells remaining in PIF rehydrated with 30% kefir supernatant solution for 1 h, demonstrating that the antimicrobial activity of kefir supernatant against C. sakazakii could be applied in real food samples.

  3. Cronobacter sakazakii in foods and factors affecting its survival, growth, and inactivation.

    PubMed

    Beuchat, Larry R; Kim, Hoikyung; Gurtler, Joshua B; Lin, Li-Chun; Ryu, Jee-Hoon; Richards, Glenner M

    2009-12-31

    Cronobacter sakazakii has been isolated from a wide range of environmental sources and from several foods of animal and plant origin. While infections caused by C. sakazakii have predominantly involved neonates and infants, its presence on or in foods other than powdered infant formula raises concern about the safety risks these foods pose to immunocompromised consumers. We have done a series of studies to better understand the survival and growth characteristics of C. sakazakii in infant formula, infant cereal, fresh-cut produce, and juices made from fresh produce. Over a 12-month storage period, the pathogen survived better in dried formula and cereal at low a(w) (0.25-0.30) than at high a(w) (0.69-0.82) and at 4 degrees C compared to 30 degrees C. C. sakazakii grows in formulas and cereals reconstituted with water or milk and held at 12-30 degrees C. The composition of formulas or cereals does not markedly affect the rate of growth. C. sakazakii grows well on fresh-cut apple, cantaloupe, watermelon, cabbage, carrot, cucumber, lettuce, and tomato at 25 degrees C and in some types of produce at 12 degrees C. Treatment of fresh fruits and vegetables with sanitizers such as chlorine, chlorine dioxide, and a peroxyacetic acid-based solution causes reductions of 1.6-5.4 log CFU/apple, tomato, and lettuce. Cells of C. sakazakii in biofilms formed on stainless steel and enteral feeding tubes or dried on the surface of stainless steel have increased resistance to disinfectants. Death of cells in biofilms is affected by atmospheric relative humidity. These studies have contributed to a better understanding of the behavior of C. sakazakii in and on foods and on food-contact surfaces, thereby enabling the development of more effective strategies and interventions for its control.

  4. Inhibition of Cronobacter sakazakii Adhesion to Caco-2 Cells by Commercial Dairy Powders and Raw Buttermilk.

    PubMed

    Ripollés, Daniel; Harouna, Saidou; Parrón, José A; Arenales, Irene; Calvo, Miguel; Pérez, María D; Sánchez, Lourdes

    2017-02-08

    Cronobacter sakazakii is a foodborne pathogen that has been associated with severe infections, mainly in neonates. The binding of this bacterium to host cell surfaces represents the first step in the pathogenesis of disease. An ELISA-based assay has been developed using a polyclonal antiserum against C. sakazakii to determine its adhesion to Caco-2 cells. The antiserum used recognized many of the outer membrane proteins of C. sakazakii. A positive correlation was found between the absorbance values obtained by ELISA and the number of bacteria adhered to cells determined by plate counting. The inhibitory effect on bacterial adhesion to cells observed with some dairy products was concentration-dependent. Commercial buttermilk caused the maximal reduction of the adhesion percentage (33.0 ± 5.07) at the highest concentration assayed (20 mg/mL), followed by butter serum (31.9 ± 5.36), skim milk (30.4 ± 5.07), and raw buttermilk (25.6 ± 3.80). In some cases, significant differences (p < 0.05) were found in the inhibition exerted by the different products evaluated. The results obtained in this study demonstrate that dairy products contain some components with the ability to inhibit the adhesion of C. sakazakii to Caco-2 cells.

  5. Antibiotic and Desiccation Resistance of Cronobacter sakazakii and C. malonaticus Isolates from Powdered Infant Formula and Processing Environments

    PubMed Central

    Fei, Peng; Jiang, Yujun; Feng, Jing; Forsythe, Stephen J.; Li, Ran; Zhou, Yanhong; Man, Chaoxin

    2017-01-01

    This study evaluated the antimicrobial and desiccation resistance of Cronobacter sakazakii and Cronobacter malonaticus isolates from powdered infant formula and processing environments. The antimicrobial susceptibility tests showed that the 70 Cronobacter strains, representing 19 sequence types, were susceptible to the most of the antibiotics except for amoxicillin-clavulanate, ampicillin, and cefazolin. Furthermore, the growth of six C. sakazakii and two C. malonaticus strains from different sequence types (STs) in hyperosmotic media was measured. The growth of the two C. sakazakii strains (CE1 and CE13) from the neonatal pathovars ST4 and ST8, were significantly higher (p < 0.05) than that of other strains. C. malonaticus strain CM35 (ST201) was the slowest grower in all strains, and most could not grow in more than 8% NaCl solution. Also the survival of these strains under desiccation conditions was followed for 1 year. The viable count of Cronobacter spp. under desiccation conditions was reduced on average by 3.02 log cycles during 1 year, with CE13 (ST8) being the most desiccation resistant strain. These results will improve our understanding of the persistence of the two closely related species C. sakazakii and C. malonaticus which are of concern for neonatal and adult health. PMID:28303125

  6. Antibiotic and Desiccation Resistance of Cronobacter sakazakii and C. malonaticus Isolates from Powdered Infant Formula and Processing Environments.

    PubMed

    Fei, Peng; Jiang, Yujun; Feng, Jing; Forsythe, Stephen J; Li, Ran; Zhou, Yanhong; Man, Chaoxin

    2017-01-01

    This study evaluated the antimicrobial and desiccation resistance of Cronobacter sakazakii and Cronobacter malonaticus isolates from powdered infant formula and processing environments. The antimicrobial susceptibility tests showed that the 70 Cronobacter strains, representing 19 sequence types, were susceptible to the most of the antibiotics except for amoxicillin-clavulanate, ampicillin, and cefazolin. Furthermore, the growth of six C. sakazakii and two C. malonaticus strains from different sequence types (STs) in hyperosmotic media was measured. The growth of the two C. sakazakii strains (CE1 and CE13) from the neonatal pathovars ST4 and ST8, were significantly higher (p < 0.05) than that of other strains. C. malonaticus strain CM35 (ST201) was the slowest grower in all strains, and most could not grow in more than 8% NaCl solution. Also the survival of these strains under desiccation conditions was followed for 1 year. The viable count of Cronobacter spp. under desiccation conditions was reduced on average by 3.02 log cycles during 1 year, with CE13 (ST8) being the most desiccation resistant strain. These results will improve our understanding of the persistence of the two closely related species C. sakazakii and C. malonaticus which are of concern for neonatal and adult health.

  7. Inhibition of Cronobacter sakazakii by heat labile bacteriocins produced by probiotic LAB isolated from healthy infants.

    PubMed

    Awaisheh, Saddam S; Al-Nabulsi, Anas A; Osaili, Tareq M; Ibrahim, Salam; Holley, Richard

    2013-09-01

    Cronobacter sakazakii is an opportunistic pathogen that can cause bacteremia, meningitis, and necrotizing enterocolitis, most often in neonates with case-fatality rates that may reach 80%. The antimicrobial activity of lactic acid bacteria against a wide range of foodborne pathogens is well-established in different types of food products. The objective of the current study was to investigate the antibacterial activity of Lactobacillus acidophilus and L. casei isolated from feces of healthy infants against different strains of C. sakazakii in agar and a rehydrated infant milk formula (RIMF) model. The inhibition zones of C. sakazakii around L. acidophilus or L. casei ranged from 22 to 32 mm on eMan Rogosa Sharpe (MRS) agar under aerobic conditions, while a slight reduction in antibacterial activity was noted on modified MRS (0.2% glucose) under anaerobic conditions. It was observed that pH-neutralized cell-free supernatant (CFS) of L. acidophilus or L. casei was inhibitory against tested C. sakazakii strains. The inhibition zones of neutralized CFS were lower than the antibacterial activities of live cultures. The antibacterial activity of CFS was abolished when CFS from L. acidophilus or L. casei was heated at 60 or 80 °C for either 10 min or 2 h, or treated with trypsin or pepsin. This was considered strong evidence that the inhibition was due to the production of bacteriocins by L. casei and L. acidophilus. Both the CFS and active growing cells of L. casei and L. acidophilus were able to reduce the viability of C. sakazakii in the RIMF model. The results may extend the use of natural antimicrobials instead of conventional preservation methods to improve the safety of RIMF.

  8. Rapid biodegradation of polycyclic aromatic hydrocarbons (PAHs) using effective Cronobacter sakazakii MM045 (KT933253).

    PubMed

    Umar, Zubairu Darma; Aziz, Nor Azwady Abd; Zulkifli, Syaizwan Zahmir; Mustafa, Muskhazli

    2017-01-01

    Polycyclic Aromatic Hydrocarbons (PAHs) are complex and widely distributed environmental pollutants that can affect living ecosystems. This study was conducted to rapidly degrade phenanthrene and pyrene representing low and high molecular weight of PAHs, respectively. Cronobacter sakazakii MM045 (KT933253) was identified from used engine oil of contaminated soil. PAHs biodegradation was carried out using 2,6-dichlorophenol indophenol (DCPIP) assay. Biodegradation influencing factors including agitation, temperature, pH, inoculums volume and salinity were enhanced using Response Surface Methodology (RSM) by Central Composite Design (CCD). Phenanthrene and pyrene biodegrading metabolites were identified using gas chromatography mass spectrophotometer (GCMS). •Initial biodegradation indicated 75.2% and 54.3% phenanthrene and pyrene degraded by C. sakazakii MM045 within 24 h. After CCD optimisation, 100% degradation was achieved for each of the phenanthrene and pyrene, resulting in the formation of intermediate metabolites.•The identified phenanthrene metabolites were 3,4-dihydroxyphenathrene, phthalic acid, pyruvic acid, acetic acid and oxalic acid. Pyrene intermediates comprised pyrene cis-4,5-dihydrodiol, 3,4-dihydroxyphenanthrene, phthalic acid, pyruvic acid, acetic acid and lactic acid.•Cronbacter sakazakii MM045 was proven to be rapid and effective in degrading PAHs within 24 h despite the unavailability of existing literatures on PAHs biodegradation.

  9. Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems.

    PubMed

    Zuber, Sophie; Boissin-Delaporte, Catherine; Michot, Lise; Iversen, Carol; Diep, Benjamin; Brüssow, Harald; Breeuwer, Pieter

    2008-11-01

    Enterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen, which can cause rare, but life-threatening infections in neonates and infants through feeding of a contaminated milk formula. We isolated 67 phages from environmental samples and tested their lytic host range on a representative collection of 40 E. sakazakii strains. A cocktail of five phages prevented the outgrowth of 35 out of 40 test strains in artificially contaminated infant formula. Two E. sakazakii phages represented prolate head Myoviridae. Molecular tests identified them as close relatives of Escherichia coli phage T4. The remaining three phages represented isometric head Myoviridae with large genome size of 140 and 200 kb, respectively, which belonged to two different DNA hybridization groups. A high dose of 10(8) pfu ml(-1) of phage could effectively sterilize a broth contaminated with both high and low pathogen counts (10(6) and 10(2) cfu ml(-1)). In contrast, broth inoculated with 10(4) phage and 10(2) bacteria per ml first showed normal bacterial growth until reaching a cell titre of 10(5) cfu ml(-1). Only when crossing this threshold, phage replication started, but it could not reduce the contamination level below 100 cfu ml(-1). Phages could be produced with titres of 10(10) pfu ml(-1) in broth culture, but they were not stable upon freeze-drying. Addition of trehalose or milk formula stabilized the phage preparation, which then showed excellent storage stability even at elevated temperature.

  10. Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems

    PubMed Central

    Zuber, Sophie; Boissin‐Delaporte, Catherine; Michot, Lise; Iversen, Carol; Diep, Benjamin; Brüssow, Harald; Breeuwer, Pieter

    2008-01-01

    Summary Enterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen, which can cause rare, but life‐threatening infections in neonates and infants through feeding of a contaminated milk formula. We isolated 67 phages from environmental samples and tested their lytic host range on a representative collection of 40 E. sakazakii strains. A cocktail of five phages prevented the outgrowth of 35 out of 40 test strains in artificially contaminated infant formula. Two E. sakazakii phages represented prolate head Myoviridae. Molecular tests identified them as close relatives of Escherichia coli phage T4. The remaining three phages represented isometric head Myoviridae with large genome size of 140 and 200 kb, respectively, which belonged to two different DNA hybridization groups. A high dose of 108 pfu ml−1 of phage could effectively sterilize a broth contaminated with both high and low pathogen counts (106 and 102 cfu ml−1). In contrast, broth inoculated with 104 phage and 102 bacteria per ml first showed normal bacterial growth until reaching a cell titre of 105 cfu ml−1. Only when crossing this threshold, phage replication started, but it could not reduce the contamination level below 100 cfu ml−1. Phages could be produced with titres of 1010 pfu ml−1 in broth culture, but they were not stable upon freeze‐drying. Addition of trehalose or milk formula stabilized the phage preparation, which then showed excellent storage stability even at elevated temperature. PMID:21261874

  11. Cronobacter sakazakii ATCC 29544 Autoaggregation Requires FliC Flagellation, Not Motility

    PubMed Central

    Hoeflinger, Jennifer L.; Miller, Michael J.

    2017-01-01

    Cronobacter sakazakii is an opportunistic nosocomial and foodborne pathogen that causes severe infections with high morbidity and mortality rates in neonates, the elderly, and immunocompromised individuals. Little is known about the pathogenesis mechanism of this pathogen and if there are any consequences of C. sakazakii colonization in healthy individuals. In this study, we characterized the mechanisms of autoaggregation in C. sakazakii ATCC 29544 (CS29544). Autoaggregation in CS29544 occurred rapidly, within 30 min, and proceeded to a maximum of 70%. Frameshift mutations in two flagellum proteins (FlhA and FliG) were identified in two nonautoaggregating CS29544 clonal variant isolates. Strategic gene knockouts were generated to determine if structurally intact and functional flagella were required for autoaggregation in CS29544. All structural knockouts (ΔflhA, ΔfliG, and ΔfliC) abolished autoaggregation, whereas the functional knockout (ΔmotAB) did not prevent autoaggregation. Complementation with FliC (ΔfliC/cfliC) restored autoaggregation. Autoaggregation was also disrupted by the addition of exogenous wild-type CS29544 filaments in a dose-dependent manner. Finally, filament supercoils tethering neighboring wild-type CS29544 cells together were observed by transmission electron microscopy. In silico analyses suggest that direct interactions of neighboring CS29544 FliC filaments proceed by hydrophobic bonding between the externally exposed hypervariable regions of the CS29544 FliC flagellin protein. Further research is needed to confirm if flagella-mediated autoaggregation plays a prominent role in C. sakazakii pathogenesis. PMID:28293226

  12. Environmental factors influencing the inactivation of Cronobacter sakazakii by high hydrostatic pressure.

    PubMed

    Arroyo, C; Cebrián, G; Mackey, B M; Condón, S; Pagán, R

    2011-05-27

    The effect of High Hydrostatic Pressure (HHP) on the survival of Cronobacter sakazakii was investigated. Deviations from linearity were found on the survival curves and the Mafart equation accurately described the kinetics of inactivation. Comparisons between strains and treatments were made based on the time needed for a 5-log(10) reduction in viable count. The ability of C. sakazakii to tolerate high pressure was strain-dependent with a 26-fold difference in resistance among four strains tested. Pressure resistance was greatest in the stationary growth phase and at the highest growth temperatures tested (30 and 37 °C). Cells treated in neutral pH buffer were 5-fold more resistant than those treated at pH 4.0, and 8-fold more sensitive than those treated in buffer with sucrose added (a(w)=0.98). Pressure resistance data obtained in buffer at the appropriate pH adequately estimated the resistance of C. sakazakii in chicken and vegetables soups. In contrast, a significant protective effect against high pressure was conferred by rehydrated powdered milk. As expected, treatment efficacy improved as pressure increased. z values of 112, 136 and 156 MPa were obtained for pH 4.0, pH 7.0 and a(w)=0.98 buffers, respectively. Cells with sublethal injury to their outer and cytoplasmic membranes were detected after HHP under all the conditions tested. The lower resistance of C. sakazakii cells when treated in media of pH 4.0 seemed to be due to a decreased barostability of the bacterial envelopes. Conversely, the higher resistance displayed in media of reduced water activity may relate to a higher stability of bacterial envelopes.

  13. Comprehensive approaches for molecular biomarker discovery for the detection and identification of Cronobacter spp. (Enterobacter sakazakii), Salmonella, and other foodborne pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cronobacter spp. (formerly Enterobacter sakazakii) and Salmonella are increasingly implicated as important bacterial contaminants in low-moisture food products, including powdered infant formula. Estimates indicate that 40-80% of infants infected with C. sakazakii and/or Salmonella in the U.S. may ...

  14. Detection of Cronobacter sakazakii in powdered infant formula using an immunoliposome-based immunomagnetic concentration and separation assay

    PubMed Central

    Shukla, Shruti; Lee, Gibaek; Song, Xinjie; Park, Jung Hyun; Cho, Hyunjeong; Lee, Eun Ju; Kim, Myunghee

    2016-01-01

    This study aimed to optimize the applicability of an immunoliposome-based immunomagnetic concentration and separation assay to facilitate rapid detection of Cronobacter sakazakii in powdered infant formula (PIF). To determine the detection limit, specificity, and pre-enrichment incubation time (0, 4, 6, and 8 h), assay tests were performed with different cell numbers of C. sakazakii (2 × 100 and 2 × 101 CFU/ml) inoculated in 10 g of PIF. The assay was able to detect as few as 2 cells of C. sakazakii/10 g of PIF sample after 6 h of pre-enrichment incubation with an assay time of 2 h 30 min. The assay was assessed for cross-reactivity with other bacterial strains and exhibited strong specificity to C. sakazakii. Moreover, the assay method was applied to the detection of C. sakazakii in PIF without pre-enrichment steps, and the results were compared with INC-ELISA and RT-PCR. The developed method was able to detect C. sakazakii in spiked PIF without pre-enrichment, whereas INC-ELISA failed to detect C. sakazakii. In addition, when compared with the results obtained with RT-PCR, our developed assay required lesser detection time. The developed assay was also not susceptible to any effect of the food matrix or background contaminant microflora. PMID:27721500

  15. Monitoring ultraviolet (UV) radiation inactivation of Cronobacter sakazakii in dry infant formula using Fourier transform infrared spectroscopy.

    PubMed

    Liu, Qian; Lu, Xiaonan; Swanson, Barry G; Rasco, Barbara A; Kang, Dong-Hyun

    2012-01-01

    Cronobacter sakazakii is an opportunistic pathogen associated with dry infant formula presenting a high risk to low birth weight neonates. The inactivation of C. sakazakii in dry infant formula by ultraviolet (UV) radiation alone and combined with hot water treatment at temperatures of 55, 60, and 65 °C were applied in this study. UV radiation with doses in a range from 12.1 ± 0.30 kJ/m² to 72.8 ± 1.83 kJ/m² at room temperature demonstrated significant inactivation of C. sakazakii in dry infant formula (P < 0.05). UV radiation combining 60 °C hot water treatment increased inactivation of C. sakazakii cells significantly (P < 0.05) in reconstituted infant formula. Significant effects of UV radiation on C. sakazakii inactivation kinetics (D value) were not observed in infant formula reconstituted in 55 and 65 °C water (P > 0.05). The inactivation mechanism was investigated using vibrational spectroscopy. Infrared spectroscopy detected significant stretching mode changes of macromolecules on the basis of spectral features, such as DNA, proteins, and lipids. Minor changes on cell membrane composition of C. sakazakii under UV radiation could be accurately and correctly monitored by infrared spectroscopy coupled with 2nd derivative transformation and principal component analysis.

  16. Thermal tolerance and survival of Cronobacter sakazakii in powdered infant formula supplemented with vanillin, ethyl vanillin, and vanillic acid.

    PubMed

    Yemiş, Gökçe Polat; Pagotto, Franco; Bach, Susan; Delaquis, Pascal

    2012-09-01

    The thermal tolerance Cronobacter sakazakii was examined in sterile powdered infant formula (PIF) rehydrated at 58 °C in water or apple juice supplemented with vanillin, ethyl vanillin, or vanillic acid. All three compounds decreased thermal tolerance during-rehydration and the lowest decimal reduction time (D-value, 0.19 ± 0.01 min) was measured in PIF rehydrated in apple juice supplemented with 20 mM vanillic acid. At this level of supplementation no C. sakazakii were detected in PIF stored for 48 h at 10 and 24 h at 21 °C subsequent to a sublethal heat treatment. Thermal tolerance during rehydration and survival in reconstituted PIF were influenced by compound type, concentration, and temperature. Supplementation of PIF with vanillin, ethyl vanillin, or vanillic acid could enhance the safety of PIF or other dehydrated foods contaminated with C. sakazakii.

  17. Genotyping and Source Tracking of Cronobacter sakazakii and C. malonaticus Isolates from Powdered Infant Formula and an Infant Formula Production Factory in China.

    PubMed

    Fei, Peng; Man, Chaoxin; Lou, Binbin; Forsythe, Stephen J; Chai, Yunlei; Li, Ran; Niu, Jieting; Jiang, Yujun

    2015-08-15

    Cronobacter spp. (formerly defined as Enterobacter sakazakii) are opportunistic bacterial pathogens of both infants and adults. In this study, we analyzed 70 Cronobacter isolates from powdered infant formula (PIF) and an infant formula production facility in China to determine possible contamination routes. The strains were profiled by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR-based O-antigen serotyping, and ompA and rpoB sequence analyses. The isolates were primarily Cronobacter sakazakii (66/70) or Cronobacter malonaticus (4/70). The strains were divided into 38 pulsotypes (PTs) using PFGE and 19 sequence types (STs) by MLST. In contrast, rpoB and ompA sequence analyses divided the strains into 10 overlapping clusters each. PCR serotyping of the 66 C. sakazakii and 4 C. malonaticus strains resulted in the identification of four C. sakazakii serotypes (O1, O2, O4, and O7) and a single C. malonaticus serotype, O2. The dominant C. sakazakii sequence types from PIF and an infant formula production factory in China were C. sakazakii clonal complex 4 (CC4) (n = 19), ST1 (n = 14), and ST64 (n = 11). C. sakazakii CC4 is a clonal lineage strongly associated with neonatal meningitis. In the process of manufacturing PIF, the spray-drying, fluidized-bed-drying, and packing areas were the main areas with Cronobacter contamination. C. sakazakii strains with the same pulsotypes (PT3 and PT2) and sequence types (ST1 and ST64) were isolated both from processing equipment and from the PIF finished product.

  18. Drying parameters greatly affect the destruction of Cronobacter sakazakii and Salmonella Typhimurium in standard buffer and milk.

    PubMed

    Lang, Emilie; Iaconelli, Cyril; Zoz, Fiona; Guyot, Stéphane; Alvarez-Martin, Pablo; Beney, Laurent; Perrier-Cornet, Jean-Marie; Gervais, Patrick

    2017-04-01

    Salmonella Typhimurium and Cronobacter sakazakii are two foodborne pathogens involved in neonatal infections from milk powder and infant formula. Their ability to survive in low-moisture food and during processing from the decontamination to the dried state is a major issue in food protection. In this work, we studied the effects of the drying process on Salmonella Typhimurium and Cronobacter sakazakii, with the aim of identifying the drying parameters that could promote greater inactivation of these two foodborne pathogens. These two bacteria were dried under different atmospheric relative humidities in milk and phosphate-buffered saline, and the delays in growth recovery and cultivability were followed. We found that water activity was related to microorganism resistance. C. sakazakii was more resistant to drying than was S. Typhimurium, and milk increased the cultivability and recovery of these two species. High drying rates and low final water activity levels (0.11-0.58) had a strong negative effect on the growth recovery and cultivability of these species. In conclusion, we suggest that effective use of drying processes may provide a complementary tool for food decontamination and food safety during the production of low-moisture foods.

  19. Development of Multiplex Real-time PCR with Internal Amplification Control for Simultaneous Detection of Salmonella and Cronobacter sakazakii in Powdered Infant Formula.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Contamination of powdered infant formula (PIF) by the bacteria Cronobacter sakazakii and Salmonella enterica was deemed a matter of great concern by the World Health Organization and the Food and Agriculture Organization of the United Nations in 2004. Therefore, we developed a rapid and sensitive m...

  20. Validation of radio-frequency dielectric heating system for destruction of Cronobacter sakazakii and Salmonella species in nonfat dry milk.

    PubMed

    Michael, M; Phebus, R K; Thippareddi, H; Subbiah, J; Birla, S L; Schmidt, K A

    2014-12-01

    Cronobacter sakazakii and Salmonella species have been associated with human illnesses from consumption of contaminated nonfat dry milk (NDM), a key ingredient in powdered infant formula and many other foods. Cronobacter sakazakii and Salmonella spp. can survive the spray-drying process if milk is contaminated after pasteurization, and the dried product can be contaminated from environmental sources. Compared with conventional heating, radio-frequency dielectric heating (RFDH) is a faster and more uniform process for heating low-moisture foods. The objective of this study was to design an RFDH process to achieve target destruction (log reductions) of C. sakazakii and Salmonella spp. The thermal destruction (decimal reduction time; D-value) of C. sakazakii and Salmonella spp. in NDM (high-heat, HH; and low-heat, LH) was determined at 75, 80, 85, or 90 °C using a thermal-death-time (TDT) disk method, and the z-values (the temperature increase required to obtain a decimal reduction of the D-value) were calculated. Time and temperature requirements to achieve specific destruction of the pathogens were calculated from the thermal destruction parameters, and the efficacy of the RFDH process was validated by heating NDM using RFDH to achieve the target temperatures and holding the product in a convection oven for the required period. Linear regression was used to determine the D-values and z-values. The D-values of C. sakazakii in HH- and LH-NDM were 24.86 and 23.0 min at 75 °C, 13.75 and 7.52 min at 80 °C, 8.0 and 6.03 min at 85 °C, and 5.57 and 5.37 min at 90 °C, respectively. The D-values of Salmonella spp. in HH- and LH-NDM were 23.02 and 24.94 min at 75 °C, 10.45 and 12.54 min at 80 °C, 8.63 and 8.68 min at 85 °C, and 5.82 and 4.55 min at 90 °C, respectively. The predicted and observed destruction of C. sakazakii and Salmonella spp. were in agreement, indicating that the behavior of the organisms was similar regardless of the heating system (conventional vs

  1. Rapid detection of Cronobacter sakazakii by real-time PCR based on the cgcA gene and TaqMan probe with internal amplification control.

    PubMed

    Hu, Shuangfang; Yu, Yigang; Li, Rong; Wu, Xinwei; Xiao, Xinglong; Wu, Hui

    2016-03-01

    Cronobacter sakazakii is a severe virulent strain that is frequently detected in powdered infant formula (PIF). Therefore, it is necessary to develop a fast and specific detection method. The specificity of our newly developed quantitative real-time PCR (qRT-PCR) was validated with DNA from 46 strains. Among them, 12 C. sakazakii strains were correctly amplified, whereas no positive florescent signal was observed from 34 nontarget controls. The detection limit of C. sakazakii was about 110 CFU/mL in broth and 1100 CFU/g in PIF. After enrichment in buffered peptone water for 6 h, our developed qRT-PCR assay could reliably detect C. sakazakii when the inoculation level was as low as 2 CFU/25 g (0.08 CFU/g) in PIF. The growth of C. sakazakii could be inhibited by the presence of Lactobacillus pentosus and Bacillus cereus, which used a longer enrichment period before the isolation was accomplished. However, at 5 and 50 CFU/25 g inoculation levels of C. sakazakii in the presence of 4 × 10(6) CFU L. pentosus/25 g or of 2 × 10(4) CFU B. cereus/25 g, the qRT-PCR assay could detect the presence of Cronobacter even though these artificially spiked samples were negative in culture. Therefore, our results indicated that the qRT-PCR assay could detect samples containing inhibitors and could avoid false negatives by using an internal amplification control.

  2. Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific Monoclonal Antibodies

    PubMed Central

    Scharinger, Eva J.; Dietrich, Richard; Kleinsteuber, Ina; Märtlbauer, Erwin

    2016-01-01

    Cronobacter sakazakii is a foodborne pathogen associated with rare but often lethal infections in neonates. Powdered infant formula (PIF) represents the most frequent source of infection. Out of the identified serotypes (O1 to O7), O1, O2, and O3 are often isolated from clinical and PIF samples. Serotype-specific monoclonal antibodies (MAbs) suitable for application in enzyme immunoassays (EIAs) for the rapid detection of C. sakazakii have not yet been developed. In this study, we created specific MAbs with the ability to bind to C. sakazakii of serotypes O1, O2, and O3. Characterization by indirect EIAs, immunofluorescence, motility assays, and immunoblotting identified lipopolysaccharide (LPS) and exopolysaccharide (EPS) as the antigenic determinants of the MAbs. The established sandwich EIAs were highly sensitive and were able to detect between 2 × 103 and 9 × 106 CFU/ml. Inclusivity tests confirmed that 93% of serotype O1 strains, 100% of O2 strains, and 87% of O3 strains were detected at low cell counts. No cross-reactivity with >100 strains of Cronobacter spp. and other Enterobacteriaceae was observed, except for that with C. sakazakii serotype O3 and Cronobacter muytjensii serotype O1. Moreover, the sandwich EIAs detected C. sakazakii in PIF samples artificially contaminated with 1 to 10 bacterial cells per 10 g of sample after 15 h of preenrichment. The use of these serotype-specific MAbs not only allows the reliable detection of C. sakazakii strains but also enables simultaneous serotyping in a simple sandwich EIA method. PMID:26850303

  3. Variability in Cell Response of Cronobacter sakazakii after Mild-Heat Treatments and Its Impact on Food Safety.

    PubMed

    Parra-Flores, Julio; Juneja, Vijay; Garcia de Fernando, Gonzalo; Aguirre, Juan

    2016-01-01

    Cronobacter spp. have been responsible for severe infections in infants associated with consumption of powdered infant formula and follow-up formulae. Despite several risk assessments described in published studies, few approaches have considered the tremendous variability in cell response that small micropopulations or single cells can have in infant formula during storage, preparation or post process/preparation before the feeding of infants. Stochastic approaches can better describe microbial single cell response than deterministic models as we prove in this study. A large variability of lag phase was observed in single cell and micropopulations of ≤50 cells. This variability increased as the heat shock increased and growth temperature decreased. Obviously, variability of growth of individual Cronobacter sakazakii cell is affected by inoculum size, growth temperature and the probability of cells able to grow at the conditions imposed by the experimental conditions should be taken into account, especially when errors in bottle-preparation practices, such as improper holding temperatures, or manipulation, may lead to growth of the pathogen to a critical cell level. The mean probability of illness from initial inoculum size of 1 cell was below 0.2 in all the cases and for inoculum size of 50 cells the mean probability of illness, in most of the cases, was above 0.7.

  4. Variability in Cell Response of Cronobacter sakazakii after Mild-Heat Treatments and Its Impact on Food Safety

    PubMed Central

    Parra-Flores, Julio; Juneja, Vijay; Garcia de Fernando, Gonzalo; Aguirre, Juan

    2016-01-01

    Cronobacter spp. have been responsible for severe infections in infants associated with consumption of powdered infant formula and follow-up formulae. Despite several risk assessments described in published studies, few approaches have considered the tremendous variability in cell response that small micropopulations or single cells can have in infant formula during storage, preparation or post process/preparation before the feeding of infants. Stochastic approaches can better describe microbial single cell response than deterministic models as we prove in this study. A large variability of lag phase was observed in single cell and micropopulations of ≤50 cells. This variability increased as the heat shock increased and growth temperature decreased. Obviously, variability of growth of individual Cronobacter sakazakii cell is affected by inoculum size, growth temperature and the probability of cells able to grow at the conditions imposed by the experimental conditions should be taken into account, especially when errors in bottle-preparation practices, such as improper holding temperatures, or manipulation, may lead to growth of the pathogen to a critical cell level. The mean probability of illness from initial inoculum size of 1 cell was below 0.2 in all the cases and for inoculum size of 50 cells the mean probability of illness, in most of the cases, was above 0.7. PMID:27148223

  5. Characterization of Putative Virulence Genes on the Related RepFIB Plasmids Harbored by Cronobacter spp. ▿ †

    PubMed Central

    Franco, A. A.; Hu, L.; Grim, C. J.; Gopinath, G.; Sathyamoorthy, V.; Jarvis, K. G.; Lee, C.; Sadowski, J.; Kim, J.; Kothary, M. H.; McCardell, B. A.; Tall, B. D.

    2011-01-01

    Cronobacter spp. are emerging neonatal pathogens that cause meningitis, sepsis, and necrotizing enterocolitis. The genus Chronobacter consists of six species: C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, and Cronobacter genomospecies group 1. Whole-genome sequencing of C. sakazakii BAA-894 and C. turicensis z3032 revealed that they harbor similarly sized plasmids identified as pESA3 (131 kb) and pCTU1 (138 kb), respectively. In silico analysis showed that both plasmids encode a single RepFIB-like origin of replication gene, repA, as well as two iron acquisition systems (eitCBAD and iucABCD/iutA). In a chrome azurol S agar diffusion assay, it was demonstrated that siderophore activity was associated with the presence of pESA3 or pCTU1. Additionally, pESA3 contains a cpa (Cronobacter plasminogen activator) gene and a 17-kb type 6 secretion system (T6SS) locus, while pCTU1 contains a 27-kb region encoding a filamentous hemagglutinin gene (fhaB), its specifc transporter gene (fhaC), and associated putative adhesins (FHA locus), suggesting that these are virulence plasmids. In a repA-targeted PCR assay, 97% of 229 Cronobacter species isolates were found to possess a homologous RepFIB plasmid. All repA PCR-positive strains were also positive for the eitCBAD and iucABCD/iutA iron acquisition systems. However, the presence of cpa, T6SS, and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type, and species. These results support the hypothesis that these plasmids have evolved from a single archetypical plasmid backbone through the cointegration, or deletion, of specific virulence traits in each species. PMID:21421789

  6. Genes Involved in Yellow Pigmentation of Cronobacter sakazakii ES5 and Influence of Pigmentation on Persistence and Growth under Environmental Stress▿

    PubMed Central

    Johler, Sophia; Stephan, Roger; Hartmann, Isabel; Kuehner, Kirsten A.; Lehner, Angelika

    2010-01-01

    Cronobacter spp. are opportunistic food-borne pathogens that are responsible for rare but highly fatal cases of meningitis and necrotizing enterocolitis in neonates. While the operon responsible for yellow pigmentation in Cronobacter sakazakii strain ES5 was described recently, the involvement of additional genes in pigment expression and the influence of pigmentation on the fitness of Cronobacter spp. have not been investigated. Thus, the aim of this study was to identify further genes involved in pigment expression in Cronobacter sakazakii ES5 and to assess the influence of pigmentation on growth and persistence under conditions of environmental stress. A knockout library was created using random transposon mutagenesis. The screening of 9,500 mutants for decreased pigment production identified 30 colorless mutants. The mapping of transposon insertion sites revealed insertions in not only the carotenoid operon but also in various other genes involved in signal transduction, inorganic ions, and energy metabolism. To determine the effect of pigmentation on fitness, colorless mutants (ΔcrtE, ΔcrtX, and ΔcrtY) were compared to the yellow wild type using growth and inactivation experiments, a macrophage assay, and a phenotype array. Among other findings, the colorless mutants grew at significantly increased rates under osmotic stress compared to that of the yellow wild type while showing increased susceptibility to desiccation. Moreover, ΔcrtE and ΔcrtY exhibited increased sensitivity to UVB irradiation. PMID:20038705

  7. Sub-lethal heat treatment affects the tolerance of Cronobacter sakazakii BCRC 13988 to various organic acids, simulated gastric juice and bile solution.

    PubMed

    Hsiao, Wan-Ling; Ho, Wei-Li; Chou, Cheng-Chun

    2010-12-15

    Cronobacter spp., formerly Enterobacter sakazakii, are considered emerging opportunistic pathogens and the etiological agent of life-threatening bacterial infections in infants. In the present study, C. sakazakii BCRC 13988 was first subjected to sub-lethal heat treatment at 47°C for 15min. Survival rates of the heat-shocked and non-shocked C. sakazakii cells in phosphate buffer solution (PBS, pH 4.0) containing organic acids (e.g. acetic, propionic, citric, lactic or tartaric acid), simulated gastric juice (pH 2.0-4.0), and bile solution (0.5 and 2.0%) were examined. Results revealed that sub-lethal heat treatment enhanced the test organism's tolerance to organic acids, although the extent of increased acid tolerance varied with the organic acid examined. Compared with the control cells, heat-shocked C. sakazakii cells after 120-min of exposure, exhibited the largest increase in tolerance in the lactic acid-containing PBS. Furthermore, although heat shock did not affect the behavior of C. sakazakii in bile solution, it increased the test organism's survival when exposed to simulated gastric juice with a pH of 3.0-4.0.

  8. Survival and growth of Cronobacter sakazakii on fresh-cut fruit and the effect of UV-C illumination and electrolyzed water in the reduction of its population.

    PubMed

    Santo, David; Graça, Ana; Nunes, Carla; Quintas, Célia

    2016-08-16

    Cronobacter sakazakii, found in foods such as powdered infant formula and plant origin ready-to-eat food, is an opportunistic pathogen to infants, neonates and vulnerable adults. The objective of this study was to monitor the growth of C. sakazakii in fresh-cut 'Royal gala' apple, 'Rocha' pear, and 'Piel de sapo' melon, and the effect of UV-C illumination, acidic electrolyzed water (AEW) and neutral electrolyzed water (NEW) in the reduction of its population. Fresh-cut fruits were inoculated and incubated at different temperatures during 10days while monitoring C. sakazakii. The inhibitory activity of different doses of UV-C (0-10kJ.m(2)), electrolyzed water and sodium hypochlorite (SH) (100ppm chlorine) was evaluated on the fruits inoculated with C. sakazakii. The bacterium showed a significant growth in the fruits at 12 and 20°C, but did not grow at 4°C, despite having survived for 10days. At 8°C, adaptation phases of 0.6-3.9days were estimated in the fruits before exponential growth. The UV-C 7.5 and 10kJ/m(2) produced greater C. sakazakii population decreases (2-2.4logcfu/g) than AEW (1.3-1.8logcfu/g), NEW (1-1.2logcfu/g) and SH (0.8-1.4logcfu/g). The UV-C decontamination system and refrigeration at 4°C, may contribute to the product's safety and quality. The results help better understand the behavior of C. sakazakii on fresh-cut fruit alerting producers of the necessity to respect the high hygienic practices, adequate refrigerating temperature maintenance and caution with the tendency to prolong the validity of this kind of ready-to-eat food.

  9. Variability in cell response of Cronobacter sakazakii after mild-heat treatments and its impact on food safety

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cronobacter spp. have been responsible for severe infections in infants associated with consumption of powdered infant formula (PIF) and follow-up formulae (FUF). Despite several risk assessments described in published studies, few approaches have considered the tremendous variability in cell respo...

  10. Investigating the Responses of Cronobacter sakazakii to Garlic-Drived Organosulfur Compounds: a Systematic Study of Pathogenic-Bacterium Injury by Use of High-Throughput Whole-Transcriptome Sequencing and Confocal Micro-Raman Spectroscopy

    PubMed Central

    Feng, Shaolong; Eucker, Tyson P.; Holly, Mayumi K.; Konkel, Michael E.

    2014-01-01

    We present the results of a study using high-throughput whole-transcriptome sequencing (RNA-seq) and vibrational spectroscopy to characterize and fingerprint pathogenic-bacterium injury under conditions of unfavorable stress. Two garlic-derived organosulfur compounds were found to be highly effective antimicrobial compounds against Cronobacter sakazakii, a leading pathogen associated with invasive infection of infants and causing meningitis, necrotizing entercolitis, and bacteremia. RNA-seq shows changes in gene expression patterns and transcriptomic response, while confocal micro-Raman spectroscopy characterizes macromolecular changes in the bacterial cell resulting from this chemical stress. RNA-seq analyses showed that the bacterial response to ajoene differed from the response to diallyl sulfide. Specifically, ajoene caused downregulation of motility-related genes, while diallyl sulfide treatment caused an increased expression of cell wall synthesis genes. Confocal micro-Raman spectroscopy revealed that the two compounds appear to have the same phase I antimicrobial mechanism of binding to thiol-containing proteins/enzymes in bacterial cells generating a disulfide stretching band but different phase II antimicrobial mechanisms, showing alterations in the secondary structures of proteins in two different ways. Diallyl sulfide primarily altered the α-helix and β-sheet, as reflected in changes in amide I, while ajoene altered the structures containing phenylalanine and tyrosine. Bayesian probability analysis validated the ability of principal component analysis to differentiate treated and control C. sakazakii cells. Scanning electron microscopy confirmed cell injury, showing significant morphological variations in cells following treatments by these two compounds. Findings from this study aid in the development of effective intervention strategies to reduce the risk of C. sakazakii contamination in the food production environment and on food contact surfaces

  11. The effect of temperature and length of heat shock treatment on the thermal tolerance and cell leakage of Cronobacter sakazakii BCRC 13988.

    PubMed

    Chang, Chia-Hsiang; Chiang, Ming-Lun; Chou, Cheng-Chun

    2009-09-15

    Enterobacter sakazakii is an emerging opportunistic pathogen associated with life-threatening illnesses in infants, with infant formula serving as the principal mode of transmission. In the present study, C. sakazakii (formely E. sakazakii) BCRC 13988 was subjected to various heat shock treatments (42-48 degrees C for 5-15 min). Its subsequent survival at 51 degrees C and the leakage of intracellular materials was investigated. It was found that 47 degrees C was the maximum growth temperature of the test organism. In addition, heat shock enhanced the thermal tolerance of C. sakazakii BCRC 13988. Within heat shock temperatures between 42 and 47 degrees C, the thermal tolerance enhancing effect increased as the length or temperature of the heat shock treatment was increased. However, increasing the heat shock temperature to 48 degrees C reduced the thermal tolerance enhancing effect. Among the various heat shocked cells examined, the 47 degrees C-15 min-heat shocked C. sakazakii exhibited the highest thermal tolerance. Moreover, electron micrograph analysis showed that heat shock treatment caused damage and disruption in C. sakazakii cells. There was a significant increase (P<0.05) in the leakage of nucleic acid and protein in the supernatant of the heat shocked cell suspension that increased as the temperature and duration of heat shock increased.

  12. Genotypic and phenotypic characteristics of Cronobacter species, with particular attention to the newly reclassified species Cronobacter helveticus, Cronobacter pulveris, and Cronobacter zurichensis.

    PubMed

    Jackson, E E; Sonbol, H; Masood, N; Forsythe, S J

    2014-12-01

    In 2013, Enterobacter helveticus, Enterobacter pulveris and Enterobacter turicensis, were reclassified as Cronobacter helveticus, Cronobacter pulveris and Cronobacter zurichensis, respectively. Previously these species had been used as negative controls for some Cronobacter detection assays. This study examined cultural, biochemical and molecular Cronobacter detection and identification assays, with emphasis on the new species. Additionally, 32 Cronobacter genomes were examined for the presence of PCR target genes using the BLAST function of the online Cronobacter PubMLST facility. The results of the cultural methods varied and no single medium was able to correctly detect all Cronobacter spp. Since the supporting databases have not been updated to include the Cronobacter genus, Enterobacter sakazakii was returned for four strains of the newly reclassified species with ID32E and none with API 20E. PCR probes targeting rpoB and ompA could not correctly identify the new Cronobacter spp., due to primer specificity or absent target genes. As neonates have been identified as a high-risk group for infection, international standards require the absence of all Cronobacter species in powdered infant formula. However, many conventional detection methods cannot correctly identify the newly recognized species. Conversely, DNA sequence-based methods can adapt to taxonomic revisions and will likely become more common.

  13. Two cases of hemorrhagic diarrhea caused by Cronobacter sakazakii in hospitalized nursing infants associated with the consumption of powdered infant formula.

    PubMed

    Flores, J Parra; Medrano, S Arvizu; Sánchez, J Silva; Fernández-Escartín, E

    2011-12-01

    Two cases of acute gastroenteritis occurred in 5-month-old infants hospitalized in a mother-and-child hospital in Queretaro, Mexico, on 24 January 2010. C. sakazakii was recovered from the powdered infant formula (PIF), rehydrated PIF (R-PIF) fed to infants, and their fecal samples. The microorganism was present at levels of 0.33 most probable number (MPN)/g and 24 MPN/ml in PIF and R-PIF, respectively. The total ingested dose for the day before the onset of the diarrheic syndrome ranged between 2,160 and 3,600 MPN/ml. All strains of C. sakazakii recovered from the three sources (R-PIF, PIF, and fecal matter) showed identical biotypes, adhesion and invasiveness factors, and pulsed-field gel electrophoresis profiles. No deaths were observed. Salmonella, Shigella, and enterotoxigenic Escherichia coli were not found in food or fecal samples.

  14. Identification and classification of Cronobacter spp. isolated from powdered food in Korea.

    PubMed

    Park, Jong-Hyun; Lee, Young-Duck; Ryu, Tae-Wha; Chang, Hyo-Ihl

    2010-04-01

    Cronobacter is a major food-borne pathogen in powdered infant formula and can lead to serious developmental aftereffect and death to infants. The contamination of Cronobacter may be a high risk for the powdered foods. To isolate and identify Korean Cronobacter from the powdered foods such as powdered infant formula and Saengsik, conventional culture method, rapid identification system, PCR, 16S rDNA sequencing were performed. As the results of isolation, seven Cronobacter were isolated from seven out of 102 powdered infant formulas and 41 Cronobacter were isolated from 41 out of 86 Saengsiks. Forty eight Cronobacter isolates were identified into C.sakazakii and C.dublenisis by 16s rDNA sequence analysis. Almost the isolates were C.sakazakii and 13% of the isolates were C. dublinesis. One fourth of the C.sakazakii showed different biochemical characteristics of negative nitrate reduction and non-motility activities with the other strains reported previously.

  15. Molecular epidemiological survey of Citrobacter freundii misidentified as Cronobacter spp. (Enterobacter sakazakii) and Enterobacter hormaechei isolated from powdered infant milk formula.

    PubMed

    Giammanco, Giovanni M; Aleo, Aurora; Guida, Ivana; Mammina, Caterina

    2011-04-01

    A total of 75 powdered infant milk formula (PIF) samples collected from pharmacies and drugstores in Western Sicily, Italy, and representative of 12 different brands were analyzed in this study to evaluate their microbiological quality. According to the U.S. Food and Drug Administration protocol, 32 samples out of 75 were contaminated by enterobacteria. Commercial biochemical API(r) 20E-system identification method indicated that six PIF samples were presumptively contaminated by Cronobacter spp., but further characterization by alpha-glucosidase based polymerase chain reaction (PCR) assay identification strongly suggested that these strains did not belong to the genus Cronobacter. Phylogenetic analysis of partial 16S rRNA (rrs) sequences combined with the results of biochemical tests allowed to identify the six strains as Citrobacter freundii. Similarly, rrs sequence analysis identified as Enterobacter hormaechei 23 strains originally ascribed to Enterobacter cloacae by the API 20E system. Characterization of C. freundii and E. hormaechei PIF isolates by the DiversiLab(r) repetitive sequence-based PCR (rep-PCR) typing method revealed a variety of amplification patterns, but the recovery of the same rep-PCR genotype in several products might indicate a special adaptation of genetic clones to this food or cross-contamination through common ingredients. Antibiotic-resistance profiles were also determined, but none of the strains tested was resistant to third-generation cephalosporins or fluoroquinolones and extended-spectrum beta-lactamase activity was not detected. Our results confirm that E. hormaechei contamination of PIF is widespread, thus making it a cause for concern. Similarly to what was demonstrated for E. hormaechei, we suggest that C. freundii also may be an under-reported cause of bacterial infection, especially in high-risk neonates, due to misidentification.

  16. Comparative Analysis of Genome Sequences Covering the Seven Cronobacter Species

    PubMed Central

    Cummings, Craig A.; Shih, Rita; Degoricija, Lovorka; Rico, Alain; Brzoska, Pius; Hamby, Stephen E.; Masood, Naqash; Hariri, Sumyya; Sonbol, Hana; Chuzhanova, Nadia; McClelland, Michael; Furtado, Manohar R.; Forsythe, Stephen J.

    2012-01-01

    Background Species of Cronobacter are widespread in the environment and are occasional food-borne pathogens associated with serious neonatal diseases, including bacteraemia, meningitis, and necrotising enterocolitis. The genus is composed of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. universalis, and C. condimenti. Clinical cases are associated with three species, C. malonaticus, C. turicensis and, in particular, with C. sakazakii multilocus sequence type 4. Thus, it is plausible that virulence determinants have evolved in certain lineages. Methodology/Principal Findings We generated high quality sequence drafts for eleven Cronobacter genomes representing the seven Cronobacter species, including an ST4 strain of C. sakazakii. Comparative analysis of these genomes together with the two publicly available genomes revealed Cronobacter has over 6,000 genes in one or more strains and over 2,000 genes shared by all Cronobacter. Considerable variation in the presence of traits such as type six secretion systems, metal resistance (tellurite, copper and silver), and adhesins were found. C. sakazakii is unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have clinical significance. The C. sakazakii ST4 strain 701 contained additional genes as compared to other C. sakazakii but none of them were known specific virulence-related genes. Conclusions/Significance Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species, and also suggested various degrees of divergence. Sets of universal core genes and accessory genes unique to each strain were identified. These gene sequences can be used for designing genus/species specific detection assays. Genes encoding adhesins, T6SS, and metal resistance genes as well as prophages are found in only subsets of genomes and have contributed considerably to the variation of

  17. Evaluation and implementation of a membrane filter method for Cronobacter detection in drinking water.

    PubMed

    Liu, Hui; Yang, Yuelian; Cui, Jinghua; Liu, Lanzheng; Liu, Huiyuan; Hu, Guangchun; Shi, Yuwen; Li, Jian

    2013-07-01

    A membrane filter (MF) method was evaluated for its suitability for qualitative and quantitative analyses of Cronobacter spp. in drinking water by pure strains of Cronobacter and non-Cronobacter, and samples spiked with chlorinated Cronobacter sakazakii ATCC 29544. The applicability was verified by the tests: for pure strains, the sensitivity and the specificity were both 100%; for spiked samples, the MF method recovered 82.8 ± 10.4% chlorinated ATCC 29544 cells. The MF method was also applied to screen Cronobacter spp. in drinking water samples from municipal water supplies on premises (MWSP) and small community water supplies on premises (SCWSP). The isolation rate of Cronobacter spp. from SCWSP samples was 31/114, which was significantly higher than that from MWSP samples which was 1/131. Besides, the study confirmed the possibility of using total coliform as an indicator of contamination level of Cronobacter spp. in drinking water, and the acquired correct positive rate was 96%.

  18. Thermobacteriological characterization of Enterobacter sakazakii.

    PubMed

    Arroyo, C; Condón, S; Pagán, R

    2009-11-30

    In the present study the influence of various environmental and physiological factors on the heat resistance of Enterobacter sakazakii (Cronobacter) have been investigated. Our results demonstrated that the heat resistance of E. sakazakii depended on the strain studied, the growth conditions - phase and temperature - the characteristics of treatment medium and the recovery conditions. The strain STCC 858 (ATCC type strain 29544) showed maximum heat resistance among the strains tested and it was selected for the further study. Stationary-phase cells grown between 20 and 37 degrees C (mean D(60)=0.9 min) resulted to be more resistant than cells grown at 10 degrees C (D(60)=0.2 min). Resistance decreased when the treatment medium pH was lower than pH 6.0, and it increased with decreasing water activity of the treatment medium, with a 32-fold increase in resistance when lowering water activity to 0.96. z value at pH 4.0 (z=4.79 degrees C) was significantly higher than at pH 7.0 (z=4.06 degrees C), although E. sakazakii cells were approximately 10 times more heat resistant at pH 7.0 than at pH 4.0 within the range of temperatures tested. Contrary to pH, the magnitude of the influence of a(w) on heat resistance did not significantly change with treatment temperature. The proportion of sublethally damaged cells was similar regardless of the treatment medium pH, but it decreased when lowering the water activity. Nevertheless, increasing treatment temperature would not result in a decreased proportion of sublethally injured E. sakazakii cells within the surviving population. Thus, the design of a theoretical combined process that could take advantage of the occurrence of sublethally injured cells would be similarly effective at low and high temperatures. E. sakazakii proved to be more heat resistant in four different liquid food matrixes than in buffers at the same pH, and this disagreement was especially higher in orange juice, which resulted to be the product that induced

  19. Development and validation of a PulseNet standardized protocol for subtyping isolates of Cronobacter species.

    PubMed

    Brengi, Silvina P; O'Brien, Stephen B; Pichel, Mariana; Iversen, Carol; Arduino, Matthew; Binsztein, Norma; Jensen, Bette; Pagotto, Franco; Ribot, Efrain M; Stephan, Roger; Cernela, Nicole; Cooper, Kara; Fanning, Séamus

    2012-09-01

    Cronobacter (formerly known as Enterobacter sakazakii) is a genus comprising seven species regarded as opportunistic pathogens that can be found in a wide variety of environments and foods, including powdered infant formula (PIF). Cronobacter sakazakii, the major species of this genus, has been epidemiologically linked to cases of bacteremia, meningitis in neonates, and necrotizing enterocolitis, and contaminated PIF has been identified as an important source of infection. Robust and reproducible subtyping methods are required to aid in the detection and investigation, of foodborne outbreaks. In this study, a pulsed-field gel electrophoresis (PFGE) protocol was developed and validated for subtyping Cronobacter species. It was derived from an existing modified PulseNet protocol, wherein XbaI and SpeI were the primary and secondary restriction enzymes used, generating an average of 14.7 and 20.3 bands, respectively. The PFGE method developed was both reproducible and discriminatory for subtyping Cronobacter species.

  20. Isolation, molecular and phenotypic characterization, and antibiotic susceptibility of Cronobacter spp. from Brazilian retail foods.

    PubMed

    Brandão, Marcelo Luiz Lima; Umeda, Natália Scudeller; Jackson, Emily; Forsythe, Stephen James; de Filippis, Ivano

    2017-05-01

    Several Cronobacter species are opportunistic pathogens that cause infections in humans. The aim of this study was to detect Cronobacter spp. from 90 samples of retail foods in Brazil, and characterize the strains by phenotypic tests, molecular assays and antibiotic susceptibility. Three isolation methodologies were evaluated using different selective enrichments and the isolates were identified using Vitek 2.0, PCRs protocols, fusA allele sequencing and multilocus sequence typing (MLST). Thirty-eight samples (42.2%) contained Cronobacter spp., and the highest percentage was found in flours (66.7%, 20/30), followed by spices and herbs (36.7%, 11/30), and cereal mixes for children (23.3%, 7/30). The 45 isolates included four species: C. sakazakii (n = 37), C. malonaticus (n = 3), C. dublinensis (n = 3), and C. muytjensii (n = 2); that presented 20 different fusA alleles. MLST analysis revealed 32 sequence types (STs), 13 of which were newly identified. All strains were sensitive to all antibiotics (n = 10) tested. The combination of CSB/v enrichment with DFI plating was considered the most efficient for Cronobacter spp. isolation. This study revealed the presence of Cronobacter spp. in foods commercialized in Brazil and the isolates showed a high diversity after MLST analysis and included two strains of the C. sakazakii ST4 neonatal meningitic pathovar.

  1. My 40-Year History with Cronobacter/Enterobacter sakazakii – Lessons Learned, Myths Debunked, and Recommendations

    PubMed Central

    Farmer, John J.

    2015-01-01

    Much has been learned about organism in the Cronobacter/Enterobacter sakazakii complex since I first named and described Enterobacter sakazakii in 1980. However, there are still wide knowledge gaps. One of the most serious is that are still many uncertainties associated with assessing the public health risk posed by these bacteria, particularly in neonatal meningitis. Over the last few decades, Cronobacter contamination of commercial powdered infant formula products has apparently been reduced, but it is still an ongoing problem. The powdered infant formula industry still cannot produce powdered formula that is free of bacterial contamination with Cronobacter, other Enterobacteriaceae, other pathogenic bacteria, and other microorganisms. Until this happens, infants and other will be at risk of becoming infected when they ingest contaminated formula. PMID:26640778

  2. Diversity of the Cronobacter Genus as Revealed by Multilocus Sequence Typing

    PubMed Central

    Joseph, S.; Sonbol, H.; Hariri, S.; Desai, P.; McClelland, M.

    2012-01-01

    Cronobacter (previously known as Enterobacter sakazakii) is a diverse bacterial genus consisting of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. universalis, C. muytjensii, C. dublinensis, and C. condimenti. In this study, we have used a multilocus sequence typing (MLST) approach employing the alleles of 7 genes (atpD, fusA, glnS, gltB, gyrB, infB, and ppsA; total length, 3,036 bp) to investigate the phylogenetic relationship of 325 Cronobacter species isolates. Strains were chosen on the basis of their species, geographic and temporal distribution, source, and clinical outcome. The earliest strain was isolated from milk powder in 1950, and the earliest clinical strain was isolated in 1953. The existence of seven species was supported by MLST. Intraspecific variation ranged from low diversity in C. sakazakii to extensive diversity within some species, such as C. muytjensii and C. dublinensis, including evidence of gene conversion between species. The predominant species from clinical sources was found to be C. sakazakii. C. sakazakii sequence type 4 (ST4) was the predominant sequence type of cerebral spinal fluid isolates from cases of meningitis. PMID:22785185

  3. Analysis and Characterization of Proteins Associated with Outer Membrane Vesicles Secreted by Cronobacter spp.

    PubMed Central

    Kothary, Mahendra H.; Gopinath, Gopal R.; Gangiredla, Jayanthi; Rallabhandi, Prasad V.; Harrison, Lisa M.; Yan, Qiong Q.; Chase, Hannah R.; Lee, Boram; Park, Eunbi; Yoo, YeonJoo; Chung, Taejung; Finkelstein, Samantha B.; Negrete, Flavia J.; Patel, Isha R.; Carter, Laurenda; Sathyamoorthy, Venugopal; Fanning, Séamus; Tall, Ben D.

    2017-01-01

    Little is known about secretion of outer membrane vesicles (OMVs) by Cronobacter. In this study, OMVs isolated from Cronobacter sakazakii, Cronobacter turicensis, and Cronobacter malonaticus were examined by electron microscopy (EM) and their associated outer membrane proteins (OMP) and genes were analyzed by SDS-PAGE, protein sequencing, BLAST, PCR, and DNA microarray. EM of stained cells revealed that the OMVs are secreted as pleomorphic micro-vesicles which cascade from the cell's surface. SDS-PAGE analysis identified protein bands with molecular weights of 18 kDa to >100 kDa which had homologies to OMPs such as GroEL; OmpA, C, E, F, and X; MipA proteins; conjugative plasmid transfer protein; and an outer membrane auto-transporter protein (OMATP). PCR analyses showed that most of the OMP genes were present in all seven Cronobacter species while a few genes (OMATP gene, groEL, ompC, mipA, ctp, and ompX) were absent in some phylogenetically-related species. Microarray analysis demonstrated sequence divergence among the OMP genes that was not captured by PCR. These results support previous findings that OmpA and OmpX may be involved in virulence of Cronobacter, and are packaged within secreted OMVs. These results also suggest that other OMV-packaged OMPs may be involved in roles such as stress response, cell wall and plasmid maintenance, and extracellular transport. PMID:28232819

  4. Strategies for the identification and tracking of cronobacter species: an opportunistic pathogen of concern to neonatal health.

    PubMed

    Yan, Qiongqiong; Fanning, Séamus

    2015-01-01

    Cronobacter species are emerging opportunistic food-borne pathogens, which consists of seven species, including C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, C. universalis, and C. condimenti. The organism can cause severe clinical infections, including necrotizing enterocolitis, septicemia, and meningitis, predominately among neonates <4 weeks of age. Cronobacter species can be isolated from various foods and their surrounding environments; however, powdered infant formula (PIF) is the most frequently implicated food source linked with Cronobacter infection. This review aims to provide a summary of laboratory-based strategies that can be used to identify and trace Cronobacter species. The identification of Cronobacter species using conventional culture method and immuno-based detection protocols were first presented. The molecular detection and identification at genus-, and species-level along with molecular-based serogroup approaches are also described, followed by the molecular sub-typing methods, in particular pulsed-field gel electrophoresis and multi-locus sequence typing. Next generation sequence approaches, including whole genome sequencing, DNA microarray, and high-throughput whole-transcriptome sequencing, are also highlighted. Appropriate application of these strategies would contribute to reduce the risk of Cronobacter contamination in PIF and production environments, thereby improving food safety and protecting public health.

  5. Use of a Pan–Genomic DNA Microarray in Determination of the Phylogenetic Relatedness among Cronobacter spp. and Its Use as a Data Mining Tool to Understand Cronobacter Biology

    PubMed Central

    Tall, Ben D.; Gangiredla, Jayanthi; Grim, Christopher J.; Patel, Isha R.; Jackson, Scott A.; Mammel, Mark K.; Kothary, Mahendra H.; Sathyamoorthy, Venugopal; Carter, Laurenda; Fanning, Séamus; Iversen, Carol; Pagotto, Franco; Stephan, Roger; Lehner, Angelika; Farber, Jeffery; Yan, Qiong Q.; Gopinath, Gopal R.

    2017-01-01

    Cronobacter (previously known as Enterobacter sakazakii) is a genus of Gram-negative, facultatively anaerobic, oxidase-negative, catalase-positive, rod-shaped bacteria of the family Enterobacteriaceae. These organisms cause a variety of illnesses such as meningitis, necrotizing enterocolitis, and septicemia in neonates and infants, and urinary tract, wound, abscesses or surgical site infections, septicemia, and pneumonia in adults. The total gene content of 379 strains of Cronobacter spp. and taxonomically-related isolates was determined using a recently reported DNA microarray. The Cronobacter microarray as a genotyping tool gives the global food safety community a rapid method to identify and capture the total genomic content of outbreak isolates for food safety, environmental, and clinical surveillance purposes. It was able to differentiate the seven Cronobacter species from one another and from non-Cronobacter species. The microarray was also able to cluster strains within each species into well-defined subgroups. These results also support previous studies on the phylogenic separation of species members of the genus and clearly highlight the evolutionary sequence divergence among each species of the genus compared to phylogenetically-related species. This review extends these studies and illustrates how the microarray can also be used as an investigational tool to mine genomic data sets from strains. Three case studies describing the use of the microarray are shown and include: (1) the determination of allelic differences among Cronobacter sakazakii strains possessing the virulence plasmid pESA3; (2) mining of malonate and myo-inositol alleles among subspecies of Cronobacter dublinensis strains to determine subspecies identity; and (3) lastly using the microarray to demonstrate sequence divergence and phylogenetic relatedness trends for 13 outer-membrane protein alleles among 240 Cronobacter and phylogenetically-related strains. The goal of this review is to

  6. Efficacy of organic acids, bacteriocins, and the lactoperoxidase system in inhibiting the growth of Cronobacter spp. in rehydrated infant formula.

    PubMed

    Oshima, Satoru; Rea, Mary C; Lothe, Sheba; Morgan, Sheila; Begley, Maire; O'Connor, Paula M; Fitzsimmons, Aidan; Kamikado, Hideaki; Walton, Richard; Ross, R Paul; Hill, Colin

    2012-10-01

    Thirty-three antimicrobial agents, including antimicrobial peptides (nisin, lacticin 3147, isracidin), organic acids, emulsifiers (organic acid esters), glycine, lysozyme, tocopherol, EDTA, milk fat globule membrane, and the lactoperoxidase system (LPOS) were screened for anti-Cronobacter sakazakii activity. The compounds were initially screened individually in parallel in synthetic media. Those showing antimicrobial activity were then tested in reconstituted whole milk and finally in reconstituted powdered infant formula (PIF), using mild temperatures of reconstitution and prolonged storage at room temperature. Propionic acid and monocaprylin (as POEM M-100) in combination showed inhibitory activity at sufficiently low concentrations (0.1 to 0.2%) in milk to be considered as potential antimicrobial additives for the inhibition of C. sakazakii in reconstituted PIF. More interestingly, LPOS, when combined with the broad-spectrum bacteriocins nisin or lacticin 3147, inhibited outgrowth of C. sakazakii at 37°C for 8 h. The combined effects of POEM M-100 and either acetate or propionate and LPOS with lacticin 3147 or nisin were evaluated under the Food and Agriculture Organization of the United Nations-World Health Organization high-risk scenario for PIF, i.e., low temperature of reconstitution and long storage or feeding times at ambient temperature. In the presence of LPOS and lacticin 3147, growth of Cronobacter spp. was inhibited for up to 12 h when the PIF was rehydrated at 40 or 50°C. These results highlight the potential of combinatory approaches to improving the safety of infant milk formula.

  7. Flagella from Five Cronobacter Species Induce Pro-Inflammatory Cytokines in Macrophage Derivatives from Human Monocytes

    PubMed Central

    Cruz-Córdova, Ariadnna; Rocha-Ramírez, Luz M.; Ochoa, Sara A.; Gónzalez-Pedrajo, Bertha; Espinosa, Norma; Eslava, Carlos; Hernández-Chiñas, Ulises; Mendoza-Hernández, Guillermo; Rodríguez-Leviz, Alejandra; Valencia-Mayoral, Pedro; Sadowinski-Pine, Stanislaw; Hernández-Castro, Rigoberto; Estrada-García, Iris; Muñoz-Hernández, Onofre; Rosas, Irma; Xicohtencatl-Cortes, Juan

    2012-01-01

    Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10) in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ∼28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng) induced the release of IL-8 (3314–6025 pg/ml), TNF-α (39–359 pg/ml), and IL-10 (2–96 pg/ml), in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1∶10, 1∶100, and 1∶200) suppressed the secretion of IL-8, TNF-α, and IL-10 between 95–100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria. PMID:23284883

  8. Flagella from five Cronobacter species induce pro-inflammatory cytokines in macrophage derivatives from human monocytes.

    PubMed

    Cruz-Córdova, Ariadnna; Rocha-Ramírez, Luz M; Ochoa, Sara A; González-Pedrajo, Bertha; Gónzalez-Pedrajo, Bertha; Espinosa, Norma; Eslava, Carlos; Hernández-Chiñas, Ulises; Mendoza-Hernández, Guillermo; Rodríguez-Leviz, Alejandra; Valencia-Mayoral, Pedro; Sadowinski-Pine, Stanislaw; Hernández-Castro, Rigoberto; Estrada-García, Iris; Muñoz-Hernández, Onofre; Rosas, Irma; Xicohtencatl-Cortes, Juan

    2012-01-01

    Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10) in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ∼28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng) induced the release of IL-8 (3314-6025 pg/ml), TNF-α (39-359 pg/ml), and IL-10 (2-96 pg/ml), in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1∶10, 1∶100, and 1∶200) suppressed the secretion of IL-8, TNF-α, and IL-10 between 95-100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria.

  9. Pan-genome analysis of the emerging foodborne pathogen Cronobacter spp. suggests a species-level bidirectional divergence driven by niche adaptation

    PubMed Central

    2013-01-01

    Background Members of the genus Cronobacter are causes of rare but severe illness in neonates and preterm infants following the ingestion of contaminated infant formula. Seven species have been described and two of the species genomes were subsequently published. In this study, we performed comparative genomics on eight strains of Cronobacter, including six that we sequenced (representing six of the seven species) and two previously published, closed genomes. Results We identified and characterized the features associated with the core and pan genome of the genus Cronobacter in an attempt to understand the evolution of these bacteria and the genetic content of each species. We identified 84 genomic regions that are present in two or more Cronobacter genomes, along with 45 unique genomic regions. Many potentially horizontally transferred genes, such as lysogenic prophages, were also identified. Most notable among these were several type six secretion system gene clusters, transposons that carried tellurium, copper and/or silver resistance genes, and a novel integrative conjugative element. Conclusions Cronobacter have diverged into two clusters, one consisting of C. dublinensis and C. muytjensii (Cdub-Cmuy) and the other comprised of C. sakazakii, C. malonaticus, C. universalis, and C. turicensis, (Csak-Cmal-Cuni-Ctur) from the most recent common ancestral species. While several genetic determinants for plant-association and human virulence could be found in the core genome of Cronobacter, the four Cdub-Cmuy clade genomes contained several accessory genomic regions important for survival in a plant-associated environmental niche, while the Csak-Cmal-Cuni-Ctur clade genomes harbored numerous virulence-related genetic traits. PMID:23724777

  10. Isolation and Antimicrobial Testing of Aeromonas spp., Citrobacter spp., Cronobacter spp., Enterobacter spp., Escherichia spp., Klebsiella spp., and Trabulsiella spp. from the Gallbladder of Pigs.

    PubMed

    Evangelopoulou, Grammato; Filioussis, Georgios; Kritas, Spyridon; Kantere, Maria; Burriel, Angeliki R

    2015-01-01

    The presence of Gram-negative bacteria species, other than Salmonella spp., in the gallbladder of pigs was examined. Isolated Gram-negative bacteria were assigned to species using the Microgen™ GnA+B-ID Systems. Of the 64 isolated strains 43 were identified as Escherichia coli, seven as Enterobacter spp., three each as Klebsiella spp., Citrobacterfreundii, Aeromonas hydrophila and Cronobacter sakazakii and one each as Escherichiafergusonii and Trabulsiella guamensis. Their antibiograms showed very high resistance to ampicillin, amoxicillin, tetracycline, chloramphenicol and sulfamethoxazole/trimethoprim. It was concluded that the pigs' gallbladder is a reservoir of potentially pathogenic Gram-negative bacteria for pork consumers.

  11. Characterisation of the antibacterial properties of a bacterial derived peptidoglycan hydrolase (LysCs4), active against C. sakazakii and other Gram-negative food-related pathogens.

    PubMed

    Endersen, Lorraine; Coffey, Aidan; Ross, R Paul; McAuliffe, Olivia; Hill, Colin; O'Mahony, Jim

    2015-12-23

    Illness caused by the consumption of contaminated food products continues to represent one of the main challenges facing food manufacturers worldwide. Even with current intervention technologies and increased hygiene measures, foodborne illness remains a significant threat to public health. This coupled with the increasing emergence of multidrug resistant pathogens has increased the need for the development of novel technologies for pathogen control. Bacterial derived peptidoglycan hydrolases represent a vast and highly diverse group of enzymes with potential for biocontrol of a range of Gram-positive and Gram-negative foodborne pathogens. In this study, we describe the identification, cloning, expression and purification of a peptidoglycan hydrolase (LysCs4) derived from Cronobacter sakazakii for biocontrol of the aforementioned infant formula pathogen itself. In silico analysis of LysCs4 revealed the gene to display greatest sequence similarity to a putative lysozyme encoded by the lytic Cronobacter phage ES2. Conserved domain analysis of LysCs4 revealed the presence of a single catalytic domain predicted to display O-Glycosyl hydrolase activity and to be a member of the GH24 family. The ability of this enzyme to hydrolyse the peptidoglycan of 25 Gram-negative strains, across 4 different genera, highlights its potential as a novel candidate for biocontrol of C. sakazakii and other Gram-negative food related pathogens.

  12. Effect of vanillin, ethyl vanillin, and vanillic acid on the growth and heat resistance of Cronobacter species.

    PubMed

    Yemiş, Gökçe Polat; Pagotto, Franco; Bach, Susan; Delaquis, Pascal

    2011-12-01

    Preservatives could be part of an effective intervention strategy for the control of Cronobacter species in foods, but few compounds with the desired antimicrobial properties have been identified to date. We examined the antibacterial activity of vanillin, ethyl vanillin, and vanillic acid against seven Cronobacter spp. in quarter-strength tryptic soy broth with 5 g/liter yeast extract (TSBYE) adjusted to pH 5.0, 6.0, and 7.0 at 10, 21, and 37°C. All compounds exhibited pH- and temperature-dependant bacteriostatic and bactericidal activity. MICs of vanillin and ethyl vanillin consistently increased with decreasing pH and temperature, but vanillic acid had little activity at pH values of 6.0 and 7.0. The MICs for all temperatures, pH values, and bacterial strains tested were 2 mg/ml ethyl vanillin, 3 mg/ml vanillin, and >8 mg/ml vanillic acid. MBCs also were influenced by pH, although significantly higher concentrations were needed to inactivate the bacteria at 21°C than at 10 or 37°C. Survivor curves for Cronobacter sakazakii strains at the MBCs of each compound revealed that all treatments resulted in immediate loss of cell viability at 37°C. Measurements of propidium iodide uptake indicated that the cell membranes were damaged by exposure to all three compounds. The thermal resistance of C. sakazakii was examined at 58°C in TSBYE supplemented with MBCs of each compound at pH 5.0 and 6.0. D-values at pH 5.0 were reduced from 14.56 ± 0.60 min to 0.93 ± 0.01, 0.63 ± 0.01, and 0.98 ± 0.02 min for vanillin, ethyl vanillin, and vanillic acid, respectively. These results suggest that vanillin, ethyl vanillin, and vanillic acid may be useful for the control of Cronobacter spp. in food during preparation and storage.

  13. The structure of the O-antigen in the endotoxin of the emerging food pathogen Cronobacter (Enterobacter) muytjensii strain 3270.

    PubMed

    MacLean, Leann L; Pagotto, Franco; Farber, Jeffrey M; Perry, Malcolm B

    2009-03-31

    Strains of the Gram-negative bacterium Cronobacter (formerly known as Enterobacter) sakazakii have been identified as emerging opportunistic pathogens that can cause enterocolitis, bacteraemia, meningitis, and brain abscess, and they have been particularly associated with meningitis in neonates where infant milk formulae have been epidemiologically linked to the disease. A study of the lipopolysaccharides produced by clinical isolates using chemical, 2D 1H and 13C NMR, and MS methods revealed that the O-polysaccharide produced by Cronobacter muytjensii strain 3270, isolated from powdered infant formula from Denmark, was a linear unbranched polymer of a repeating pentasaccharide unit composed of 2-acetamido-2-deoxy-d-galactose (d-GalNAc), 2-acetamido-2-deoxy-d-glucose (d-GlcNAc), 3-acetamido-3-deoxy-d-quinovose (d-Qui3NAc), l-rhamnose (l-Rha), and d-glucuronic acid (d-GlcA) in equimolar ratio, and has the structure -->3)-alpha-D-GalpNAc-(1-->4)-alpha-D-Quip3NAc-(1-->3)-alpha-L-Rhap-(1-->6)-alpha-D-GlcpNAc-(1-->4)-beta-D-GlcpA-(1--> The specific structural characteristics of the O-polysaccharides of C. muytjensii may be of value in the identification and tracking of the bacterial pathogen.

  14. Detection of Cronobacter Genus in Powdered Infant Formula by Enzyme-linked Immunosorbent Assay Using Anti-Cronobacter Antibody

    PubMed Central

    Song, Xinjie; Shukla, Shruti; Lee, Gibaek; Park, Sunhyun; Kim, Myunghee

    2016-01-01

    Cronobacter species (Cronobacter spp.) are hazardous foodborne pathogens associated with baby food, powdered infant formula (PIF). To develop a rapid and sensitive method for simultaneous detection of seven Cronobacter spp. in PIF, an indirect non-competitive enzyme-linked immunosorbent assay (INC-ELISA) was developed based on a novel immunoglobulin G (IgG), anti-Cronobacter IgG. The developed INC-ELISA was able to detect seven Cronobacter spp. at concentrations ranging from (5.6 ± 0.30) × 103 to (2.1 ± 0.01) × 105 colony forming unit (CFU)/mL in pure culture. Further, INC-ELISA employing anti-Cronobacter IgG was applicable for analysis of PIF samples contaminated with less than <10 cells of Cronobacter spp. per 25 g of PIF in 36 h. The developed antibody showed slight cross-reactivity with Franconibacter pulveris (LMG 24057) at high concentration (108 CFU/mL). The INC-ELISA method displayed excellent specificity without compromising cross-reactivity with other foodborne pathogens. The INC-ELISA assay method developed in this study using a novel anti-Cronobacter IgG facilitated highly sensitive, efficient, and rapid detection of Cronobacter spp. in baby food. PMID:27493642

  15. Isolation and identification of Enterobacter sakazakii in infant milk formulas.

    PubMed

    Torres-Chavolla, Edith; Ramírez-Cerda, Elsa; Gutiérrez-Rojo, Rosalba

    2007-01-01

    Enterobacter sakazakii is a pathogen of increasing medical concern, due to it being implicated in cases of meningitis, sepis, and necrotizing enterocolitis associated with the consumption of contaminated infant milk formula. At present, the method adopted by the Mexican food industry for the isolation and identification of E. sakazakii is based on the methodology of the United States Food and Drug Administration (FDA). However, this procedure is laborious and requires 7 days to obtain a confirmative result. The objective of this study was to determine the presence of E. sakazakii in two types of powdered infant milk formula, using an alternative method that requires less time and a smaller sample size than the FDA protocol. We adapted Leuschner's procedure by eliminating violet red bile glucose agar (VRBG) plates and instead adopting white light incubation to stimulate yellow pigment development. This allowed for isolation of E. sakazakii from powdered infant milk formula using a smaller sample and requiring only 5 days for analysis. Results showed that 92% of formula 1 and 32% of formula 2 was positive for E. sakazakii. The high contamination level of E. sakazakii suggests the need for monitoring hygienic conditions in the manufacturing plant and to assess the prevalence of E. sakazakii in powdered infant milk formulas sold in México.

  16. Powder infant formula milk contaminated with Enterobacter sakazakii.

    PubMed

    Oonaka, Kenji; Furuhata, Katsunori; Hara, Motonobu; Fukuyama, Masafumi

    2010-03-01

    To clarify the route and source of Enterobacter sakazakii infection in a basic study, we analyzed powder infant formula milk (PIF), which may be an important source of infantile infection, regarding contamination with Enterobacteriaceae including this type of bacteria, and conducted drug sensitivity tests with various antimicrobial agents. Enterobacteriaceae was isolated 36 (24.2%) of 149 PIF samples. These comprised of 12 (19.7%) of 61 domestically produced samples and 24 (27.3%) of 88 imported samples. E. sakazakii was isolated in 9 (6.6%) of the 149 PIF samples. These comprised 4 (6.6%) of 61 domestically produced samples and 5 (5.7%) of 88 imported samples. In 8 of the 9 samples in which E. sakazakii was isolated, the bacterial levels were estimated to be 0.36 MPN/100 g. However, one imported sample showed a bacterial level of 0.91 MPN/100 g. In the drug sensitivity tests of E. sakazakii isolated from PIF, we compared the MIC(90) values. E. sakazakii was highly sensitive to 9 agents: cefotaxime, ceftriaxone, cefoperazone, ceftazidime, cefpirome, cefozopran, gentamicin, meropenem, and ciprofloxacin, and moderately sensitive to 5 agents: piperacillin, erythromycin, minocycline, chloramphenicol, and rifampicin. However, it was resistant to 2 agents, ampicillin and lincomycin.

  17. [Taxonomy of Enterobacter sakazakii and the biological characteristics of the new species and genus].

    PubMed

    Zhao, Guiming; Yuan, Fei; Yang, Hairong; Zhao, Yongsheng; Chen, Ying

    2010-03-01

    Enterobacter sakazakii, one of the major pathogens affecting the safety of infant formula powder was defined as a species in 1980. However, the new names and new combinations about Enterobacter sakazakii notified in volume 58, part 6, of the International Journal of Systematic and Evolutionary Microbiology (IJSB). The taxonomic relationship of strains described as E. sakazakii, biological characteristics of its new genus and species, the development related to its isolation and identification were reviewed in this paper, in order to facilitate the related personnel to keep in touch with the latest developments on E. sakazakii. It's also conducive to unify and standardize the Chinese name for E. sakazakii.

  18. Prevalence and Relative Risk of Cronobacter spp., Salmonella spp., and Listeria monocytogenes Associated with the Body Surfaces and Guts of Individual Filth Flies

    PubMed Central

    Pearson, Rachel E. Goeriz; Miller, Amy K.; Ziobro, George C.

    2012-01-01

    Although flies are important vectors of food-borne pathogens, there is little information to accurately assess the food-related health risk of the presence of individual flies, especially in urban areas. This study quantifies the prevalence and the relative risk of food-borne pathogens associated with the body surfaces and guts of individual wild flies. One hundred flies were collected from the dumpsters of 10 randomly selected urban restaurants. Flies were identified using taxonomic keys before being individually dissected. Cronobacter spp., Salmonella spp., and Listeria monocytogenes were detected using the PCR-based BAX system Q7. Positive samples were confirmed by culture on specific media and through PCR amplification and sequencing or ribotyping. Among collected flies were the housefly, Musca domestica (47%), the blowflies, Lucilia cuprina (33%) and Lucilia sericata (14%), and others (6%). Cronobacter species were detected in 14% of flies, including C. sakazakii, C. turicensis, and C. universalis, leading to the proposal of flies as a natural reservoir of this food-borne pathogen. Six percent of flies carried Salmonella enterica, including the serovars Poona, Hadar, Schwarzengrund, Senftenberg, and Brackenridge. L. monocytogenes was detected in 3% of flies. Overall, the prevalence of food-borne pathogens was three times greater in the guts than on the body surfaces of the flies. The relative risk of flies carrying any of the three pathogens was associated with the type of pathogen, the body part of the fly, and the ambient temperature. These data enhance the ability to predict the microbiological risk associated with the presence of individual flies in food and food facilities. PMID:22941079

  19. Identification and phylogeny of Enterobacter sakazakii relative to Enterobacter and Citrobacter Species.

    PubMed

    Iversen, Carol; Waddington, Michael; On, Stephen L W; Forsythe, Stephen

    2004-11-01

    The phylogenetic relationships of Enterobacter sakazakii strains were investigated using 16S ribosomal DNA (rDNA) and hsp60 sequencing. Each analysis distributed E. sakazakii strains among four clusters, indicating substantial taxonomic heterogeneity. The E. sakazakii type strain 16S rDNA sequence was 97.8% similar to that of Citrobacter koseri but 97.0% similar to that of Enterobacter cloacae.

  20. Enterobacter sakazakii in food and beverages (other than infant formula and milk powder).

    PubMed

    Friedemann, Miriam

    2007-05-01

    The ubiqitous microorganism Enterobacter sakazakii is a rare contaminant of infant formula and may cause severe systemic infection in neonates. So far, other food is not known to cause E. sakazakii-infections. The scarce information about the ecology of E. sakazakii and the uncertainty concerning the source of infection in children and adults warrant a summary of the current knowledge about the presence of this opportunistic microorganism in food other than infant formula. This review systematizes publications on the presence of E. sakazakii in food and beverages until June 2006. Food other than infant formula has been rarely investigated for the presence of E. sakazakii. Nevertheless, this microorganism could be isolated from a wide spectrum of food and food ingredients. E. sakazakii was isolated from plant food and food ingredients like cereal, fruit and vegetables, legume products, herbs and spices as well as from animal food sources like milk, meat and fish and products made from these foods. The spectrum of E. sakazakii-contaminated food covers both raw and processed food. The kind of processing of E. sakazakii-contaminated food was not restricted to dry products. Fresh, frozen, ready-to-eat, fermented and cooked food products as well as beverages and water suitable for the preparation of food, were found to be contaminated by E. sakazakii. Although E. sakazakii-contaminated food do not have general public health significance, measures for prevention should consider the presence of E. sakazakii in food, food ingredients, their processing and preparation as possible source of contamination, colonization or infection.

  1. Diversity of Cronobacter spp. isolates from the vegetables in the middle-east coastline of China.

    PubMed

    Chen, Wanyi; Yang, Jielin; You, Chunping; Liu, Zhenmin

    2016-06-01

    Cronobacter spp. has caused life-threatening neonatal infections mainly resulted from consumption of contaminated powdered infant formula. A total of 102 vegetable samples from retail markets were evaluated for the presence of Cronobacter spp. Thirty-five presumptive Cronobacter isolates were isolated and identified using API 20E and 16S rDNA sequencing analyses. All isolates and type strains were characterized using enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and genetic profiles of cluster analysis from this molecular typing test clearly showed that there were differences among isolates from different vegetables. A polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) based on the amplification of the gyrB gene (1258 bp) was developed to differentiate among Cronobacter species. A new PCR-RFLP assay based on the amplification of the gyrB gene using Alu I and Hinf I endonuclease combination is established and it has been confirmed an accurate and rapid subtyping method to differentiate Cronobacter species. Sequence analysis of the gyrB gene was proven to be suitable for the phylogenetic analysis of the Cronobacter strains, which has much better resolution based on SNPs in the identification of Cronobacter species specificity than PCR-RFLP and ERIC-PCR. Our study further confirmed that vegetables are one of the most common habitats or sources of Cronobacter spp. contamination in the middle-east coastline of China.

  2. Novel PCR-RFLP system based on rpoB gene for differentiation of Cronobacter species.

    PubMed

    Vlach, Jiří; Javůrková, Barbora; Karamonová, Ludmila; Blažková, Martina; Fukal, Ladislav

    2017-04-01

    Bacteria from the genus Cronobacter are opportunistic foodborne pathogens that can cause severe infections. More rapid, cost-effective and reliable methods are still required for the species identification of Cronobacter spp. In this study, we present a novel PCR-RFLP-based method that uses a newly designed pair of primers for the PCR-amplification of a partial rpoB gene sequence (1635 bp). The amplified products of DNA from 80 Cronobacter strains were separately digested with three restriction endonucleases (Csp6I, HinP1I, MboI). Using the obtained restriction patterns, a PCR-RFLP identification system was created to enable differentiation between all seven currently-known Cronobacter species. The functionality of our method was successfully verified on real food samples. Moreover, the relationships between the Cronobacter species were determined via a phylogenetic tree created from the RFLP patterns.

  3. Characterization of surface proteins of Cronobacter muytjensii using monoclonal antibodies and MALDI-TOF Mass spectrometry

    PubMed Central

    2011-01-01

    Background Cronobacter spp. is a newly emerging pathogen that causes meningitis in infants and other diseases in elderly and immunocompromised individuals. This study was undertaken to investigate surface antigenic determinants in Cronobacter spp. using monoclonal antibodies (MAbs) and MALDI-TOF Mass spectrometry. Results Spleenocytes from mice that were immunized with heat-killed (20 min, 80°C) Cronobacter cells were fused with SP2 myeloma cells. Five desirable MAbs (A1, B5, 2C2, C5 and A4) were selected. MAbs A1, B5, 2C2 and C5 were of IgG2a isotype while A4 was an IgM. Specificity of the MAbs was determined by using immunoblotting with outer membrane protein preparations (OMPs) extracted from 12 Cronobacter and 6 non-Cronobacter bacteria. All MAbs recognized proteins with molecular weight ranging between 36 and 49 kDa except for one isolate (44) in which no OMPs were detected. In addition, MAbs recognized two bands (38-41 kDa) in four of the non-Cronobacter bacteria. Most of the proteins recognized by the MAbs were identified by MALDI-TOF peptide sequencing and appeared to be heterogeneous with the identities of some of them are still unknown. All MAbs recognized the same epitope as determined by an additive Index ELISA with their epitopes appeared to be conformational rather than sequential. Further, none of the MAbs recognized purified LPS from Cronobacter spp. Specificity of the MAbs toward OMPs was further confirmed by transmission electron microscopy. Conclusions Results obtained in this study highlight the immunological cross-reactivity among Cronobacter OMPs and their Enterobacteriaceae counterparts. Nevertheless, the identity of the identified proteins appeared to be different as inferred from the MALDI-TOF sequencing and identification. PMID:21702985

  4. Survival of Enterobacter sakazakii in infant cereal as affected by composition, water activity, and temperature.

    PubMed

    Lin, Li-Chun; Beuchat, Larry R

    2007-01-01

    Enterobacter sakazakii infections in preterm neonates and infants have been epidemiologically associated with consumption of reconstituted powdered infant formula. The bacterium has been isolated from grain, infant cereals, and cereal factory environments. A study was done to determine the survival characteristics of E. sakazakii initially at populations of 0.31 and 5.03 logCFU/g of infant rice cereal (a(w) 0.30, 0.45-0.46, and 0.68-0.69). Cereal was stored at 4, 21, and 30 degrees C and populations were monitored for up to 12 months. Survival of the pathogen in infant rice, barley, oatmeal, and mixed grain cereals (a(w) 0.63-0.66, 0.76, or 0.82-0.83) initially containing a population of 4.93-5.64 logCFU/g and held at 4, 21, and 30 degrees C up to 24 weeks was determined. Populations decreased significantly (p < or = 0.05) in all cereals stored at 21 and 30 degrees C regardless of a(w). Increases in a(w) or storage temperature accelerated the rate of death of E. sakazakii in dry infant cereals. However, at an initial population of 0.31 logCFU/g, E. sakazakii survived in rice cereal (a(w) 0.30-0.69) for up to 12 months at all storage temperatures. Survival of E. sakazakii was not affected by the composition of dry infant rice, barley, mixed grain, and oatmeal cereals (initial a(w) 0.63-0.83) stored for up to 24 weeks at 4, 21, or 30 degrees C. This study demonstrated that E. sakazakii can survive for up to 12 months in infant cereals having a wide range of a(w) when storage is at temperatures simulating those to which they may be exposed during distribution, at retail, and in the home.

  5. Inactivation of Enterobacter sakazakii in reconstituted infant formula by trans-cinnamaldehyde.

    PubMed

    Amalaradjou, Mary Anne Roshni; Hoagland, Thomas A; Venkitanarayanan, Kumar

    2009-02-15

    Enterobacter sakazakii is an emerging pathogen which causes a life-threatening form of meningitis, necrotizing colitis and meningoencephalitis in neonates and children. Epidemiological studies implicate dried infant formula as the principal source of the pathogen. Trans-cinnamaldehyde is a major component of bark extract of cinnamon. It is classified as generally recognized as safe (GRAS) by the U.S. Food and Drug Administration, and is approved for use in food (21 CFR 182.60). The objective of this study was to determine the antibacterial effect of trans-cinnamaldehyde on E. sakazakii in reconstituted infant formula. A 5-strain mixture of E. sakazakii was inoculated into 10 ml samples of reconstituted infant formula (at 6.0 log CFU/ml) containing 0%, 0.15%, 0.3% or 0.5% trans-cinnamaldehyde. The samples were incubated at 37, 23, 8 or 4 degrees C for 0, 6, 10 and 24 h, and the surviving populations of E. sakazakii at each sampling time were enumerated. In addition, potential cytotoxicity of trans-cinnamaldehyde, if any, was determined on human embryonic intestinal cells (INT-407). The treatments containing trans-cinnamaldehyde significantly reduced (P<0.05) the population of E. sakazakii, compared to the controls. Trans-cinnamaldehyde (0.5%) reduced the pathogen to undetectable levels by 4 h of incubation at 37 or 23 degrees C and 10 h of incubation at 8 or 4 degrees C, respectively. Trans-cinnamaldehyde produced no cytotoxic effects on human embryonic intestinal cells at the tested concentrations. Results indicate that trans-cinnamaldehyde could potentially be used to kill E. sakazakii in reconstituted infant formula, however sensory studies are warranted before recommending its use.

  6. Enterobacter sakazakii: An Emerging Pathogen in Infants and Neonates

    PubMed Central

    Petrosyan, Mikael; Ford, Henri R.; Prasadarao, Nemani V.

    2008-01-01

    Abstract Background Enterobacter sakazakii (ES) is an emerging pathogen associated with the ingestion of contaminated reconstituted formula that causes necrotizing enterocolitis, sepsis, and meningitis in low-birth-weight preterm neonatal infants. Necrotizing enterocolitis remains the most common gastrointestinal surgical emergency in these infants. In recent years, the International Commission on Microbiological Specifications for Foods has ranked ES a “severe hazard for restricted populations.” Because of its resistance to certain antibiotics, better understanding of ES pathogenesis is needed to aid in the development of new preventive strategies. Methods Review of pertinent English-language literature. Results Neonatal and older infants appear to be at the highest risk, although adult ES infections have been reported. We discuss the origins of ES, the detection and pathogenesis of the disease, and potential prevention strategies. Conclusions The precise pathogenesis of ES remains a mystery. Appropriate measures by parents, infant formula manufacturers, and health care providers, as well as understanding of the pathogenesis, are important in the prevention of ES-related infections. PMID:18687047

  7. Growth inhibition of Cronobacter spp. strains in reconstituted powdered infant formula acidified with organic acids supported by natural stomach acidity.

    PubMed

    Zhu, S; Schnell, S; Fischer, M

    2013-09-01

    Cronobacter is associated with outbreaks of rare, but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in newborns. This study was conducted to determine the effect of organic acids on growth of Cronobacter in laboratory medium and reconstituted powdered infant formula (PIF) as well as the bacteriostatic effect of slightly acidified infant formula when combined with neonatal gastric acidity. Inhibitory effect of seven organic acids on four acid sensitive Cronobacter strains was determined in laboratory medium with broth dilution method at pH 5.0, 5.5 and 6.0. Acetic, butyric and propionic acids were most inhibitive against Cronobacter in the laboratory medium. The killing effect of these three acids was partially buffered in reconstituted PIF. Under neonatal gastric acid condition of pH 5.0, the slightly acidified formula which did not exert inhibition effect solely reduced significantly the Cronobacter populations. A synergistic effect of formula moderately acidified with organic acid combined with the physiological infant gastric acid was visible in preventing the rapid growth of Cronobacter in neonatal stomach. The study contributed to a better understanding of the inhibitory effect of organic acids on Cronobacter growth in different matrixes and provided new ideas in terms of controlling bacteria colonization and translocation by acidified formula.

  8. Inactivation of Enterobacter sakazakii, Bacillus cereus, and Salmonella typhimurium in powdered weaning food by electron-beam irradiation

    NASA Astrophysics Data System (ADS)

    Hong, Yun-Hee; Park, Ji-Yong; Park, Jong-Hyun; Chung, Myong-Soo; Kwon, Ki-Sung; Chung, Kyungsook; Won, Misun; Song, Kyung-Bin

    2008-09-01

    Inactivation of Enterobacter sakazakii, Bacillus cereus, and Salmonella typhimurium were evaluated in powdered weaning food using electron-beam irradiation. E. sakazakii, B. cereus, and S. typhimurium were eliminated by irradiation at 16, 8, and 8 kGy, respectively. The D10-vlaues of E. sakazakii, B. cereus, and S. typhimurium inoculated on powdered weaning food were 4.83, 1.22, and 0.98 kGy, respectively. The results suggest that electron-beam irradiation should inhibit the growth of pathogenic bacteria on baby food without impairing qualities.

  9. Inactivation of Enterobacter sakazakii of dehydrated infant formula by gamma-irradiation

    NASA Astrophysics Data System (ADS)

    Lee, Ju-Woon; Oh, Sang-Hee; Byun, Eui-Baek; Kim, Jae-Hun; Kim, Jang-Ho; Woon, Jae-Ho; Byun, Myung-Woo

    2007-11-01

    Enterobacter sakazakii has been implicated as a causal organism in a severe form of neonatal meningitis, with reported mortality rates of 20%. The population at greatest risk is immunocompromised infants of any age. Dried infant formula has been identified as a potential source of the organism in both outbreaks and sporadic cases. The objective of this study was to investigate theirradiation effect of the inactivation on E. sakazakii (ATCC 29544) of a dehydrated infant formula. The D10-values were 0.22-0.27 and 0.76 kGy for broth and dehydrated infant formula, respectively. The irradiation at 5.0 kGy was able to completely eliminate the E. sakazakii inoculated at 8.0 to 9.0 log CFU g -1 onto a dehydrated infant formula. There was no regrowth for all samples during the time they were stored at 10 °C for 6 h after rehydration. The present results indicated that a gamma-irradiation could potentially be used to inactivate E. sakazakii in a dehydrated powdered infant formula.

  10. Inactivation of Enterobacter sakazakii by Water-soluble Muscadine Seed Extracts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hot and cold water-soluble muscadine (Vitis rotundifolia) seed extracts and their polar and polyphenol fractions from two Muscadine cultivars (‘Ison’, purple and ‘Carlos’, bronze) were investigated for their inhibition of Enterobacter sakazakii. The heat treatment on each seed extract not only incre...

  11. Microbiological quality of selected spices and herbs including the presence of Cronobacter spp.

    PubMed

    Garbowska, M; Berthold-Pluta, A; Stasiak-Różańska, L

    2015-08-01

    The cultivation of spices and herbs in parts of the world characterized by warm climate and high humidity provides excellent conditions for the development of microorganisms, including the undesirable ones. The aim of this study was to determine the microbiological quality of spices and herbs available on the Polish market, considering the occurrence of Cronobacter species bacteria. Analyses covered 60 samples of commercial spices and herbs, including 38 samples of dried herbs (basil, bay leaves, thyme, oregano, tarragon, marjoram, dill, parsley, rosemary, lovage) and 16 samples of seasoning blends as well as 6 samples of spices seeds and fruits (pimento, black pepper, coriander). All samples were tested for the total count of aerobic mesophilic bacteria (TAMB) and for the presence of Cronobacter spp. In most of the samples of spices and herbs (60.0%), the TAMB did not exceed 10(4) CFU/g, and the level regarded as unacceptable (>10(6) CFU/g) was not identified in any of the samples. The presence of Cronobacter spp. was demonstrated in 10 (16.7%) samples of the analyzed products, however these were mainly samples of herbs (basil, tarragon, parsley) and one sample of a seasoning blend (Provence herbs). The highest microbiological contamination (TAMB) was found in samples of herbs (oregano, tarragon, basil) and in ready seasoning blends, in 21.1% and 25.0% of which the total count of aerobic mesophiles was in the range of 10(5)-10(6) CFU/g. In all samples of spices seeds and fruits (coriander, black pepper and pimento), the total count of aerobic bacteria reached <10(4) CFU/g. Results achieved in the study indicate good hygienic conditions in the production process of spices and herbs available on the Polish market. The study demonstrated also that dried spices and herbs may be carriers of Cronobacter species bacteria, though their presence in not often detected in products of this type.

  12. Evaluation of zebrafish as a model to study the pathogenesis of the opportunistic pathogen Cronobacter turicensis.

    PubMed

    Fehr, Alexander; Eshwar, Athmanya K; Neuhauss, Stephan C F; Ruetten, Maja; Lehner, Angelika; Vaughan, Lloyd

    2015-05-01

    Bacteria belonging to the genus Cronobacter spp. have been recognized as causative agents of life-threatening systemic infections, primarily in premature, low-birth weight and/or immune-compromised neonates. Knowledge remains scarce regarding the underlying molecular mechanisms of disease development. In this study, we evaluated the use of a zebrafish model to study the pathogenesis of Cronobacter turicensis LMG 23827(T), a clinical isolate responsible for two fatal sepsis cases in neonates. Here, the microinjection of approximately 50 colony forming units (CFUs) into the yolk sac resulted in the rapid multiplication of bacteria and dissemination into the blood stream at 24 h post infection (hpi), followed by the development of a severe bacteremia and larval death within 3 days. In contrast, the innate immune response of the embryos was sufficiently developed to control infection after the intravenous injection of up to 10(4) CFUs of bacteria. Infection studies using an isogenic mutant devoid of surviving and replicating in human macrophages (ΔfkpA) showed that this strain was highly attenuated in its ability to kill the larvae. In addition, the suitability of the zebrafish model system to study the effectiveness of antibiotics to treat Cronobacter infections in zebrafish embryos was examined. Our data indicate that the zebrafish model represents an excellent vertebrate model to study virulence-related aspects of this opportunistic pathogen in vivo.

  13. Temporal and spatial distribution of Cronobacter isolates in a milk powder processing plant determined by pulsed-field gel electrophoresis.

    PubMed

    Hein, Ingeborg; Gadzov, Boris; Schoder, Dagmar; Foissy, Helmut; Malorny, Burkhard; Wagner, Martin

    2009-03-01

    A milk powder processing line was sampled for the presence of Enterobacteriaceae and the opportunistic neonatal pathogen Cronobacter at six different sampling sites during an 11-month period. The highest number of Enterobacteriaceae-positive samples was recovered from the raw milk concentrate before pasteurization (78.2%) and from nonproduct samples of the processing line (86.5%), which included swabs from the drying tower and screw conveyers, swabs from the explosion chamber, waste water after the automated cleaning-in-place procedure, air filter cut-outs, and floor samples underneath the outlet of the packaging machine. The prepackaged and packaged final product was contaminated at a rate of 6.6% and 7.1%, respectively. The prevalence of Cronobacter in the prefinal product and the prepackaged and packaged final product was 14.3%, 3.8%, and 2.1%, respectively. Pulsed-field gel electrophoresis (PFGE) analysis of 133 Cronobacter isolates yielded 40 different PFGE profiles. Long-term persistence in the processing line of some of these PFGE profiles was observed. Comparison of the PFGE profiles recovered at different sampling sites revealed the supply air as a potential source for extrinsic Cronobacter contamination. In addition, recovery of the same PFGE profiles before and after CIP events followed by heat treatment indicated the inefficiency of these hygiene measures to completely eliminate Cronobacter from all areas of the processing line. This information provides an essential basis for developing control and prevention strategies concerning this opportunistic pathogen.

  14. Growth of Enterobacter sakazakii in reconstituted infant formula as affected by composition and temperature.

    PubMed

    Gurtler, Joshua B; Beuchat, Larry R

    2007-09-01

    The ability of Enterobacter sakazakii to cause infections in infants, coupled with its documented presence in some lots of commercially manufactured powdered infant formula, raises a concern about the potential for its growth in reconstituted formula, with consequent increased safety risk. A study was done to determine these characteristics in four commercial milk-based powdered infant formulas and two soy-based formulas reconstituted with water and inoculated with a 10-strain mixture of E. sakazakii at populations of 0.02 and 0.53 CFU/ml (ca. 13 CFU/100 g and ca. 409 CFU/100 g of powdered formula, respectively). Reconstituted formulas were stored at 4, 12, 21, and 30 degrees C, and populations were monitored up to 72 h. E. sakazakii did not grow in formulas stored at 4 degrees C, although it was detected by enrichment of all formulas 72 h after reconstitution. Initially at a population of 0.02 CFU/ml, E. sakazakii grew to populations > or = 1 log CFU/ml of reconstituted formulas held at 12, 21, and 30 degrees C for 48, 12, and 8 h, respectively. At an initial population of 0.53 CFU/ml, the pathogen grew to populations > or = 1 log CFU/ml in reconstituted infant formula held at 12 and 21 degrees C for 24 and 8 h, respectively, and to populations 2.55 to 3.14 log CFU/ml when held at 30 degrees C for 8 h. Populations initially at 0.02 and 0.53 CFU/ml of reconstituted formula increased to < or = 0.25 and 0.4 log CFU/ml, respectively, when formulas were held at 30 degrees C for 4 h. Growth was not greatly influenced by the composition of formulas. Results show that the hang time for reconstituted infant formula held at temperatures in neonatal intensive care units should be no longer than 4 h. Portions of reconstituted infant formula not fed to infants should be stored at < or = 4 degrees C, a temperature at which E. sakazakii will not grow.

  15. Survival of Enterobacter sakazakii on fresh produce as affected by temperature, and effectiveness of sanitizers for its elimination.

    PubMed

    Kim, Hoikyung; Ryu, Jee-Hoon; Beuchat, Larry R

    2006-09-01

    A study was done to determine the survival characteristics of Enterobacter sakazakii on the surface of apples, cantaloupes, strawberries, lettuce, and tomatoes stored at 4, 12, and 25 degrees C for 8-28 days. Populations significantly decreased (por=50 microg/ml, were equivalent in killing E. sakazakii on apples. Populations of E. sakazakii on apples treated with 10 microg/ml chlorine dioxide for 1 or 5 min were significantly reduced (por=4.00 log CFU/apple. Reductions of >or=3.70 log CFU/tomato were achieved by treatment with 10 microg/ml chlorine or chlorine dioxide or 40 microg/ml Tsunami 200 for 5 min. Reductions in populations of E. sakazakii on lettuce treated with chlorine at 10, 50, and 100 microg/ml for 1 min ranged from 1.61 to 2.50 log CFU/sample (26+/-4 g), compared to populations remaining on lettuce washed with water. Chlorine was less effective in killing E. sakazakii on lettuce than on apples or tomatoes. Treatment of lettuce with Tsunami 200 (40 and 80 microg/ml) for 5 min caused a reduction of >or=5.31 log CFU/sample. Results provide insights to predicting survival characteristics of E. sakazakii on produce and the efficacy of sanitizers in killing the bacterium.

  16. Linking Genomo- and Pathotype: Exploiting the Zebrafish Embryo Model to Investigate the Divergent Virulence Potential among Cronobacter spp.

    PubMed Central

    Eshwar, Athmanya K.; Tall, Ben D.; Gangiredla, Jayanthi; Gopinath, Gopal R.; Patel, Isha R.; Neuhauss, Stephan C. F.; Stephan, Roger; Lehner, Angelika

    2016-01-01

    Bacteria belonging to the genus Cronobacter have been recognized as causative agents of life-threatening systemic infections primarily in premature, low-birth weight and immune-compromised neonates. Apparently not all Cronobacter species are linked to infantile infections and it has been proposed that virulence varies among strains. Whole genome comparisons and in silico analysis have proven to be powerful tools in elucidating potential virulence determinants, the presence/absence of which may explain the differential virulence behaviour of strains. However, validation of these factors has in the past been hampered by the availability of a suitable neonatal animal model. In the present study we have used zebrafish embryos to model Cronobacter infections in vivo using wild type and genetically engineered strains. Our experiments confirmed the role of the RepF1B-like plasmids as “virulence plasmids” in Cronobacter and underpinned the importantce of two putative virulence factors—cpa and zpx—in in vivo pathogenesis. We propose that by using this model in vivo infection studies are now possible on a large scale level which will boost the understanding on the virulence strategies employed by these pathogens. PMID:27355472

  17. Fate of Enterobacter sakazakii attached to or in biofilms on stainless steel upon exposure to various temperatures or relative humidities.

    PubMed

    Kim, Hoikyung; Bang, Jihyun; Beuchat, Larry R; Ryu, Jee-Hoon

    2008-05-01

    Survival of Enterobacter sakazakii dried on the surface of stainless steel and exposed to 43% relative humidity, as affected by temperature, was studied. Populations of E. sakazakii (7.4 to 8.6 log CFU per coupon) on coupons dried for 2 h at 22 degrees C decreased significantly (P < or = 0.05) at 4, 25, and 37 degrees C within 10, 3, and 1 day(s), respectively, but the pathogen remained viable for up to 60 days. At a given storage temperature and time, reductions were significantly greater when cells had been suspended in water rather than in infant formula before drying. Formation of biofilm by E. sakazakii on stainless steel immersed in M9 medium, which contains minimal concentrations of nutrients, and infant formula at 25 degrees C and subsequent survival of cells at 25 degrees C as affected by exposure to 23, 43, 68, 85, and 100% relative humidity were investigated. Some of the cells in these biofilms survived under all test relative humidities for up to 42 days. The overall order of survival as affected by relative humidity was 100 > 23 = 43 = 68 > 85% relative humidity, regardless of the medium in which the biofilm was formed. Reduction in viability of cells was significantly greater in biofilm that had formed in M9 medium than in biofilm formed in infant formula. Results indicate that infant formula provides protection for attached cells, as well as cells in biofilm, against lethality on exposure to desiccation. These results are useful when predicting the survival characteristics of E. sakazakii on stainless steel surfaces in processing and preparation kitchen environments.

  18. Actual distribution of Cronobacter spp. in industrial batches of powdered infant formula and consequences for performance of sampling strategies.

    PubMed

    Jongenburger, I; Reij, M W; Boer, E P J; Gorris, L G M; Zwietering, M H

    2011-11-15

    The actual spatial distribution of microorganisms within a batch of food influences the results of sampling for microbiological testing when this distribution is non-homogeneous. In the case of pathogens being non-homogeneously distributed, it markedly influences public health risk. This study investigated the spatial distribution of Cronobacter spp. in powdered infant formula (PIF) on industrial batch-scale for both a recalled batch as well a reference batch. Additionally, local spatial occurrence of clusters of Cronobacter cells was assessed, as well as the performance of typical sampling strategies to determine the presence of the microorganisms. The concentration of Cronobacter spp. was assessed in the course of the filling time of each batch, by taking samples of 333 g using the most probable number (MPN) enrichment technique. The occurrence of clusters of Cronobacter spp. cells was investigated by plate counting. From the recalled batch, 415 MPN samples were drawn. The expected heterogeneous distribution of Cronobacter spp. could be quantified from these samples, which showed no detectable level (detection limit of -2.52 log CFU/g) in 58% of samples, whilst in the remainder concentrations were found to be between -2.52 and 2.75 log CFU/g. The estimated average concentration in the recalled batch was -2.78 log CFU/g and a standard deviation of 1.10 log CFU/g. The estimated average concentration in the reference batch was -4.41 log CFU/g, with 99% of the 93 samples being below the detection limit. In the recalled batch, clusters of cells occurred sporadically in 8 out of 2290 samples of 1g taken. The two largest clusters contained 123 (2.09 log CFU/g) and 560 (2.75 log CFU/g) cells. Various sampling strategies were evaluated for the recalled batch. Taking more and smaller samples and keeping the total sampling weight constant, considerably improved the performance of the sampling plans to detect such a type of contaminated batch. Compared to random sampling

  19. Molecular characterization of the alpha-glucosidase activity in Enterobacter sakazakii reveals the presence of a putative gene cluster for palatinose metabolism.

    PubMed

    Lehner, Angelika; Riedel, Kathrin; Rattei, Thomas; Ruepp, Andreas; Frishman, Dimitrij; Breeuwer, Pieter; Diep, Benjamin; Eberl, Leo; Stephan, Roger

    2006-12-01

    Enterobacter sakazakii is considered an opportunistic pathogen for premature infants and neonates. Although E. sakazakii has been isolated from various types of food, recontaminated dried infant formula has been epidemiologically identified as the major source of infection. Amongst others, alpha-glucosidase activity is one of the most important biochemical features, which differentiates E. sakazakii from other species in the family Enterobacteriaceae and has therefore been used as a selective marker in the development of differential media. However, it has been shown, that methods based on this biochemical feature are prone to producing false-positive results for presumptive E. sakazakii colonies due to the presence of this enzymatic activity in other species of the Enterobacteriaceae. Therefore, elucidation of the molecular basis responsible for the biochemical feature in E. sakazakii would provide novel targets suitable for the development of more specific and direct identification systems for this organism. By applying the bacterial artificial chromosome (BAC) approach, along with heterologous gene expression in Escherichia coli, the molecular basis of the alpha-glucosidase activity in E. sakazakii was characterized. Here we report the identification of two different alpha-glucosidase encoding genes. Homology searches of the deduced amino acid sequences revealed that the proteins belong to a cluster of gene products putatively responsible for the metabolism of isomaltulose (palatinose; 6-O-alpha-d-glucopyranosyl-d-fructose). The glycosyl-hydrolyzing activity of each protein was demonstrated by subcloning the respective open reading frames and screening of E. coli transformants for their ability to hydrolyze 4-methyl-umbelliferyl-alpha-d-glucoside. Analysis at the protein level revealed that both enzymes belong to the intracellular fraction of cell proteins. The presence of the postulated palatinose metabolism was proven by growth experiments using this sugar as

  20. Isolation of Enterobacter sakazakii from ass' milk in Sicily: case report, safety and legal issues.

    PubMed

    Conte, F; Passantino, A

    2008-07-01

    Enterobacter sakazakii (Es) infections are likely to involve newborns and infants, causing meningitis and necrotizing enterocolitis and sepsis. Contamination of infant formulae milk during factory production or bottle preparation is implicated. Es has been isolated from environmental sources and from food other than infant formula and milk powder, but why it is associated only with the consumption of infant formulae, is unclear. According to Regulation (EC) No. 2073/2005 on the microbiological criteria for foodstuffs, Es is considered a microorganisms of greatest concern in infant formulae and follow-on formulae. Es is included between "safety criteria". The isolation of two strains of Es from 50 samples of ass' milk in Sicily is described. The antibiotic resistance profile of the isolates revealed a multiple resistance profile, including fluoroquinolones, commonly used to treat the infections. The authors underline the importance of survey because in Italy ass' milk is considered one of the solutions for infants suffering from hypersensitivity to milk protein of some animal species. There is scarce information about the ecology and the uncertainty concerning the source of infection in the children and adults; the authors are concerned that ass' milk could become a high-risk food.

  1. Effects of preculturing conditions on lag time and specific growth rate of Enterobacter sakazakii in reconstituted powdered infant formula.

    PubMed

    Kandhai, M C; Reij, M W; Grognou, C; van Schothorst, M; Gorris, L G M; Zwietering, M H

    2006-04-01

    Enterobacter sakazakii can be present, although in low levels, in dry powdered infant formulae, and it has been linked to cases of meningitis in neonates, especially those born prematurely. In order to prevent illness, product contamination at manufacture and during preparation, as well as growth after reconstitution, must be minimized by appropriate control measures. In this publication, several determinants of the growth of E. sakazakii in reconstituted infant formula are reported. The following key growth parameters were determined: lag time, specific growth rate, and maximum population density. Cells were harvested at different phases of growth and spiked into powdered infant formula. After reconstitution in sterile water, E. sakazakii was able to grow at temperatures between 8 and 47 degrees C. The estimated optimal growth temperature was 39.4 degrees C, whereas the optimal specific growth rate was 2.31 h(-1). The effect of temperature on the specific growth rate was described with two secondary growth models. The resulting minimum and maximum temperatures estimated with the secondary Rosso equation were 3.6 degrees C and 47.6 degrees C, respectively. The estimated lag time varied from 83.3 +/- 18.7 h at 10 degrees C to 1.73 +/- 0.43 h at 37 degrees C and could be described with the hyperbolic model and reciprocal square root relation. Cells harvested at different phases of growth did not exhibit significant differences in either specific growth rate or lag time. Strains did not have different lag times, and lag times were short given that the cells had spent several (3 to 10) days in dry powdered infant formula. The growth rates and lag times at various temperatures obtained in this study may help in calculations of the period for which reconstituted infant formula can be stored at a specific temperature without detrimental impact on health.

  2. Evaluation of the International Organization for Standardization-International Dairy Federation (ISO-IDF) draft standard method for detection of Enterobacter sakazakii in powdered infant food formulas.

    PubMed

    Besse, Nathalie Gnanou; Leclercq, Alexandre; Maladen, Véronique; Tyburski, Corinne; Bertrand, Lombard

    2006-01-01

    As a result of the growing recognition of Enterobacter sakazakii as an emergent pathogen, the International Dairy Federation (IDF) and the International Organization for Standardization (ISO) have standardized a reference method for the detection of E. sakazakii in milk powder products and powdered infant food formulas (IFF). The objectives of this study were to assess the applicability of the ISO-IDF draft standard, and to compare several chromogenic selective media for E. sakazakii [ready-to-use ESIATM, homemade E. sakazakii isolation agar, and Druggan-Forsythe-lversen (DFI) agar], and a selective media for Enterobacteriaceae Violet Red Bile Glucose (VRBG). We found that the method is sensitive, selective, and applicable to the analysis of powdered IFF, provided that some modifications are added. In particular, definition of typical colonies on chromogenic media should be less restrictive, and the possibility of using chromogenic media other than ESIA should be introduced. Any of the chromogenic media tested here could be used initially, since their performances were similar. In these media, alpha-glucosidase-positive but non-yellow-pigmented isolates should be also considered. Consequently, the yellow pigmentation test should be abandoned, or completed with another test in order to select colonies to confirm. Although the specificity of VRBG was relatively poor, it could be used as a second nonchromogenic medium.

  3. Effect of heat shock and recovery temperature on variability of single cell lag time of Cronobacter turicensis.

    PubMed

    Xu, Y Zh; Métris, A; Stasinopoulos, D M; Forsythe, S J; Sutherland, J P

    2015-02-01

    The effect of heat stress and subsequent recovery temperature on the individual cellular lag of Cronobacter turicensis was analysed using optical density measurements. Low numbers of cells were obtained through serial dilution and the time to reach an optical density of 0.035 was determined. Assuming the lag of a single cell follows a shifted Gamma distribution with a fixed shape parameter, the effect of recovery temperature on the individual lag of untreated and sublethally heat treated cells of Cr. turicensis were modelled. It was found that the shift parameter (Tshift) increased asymptotically as the temperature decreased while the logarithm of the scale parameter (θ) decreased linearly with recovery temperature. To test the validity of the model in food, growth of low numbers of untreated and heat treated Cr. turicensis in artificially contaminated infant first milk was measured experimentally and compared with predictions obtained by Monte Carlo simulations. Although the model for untreated cells slightly underestimated the actual growth in first milk at low temperatures, the model for heat treated cells was in agreement with the data derived from the challenge tests and provides a basis for reliable quantitative microbiological risk assessments for Cronobacter spp. in infant milk.

  4. Taxonomic evaluation of the genus Enterobacter based on multilocus sequence analysis (MLSA): proposal to reclassify E. nimipressuralis and E. amnigenus into Lelliottia gen. nov. as Lelliottia nimipressuralis comb. nov. and Lelliottia amnigena comb. nov., respectively, E. gergoviae and E. pyrinus into Pluralibacter gen. nov. as Pluralibacter gergoviae comb. nov. and Pluralibacter pyrinus comb. nov., respectively, E. cowanii, E. radicincitans, E. oryzae and E. arachidis into Kosakonia gen. nov. as Kosakonia cowanii comb. nov., Kosakonia radicincitans comb. nov., Kosakonia oryzae comb. nov. and Kosakonia arachidis comb. nov., respectively, and E. turicensis, E. helveticus and E. pulveris into Cronobacter as Cronobacter zurichensis nom. nov., Cronobacter helveticus comb. nov. and Cronobacter pulveris comb. nov., respectively, and emended description of the genera Enterobacter and Cronobacter.

    PubMed

    Brady, Carrie; Cleenwerck, Ilse; Venter, Stephanus; Coutinho, Teresa; De Vos, Paul

    2013-07-01

    The taxonomy of Enterobacter has a complicated history, with several species transferred to and from this genus. Classification of strains is difficult owing to its polyphyletic nature, based on 16S rRNA gene sequences. It has been previously acknowledged that Enterobacter contains species which should be transferred to other genera. In an attempt to resolve the taxonomy of Enterobacter, MLSA based on partial sequencing of protein-encoding genes (gyrB, rpoB, infB and atpD) was performed on the type strains and reference strains of Enterobacter, Cronobacter and Serratia species, as well as members of the closely related genera Citrobacter, Klebsiella, Kluyvera, Leclercia, Mangrovibacter, Raoultella and Yokenella. Phylogenetic analyses of the concatenated nucleotide sequences revealed that Enterobacter can be divided into five strongly supported MLSA groups, suggesting that the species should be reclassified into five different genera. Further support for this was provided by a concatenated amino acid tree, phenotypic characteristics and fatty acid profiles, enabling differentiation of the MLSA groups. Three novel genera are proposed: Lelliottia gen. nov., Pluralibacter gen. nov. and Kosakonia gen. nov. and the following new combinations: Lelliottia nimipressuralis comb. nov., Lelliottia amnigena comb. nov., Pluralibacter gergoviae comb. nov., Pluralibacter pyrinus comb. nov., Kosakonia cowanii comb. nov., Kosakonia radicincitans comb. nov., Kosakonia oryzae comb. nov., Kosakonia arachidis comb. nov., Cronobacter helveticus comb. nov. and Cronobacter pulveris comb. nov. Additionally, the novel epithet Cronobacter zurichensis nom. nov. is proposed for the reclassification of Enterobacter turicensis into the genus Cronobacter, as Cronobacter turicensis (Iversen et al., 2008) is already in use.

  5. Genome analysis of Cronobacter phage vB_CsaP_Ss1 reveals an endolysin with potential for biocontrol of Gram-negative bacterial pathogens.

    PubMed

    Endersen, Lorraine; Guinane, Caitriona M; Johnston, Christopher; Neve, Horst; Coffey, Aidan; Ross, R Paul; McAuliffe, Olivia; O'Mahony, Jim

    2015-02-01

    Bacteriophages and their derivatives are continuously gaining impetus as viable alternative therapeutic agents to control harmful multidrug-resistant bacterial pathogens, particularly in the food industry. The reduced efficacy of conventional antibiotics has resulted in a quest to find novel alternatives in the war against infectious disease. This study describes the full-genome sequence of Cronobacter phage vB_CsaP_Ss1, with subsequent cloning and expression of its endolysin, capable of hydrolysing Gram-negative peptidoglycan. Cronobacter phage vB_CsaP_Ss1 is composed of 42 205 bp of dsDNA with a G+C content of 46.1 mol%. A total of 57 ORFs were identified of which 18 could be assigned a putative function based on similarity to characterized proteins. The genome of Cronobacter phage vB_CsaP_Ss1 showed little similarity to any other bacteriophage genomes available in the database and thus was considered unique. In addition, functional analysis of the predicted endolysin (LysSs1) was also investigated. Zymographic experiments demonstrated the hydrolytic activity of LysSs1 against Gram-negative peptidoglycan, and this endolysin thus represents a novel candidate with potential for use against Gram-negative pathogens.

  6. The DSF type quorum sensing signalling system RpfF/R regulates diverse phenotypes in the opportunistic pathogen Cronobacter.

    PubMed

    Suppiger, Angela; Eshwar, Athmanya Konegadde; Stephan, Roger; Kaever, Volkhard; Eberl, Leo; Lehner, Angelika

    2016-01-04

    Several bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals to control biofilm formation and virulence. Previous work showed that in Burkholderia cenocepacia the RpfFBc/RpfR system is involved in sensing and responding to DSF signals and that this signal/sensor gene pair is highly conserved in several bacterial species including Cronobacter spp. Here we show that C. turicensis LMG 23827(T) possesses a functional RpfF/R system that is involved in the regulation of various phenotypes, including colony morphology, biofilm formation and swarming motility. In vivo experiments using the zebrafish embryo model revealed a role of this regulatory system in virulence of this opportunistic pathogen. We provide evidence that the RpfF/R system modulates the intracellular c-di-GMP level of the organism, an effect that may underpin the alteration in phenotype and thus the regulated phenotypes may be a consequence thereof. This first report on an RpfF/R-type QS system of an organism outside the genus Burkholderia revealed that both the underlying molecular mechanisms as well as the regulated functions show a high degree of conservation.

  7. Learn About Cronobacter Infection

    MedlinePlus

    ... Refrigeration slows the growth of germs and increases safety. Remember, when in doubt, throw it out. Practice proper hygiene! Always wash your hands carefully with soap and water during key times. Before preparing and feeding bottles ...

  8. Re-examination of the taxonomic status of Enterobacter helveticus, Enterobacter pulveris and Enterobacter turicensis as members of the genus Cronobacter and their reclassification in the genera Franconibacter gen. nov. and Siccibacter gen. nov. as Franconibacter helveticus comb. nov., Franconibacter pulveris comb. nov. and Siccibacter turicensis comb. nov., respectively.

    PubMed

    Stephan, Roger; Grim, Christopher J; Gopinath, Gopal R; Mammel, Mark K; Sathyamoorthy, Venugopal; Trach, Larisa H; Chase, Hannah R; Fanning, Séamus; Tall, Ben D

    2014-10-01

    Recently, a taxonomical re-evaluation of the genus Enterobacter, based on multi-locus sequence typing (MLST) analysis, has led to the proposal that the species Enterobacter pulveris, Enterobacter helveticus and Enterobacter turicensis should be reclassified as novel species of the genus Cronobacter. In the present work, new genome-scale analyses, including average nucleotide identity, genome-scale phylogeny and k-mer analysis, coupled with previously reported DNA-DNA hybridization values and biochemical characterization strongly indicate that these three species of the genus Enterobacter are not members of the genus Cronobacter, nor do they belong to the re-evaluated genus Enterobacter. Furthermore, data from this polyphasic study indicated that all three species constitute two new genera. We propose reclassifying Enterobacter pulveris and Enterobacter helveticus in the genus Franconibacter gen. nov. as Franconibacter pulveris comb. nov. (type strain 601/05(T) = LMG 24057(T) = DSM 19144(T)) and Franconibacter helveticus comb. nov. (type strain 513/05(T) = LMG 23732(T) = DSM 18396(T)), respectively, and Enterobacter turicensis in the genus Siccibacter gen. nov. as Siccibacter turicensis comb. nov. (type strain 508/05(T) = LMG 23730(T) = DSM 18397(T)).

  9. Selected Pathogens of Concern to Industrial Food Processors: Infectious, Toxigenic, Toxico-Infectious, Selected Emerging Pathogenic Bacteria

    NASA Astrophysics Data System (ADS)

    Behling, Robert G.; Eifert, Joseph; Erickson, Marilyn C.; Gurtler, Joshua B.; Kornacki, Jeffrey L.; Line, Erick; Radcliff, Roy; Ryser, Elliot T.; Stawick, Bradley; Yan, Zhinong

    This chapter, written by several contributing authors, is devoted to discussing selected microbes of contemporary importance. Microbes from three categories are described by the following: (1) infectious invasive agents like Salmonella, Listeria monocytogenes, and Campylobacter; (2) toxigenic pathogens such as Staphylococcus aureus, Bacillus cereus, and Clostridium botulinum; and (3) toxico-infectious agents like enterohemorrhagic Escherichia coli and Clostridium perfringens. In addition, emerging pathogens, like Cronobacter (Enterobacter) sakazakii, Arcobacter spp., and Mycobacterium avium subspecies paratuberculosis are also described.

  10. Antimicrobial Activity of Kefir against Various Food Pathogens and Spoilage Bacteria

    PubMed Central

    Kim, Hyunsook

    2016-01-01

    Kefir is a unique fermented dairy product produced by a mixture of lactic acid bacteria, acetic acid bacteria, and yeast. Here, we compared the antimicrobial spectra of four types of kefirs (A, L, M, and S) fermented for 24, 36, 48, or 72 h against eight food-borne pathogens. Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Escherichia coli, Salmonella Enteritidis, Pseudomonas aeruginosa, and Cronobacter sakazakii were used as test strains, and antibacterial activity was investigated by the spot on lawn method. The spectra, potencies, and onsets of activity varied according to the type of kefir and the fermentation time. The broadest and strongest antimicrobial spectrum was obtained after at least 36-48 h of fermentation for all kefirs, although the traditional fermentation method of kefir is for 18-24 h at 25℃. For kefir A, B. cereus, E. coli, S. Enteritidis, P. aeruginosa, and C. sakazakii were inhibited, while B. cereus, S. aureus, E. coli, S. Enteritidis, P. aeruginosa, and C. sakazakii were inhibited to different extents by kefirs L, M, and S. Remarkably, S. aureus, S. Enteritidis, and C. sakazakii were only inhibited by kefirs L, M, and S, and L. monocytogenes by kefir M after fermentation for specific times, suggesting that the antimicrobial activity is attributable not only to a low pH but also to antimicrobial substances secreted during the fermentation. PMID:28115890

  11. Assessment of a regulatory sanitization process in Egyptian dairy plants in regard to the adherence of some food-borne pathogens and their biofilms.

    PubMed

    Bayoumi, Mohamed A; Kamal, Rania M; Abd El Aal, Salah F; Awad, Esmat I

    2012-09-03

    Food-borne pathogens may develop certain strategies that enable them to defy harsh conditions such as chemical sanitization. Biofilm formation represents a prominent one among those adopted strategies, by which food-borne pathogens protect themselves against external threats. Thus, bacterial biofilm is considered as a major hazard for safe food production. This study was designed to investigate the adherence and the biofilm formation ability of some food-borne pathogens on stainless steel and polypropylene surfaces using chip assay, and to validate regular sanitizing process (sodium hypochlorite 250 mg/L) for effective elimination of those pathogens. Sixteen pathogenic bacterial strains, previously isolated from raw milk and dairy products at Zagazig city, Egypt (9 Staphylococcus aureus, 4 Cronobacter sakazakii and 3 Salmonella enterica serovar Typhimurium), were chosen for this study. Strains showed different patterns of adherence and biofilm formation on tested surfaces with minor significance between surfaces. The ability of sodium hypochlorite to completely eradicate either adhered or biofilm-embedded pathogens varied significantly depending on the strain and type of surface used. Whilst, sodium hypochlorite reduced tested pathogens counts per cm² of produced biofilms, but it was not able to entirely eliminate neither them nor adherent Cronobacter sakazakii to stainless steel surface. This study revealed that biofilm is considered as a sustainable source of contamination of dairy products with these pathogens, and also emphasized the need of paying more attention to the cleaning and sanitizing processes of food contact surfaces.

  12. Microbial growth in dry grain food (Sunsik) beverages prepared with water, milk, soymilk, or honey-water.

    PubMed

    Jung, Jin-Ho; Lee, Sun-Young

    2010-05-01

    This study was conducted to investigate the growth of microorganisms, including pathogenic bacteria such as Cronobacter sakazakii and Bacillus cereus, in Sunsik beverages made of water, milk, soymilk, or honey-water during storage at room temperature. Prepared Sunsik beverages were stored at room temperature and the growth of total aerobic counts, Escherichia coli/coliforms, and yeast and mold were measured. Also, samples inoculated with a cocktail of C. sakazakii or B. cereus spores were stored at room temperature and their growths were determined during storage. Populations of total aerobic counts and coliforms significantly increased with increasing storage time at room temperature, which resulted in higher than 8 log and 7 log after 24 h in all samples except for the honey-water sample, respectively. Levels of total aerobic counts and coliforms were significantly lower in the honey-water sample than in the other samples after 6 and 9 h of storage, respectively. Initial populations of C. sakazakii and B. cereus ranged from 0 to 1 log CFU/mL, respectively, and these populations significantly increased with increasing storage time at room temperature. Therefore, populations of C. sakazakii and B. cereus were approximately 7 to 8 log CFU/mL after 24 h of storage. However, after 12 and 9 h of storage, there were significant differences in levels of C. sakazakii and B. cereus between the honey-water sample and the other samples, respectively. Based on these results, the addition of honey can inhibit microbial growth in Sunsik beverages; however, the best way to avoid pathogen infection would be to consume Sunsik beverages as soon as possible after preparation.

  13. Molecular identification of potential denitrifying bacteria and use of D-optimal mixture experimental design for the optimization of denitrification process.

    PubMed

    Ben Taheur, Fadia; Fdhila, Kais; Elabed, Hamouda; Bouguerra, Amel; Kouidhi, Bochra; Bakhrouf, Amina; Chaieb, Kamel

    2016-04-01

    Three bacterial strains (TE1, TD3 and FB2) were isolated from date palm (degla), pistachio and barley. The presence of nitrate reductase (narG) and nitrite reductase (nirS and nirK) genes in the selected strains was detected by PCR technique. Molecular identification based on 16S rDNA sequencing method was applied to identify positive strains. In addition, the D-optimal mixture experimental design was used to optimize the optimal formulation of probiotic bacteria for denitrification process. Strains harboring denitrification genes were identified as: TE1, Agrococcus sp LN828197; TD3, Cronobacter sakazakii LN828198 and FB2, Pedicoccus pentosaceus LN828199. PCR results revealed that all strains carried the nirS gene. However only C. sakazakii LN828198 and Agrococcus sp LN828197 harbored the nirK and the narG genes respectively. Moreover, the studied bacteria were able to form biofilm on abiotic surfaces with different degree. Process optimization showed that the most significant reduction of nitrate was 100% with 14.98% of COD consumption and 5.57 mg/l nitrite accumulation. Meanwhile, the response values were optimized and showed that the most optimal combination was 78.79% of C. sakazakii LN828198 (curve value), 21.21% of P. pentosaceus LN828199 (curve value) and absence (0%) of Agrococcus sp LN828197 (curve value).

  14. Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases.

    PubMed

    Manthei, Kelly A; Hill, Morgan C; Burke, Jordan E; Butcher, Samuel E; Keck, James L

    2015-04-07

    RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. Here, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ∼90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. This bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.

  15. Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

    PubMed Central

    Manthei, Kelly A.; Hill, Morgan C.; Burke, Jordan E.; Butcher, Samuel E.; Keck, James L.

    2015-01-01

    RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. Here, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3′ single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ∼90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. This bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures. PMID:25831501

  16. Antibacterial effectiveness of chitosan-propolis coated polypropylene films against foodborne pathogens.

    PubMed

    Torlak, Emrah; Sert, Durmuş

    2013-09-01

    Antibacterial properties of chitosan are well documented in the literature. However its antibacterial effectiveness in the film form is controversial due to the methodological differences in test methods used. In this study, antibacterial effectiveness of chitosan-coated polypropylene films alone and incorporating ethanolic extract of propolis (EEP) were evaluated against six foodborne pathogens (Bacillus cereus, Cronobacter sakazakii, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium and Staphylococcus aureus) using the ISO 22196 method designed for the antibacterial treated plastic products. The results demonstrated that chitosan coated film exhibited the broad-spectrum antibacterial activity. Incorporation of EPP to coating at 10% (propolis resin/chitosan) enhanced antibacterial activity against all pathogens tested. Results of this study revealed that chitosan has antibacterial activity in the film form and that propolis is a promising antimicrobial for the food packaging applications.

  17. Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

    SciTech Connect

    Manthei, Kelly A.; Hill, Morgan C.; Burke, Jordan E.; Butcher, Samuel E.; Keck, James L.

    2015-03-23

    RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. In this paper, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ~90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. Finally, this bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.

  18. Time for the 70°C water precautionary option in the home dilution of powdered infant formula.

    PubMed

    Silano, Marco; Paganin, Paola; Davanzo, Riccardo

    2016-02-19

    Powdered infant formulas (PIF) are usually not sterile and may frequently be contaminated by several bacteria strains. Among them, Cronobacter species, previously known as Enterobacter sakazakii, is one of the most harmful, since it might be the causative agent of sepsis and meningitis in newborns and preterm infants during the first weeks of life. The mortality rate of these infections is up to 80 %. Therefore, some precautions are required in the home handling and dilution of PIF. Whereas there is wide consensus about the need that a PIF should be used immediately after being diluted or, if not, stored at < "5 °C", still recently the optimal temperature of the water used to dilute PIF is controversial among scientific societies and health agencies. The current knowledge is reviewed in this paper and provides sufficient evidence to cautiously advise the use of hot water at a temperature of "70 °C" in the dilution of PIF in order to prevent the Cronobacter sp. contamination and growth.

  19. Synergistic effects of ethanol and UV radiation to reduce levels of selected foodborne pathogenic bacteria.

    PubMed

    Ha, Ji-Hyoung; Ha, Sang-Do

    2010-03-01

    The purpose of this study was to determine whether combined treatments would produce synergistic disinfection effects on food products during food processing compared with single treatments. We investigated the bactericidal effects of a commercial chemical disinfectant (ethanol) and of UV radiation on Bacillus cereus F4810/72, Cronobacter sakazakii KCTC 2949, Staphylococcus aureus ATCC 35556, Escherichia coli ATCC 10536, and Salmonella enterica Typhimurium NO/NA in vitro. Various concentrations of ethanol (10, 30, 40, and 50%) were tested with various exposure doses of UV radiation (6, 96, 216, 360, and 504 mWs/cm(2)) with a UV lamp. The combined ethanol-UV treatments resulted in greater reductions in bacterial counts than did either treatment alone. The synergistic effect values for B. cereus, C. sakazakii, S. aureus, S. enterica Typhimurium NO/NA, and E. coli were 0.40 to 1.52, 0.52 to 1.74, 0.20 to 2.32, 0.07 to 1.14, and 0.02 to 1.75 log CFU/ml, respectively. The results of this study suggest that a significant synergistic benefit results from combining ethanol and UV treatments against foodborne pathogens in vitro.

  20. Microbiological Examination of Bulk Tank Goat's Milk in the Castilla y León Region in Northern Spain.

    PubMed

    Álvarez-Suárez, María-Elena; Otero, Andrés; García-López, María-Luisa; Santos, Jesús A

    2015-12-01

    The purpose of the study was to evaluate the microbiological status (mesophilic aerobic microorganism counts) of 68 samples of bulk tank goat's milk and determine the risk associated with the foodborne pathogens Staphylococcus aureus, enteropathogenic and Shiga toxin-producing Escherichia coli, and Cronobacter sakazakii. Most samples (83.8%) complied with the limits of mesophilic aerobe counts set in the European Union for milk of species other than cows. A total of 144 isolates of coagulase-positive staphylococci were characterized, and 11 (7.6%) of them carried staphylococcal enterotoxin (SE) genes of the classical types (encoding SEA to SEE), distributed as follows: 4 carried the SEA gene, 1 the SEB gene, and 6 the SED gene. C. sakazakii was not detected in any sample. Regarding detection of E. coli virulence-related genes in enriched milk samples, 12 milk samples were positive only for the presence of stx genes, 4 were positive for both stx and eae genes, and 20 were negative for stx amplification and positive for eae amplification. Seven enteropathogenic E. coli and 9 Shiga toxin-producing E. coli isolates (one of them of serogroup O157) were recovered. In conclusion, goat's milk produced on farms in Castilla y León is generally in accordance with European Union standards, but the presence of pathogenic E. coli isolates indicates that the consumption of raw goat's milk may pose a risk to public health.

  1. Innovative use of platinum compounds to selectively detect live microorganisms by polymerase chain reaction.

    PubMed

    Soejima, Takashi; Minami, Jun-Ichi; Xiao, Jin-Zhong; Abe, Fumiaki

    2016-02-01

    PCR cannot distinguish live microorganisms from dead ones. To circumvent this disadvantage, ethidium/propidium-monoazide (EMA/PMA) and psoralen to discriminate live from dead bacteria have been used for 2 decades. These methods require the use of numerous laborious procedures. We introduce an innovative method that uses platinum compounds, which are primarily used as catalysts in organic chemistry and partly used as anti-cancer drugs. Microorganisms are briefly exposed to platinum compounds in vivo, and these compounds penetrate dead (compromised) microorganisms but not live ones and are chelated by chromosomal DNA. The use of platinum compounds permits clear discrimination between live and dead microorganisms in water and milk (including Cronobacter sakazakii and Escherichia coli) via PCR compared with typically used PMA. This platinum-PCR method could enable the specific detection of viable coliforms in milk at a concentration of 5-10 CFU mL(-1) specified by EU/USA regulations after a 4-h process. For sample components, environmental water contains lower levels of PCR inhibitors than milk does, and milk is similar to infant formula, skim milk and blood; thus, the use of the platinum-PCR method could also prevent food poisoning due to the presence of C. sakazakii in dairy products. This method could provide outstanding rapidity for use in environmental/food/clinical tests. Platinum-PCR could also be a substitute for the typical culture-based methods currently used.

  2. Pasteurization of milk: the heat inactivation kinetics of milk-borne dairy pathogens under commercial-type conditions of turbulent flow.

    PubMed

    Pearce, L E; Smythe, B W; Crawford, R A; Oakley, E; Hathaway, S C; Shepherd, J M

    2012-01-01

    This is the first study to report kinetic data on the survival of a range of significant milk-borne pathogens under commercial-type pasteurization conditions. The most heat-resistant strain of each of the milk-borne pathogens Staphylococcus aureus, Yersinia enterocolitica, pathogenic Escherichia coli, Cronobacter sakazakii (formerly known as Enterobacter sakazakii), Listeria monocytogenes, and Salmonella was selected to obtain the worst-case scenario in heat inactivation trials using a pilot-plant-scale pasteurizer. Initially, approximately 30 of each species were screened using a submerged coil unit. Then, UHT milk was inoculated with the most heat-resistant pathogens at ~10(7)/mL and heat treated in a pilot-plant-scale pasteurizer under commercial-type conditions of turbulent flow for 15s over a temperature range from 56 to 66°C and at 72°C. Survivors were enumerated on nonselective media chosen for the highest efficiency of plating of heat-damaged bacteria of each of the chosen strains. The mean log(10) reductions and temperatures of inactivation of the 6 pathogens during a 15-s treatment were Staph. aureus >6.7 at 66.5°C, Y. enterocolitica >6.8 at 62.5°C, pathogenic E. coli >6.8 at 65°C, C. sakazakii >6.7 at 67.5°C, L. monocytogenes >6.9 at 65.5°C, and Salmonella ser. Typhimurium >6.9 at 61.5°C. The kinetic data from these experiments will be used by the New Zealand Ministry of Agriculture and Forestry to populate the quantitative risk assessment model being developed to investigate the risks to New Zealand consumers from pasteurized, compared with nonpasteurized, milk and milk products.

  3. The Pathogen-annotated Tracking Resource Network (PATRN) system: a web-based resource to aid food safety, regulatory science, and investigations of foodborne pathogens and disease.

    PubMed

    Gopinath, G; Hari, K; Jain, R; Mammel, M K; Kothary, M H; Franco, A A; Grim, C J; Jarvis, K G; Sathyamoorthy, V; Hu, L; Datta, A R; Patel, I R; Jackson, S A; Gangiredla, J; Kotewicz, M L; LeClerc, J E; Wekell, M; McCardell, B A; Solomotis, M D; Tall, B D

    2013-06-01

    Investigation of foodborne diseases requires the capture and analysis of time-sensitive information on microbial pathogens that is derived from multiple analytical methods and sources. The web-based Pathogen-annotated Tracking Resource Network (PATRN) system (www.patrn.net) was developed to address the data aggregation, analysis, and communication needs important to the global food safety community for the investigation of foodborne disease. PATRN incorporates a standard vocabulary for describing isolate metadata and provides a representational schema for a prototypic data exchange standard using a novel data loading wizard for aggregation of assay and attribution information. PATRN currently houses expert-curated, high-quality "foundational datasets" consisting of published experimental results from conventional assays and next generation analysis platforms for isolates of Escherichia coli, Listeria monocytogenes, and Salmonella, Shigella, Vibrio and Cronobacter species. A suite of computational tools for data mining, clustering, and graphical representation is available. Within PATRN, the public curated data repository is complemented by a secure private workspace for user-driven analyses, and for sharing data among collaborators. To demonstrate the data curation, loading wizard features, and analytical capabilities of PATRN, three use-case scenarios are presented. Use-case scenario one is a comparison of the distribution and prevalence of plasmid-encoded virulence factor genes among 249 Cronobacter strains with similar attributes to that of nine Cronobacter isolates from recent cases obtained between March and October, 2010-2011. To highlight PATRN's data management and trend finding tools, analysis of datasets, stored in PATRN as part of an ongoing surveillance project to identify the predominant molecular serogroups among Cronobacter sakazakii isolates observed in the USA is shown. Use-case scenario two demonstrates the secure workspace available for private

  4. Atypical internal yellowing of papaya fruit in Hawaii caused by Enterobacter sakazakii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Internal yellowing (IY), characterized by yellow discolored tissue around the papaya (Carica papaya) seed cavity, diffuse margins and the presence of a distinctly rotten odor, was first reported in 1987. These symptoms were associated with the causal agent Enterobacter cloacae. Here we report the fo...

  5. Factors influencing the accuracy of the plating method used to enumerate low numbers of viable micro-organisms in food.

    PubMed

    Jongenburger, I; Reij, M W; Boer, E P J; Gorris, L G M; Zwietering, M H

    2010-09-30

    This study aims to assess several factors that influence the accuracy of the plate count technique to estimate low numbers of micro-organisms in liquid and solid food. Concentrations around 10CFU/mL or 100CFU/g in the original sample, which can still be enumerated with the plate count technique, are considered as low numbers. The impact of low plate counts, technical errors, heterogeneity of contamination and singular versus duplicate plating were studied. Batches of liquid and powdered milk were artificially contaminated with various amounts of Cronobacter sakazakii strain ATCC 29544 to create batches with accurately known levels of contamination. After thoroughly mixing, these batches were extensively sampled and plated in duplicate. The coefficient of variation (CV) was calculated for samples from both batches of liquid and powdered product as a measure of the dispersion within the samples. The impact of technical errors and low plate counts were determined theoretically, experimentally, as well as with Monte Carlo simulations. CV-values for samples of liquid milk batches were found to be similar to their theoretical CV-values established by assuming Poisson distribution of the plate counts. However, CV-values of samples of powdered milk batches were approximately five times higher than their theoretical CV-values. In particular, powdered milk samples with low numbers of Cronobacter spp. showed much more dispersion than expected which was likely due to heterogeneity. The impact of technical errors was found to be less prominent than that of low plate counts or of heterogeneity. Considering the impact of low plate counts on accuracy, it would be advisable to keep to a lower limit for plate counts of 25 colonies/plate rather than to the currently advocated 10 colonies/plate. For a powdered product with a heterogeneous contamination, it is more accurate to use 10 plates for 10 individual samples than to use the same 10 plates for 5 samples plated in duplicate.

  6. Surface-enhanced Raman spectroscopy introduced into the International Standard Organization (ISO) regulations as an alternative method for detection and identification of pathogens in the food industry.

    PubMed

    Witkowska, Evelin; Korsak, Dorota; Kowalska, Aneta; Księżopolska-Gocalska, Monika; Niedziółka-Jönsson, Joanna; Roźniecka, Ewa; Michałowicz, Weronika; Albrycht, Paweł; Podrażka, Marta; Hołyst, Robert; Waluk, Jacek; Kamińska, Agnieszka

    2017-02-01

    We show that surface-enhanced Raman spectroscopy (SERS) coupled with principal component analysis (PCA) can serve as a fast, reliable, and easy method for detection and identification of food-borne bacteria, namely Salmonella spp., Listeria monocytogenes, and Cronobacter spp., in different types of food matrices (salmon, eggs, powdered infant formula milk, mixed herbs, respectively). The main aim of this work was to introduce the SERS technique into three ISO (6579:2002; 11290-1:1996/A1:2004; 22964:2006) standard procedures required for detection of these bacteria in food. Our study demonstrates that the SERS technique is effective in distinguishing very closely related bacteria within a genus grown on solid and liquid media. The advantages of the proposed ISO-SERS method for bacteria identification include simplicity and reduced time of analysis, from almost 144 h required by standard methods to 48 h for the SERS-based approach. Additionally, PCA allows one to perform statistical classification of studied bacteria and to identify the spectrum of an unknown sample. Calculated first and second principal components (PC-1, PC-2) account for 96, 98, and 90% of total variance in the spectra and enable one to identify the Salmonella spp., L. monocytogenes, and Cronobacter spp., respectively. Moreover, the presented study demonstrates the excellent possibility for simultaneous detection of analyzed food-borne bacteria in one sample test (98% of PC-1 and PC-2) with a goal of splitting the data set into three separated clusters corresponding to the three studied bacteria species. The studies described in this paper suggest that SERS represents an alternative to standard microorganism diagnostic procedures. Graphical Abstract New approach of the SERS strategy for detection and identification of food-borne bacteria, namely S. enterica, L. monocytogenes, and C. sakazakii in selected food matrices.

  7. Prevalence and Antimicrobial Susceptibility of Enterobacteriaceae Isolated from Retail Pepper in Vietnam.

    PubMed

    Harada, Tetsuya; Yamane, Ryoko; Dang, Van Chinh; Nguyen, Do Phuc; Nguyen, Thi Anh Dao; Jinnai, Michio; Yonogi, Shinya; Kawahara, Ryuji; Kanki, Masashi; Kawai, Takao; Kawatsu, Kentaro; Kumeda, Yuko; Isegawa, Yuji; Yamamoto, Yoshimasa

    2017-03-28

    To investigate the microbial quality of retail pepper in Vietnam, the enumeration and detection of Enterobacteriaceae and the screening of cefotaxime (CTX)-resistant coliforms were performed by using 84 commercial samples. Although Enterobacteriaceae were isolated from 78 samples, the number of Enterobacteriaceae was lower than 1.0 log CFU/g in 46 samples. For the detection of Enterobacteriaceae with the International Organization for Standardization methods, Salmonella spp., Escherichia coli , Klebsiella pneumoniae , Cronobacter sakazakii , and Enterobacter cloacae complex were isolated from 5, 12, 36, 19, and 30 samples, respectively. During screening of CTX-resistant coliforms, K. pneumoniae , C. sakazakii , and E. cloacae complex were isolated from 8, 1, and 21 samples, respectively. Seven K. pneumoniae and seven E. cloacae complex isolates obtained in the screening of CTX-resistant coliforms were resistant to at least one of the three third-generation cephalosporins (CTX, ceftazidime, and cefpodoxime). Moreover, one E. cloacae complex cluster IV and all K. pneumoniae isolates were positive for extended-spectrum β-lactamase genes or plasmid-mediated AmpC β-lactamase genes or both. Additionally, two extended-spectrum β-lactamase-producing K. pneumoniae isolates and one AmpC β-lactamase-producing E. cloacae complex cluster IV isolate were positive for the plasmid-mediated quinolone resistance determinants and also had amino acid alterations in the quinolone resistance-determining regions of GyrA and ParC. Furthermore, 10 E. cloacae complex isolates were positive for the plasmid-mediated fosfomycin resistance gene fosA. As pepper is often consumed without a heating process, the possible spread to humans of foodborne, opportunistic, and nosocomial infection pathogens or resistance genes from foods prepared or seasoned with pepper cannot be excluded. Therefore, it is necessary to handle pepper by using hygienic conditions during the cultivation, harvesting and

  8. Disinfection methods used in decontamination of bottles used for feeding powdered infant formula.

    PubMed

    Redmond, Elizabeth; Griffith, Christopher J

    2009-01-01

    Infant susceptibility and the risks posed by infections associated with bottle-fed powdered infant formula (PIF) have received increased attention in recent years. Intrinsic contamination of PIF with pathogens has been reported and extrinsic contamination can be introduced from the handler or the environment during reconstitution. Recommended disinfection advice and bottle decontamination have changed in recent years and the aim of this study was to validate the efficacy of four current disinfection methods using bottles that had contained reconstituted PIF spiked with either a representative mixed bacterial culture or specific pathogens. Initially, bottles (n = 6) of reconstituted formula were spiked with 10(5) cfu/ml representative mixed culture. For subsequent experiments, reconstituted formula was spiked with either 10(2) and 10(4) cfu/ml of Enterobacter sakazakii (Cronobacter), Bacillus cereus and Staphylococcus aureus. Before disinfection, bottles were cleaned according to recommended guidelines. Disinfection procedures tested included a hypochlorite-based chemical solution and three heat-based methods. Bottles were sampled in four sites. Before cleaning and disinfection, the inner screw cap and inner-teat were the most heavily contaminated sites with 1.6-7.4 x 10(3) cfu/per-area-sampled; the bottle interior was more contaminated overall with 1.2 x 10(4) cfu/per-area-sampled. After disinfection, adherence to recommended procedures (combined with good hygiene) enabled effective decontamination to be achieved using all methods. Small differences in disinfection ability were not significant (p > 0.05). Cumulatively, 800 sites were sampled and no B. cereus or E. sakazakii were isolated. S. aureus was isolated from 0.1% of sites with one site exceeding 1 cfu/ml. Findings indicate the potential for bottle contamination and that strict adherence to four currently used methods allowed effective decontamination. This highlights the importance of effective consumer

  9. Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae Isolated from Vegetables Imported from the Dominican Republic, India, Thailand, and Vietnam

    PubMed Central

    Zurfluh, Katrin; Nüesch-Inderbinen, Magdalena; Morach, Marina; Zihler Berner, Annina; Hächler, Herbert

    2015-01-01

    To examine to what extent fresh vegetables imported into Switzerland represent carriers of extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae, 169 samples of different types of fresh vegetables imported into Switzerland from the Dominican Republic, India, Thailand, and Vietnam were analyzed. Overall, 25.4% of the vegetable samples yielded one or more ESBL-producing Enterobacteriaceae, 78.3% of which were multidrug resistant. Sixty isolates were obtained: Escherichia coli, 26; Klebsiella pneumoniae, 26; Enterobacter cloacae, 6; Enterobacter aerogenes, 1; and Cronobacter sakazakii, 1. We found 29 isolates producing CTX-M-15, 8 producing CTX-M-14, 7 producing CTX-M-55, 3 producing CTX-M-65, 1 each producing CTX-M-1, CTX-M-3, CTX-M-27, and CTX-M-63, 5 producing SHV-2, 3 producing SHV-12, and 1 producing SHV-2a. Four of the E. coli isolates belonged to epidemiologically important clones: CTX-M-15-producing B2:ST131 (1 isolate), D:ST405 (1 isolate), and D:ST38 (2 isolates). One of the D:ST38 isolates belonged to the extraintestinal enteroaggregative E. coli (EAEC) D:ST38 lineage. Two of the K. pneumoniae isolates belonged to the epidemic clones sequence type 15 (ST15) and ST147. The occurrence of antibiotic-resistant pathogenic and commensal Enterobacteriaceae in imported agricultural foodstuffs constitutes a source of ESBL genes and a concern for food safety. PMID:25724954

  10. Evaluation of the microbial safety of child food of animal origin in Greece.

    PubMed

    Liandris, Emmanouil; Gazouli, Maria; Taka, Styliani; Andreadou, Margarita; Vaiopoulou, Anna; Tzimotoudis, Nikolaos; Kasampalidis, Ioannis; Mpaseas, Dionysis; Fyliousis, George; Poltronieri, Palmiro; Poltrionieri, Palmiro; Cook, Nigel; Ikonomopoulos, John

    2014-03-01

    Foodborne illness is a major cause of morbidity and mortality especially for children, even in the developed world. The aim of this study was to assess the microbial safety of food of animal origin intended for consumption by children in Greece. Sampling involved 8 categories of retail products and was completed with a collection of 850 samples. These were tested by PCR and/or culture for Listeria monocytogenes, Campylobacter spp., Escherichia coli O157, Salmonella spp., Cronobacter sakazakii, Brucella spp., and Mycobacterium avium subsp paratuberculosis (MAP). The number of positive results recorded collectively for the pathogens under investigation over the total number of samples tested was 3.52% and 0.12% by PCR and culture, respectively. The most frequently detected pathogen was enterohemorrhagic E. coli (1.29%) followed by Brucella (0.82%) and Listeria (0.82%). DNA belonging to MAP was detected in 0.35% of samples, which was also the percentage of positivity recorded for Campylobacter. The percentage for Salmonella was 0.12%. It can be concluded from the results that there is no indication of noncompliance for the tested food samples. However, detection of DNA belonging to pathogens that are transmissible to humans through food is indicative that constant vigilance regarding food safety is an absolute necessity.

  11. Hygienic characteristics and microbiological hazard identification in horse and donkey raw milk.

    PubMed

    Colavita, Giampaolo; Amadoro, Carmela; Rossi, Franca; Fantuz, Francesco; Salimei, Elisabetta

    2016-01-01

    Today the interest toward horse (Equus caballus) and donkey (Equus asinus) milk for human consumption is receiving a renewed attention because of its particular composition, hypoallergenicity, and nutraceutical properties. The realistic perspective of global use of this aliment in balanced diets, especially for infancy and geriatrics, poses the need for a more in depth knowledge on milk hygiene and on the health status of dairy animals, as a prerequisite of consumers' safety. The aim of this paper was to review the available literature on the health and hygiene parameters as well as on the potential microbiological hazards in horse and donkey milk and the risks related to their consumption. Both microbial contamination and somatic cell count are reasonably low in equine milk and also the presence of pathogens, like Escherichia coli O157, Salmonella spp., Campylobacter spp., Yersinia enterocolitica, Brucella spp., Mycobacterium spp., Bacillus cereus, Cronobacter sakazakii, Streptococcus equi subsp. zooepidemicus, Rhodococcus equi, Streptococcus dysgalactiae subsp. equisimilis, Clostridium difficile and Burkholderia mallei is low. However, in those regions of the world where the prevalence of Brucella spp. and Rhodococcus equi is high, the alimentary risks could increase. Similarly, in areas with higher incidence of immunocompromised people, the increased risks should be warned not only for pathogens but also for opportunistic microbiota.

  12. Cultivable endophytic bacteria from leaf bases of Agave tequilana and their role as plant growth promoters

    PubMed Central

    Martínez-Rodríguez, Julia del C.; la Mora-Amutio, Marcela De; Plascencia-Correa, Luis A.; Audelo-Regalado, Esmeralda; Guardado, Francisco R.; Hernández-Sánchez, Elías; Peña-Ramírez, Yuri J.; Escalante, Adelfo; Beltrán-García, Miguel J.; Ogura, Tetsuya

    2014-01-01

    Agave tequilana Weber var. ‘Azul’ is grown for the production of tequila, inulin and syrup. Diverse bacteria inhabit plant tissues and play a crucial role for plant health and growth. In this study culturable endophytic bacteria were extracted from leaf bases of 100 healthy Agave tequilana plants. In plant tissue bacteria occurred at mean population densities of 3 million CFU/g of fresh plant tissue. Three hundred endophytic strains were isolated and 16s rDNA sequences grouped the bacteria into eight different taxa that shared high homology with other known sequences. Bacterial endophytes were identified as Acinectobacter sp., A. baumanii, A. bereziniae, Cronobacter sakazakii, Enterobacter hormaechei, Bacillus sp. Klebsiella oxytoca, Pseudomonas sp., Enterococcus casseliflavus, Leuconostoc mesenteroides subsp. mesenteroides and Gluconobacter oxydans. Isolates were confirmed to be plant growth promoting bacteria (PGPB) by their capacities for nitrogen fixation, auxin production, phosphate solubilization, or antagonism against Fusarium oxysporum AC132. E. casseliflavus JM47 and K. oxytoca JM26 secreted the highest concentrations of IAA. The endophyte Acinectobacter sp. JM58 exhibited the maximum values for nitrogen fixation and phosphate solubilization index (PSI). Inhibition of fungi was found in Pseudomonas sp. JM9p and K. oxytoca JM26. Bacterial endophytes show promise for use as bio-inoculants for agave cultivation. Use of endophytes to enhance cultivation of agave may be particularly important for plants produced by micropropagation techniques, where native endophytes may have been lost. PMID:25763038

  13. Microbial examination of nonheated foods served in feeding programs of elementary schools, Iksan City, Jeonbuk Province, Korea.

    PubMed

    Ryu, Jee-Hoon; Ko, Jinyoung; Park, Hyoseok; Yang, Soonwook; Kim, Hoikyung

    2011-09-01

    More than 90% of elementary school students in Korea have lunch provided by a school feeding program. This study examined nonheated foods, foods in which final ingredients were added after cooking ("heated/nonheated foods"), and desserts for microbial contamination levels and the presence of foodborne pathogens. We obtained a total of 77 food samples belonging to the above three groups from four elementary schools located in Iksan, Jeonbuk, Korea, from June to July 2010. Among the samples, 15% of nonheated foods and 9% of heated/nonheated foods contained > 6 log CFU of aerobic bacteria per g. Unacceptable coliform counts according to Korean national standards (3 log CFU/g) were also observed in 30, 4.5, and 26% of nonheated foods, heated/nonheated foods, and desserts, respectively. The foodborne pathogens Escherichia coli O157:H7, Bacillus cereus, and Cronobacter sakazakii were found in two, one, and two of the total samples, respectively. Detection of E. coli O157:H7 indicates a low level of safety in the school lunches served in Korean elementary schools. To improve food safety, hazard analysis critical control point guidelines should be applied to school food service establishments to lower the microbial risks in foods served to children.

  14. Plant growth promoting potential and phylogenetic characteristics of a lichenized nitrogen fixing bacterium, Enterobacter cloacae.

    PubMed

    Swamy, Chidanandamurthy Thippeswamy; Gayathri, Devaraja; Devaraja, Thimmalapura Neelakantaiah; Bandekar, Mandar; D'Souza, Stecy Elvira; Meena, Ram Murti; Ramaiah, Nagappa

    2016-12-01

    Lichens are complex symbiotic association of mycobionts, photobionts, and bacteriobionts, including chemolithotropic bacteria. In the present study, 46 lichenized bacteria were isolated by conventional and enrichment culture methods on nitrogen-free bromothymol blue (NFb) medium. Only 11 of the 46 isolates fixed nitrogen on NFb and had reduced acetylene. All these 11 isolates had also produced siderophore and 10 of them the IAA. Further, ammonia production was recorded from nine of these nitrogen fixers (NF). On molecular characterization, 16 S rRNA sequencing recorded that, nine NF belonged to Proteobacteria, within Gammaproteobacteria, and were closely related to Enterobacter sp. with a maximum similarity to Enterobacter cloacae. Each one of our NF isolates was aligned closely to Enterobacter pulveris strain E443, Cronobacter sakazakii strain PNP8 and Providencia rettgeri strain ALK058. Notably, a few strains we examined found to possess plant growth promoting properties. This is the first report of Enterobacter sp. from lichens which may be inhabit lichen thalli extrinsically or intrinsically.

  15. Recovery Estimation of Dried Foodborne Pathogens Is Directly Related to Rehydration Kinetics

    PubMed Central

    Lang, Emilie; Zoz, Fiona; Iaconelli, Cyril; Guyot, Stéphane; Alvarez-Martin, Pablo; Beney, Laurent; Perrier-Cornet, Jean-Marie; Gervais, Patrick

    2016-01-01

    Drying is a common process which is used to preserve food products and technological microorganisms, but which is deleterious for the cells. The aim of this study is to differentiate the effects of drying alone from the effects of the successive and necessary rehydration. Rehydration of dried bacteria is a critical step already studied in starter culture but not for different kinetics and not for pathogens. In the present study, the influence of rehydration kinetics was investigated for three foodborne pathogens involved in neonatal diseases caused by the consumption of rehydrated milk powder: Salmonella enterica subsp. enterica serovar Typhimurium, Salmonella enterica subsp. enterica serovar Senftenberg and Cronobacter sakazakii. Bacteria were dried in controlled relative humidity atmospheres and then rehydrated using different methods. Our results showed that the survival of the three pathogens was strongly related to rehydration kinetics. Consequently, rehydration is an important step to consider during food safety assessment or during studies of dried foodborne pathogens. Also, it has to be considered with more attention in consumers’ homes during the preparation of food, like powdered infant formula, to avoid pathogens recovery. PMID:27494169

  16. Effect of gamma irradiation on microbial quality of minimally processed carrot and lettuce: A case study in Greater Accra region of Ghana

    NASA Astrophysics Data System (ADS)

    Frimpong, G. K.; Kottoh, I. D.; Ofosu, D. O.; Larbi, D.

    2015-05-01

    The effect of ionizing radiation on the microbiological quality on minimally processed carrot and lettuce was studied. The aim was to investigate the effect of irradiation as a sanitizing agent on the bacteriological quality of some raw eaten salad vegetables obtained from retailers in Accra, Ghana. Minimally processed carrot and lettuce were analysed for total viable count, total coliform count and pathogenic organisms. The samples collected were treated and analysed for a 15 day period. The total viable count for carrot ranged from 1.49 to 14.01 log10 cfu/10 g while that of lettuce was 0.70 to 8.5 7 log10 cfu/10 g. It was also observed that total coliform count for carrot was 1.46-7.53 log10 cfu/10 g and 0.14-7.35 log10 cfu/10 g for lettuce. The predominant pathogenic organisms identified were Bacillus cereus, Cronobacter sakazakii, Staphylococcus aureus, and Klebsiella spp. It was concluded that 2 kGy was most effective for medium dose treatment of minimally processed carrot and lettuce.

  17. Simultaneous Identification of 13 Foodborne Pathogens by Using Capillary Electrophoresis-Single Strand Conformation Polymorphism Coupled with Multiplex Ligation-Dependent Probe Amplification and Its Application in Foods.

    PubMed

    Kim, So-Young; Chung, Boram; Chang, Jin-Hee; Jung, Gyoo Yeol; Kim, Hyoun Wook; Park, Beom-Young; Oh, Sang Suk; Oh, Mi-Hwa

    2016-10-01

    Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) coupled with stuffer-free multiplex ligation-dependent probe amplification (MLPA) was developed to identify 13 species of foodborne pathogens simultaneously. Species-specific MLPA probes were designed for nine of these species. These probes were targeted to the groEL, glyA, MMS, tuf, inv, ipaH, nuc, vvh, and 16S rRNA genes, which corresponded to Bacillus cereus, Campylobacter coli, Cronobacter sakazakii, Enterococcus spp., Salmonella spp., Shigella spp., Staphylococcus aureus, Vibrio vulnificus, and Yersinia enterocolitica, respectively. MLPA probes that had been previously developed by our laboratory were used for the other four species (Campylobacter jejuni, Clostridium perfringens, Escherichia coli O157:H7, and Listeria monocytogenes). The CE-SSCP method was optimized to identify all 13 foodborne microbes simultaneously in a single electrogram, in which 50-500 pg genomic DNA was detected per microbe. Twelve species were detected from animal-derived food samples (specifically, milk and sliced ham) that had been artificially inoculated with 12 of the foodborne pathogens, excluding V. vulnificus, which is not usually associated with animal foods. The method developed here could be used as an early warning system for outbreaks of foodborne diseases associated with animal-derived foods in the food industry.

  18. Cultivable endophytic bacteria from leaf bases of Agave tequilana and their role as plant growth promoters.

    PubMed

    Martínez-Rodríguez, Julia del C; De la Mora-Amutio, Marcela; Plascencia-Correa, Luis A; Audelo-Regalado, Esmeralda; Guardado, Francisco R; Hernández-Sánchez, Elías; Peña-Ramírez, Yuri J; Escalante, Adelfo; Beltrán-García, Miguel J; Ogura, Tetsuya

    2014-01-01

    Agave tequilana Weber var. 'Azul' is grown for the production of tequila, inulin and syrup. Diverse bacteria inhabit plant tissues and play a crucial role for plant health and growth. In this study culturable endophytic bacteria were extracted from leaf bases of 100 healthy Agave tequilana plants. In plant tissue bacteria occurred at mean population densities of 3 million CFU/g of fresh plant tissue. Three hundred endophytic strains were isolated and 16s rDNA sequences grouped the bacteria into eight different taxa that shared high homology with other known sequences. Bacterial endophytes were identified as Acinectobacter sp., A. baumanii, A. bereziniae, Cronobacter sakazakii, Enterobacter hormaechei, Bacillus sp. Klebsiella oxytoca, Pseudomonas sp., Enterococcus casseliflavus, Leuconostoc mesenteroides subsp. mesenteroides and Gluconobacter oxydans. Isolates were confirmed to be plant growth promoting bacteria (PGPB) by their capacities for nitrogen fixation, auxin production, phosphate solubilization, or antagonism against Fusarium oxysporum AC132. E. casseliflavus JM47 and K. oxytoca JM26 secreted the highest concentrations of IAA. The endophyte Acinectobacter sp. JM58 exhibited the maximum values for nitrogen fixation and phosphate solubilization index (PSI). Inhibition of fungi was found in Pseudomonas sp. JM9p and K. oxytoca JM26. Bacterial endophytes show promise for use as bio-inoculants for agave cultivation. Use of endophytes to enhance cultivation of agave may be particularly important for plants produced by micropropagation techniques, where native endophytes may have been lost.

  19. Evaluation of microbial quality and yeast diversity in fresh-cut apple.

    PubMed

    Graça, Ana; Santo, David; Esteves, Eduardo; Nunes, Carla; Abadias, Maribel; Quintas, Célia

    2015-10-01

    The present work's aim was to study the microbial quality of minimally processed apples commercialized in Portugal. Sixty eight samples of fresh-cut apple were analyzed before their best-before date in 2011 and 2012 for aerobic mesophilic and psychrotrophic microorganisms, total coliforms, lactic-acid bacteria (LAB), coagulase-positive staphylococci and fungi. The parameters of food safety studied were Cronobacter sakazakii, Salmonella spp. and Listeria sp. Samples were analyzed according to standard methodologies and using Chromocult Agar for coliforms and Escherichia coli. The yeasts were identified by restriction analysis of the ITS-5.8S rDNA-region and 26S rDNA partial sequencing. The mesophilic and psychrotrophic microorganisms ranged from 3.3 to 8.9 and from 4.9 to 8.4 log CFU/g, respectively. Coliforms were detected in all the samples and staphylococci in 5.8% of them. LAB numbers varied from 2.8 to 8.7 and fungi (yeast and molds) from 3.6 to 7.1 log CFU/g. The most common yeasts were Candida sake and Pichia fermentans followed by Hanseniaspora spp., Candida spp., Meyerozyma guilliermondii, Metschnikowia pulcherrima, Cryptococcus spp. and the psychrotrophic Cystofilobasidium infirmominiatum. Foodborne bacteria and opportunistic pathogenic yeasts were not detected in the apples studied. The results obtained respected the European Commission regulation regarding criteria of food hygiene and safety.

  20. Incidence of bacteria of public health interest carried by cockroaches in different food-related environments.

    PubMed

    García, F; Notario, M J; Cabanás, J M; Jordano, R; Medina, L M

    2012-11-01

    The aim of this study was to determine the incidence of bacteria of public health interest transmitted by cockroaches in different food-related environments. From April to November, cockroaches were trapped in 11 buildings in different urban areas of Western Andalusia (Spain): three hotels, four grocery stores, a catering establishment, a food-industry plant, a health center, and a care home. The presence of a number of bacterial species, including Salmonella, in these food-related environments was confirmed; these species included microorganisms listed in European Union regulations, such as Salmonella spp., Enterobacter sakazakii (Cronobacter spp.), and Escherichia coli. A wide variety of species were isolated, some belonging to different genera that have a significant impact on public health and hygiene, such as Enterobacter and Klebsiella. To ensure adequate elimination of these microorganisms in food-related environments, the control of vectors such as Blattella germanica, Periplaneta americana, and Blatta orientalis, together with a thorough review of hygiene strategies, appears to be fundamental. It is clearly essential to compare the results of hygiene regulations implemented in food-related environments.

  1. Label-Free 3D Ag Nanoflower-Based Electrochemical Immunosensor for the Detection of Escherichia coli O157:H7 Pathogens

    NASA Astrophysics Data System (ADS)

    Huang, He; Liu, Minghuan; Wang, Xiangsheng; Zhang, Wenjie; Yang, Da-Peng; Cui, Lianhua; Wang, Xiansong

    2016-11-01

    It is highly desirable to develop a rapid and simple method to detect pathogens. Combining nanomaterials with electrochemical techniques is an efficient way for pathogen detection. Herein, a novel 3D Ag nanoflower was prepared via a biomineralization method by using bovine serum albumin (BSA) as a template. It was adopted as a sensing interface to construct an electrochemical bacteria immunosensor for the rapid detection of foodborne pathogens Escherichia coli ( E. coli) O157:H7. Bacterial antibody was immobilized onto the surface of Ag nanoflowers through covalent conjugation. Electrochemical impedance spectroscopy (EIS) was used to detect and validate the resistance changes, where [Fe(CN)6]3-/4- acted as the redox probe. A linear relation between R et and E. coli concentration was obtained in the E. coli concentration range of 3.0 × 102-3.0 × 108 cfu mL-1. The as-prepared biosensor gave rise to an obvious response to E. coli but had no distinct response to Cronobacter sakazakii, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus albus, Lactobacillus easei, and Shigella flexneri, revealing a high selectivity for the detection of the pathogens down to 100 cfu mL-1 in a short time. We believe that this BSA-conjugated 3D Ag nanoflowers could be used as a powerful interface material with good conductivity and biocompatibility for improving pathogen detection and treatment in the field of medicine, environment, and food safety.

  2. Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

    DOE PAGES

    Manthei, Kelly A.; Hill, Morgan C.; Burke, Jordan E.; ...

    2015-03-23

    RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. In this paper, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ~90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATPmore » hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. Finally, this bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.« less

  3. Resistance of pathogenic bacteria on the surface of stainless steel depending on attachment form and efficacy of chemical sanitizers.

    PubMed

    Bae, Young-Min; Baek, Seung-Youb; Lee, Sun-Young

    2012-02-15

    Various bacteria including food spoilage bacteria and pathogens can form biofilms on different food processing surfaces, leading to potential food contamination or spoilage. Therefore, the survival of foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Cronobacter sakazakii) in different forms (adhered cells, biofilm producing in TSB, biofilm producing at RH 100%) on the surface of stainless steel and stored at various relative humidities (RH 23%, 43%, 68%, 85%, and 100%) at room temperature for 5 days was investigated in this study. Additionally, the efficacy of chemical sanitizers (chlorine-based and alcohol-based commercial sanitizers) on inhibiting various types of biofilms of E. coli O157:H7 and S. aureus on the surface of stainless steel was investigated. The number of pathogens on the surface of stainless steel in TSB stored at 25°C for 7 days or RH 100% at 25°C for 7 days was significantly increased and resulted in the increase of 3 log(10) CFU/coupon after 1 day, and these levels were maintained for 7 days. When stainless steel coupons were stored at 25°C for 5 days, the number of pathogens on the surface of stainless steel was significantly reduced after storage at RH 23%, 43%, 68%, and 85%, but not at 100%. When the bacteria formed biofilms on the surface of stainless steel in TSB after 6 days, the results were similar to those of the attached form. However, levels of S. aureus and C. sakazakii biofilms were more slowly reduced after storage at RH 23%, 43%, 68%, and 85% for 5 days than were those of the other pathogens. Formation of biofilms stored at RH 100% for 5 days displayed the highest levels of resistance to inactivation. Treatment with the alcohol sanitizer was very effective at inactivating attached pathogens or biofilms on the surface of stainless steel. Reduction levels of alcohol sanitizer treatment ranged from 1.91 to 4.77 log and from 4.35 to 5.35 log CFU/coupon in E. coli

  4. Characterization of IncI1 sequence type 71 epidemic plasmid lineage responsible for the recent dissemination of CTX-M-65 extended-spectrum β-lactamase in the Bolivian Chaco region.

    PubMed

    Riccobono, Eleonora; Di Pilato, Vincenzo; Di Maggio, Tiziana; Revollo, Carmen; Bartoloni, Alessandro; Pallecchi, Lucia; Rossolini, Gian Maria

    2015-09-01

    During the last decade, a significant diffusion of CTX-M-type extended-spectrum β-lactamases (ESBLs) was observed in commensal Escherichia coli from healthy children in the Bolivian Chaco region, with initial dissemination of CTX-M-2, which was then replaced by CTX-M-15 and CTX-M-65. In this work, we demonstrate that the widespread dissemination of CTX-M-65 observed in this context was related to the polyclonal spreading of an IncI1 sequence type 71 (ST71) epidemic plasmid lineage. The structure of the epidemic plasmid population was characterized by complete sequencing of four representatives and PCR mapping of the remainder (n = 16). Sequence analysis showed identical plasmid backbones (similar to that of the reference IncI1 plasmid, R64) and a multiresistance region (MRR), which underwent local microevolution. The MRR harbored genes responsible for resistance to β-lactams, aminoglycosides, florfenicol, and fosfomycin (with microevolution mainly consisting of deletion events of resistance modules). The bla CTX-M-65 module harbored by the IncI1 ST71 epidemic plasmid was apparently derived from IncN-type plasmids, likely via IS26-mediated mobilization. The plasmid could be transferred by conjugation to several different enterobacterial species (Escherichia coli, Cronobacter sakazakii, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, and Salmonella enterica) and was stably maintained without selective pressure in these species, with the exception of K. oxytoca and S. enterica. Fitness assays performed in E. coli recipients demonstrated that the presence of the epidemic plasmid was apparently not associated with a significant biological cost.

  5. Structure and Function of the PriC DNA Replication Restart Protein.

    PubMed

    Wessel, Sarah R; Cornilescu, Claudia C; Cornilescu, Gabriel; Metz, Alice; Leroux, Maxime; Hu, Kaifeng; Sandler, Steven J; Markley, John L; Keck, James L

    2016-08-26

    Collisions between DNA replication complexes (replisomes) and barriers such as damaged DNA or tightly bound protein complexes can dissociate replisomes from chromosomes prematurely. Replisomes must be reloaded under these circumstances to avoid incomplete replication and cell death. Bacteria have evolved multiple pathways that initiate DNA replication restart by recognizing and remodeling abandoned replication forks and reloading the replicative helicase. In vitro, the simplest of these pathways is mediated by the single-domain PriC protein, which, along with the DnaC helicase loader, can load the DnaB replicative helicase onto DNA bound by the single-stranded DNA (ssDNA)-binding protein (SSB). Previous biochemical studies have identified PriC residues that mediate interactions with ssDNA and SSB. However, the mechanisms by which PriC drives DNA replication restart have remained poorly defined due to the limited structural information available for PriC. Here, we report the NMR structure of full-length PriC from Cronobacter sakazakii PriC forms a compact bundle of α-helices that brings together residues involved in ssDNA and SSB binding at adjacent sites on the protein surface. Disruption of these interaction sites and of other conserved residues leads to decreased DnaB helicase loading onto SSB-bound DNA. We also demonstrate that PriC can directly interact with DnaB and the DnaB·DnaC complex. These data lead to a model in which PriC acts as a scaffold for recruiting DnaB·DnaC to SSB/ssDNA sites present at stalled replication forks.

  6. Characterization of IncI1 Sequence Type 71 Epidemic Plasmid Lineage Responsible for the Recent Dissemination of CTX-M-65 Extended-Spectrum β-Lactamase in the Bolivian Chaco Region

    PubMed Central

    Riccobono, Eleonora; Di Pilato, Vincenzo; Di Maggio, Tiziana; Revollo, Carmen; Bartoloni, Alessandro; Pallecchi, Lucia

    2015-01-01

    During the last decade, a significant diffusion of CTX-M-type extended-spectrum β-lactamases (ESBLs) was observed in commensal Escherichia coli from healthy children in the Bolivian Chaco region, with initial dissemination of CTX-M-2, which was then replaced by CTX-M-15 and CTX-M-65. In this work, we demonstrate that the widespread dissemination of CTX-M-65 observed in this context was related to the polyclonal spreading of an IncI1 sequence type 71 (ST71) epidemic plasmid lineage. The structure of the epidemic plasmid population was characterized by complete sequencing of four representatives and PCR mapping of the remainder (n = 16). Sequence analysis showed identical plasmid backbones (similar to that of the reference IncI1 plasmid, R64) and a multiresistance region (MRR), which underwent local microevolution. The MRR harbored genes responsible for resistance to β-lactams, aminoglycosides, florfenicol, and fosfomycin (with microevolution mainly consisting of deletion events of resistance modules). The blaCTX-M-65 module harbored by the IncI1 ST71 epidemic plasmid was apparently derived from IncN-type plasmids, likely via IS26-mediated mobilization. The plasmid could be transferred by conjugation to several different enterobacterial species (Escherichia coli, Cronobacter sakazakii, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, and Salmonella enterica) and was stably maintained without selective pressure in these species, with the exception of K. oxytoca and S. enterica. Fitness assays performed in E. coli recipients demonstrated that the presence of the epidemic plasmid was apparently not associated with a significant biological cost. PMID:26100713

  7. Enterobacter and Klebsiella species isolated from fresh vegetables marketed in Valencia (Spain) and their clinically relevant resistances to chemotherapeutic agents.

    PubMed

    Falomir, María Pilar; Rico, Hortensia; Gozalbo, Daniel

    2013-12-01

    Occurrence of antibiotic-resistant pathogenic or commensal enterobacteria in marketed agricultural foodstuffs may contribute to their incorporation into the food chain and constitutes an additional food safety concern. In this work, we have determined the clinically relevant resistances to 11 common chemotherapeutic agents in Enterobacter and Klebsiella isolates from fresh vegetables from various sources (supermarkets and greengrocers' shops in Valencia, Spain). A total of 96 isolates were obtained from 160 vegetables analyzed (50% positive samples): 68 Enterobacter isolates (59 E. cloacae, two E. aerogenes, two E. cancerogenus, one E. gergoviae, and four E. sakazakii, currently Cronobacter spp.), and 28 Klebsiella isolates (19 K. oxytoca and 9 K. pneumoniae). Only seven isolates were susceptible to all agents tested, and no resistances to ceftazidime, ciprofloxacin, gentamicin, and chloramphenicol were detected. Most isolates were resistant to amoxicillin/clavulanic acid (74 [58 Enterobacter and 16 Klebsiella]) or to ampicillin (80 [55/25]). Other resistances were less frequent: nitrofurantoin (13 isolates [12/1]), tetracycline (6 [5/1]), co-trimoxazole (3 [3/0]), cefotaxime (1 [1/0]), and streptomycin (2 [1/1]). Multiresistant isolates to two (56 [41/15]), three (10 E. cloacae isolates), four (one E. cloacae and one K. pneumoniae isolate), and five (two E. cloacae isolates) chemotherapeutic agents were also detected. The presence of potential pathogens points to marketed fresh produce, which often is eaten raw, as a risk factor for consumer health. In addition, these results support the usefulness of these bacterial species as indicators of the spreading of antibiotic resistances into the environment, particularly in the food chain, and suggest their role as carriers of resistance determinants from farms to consumers, which may constitute an additional "silent" food safety concern. Therefore, there is a need to improve the hygienic quality of marketed fresh

  8. Bacterial maximum non-inhibitory and minimum inhibitory concentrations of different water activity depressing solutes.

    PubMed

    Cebrián, G; Arroyo, C; Mañas, P; Condón, S

    2014-10-01

    The NaCl MNICs (maximum non-inhibitory concentrations) and MICs (minimum inhibitory concentrations) for growth of various strains of six bacterial species were determined and then compared with those obtained for seven other solutes. The influence of prior growth conditions on the MNICs and MICs was also evaluated. No significant changes on the MNICs and MICs were found among the strains studied within each species. Among all factors investigated, only growth phase -for Gram-negatives- and growth at high NaCl concentrations led to a change in the NaCl MNICs. Species could be classified depending on its NaCl MNICs and MICs (in decreasing order) as follows: Staphylococcus aureus, Listeria monocytogenes, Cronobacter sakazakii, Enterococcus faecium, Escherichia coli and Salmonella Typhimurium. Similar results were obtained for KCl, LiCl, and sodium acetate, but not for the remaining solutes investigated (sucrose, glycerol, MgCl2 and CaCl2). Results obtained indicate that, in general, Gram-negatives showed lower MNICs and MICs than Gram-positives for all the solutes, S. aureus being the most solute tolerant microorganism. When compared on a molar basis, glycerol showed the highest MNICs and MICs for all the microorganisms -except for S. aureus- and LiCl the lowest ones. NaCl MNICs and MICs were not significantly different from those of KCl when compared on a molar basis. Therefore, the inhibitory action of NaCl could not be linked to the specific action of Na(+). Results also showed that the Na(+) tolerance of some species was Cl(-) dependent whereas for others it was not, and that factors others than aw-decrease contribute to the inhibitory action of LiCl, CaCl2 and MgCl2.

  9. Advantages and challenges of increased antimicrobial copper use and copper mining.

    PubMed

    Elguindi, Jutta; Hao, Xiuli; Lin, Yanbing; Alwathnani, Hend A; Wei, Gehong; Rensing, Christopher

    2011-07-01

    Copper is a highly utilized metal for electrical, automotive, household objects, and more recently as an effective antimicrobial surface. Copper-containing solutions applied to fruits and vegetables can prevent bacterial and fungal infections. Bacteria, such as Salmonellae and Cronobacter sakazakii, often found in food contamination, are rapidly killed on contact with copper alloys. The antimicrobial effectiveness of copper alloys in the healthcare environment against bacteria causing hospital-acquired infections such as methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli O157:H7, and Clostridium difficile has been described recently. The use of copper and copper-containing materials will continue to expand and may lead to an increase in copper mining and production. However, the copper mining and manufacturing industry and the consumer do not necessarily enjoy a favorable relationship. Open pit mining, copper mine tailings, leaching products, and deposits of toxic metals in the environment often raises concerns and sometimes public outrage. In addition, consumers may fear that copper alloys utilized as antimicrobial surfaces in food production will lead to copper toxicity in humans. Therefore, there is a need to mitigate some of the negative effects of increased copper use and copper mining. More thermo-tolerant, copper ion-resistant microorganisms could improve copper leaching and lessen copper groundwater contamination. Copper ion-resistant bacteria associated with plants might be useful in biostabilization and phytoremediation of copper-contaminated environments. In this review, recent progress in microbiological and biotechnological aspects of microorganisms in contact with copper will be presented and discussed, exploring their role in the improvement for the industries involved as well as providing better environmental outcomes.

  10. Outbreak of a novel Enterobacter sp. carrying blaCTX-M-15 in a neonatal unit of a tertiary care hospital in Tanzania.

    PubMed

    Mshana, Stephen E; Gerwing, Lisa; Minde, Mercy; Hain, Torsten; Domann, Eugen; Lyamuya, Eligius; Chakraborty, Trinad; Imirzalioglu, Can

    2011-09-01

    Enterobacter hormaechei and Cronobacter sakazakii are amongst the most important causes of outbreaks of neonatal sepsis associated with powdered milk. In this study, we report for the first time an outbreak of a novel Enterobacter sp. harbouring bla(CTX-M-15) in a neonatal unit in Tanzania. Seventeen Gram-negative enteric isolates from neonatal blood cultures were studied. Antibiotic susceptibility was assessed by disc diffusion testing, and the presence of the bla(CTX-M-15) gene was established by polymerase chain reaction (PCR) and sequencing. Isolates were typed by pulsed-field gel electrophoresis (PFGE). Identification by biochemical profiling was followed by nucleotide sequencing of 16S ribosomal DNA (rDNA), rpoB and hsp60 alleles. Environmental sampling was done and control measures were established. Isolates were initially misidentified based on their fermentation characteristics and agglutination as Salmonella enterica serotype Paratyphi. All isolates were resistant to multiple antibiotics, except for ciprofloxacin and carbapenems, and were found to harbour bla(CTX-M-15) on a 291-kb narrow-range plasmid. PFGE analysis indicated the clonal outbreak of a single strain, infecting 17 neonates with a case fatality rate of 35%. The same strain was isolated from a milk bucket. Phylogenetic analysis using 16S rDNA, rpoB and hsp60 sequences permitted no definitive identification, clustering the strains in the Enterobacter cloacae complex with similarities of 92-98.8%. The data describe an outbreak of a novel bla(CTX-M-15)-positive, multiresistant Enterobacter strain in an African neonatal unit that can easily be misidentified taxonomically. These data highlight the need for constant surveillance of bacteria harbouring extended-spectrum β-lactamases as well as improvements in hygiene measures in developing countries.

  11. Analysis of microflora profile in Korean traditional nuruk.

    PubMed

    Song, Sang Hoon; Lee, Chunghee; Lee, Sulhee; Park, Jung Min; Lee, Hyong-Joo; Bai, Dong-Hoon; Yoon, Sung-Sik; Choi, Jun Bong; Park, Young Seo

    2013-01-01

    A variety of nuruk were collected from various provinces in Korea, and their microflora profiles were analyzed at the species level. A total of 42 nuruk samples were collected and when the viable cell numbers in these nuruk were enumerated, the average cell numbers of bacteria, fungi, yeast, and lactic acid bacteria from all nuruk were 7.21, 7.91, 3.49, and 4.88 log CFU/10 g, respectively. There were no significant differences in viable cell numbers of bacteria or fungi according to regions collected. Bacillus amyloliquefaciens and B. subtilis were the predominant bacterial strains in most samples. A significant portion, 13 out of 42 nuruk, contained foodborne pathogens such as B. cereus or Cronobacter sakazakii. There were various species of lactic acid bacteria such as Enterococcus faecium and Pediococcus pentosaceus in nuruk. It was unexpectedly found that only 13 among the 42 nuruk samples contained Aspergillus oryzae, the representative saccharifying fungi in makgeolli, whereas a fungi Lichtheimia corymbifera was widely distributed in nuruk. It was also found that Pichia jadinii was the predominant yeast strain in most nuruk, but the representative alcohol fermentation strain, Saccharomyces cerevisiae, was isolated from only 18 out of the 42 nuruk. These results suggested that a variety of species of fungi and yeast were distributed in nuruk and involved in the fermentation of makgeolli. In this study, a total of 64 bacterial species, 39 fugal species, and 15 yeast species were identified from nuruk. Among these strains, 37 bacterial species, 20 fungal species, and 8 yeast species were distributed less than 0.1%.

  12. Biocontrol and Rapid Detection of Food-Borne Pathogens Using Bacteriophages and Endolysins

    PubMed Central

    Bai, Jaewoo; Kim, You-Tae; Ryu, Sangryeol; Lee, Ju-Hoon

    2016-01-01

    Bacteriophages have been suggested as natural food preservatives as well as rapid detection materials for food-borne pathogens in various foods. Since Listeria monocytogenes-targeting phage cocktail (ListShield) was approved for applications in foods, numerous phages have been screened and experimentally characterized for phage applications in foods. A single phage and phage cocktail treatments to various foods contaminated with food-borne pathogens including E. coli O157:H7, Salmonella enterica, Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Cronobacter sakazakii, and Vibrio spp. revealed that they have great potential to control various food-borne pathogens and may be alternative for conventional food preservatives. In addition, phage-derived endolysins with high host specificity and host lysis activities may be preferred to food applications rather than phages. For rapid detection of food-borne pathogens, cell-wall binding domains (CBDs) from endolysins have been suggested due to their high host-specific binding. Fluorescence-tagged CBDs have been successfully evaluated and suggested to be alternative materials of expensive antibodies for various detection applications. Most recently, reporter phage systems have been developed and tested to confirm their usability and accuracy for specific detection. These systems revealed some advantages like rapid detection of only viable pathogenic cells without interference by food components in a very short reaction time, suggesting that these systems may be suitable for monitoring of pathogens in foods. Consequently, phage is the next-generation biocontrol agent as well as rapid detection tool to confirm and even identify the food-borne pathogens present in various foods. PMID:27092128

  13. Genetic determinants of heat resistance in Escherichia coli

    PubMed Central

    Mercer, Ryan G.; Zheng, Jinshui; Garcia-Hernandez, Rigoberto; Ruan, Lifang; Gänzle, Michael G.; McMullen, Lynn M.

    2015-01-01

    Escherichia coli AW1.7 is a heat resistant food isolate and the occurrence of pathogenic strains with comparable heat resistance may pose a risk to food safety. To identify the genetic determinants of heat resistance, 29 strains of E. coli that differed in their of heat resistance were analyzed by comparative genomics. Strains were classified as highly heat resistant strains, exhibiting a D60-value of more than 6 min; moderately heat resistant strains, exhibiting a D60-value of more than 1 min; or as heat sensitive. A ~14 kb genomic island containing 16 predicted open reading frames encoding putative heat shock proteins and proteases was identified only in highly heat resistant strains. The genomic island was termed the locus of heat resistance (LHR). This putative operon is flanked by mobile elements and possesses >99% sequence identity to genomic islands contributing to heat resistance in Cronobacter sakazakii and Klebsiella pneumoniae. An additional 41 LHR sequences with >87% sequence identity were identified in 11 different species of β- and γ-proteobacteria. Cloning of the full length LHR conferred high heat resistance to the heat sensitive E. coli AW1.7ΔpHR1 and DH5α. The presence of the LHR correlates perfectly to heat resistance in several species of Enterobacteriaceae and occurs at a frequency of 2% of all E. coli genomes, including pathogenic strains. This study suggests the LHR has been laterally exchanged among the β- and γ-proteobacteria and is a reliable indicator of high heat resistance in E. coli. PMID:26441869

  14. Multiplex detection of nine food-borne pathogens by mPCR and capillary electrophoresis after using a universal pre-enrichment medium.

    PubMed

    Villamizar-Rodríguez, Germán; Fernández, Javier; Marín, Laura; Muñiz, Juan; González, Isabel; Lombó, Felipe

    2015-01-01

    Routine microbiological quality analyses in food samples require, in some cases, an initial incubation in pre-enrichment medium. This is necessary in order to ensure that small amounts of pathogenic strains are going to be detected. In this work, a universal pre-enrichment medium has been developed for the simultaneous growth of Bacillus cereus, Campylobacter jejuni, Clostridium perfringens, Cronobacter sakazakii, Escherichia coli, Enterobacteriaceae family (38 species, 27 genera), Listeria monocytogenes, Staphylococcus aureus, Salmonella spp. (two species, 13 strains). Growth confirmation for all these species was achieved in all cases, with excellent enrichments. This was confirmed by plating on the corresponding selective agar media for each bacterium. This GVUM universal pre-enrichment medium could be useful in food microbiological analyses, where different pathogenic bacteria must be detected after a pre-enrichment step. Following, a mPCR reaction for detection of all these pathogens was developed, after designing a set of nine oligonucleotide pairs from specific genetic targets on gDNA from each of these bacteria, covering all available strains already sequenced in GenBank for each pathogen type. The detection limits have been 1 Genome Equivalent (GE), with the exception of the Fam. Enterobacteriaceae (5 GEs). We obtained amplification for all targets (from 70 to 251 bp, depending on the bacteria type), showing the capability of this method to detect the most important industrial and sanitary food-borne pathogens from a universal pre-enrichment medium. This method includes an initial pre-enrichment step (18 h), followed by a mPCR (2 h) and a capillary electrophoresis (30 min); avoiding the tedious and long lasting growing on solid media required in traditional analysis (1-4 days, depending on the specific pathogen and verification procedure). An external testing of this method was conducted in order to compare classical and mPCR methods. This evaluation was

  15. pDGO100, a type 1 IncC plasmid from 1981 carrying ARI-A and a Tn1696-like transposon in a novel integrating element.

    PubMed

    Harmer, Christopher J; Partridge, Sally R; Hall, Ruth M

    2016-07-01

    Most A/C plasmids sequenced to date were recovered in the last two decades. To gain insight into the evolution of this group, the IncC plasmid pDGO100, found in a multiply antibiotic-resistant Escherichia coli strain isolated in 1981, was sequenced. pDGO100 belongs to the type 1 lineage and carries an ARI-A antibiotic resistance island but not an ARI-B island. The A/C2 backbone of pDGO100 has a deletion in the rhs1 gene previously found in pRMH760 and differs by only six single base pair substitutions from pRMH760, recovered at the same hospital 16years later. This confirms that the separation of type 1 and type 2 IncC plasmids is long standing. The ARI-A islands are also closely related, but pRMH760 contains Tn4352B in tniA of Tn402, while in pDGO100, Tn4352 has inserted into merA of pDUmer. pDGO100 also carries an additional 46kb insertion that includes a Tn1696-like transposon with the dfrB3 gene cassette. This insertion was identified as a novel integrating element, with an int gene at one end, and also includes the fec iron uptake operon that has been acquired from the E. coli chromosome. Related integrating elements carrying the same int gene were found in A/C2, IncHI1, and IncHI2 plasmids, and in the chromosomes of Enterobacter cloacae, Klebsiella oxytoca, and Cronobacter sakazakii isolates. In the Enterobacteriaceae chromosomes, these integrating elements appear to target a gene encoding a radical SAM superfamily protein. In the A/C2, IncHI1, and IncHI2 plasmids, genes encoding a phosphoadenosine phosphosulfate reductase were interrupted. The extremities of the integrating element are highly conserved, whilst the internal gene content varies. The detection of integrative elements in plasmids demonstrates an increased range of locations into which this type of mobile element can integrate and insertion in plasmids is likely to assist their spread.

  16. Multiplex detection of nine food-borne pathogens by mPCR and capillary electrophoresis after using a universal pre-enrichment medium

    PubMed Central

    Villamizar-Rodríguez, Germán; Fernández, Javier; Marín, Laura; Muñiz, Juan; González, Isabel; Lombó, Felipe

    2015-01-01

    Routine microbiological quality analyses in food samples require, in some cases, an initial incubation in pre-enrichment medium. This is necessary in order to ensure that small amounts of pathogenic strains are going to be detected. In this work, a universal pre-enrichment medium has been developed for the simultaneous growth of Bacillus cereus, Campylobacter jejuni, Clostridium perfringens, Cronobacter sakazakii, Escherichia coli, Enterobacteriaceae family (38 species, 27 genera), Listeria monocytogenes, Staphylococcus aureus, Salmonella spp. (two species, 13 strains). Growth confirmation for all these species was achieved in all cases, with excellent enrichments. This was confirmed by plating on the corresponding selective agar media for each bacterium. This GVUM universal pre-enrichment medium could be useful in food microbiological analyses, where different pathogenic bacteria must be detected after a pre-enrichment step. Following, a mPCR reaction for detection of all these pathogens was developed, after designing a set of nine oligonucleotide pairs from specific genetic targets on gDNA from each of these bacteria, covering all available strains already sequenced in GenBank for each pathogen type. The detection limits have been 1 Genome Equivalent (GE), with the exception of the Fam. Enterobacteriaceae (5 GEs). We obtained amplification for all targets (from 70 to 251 bp, depending on the bacteria type), showing the capability of this method to detect the most important industrial and sanitary food-borne pathogens from a universal pre-enrichment medium. This method includes an initial pre-enrichment step (18 h), followed by a mPCR (2 h) and a capillary electrophoresis (30 min); avoiding the tedious and long lasting growing on solid media required in traditional analysis (1–4 days, depending on the specific pathogen and verification procedure). An external testing of this method was conducted in order to compare classical and mPCR methods. This evaluation was

  17. Comparative Resistance of Bacterial Foodborne Pathogens to Non-thermal Technologies for Food Preservation

    PubMed Central

    Cebrián, Guillermo; Mañas, Pilar; Condón, Santiago

    2016-01-01

    In this paper the resistance of bacterial foodborne pathogens to manosonication (MS), pulsed electric fields (PEFs), high hydrostatic pressure (HHP), and UV-light (UV) is reviewed and compared. The influence of different factors on the resistance of bacterial foodborne pathogens to these technologies is also compared and discussed. Only results obtained under harmonized experimental conditions have been considered. This has allowed us to establish meaningful comparisons and draw significant conclusions. Among the six microorganisms here considered, Staphyloccocus aureus is the most resistant foodborne pathogen to MS and HHP and Listeria monocytogenes to UV. The target microorganism of PEF would change depending on the treatment medium pH. Thus, L. monocytogenes is the most PEF resistant microorganism at neutral pH but Gram-negatives (Escherichia coli, Salmonella spp., Cronobacter sakazakii, Campylobacter jejuni) would display a similar or even higher resistance at acidic pH. It should be noted that, in acidic products, the baroresistance of some E. coli strains would be comparable to that of S. aureus. The factors affecting the resistance of bacterial foodborne pathogens, as well as the magnitude of the effect, varied depending on the technology considered. Inter- and intra-specific differences in microbial resistance to PEF and HHP are much greater than to MS and UV. Similarly, both the pH and aw of the treatment medium highly condition microbial resistance to PEF and HHP but no to MS or UV. Growth phase also drastically affected bacterial HHP resistance. Regarding UV, the optical properties of the medium are, by far, the most influential factor affecting its lethal efficacy. Finally, increasing treatment temperature leads to a significant increase in lethality of the four technologies, what opens the possibility of the development of combined processes including heat. The appearance of sublethally damaged cells following PEF and HHP treatments could also be

  18. Comparative Resistance of Bacterial Foodborne Pathogens to Non-thermal Technologies for Food Preservation.

    PubMed

    Cebrián, Guillermo; Mañas, Pilar; Condón, Santiago

    2016-01-01

    In this paper the resistance of bacterial foodborne pathogens to manosonication (MS), pulsed electric fields (PEFs), high hydrostatic pressure (HHP), and UV-light (UV) is reviewed and compared. The influence of different factors on the resistance of bacterial foodborne pathogens to these technologies is also compared and discussed. Only results obtained under harmonized experimental conditions have been considered. This has allowed us to establish meaningful comparisons and draw significant conclusions. Among the six microorganisms here considered, Staphyloccocus aureus is the most resistant foodborne pathogen to MS and HHP and Listeria monocytogenes to UV. The target microorganism of PEF would change depending on the treatment medium pH. Thus, L. monocytogenes is the most PEF resistant microorganism at neutral pH but Gram-negatives (Escherichia coli, Salmonella spp., Cronobacter sakazakii, Campylobacter jejuni) would display a similar or even higher resistance at acidic pH. It should be noted that, in acidic products, the baroresistance of some E. coli strains would be comparable to that of S. aureus. The factors affecting the resistance of bacterial foodborne pathogens, as well as the magnitude of the effect, varied depending on the technology considered. Inter- and intra-specific differences in microbial resistance to PEF and HHP are much greater than to MS and UV. Similarly, both the pH and aw of the treatment medium highly condition microbial resistance to PEF and HHP but no to MS or UV. Growth phase also drastically affected bacterial HHP resistance. Regarding UV, the optical properties of the medium are, by far, the most influential factor affecting its lethal efficacy. Finally, increasing treatment temperature leads to a significant increase in lethality of the four technologies, what opens the possibility of the development of combined processes including heat. The appearance of sublethally damaged cells following PEF and HHP treatments could also be

  19. Selected pathogens of concern to industrial food processors: infectious, toxigenic, toxico-infectious, selected emerging pathogenic bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enterobacter sakazakii is a rod-shaped bacterium that has been implicated in rare cases of neonatal sepsis, meningitis and is associated with necrotizing enterocolitis in infants. Over 80 cases of E. sakazakii-related illness have been reported, although few of these have occurred in adults. There...

  20. Escherichia coli and other Enterobacteriaceae: Food poisoning and health effects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The family Enterobactericeae consists of rod-shaped, Gram-negative, facultatively anaerobic, non-spore forming bacteria and also includes the food-borne pathogens, Cronobacter spp., Escherichia coli, Salmonella enterica, Shigella spp., and Yersinia spp. Illness caused by these pathogens is acquired...

  1. Biodegradation of organic compounds of molasses melanoidin (MM) from biomethanated distillery spent wash (BMDS) during the decolourisation by a potential bacterial consortium.

    PubMed

    Yadav, Sangeeta; Chandra, Ram

    2012-07-01

    Molasses melanoidin (MM) is a major pollutant in biomethanated distillery spent wash (BMDS) due to its recalcitrant properties. The 75% colour and 71% COD of MM (1,000 ppm) were reduced with developed bacterial consortium comprising Proteus mirabilis (IITRM5; FJ581028), Bacillus sp. (IITRM7; FJ581030), Raoultella planticola (IITRM15; GU329705) and Enterobacter sakazakii (IITRM16, FJ581031) in the ratio of 4:3:2:1 within 10 days at optimized nutrient. Bacterial consortium showed manganese peroxidase and laccase activity during MM decolourisation. The dominant growth of R. planticola and E. sakazakii was noted in consortium during MM decolourisation. The comparative GC-MS analysis of extracted compounds of control and degraded samples showed that most of the compounds present in control were completely utilized by bacterial consortium along with production of some metabolites. The developed bacterial consortium could be a tool for the decolourisation and degradation of melanoidin containing BMDS.

  2. Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

    PubMed Central

    Pava-Ripoll, Monica; Pearson, Rachel E.G.; Miller, Amy K.; Ziobro, George C.

    2015-01-01

    There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how

  3. Detection of foodborne bacterial pathogens from individual filth flies.

    PubMed

    Pava-Ripoll, Monica; Pearson, Rachel E G; Miller, Amy K; Ziobro, George C

    2015-02-13

    There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how

  4. Things Are Getting Hairy: Enterobacteria Bacteriophage vB_PcaM_CBB

    PubMed Central

    Buttimer, Colin; Hendrix, Hanne; Oliveira, Hugo; Casey, Aidan; Neve, Horst; McAuliffe, Olivia; Ross, R. Paul; Hill, Colin; Noben, Jean-Paul; O'Mahony, Jim; Lavigne, Rob; Coffey, Aidan

    2017-01-01

    Enterobacteria phage vB_PcaM_CBB is a “jumbo” phage belonging to the family Myoviridae. It possesses highly atypical whisker-like structures along the length of its contractile tail. It has a broad host range with the capability of infecting species of the genera Erwinia, Pectobacterium, and Cronobacter. With a genome of 355,922 bp, excluding a predicted terminal repeat of 22,456 bp, phage CBB is the third largest phage sequenced to date. Its genome was predicted to encode 554 ORFs with 33 tRNAs. Based on prediction and proteome analysis of the virions, 29% of its predicted ORFs could be functionally assigned. Protein comparison shows that CBB shares between 33–38% of its proteins with Cronobacter phage GAP32, coliphages PBECO4 and 121Q as well as Klebsiella phage vB_KleM_Rak2. This work presents a detailed and comparative analysis of vB_PcaM_CBB of a highly atypical jumbo myoviridae phage, contributing to a better understanding of phage diversity and biology. PMID:28174560

  5. Pyrosequencing analysis of microbial community and food-borne bacteria on restaurant cutting boards collected in Seri Kembangan, Malaysia, and their correlation with grades of food premises.

    PubMed

    Abdul-Mutalib, Noor-Azira; Amin Nordin, Syafinaz; Osman, Malina; Ishida, Natsumi; Tashiro, Kosuke; Sakai, Kenji; Tashiro, Yukihiro; Maeda, Toshinari; Shirai, Yoshihito

    2015-05-04

    This study adopts the pyrosequencing technique to identify bacteria present on 26 kitchen cutting boards collected from different grades of food premises around Seri Kembangan, a city in Malaysia. Pyrosequencing generated 452,401 of total reads of OTUs with an average of 1.4×10(7) bacterial cells/cm(2). Proteobacteria, Firmicutes and Bacteroides were identified as the most abundant phyla in the samples. Taxonomic richness was generally high with >1000 operational taxonomic units (OTUs) observed across all samples. The highest appearance frequencies (100%) were OTUs closely related to Enterobacter sp., Enterobacter aerogenes, Pseudomonas sp. and Pseudomonas putida. Several OTUs were identified most closely related to known food-borne pathogens, including Bacillus cereus, Cronobacter sakazaki, Cronobacter turisensis, Escherichia coli, E. coli O157:H7, Hafnia alvei, Kurthia gibsonii, Salmonella bongori, Salmonella enterica, Salmonella paratyphi, Salmonella tyhpi, Salmonella typhimurium and Yersinia enterocolitica ranging from 0.005% to 0.68% relative abundance. The condition and grade of the food premises on a three point cleanliness scale did not correlate with the bacterial abundance and type. Regardless of the status and grades, all food premises have the same likelihood to introduce food-borne bacteria from cutting boards to their foods and must always prioritize the correct food handling procedure in order to avoid unwanted outbreak of food-borne illnesses.

  6. Work of adhesion of dairy products on stainless steel surface

    PubMed Central

    Bernardes, Patrícia Campos; Araújo, Emiliane Andrade; dos Santos Pires, Ana Clarissa; Queiroz Fialho Júnior, José Felício; Lelis, Carini Aparecida; de Andrade, Nélio José

    2012-01-01

    The adhesion of the solids presents in food can difficult the process of surface cleaning and promotes the bacterial adhesion process and can trigger health problems. In our study, we used UHT whole milk, chocolate based milk and infant formula to evaluate the adhesion of Enterobacter sakazakii on stainless steel coupons, and we determine the work of adhesion by measuring the contact angle as well as measured the interfacial tension of the samples. In addition we evaluated the hydrophobicity of stainless steel after pre-conditioning with milk samples mentioned. E. sakazakii was able to adhere to stainless steel in large numbers in the presence of dairy products. The chocolate based milk obtained the lower contact angle with stainless steel surface, higher interfacial tension and consequently higher adhesion work. It was verified a tendency of decreasing the interfacial tension as a function of the increasing of protein content. The preconditioning of the stainless steel coupons with milk samples changed the hydrophobic characteristics of the surfaces and became them hydrophilic. Therefore, variations in the composition of the milk products affect parameters important that can influence the procedure of hygiene in surface used in food industry. PMID:24031951

  7. Microbiological examination of vegetable seed sprouts in Korea.

    PubMed

    Kim, Hoikyung; Lee, Youngjun; Beuchat, Larry R; Yoon, Bong-June; Ryu, Jee-Hoon

    2009-04-01

    Sprouted vegetable seeds used as food have been implicated as sources of outbreaks of Salmonella and Escherichia coli O157:H7 infections. We profiled the microbiological quality of sprouts and seeds sold at retail shops in Seoul, Korea. Ninety samples of radish sprouts and mixed sprouts purchased at department stores, supermarkets, and traditional markets and 96 samples of radish, alfalfa, and turnip seeds purchased from online stores were analyzed to determine the number of total aerobic bacteria (TAB) and molds or yeasts (MY) and the incidence of Salmonella, E. coli O157:H7, and Enterobacter sakazakii. Significantly higher numbers of TAB (7.52 log CFU/g) and MY (7.36 log CFU/g) were present on mixed sprouts than on radish sprouts (6.97 and 6.50 CFU/g, respectively). Populations of TAB and MY on the sprouts were not significantly affected by location of purchase. Radish seeds contained TAB and MY populations of 4.08 and 2.42 log CFU/g, respectively, whereas populations of TAB were only 2.54 to 2.84 log CFU/g and populations of MY were 0.82 to 1.69 log CFU/g on alfalfa and turnip seeds, respectively. Salmonella and E. coli O157:H7 were not detected on any of the sprout and seed samples tested. E. sakazakii was not found on seeds, but 13.3% of the mixed sprout samples contained this potentially pathogenic bacterium.

  8. Biochemical characteristics and biological properties of Annurca apple cider.

    PubMed

    Fratianni, Florinda; De Giulio, Alfonso; Sada, Alfonso; Nazzaro, Filomena

    2012-01-01

    Our work aimed to investigate the phenolic composition and antioxidant capacity of the de-alcoholized extract of cider obtained from the Annurca apple (Malus domestica var. Annurca). The antimicrobial effect of the extract against different pathogens, including Chronobacter sakazakii, was also examined. The extract's potential anti-quorum-sensing (AQS) activity was assessed using the test microorganism Chromobacterium violaceum. Biochemical analysis of the extract using ultra-performance liquid chromatography revealed catechin and caffeic acid as the most abundant polyphenols present, which represented about 35.5% and 36.6% of the total phenolics identified, respectively. An antioxidant capacity was also found (50% effective concentraiton=10 μL). The extract exhibited clear antimicrobial activity against all strains used in the experiments. Escherichia coli and Bacillus cereus were the most sensitive bacteria to the antimicrobial activity. The extract also inhibited the growth of the emergent pathogen strain C. sakazakii. The AQS activity of apple cider is reported here for the first time. In conclusion, our results demonstrate some biological properties of the apple cider and contribute to reinforcing the potential of the apple and its derivatives as functional components of the diet.

  9. Isolation and identification of bacteria associated with adult laboratory Mexican fruit flies, Anastrepha ludens (Diptera: Tephritidae).

    PubMed

    Kuzina, L V; Peloquin, J J; Vacek, D C; Miller, T A

    2001-04-01

    From the guts of new and old colonies (female and male) of Mexican fruit flies, Anastrepha ludens (Diptera: Tephritidae), we identified a total of 18 different bacterial species belonging to the family Enterobacteriaceae, Pseudomonadaceae, Vibrionaceae, Micrococcaceae, Deinococcacea, Bacillaceae, and the genus Listeria. Enterobacter, Providencia, Serratia, and Staphylococcus spp. were the most frequently isolated genera, with Citrobacter, Streptococcus, Aerococcus, and Listeria found less frequently. We found Bacillus cereus, Enterobacter sakazakii, Providencia stuartii, and Pseudomonas aeruginosa only in the new colony, Aeromonas hydrophila and Klebsiella pneumoniae spp. pneumoniae only in the old colony. We also studied resistance/sensitivity to 12 antibiotics for six bacterial isolates such as Enterobacter cloacae, E. sakazakii, K. pneumoniae spp., Providencia rettgeri, P. aeruginosa, and Bacillus cereus. Isolates on the whole were resistant to penicillin and ampicillin (five of six isolates) and sensitive to rifampin and streptomycin (six of six isolates). Antibiotic resistance profiles might be useful characteristics for distinguishing among species and strains of these bacteria, probably having ecological significance with respect to intra- and inter-specific competition within host cadavers, and could have implications for the utility of these organisms for biological control, including the alternative control strategy, paratransgenesis.

  10. Characterization of the Zinc-Containing Metalloprotease Encoded by zpx and Development of a Species-Specific Detection Method for Enterobacter sakazakii▿

    PubMed Central

    Kothary, M. H.; McCardell, B. A.; Frazar, C. D.; Deer, D.; Tall, B. D.

    2007-01-01

    Enterobacter sakazakii causes a severe form of neonatal meningitis that occurs as sporadic cases as well as outbreaks. The disease has been epidemiologically associated with consumption of reconstituted, dried infant formulas. Very little information is available regarding pathogenicity of the organism and production of virulence factors. Clinical and environmental strains were screened for production of factors which have activity against Chinese hamster ovary (CHO) cells in tissue culture. Polymyxin B lysate and sonicate preparations but not culture supernatants from the strains caused “rounding” of CHO cells. Subsequent studies showed that the CHO cell-rounding factor is a proteolytic enzyme that has activity against azocasein. The cell-bound protease was isolated by using a combination of polymyxin B lysis, followed by sonication of cells harvested from tryptone broth. The protease was purified to homogeneity by sequential ammonium sulfate precipitation, gel filtration chromatography with Sephadex G-100, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and a second gel filtration with Sephadex G-100. In addition to activity against azocasein, the purified protease also exhibits activity against azocoll and insoluble casein but not elastin. The protease has a molecular weight of 38,000 and an isoelectric point of 4.4. It is heat labile and for maximal activity against azocasein has an optimum temperature of 37°C and a pH range of 5 to 7. Proteolytic activity is inhibited by ortho-phenanthroline and Zincov but is not affected by phenylmethylsulfonyl fluoride, N-ethylmaleimide, and trypsin inhibitors, which demonstrates that the protease is a zinc-containing metalloprotease. The metalloprotease does not hemagglutinate chicken or sheep erythrocytes. Twenty-three to 27 of the first 42 N-terminal amino acid residues of the metalloprotease are identical to proteases produced by Serratia proteamaculans, Pectobacterium carotovorum, and Anabaena

  11. Microbiological assessment and evaluation of rehydration instructions on powdered infant formulas, follow-up formulas, and infant foods in Malaysia.

    PubMed

    Abdullah Sani, N; Hartantyo, S H P; Forsythe, S J

    2013-01-01

    A total of 90 samples comprising powdered infant formulas (n=51), follow-up formulas (n=21), and infant foods (n=18) from 15 domestic and imported brands were purchased from various retailers in Klang Valley, Malaysia and evaluated in terms of microbiological quality and the similarity of rehydration instructions on the product label to guidelines set by the World Health Organization. Microbiological analysis included the determination of aerobic plate count (APC) and the presence of Enterobacteriaceae and Cronobacter spp. Isolates of interest were identified using ID 32E (bioMérieux France, Craponne, France). In this study, 87% of powdered infant formulas, follow-up formulas, and infant foods analyzed had an APC below the permitted level of <10(4) cfu/g. These acceptable APC ranged between <10(2) to 7.2×10(3) cfu/g. The most frequently isolated Enterobacteriaceae was Enterobacter cloacae, which was present in 3 infant formulas and 1 infant food tested. Other Enterobacteriaceae detected from powdered infant and follow-up formulas were Citrobacter spp., Klebsiella spp., and other Enterobacter spp. No Cronobacter species were found in any samples. Rehydration instructions from the product labels were collated and it was observed that none directed the use of water with a temperature >70°C for formula preparation, as specified by the 2008 revised World Health Organization guidelines. Six brands instructed the use of water at 40 to 55°C, a temperature range that would support the survival and even growth of Enterobacteriaceae.

  12. Comparing the antimicrobial effectiveness of NaCl and KCl with a view to salt/sodium replacement.

    PubMed

    Bidlas, Eva; Lambert, Ronald J W

    2008-05-10

    A study using a small range of pathogenic bacterial species (Aeromonas hydrophila, Enterobacter sakazakii, Shigella flexneri, Yersinia enterocolitica and 3 strains of Staphylococcus aureus) has shown that potassium chloride has an equivalent antimicrobial effect on these organisms when calculated on a molar basis. Combined NaCl and KCl experiments were carried out and data was analysed using a modification to the Lambert and Lambert [Lambert, R.J.W., and Lambert, R., 2003. A model for the efficacy of combined inhibitors. Journal of Applied Microbiology 95, 734-743.] model for combined inhibitors and showed that in combination KCl is a direct 1:1 molar replacement for the antimicrobial effect of common salt. If this is a general finding then, where salt is used to help preserve a product, partial or complete replacement by KCl is possible.

  13. First report of metallo-β-lactamases producing Enterobacter spp. strains from Venezuela.

    PubMed

    Martínez, Dianny; Rodulfo, Hectorina E; Rodríguez, Lucy; Caña, Luisa E; Medina, Belkis; Guzman, Militza; Carreño, Numirin; Marcano, Daniel; De Donato, Marcos

    2014-01-01

    Clinical strains of Enterobacter were isolated from Cumana's Central Hospital in Venezuela, and classified as E. cloacae (21), E. aerogenes (7), E. intermedium (1), E. sakazakii (1) and three unclassified. The strains showed high levels of resistance, especially to SXT (58.1%), CRO (48.8%), CAZ (46.6%), PIP (46.4%), CIP (45.2%) and ATM (43.3%). This is the first report for South America of blaVIM-2 in two E. cloacae and one Enterobacter sp., which also showed multiple mechanisms of resistance. Both E. cloacae showed blaTEM-1, but only one showed blaCTX-M-15 gene, while no blaSHV was detected.

  14. Cultivation Conditions for Phytase Production from Recombinant Escherichia coli DH5α

    PubMed Central

    Ariff, Rafidah Mohd; Fitrianto, Anwar; Abd. Manap, Mohd Yazid; Ideris, Aini; Kassim, Azhar; Suhairin, Afinah; Hussin, Anis Shobirin Meor

    2013-01-01

    Response surface methodology (RSM) was used to optimize the cultivation conditions for the production of phytase by recombinant Escherichia coli DH5α. The optimum predicted cultivation conditions for phytase production were at 3 hours seed age, a 2.5% inoculum level, an L-arabinose concentration of 0.20%, a cell concentration of 0.3 (as measured at 600 nm) and 17 hours post-induction time with a predicted phytase activity of 4194.45 U/mL. The model was validated and the results showed no significant difference between the experimental and the predicted phytase activity (P = 0.305). Under optimum cultivation conditions, the phytase activity of the recombinant E. coli DH5α was 364 times higher compared to the phytase activity of the wild-type producer, Enterobacter sakazakii ASUIA279. Hence, optimization of the cultivation conditions using RSM positively increased phytase production from recombinant E. coli DH5α. PMID:24826071

  15. Decoction, infusion and hydroalcoholic extract of cultivated thyme: antioxidant and antibacterial activities, and phenolic characterisation.

    PubMed

    Martins, Natália; Barros, Lillian; Santos-Buelga, Celestino; Silva, Sónia; Henriques, Mariana; Ferreira, Isabel C F R

    2015-01-15

    Bioactivity of thyme has been described, but mostly related to its essential oils, while studies with aqueous extracts are scarce. Herein, the antioxidant and antibacterial properties of decoction, infusion and hydroalcoholic extract, as also their phenolic compounds, were evaluated and compared. Decoction showed the highest concentration of phenolic compounds (either phenolic acids or flavonoids), followed by infusion and hydroalcoholic extract. In general, the samples were effective against gram-positive (Staphylococcus aureus and Staphylococcus epidermidis) and gram-negative (Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa, Enterococcus aerogenes, Proteus vulgaris and Enterobacter sakazakii) bacteria, with decoction presenting the most pronounced effect. This sample also displayed the highest radical scavenging activity and reducing power. Data obtained support the idea that compounds with strong antioxidant and antibacterial activities are also water-soluble. Furthermore, the use of thyme infusion and decoction, by both internal and external use, at recommended doses, is safe and no adverse reactions have been described.

  16. Detection of persistent organic compounds from biomethanated distillery spent wash (BMDS) and their degradation by manganese peroxidase and laccase producing bacterial strains.

    PubMed

    Yadav, Sangeeta; Chandra, Ram

    2013-07-01

    Biomethanated distillery spent wash (BMDS) retains dark black colour with complex persistent organic pollutants even after anaerobic treatment. The specific ratio (4:3:1:1) of Proteus mirabilis (FJ581028), Bacillus sp. (FJ581030), Raoultella planticola (GU329705) and Enterobacter sakazakii (FJ581031) decolourised BMDS up to 76% within 192 hr along with degradation of persistent organic compounds in presence of glucose (1%) and peptone (0.1%). The colour removal ability was noted due to ligninolytic enzyme activity. Where the maximum manganese peroxidase was 1.93 U ml(-1) and laccase activity equalled 0.84 U ml(-1). The gas chromatography-mass spectrophotometry (GC-MS) analysis confirmed the direct correlation between colourant and persistent organic pollutants due to simultaneous reduction of colour and pollutants present in BMDS. The seed germination test showed reduction of 75% toxicity after bacterial treatment process.

  17. Essential oil from leaves of Lantana canescens and L. lopez-palacii grown in Colombia.

    PubMed

    Peralta-Bohórquez, Andrés F; Quijano-Célis, Clara; Gaviria, Mauricio; Vanegas-López, Consuelo; Pino, Jorge A

    2011-02-01

    The chemical composition of the volatile compounds from the leaves of Lantana canescens Kunth (Verbenaceae) and L. lopez-palacii Moldenke grown in Colombia were analyzed by GC and GC/MS. One hundred and thirty-nine volatile compounds were identified in L. canescens, of which the major ones were beta-caryophyllene (13.5%), germacrene D (10.3%) and 1-octen-3-ol (8.4%). In the oil obtained from L. lopez-palacii, eighty-three compounds were identified, of which the most prominent were 1-octen-3-ol (24.4%) and beta-caryophyllene (15.2%). The in vitro antibacterial activity of the L. lopez-palacii essential oil was studied against three bacterial strains using the disc diffusion method. No antimicrobial activity was found against Escherichia coli, Enterobacter sakazakii and Listeria monocytogenes.

  18. Inactivation of Gram-Negative Bacteria by Low-Pressure RF Remote Plasma Excited in N2-O2 Mixture and SF6 Gases.

    PubMed

    Al-Mariri, Ayman; Saloum, Saker; Mrad, Omar; Swied, Ghayath; Alkhaled, Bashar

    2013-12-01

    The role of low-pressure RF plasma in the inactivation of Escherichia coli O157, Klebsiella pneumoniae, Proteus mirabilis, and Enterobacter sakazakii using N2-O2 and SF6 gases was assessed. 1×10(9) colony-forming units (CFUs) of each bacterial isolate were placed on three polymer foils. The effects of pressure, power, distance from the source, and exposure time to plasma gases were optimized. The best conditions to inactivate the four bacteria were a 91%N2-9%O2 mixture and a 30-minute exposure time. SF6 gas was more efficient for all the tested isolates in as much as the treatment time was reduced to only three minutes. Therefore, low-pressure plasma could be used to sterilize heat and/or moisture-sensitive medical instruments.

  19. Application of bacteriophages in post-harvest control of human pathogenic and food spoiling bacteria.

    PubMed

    Pérez Pulido, Rubén; Grande Burgos, Maria José; Gálvez, Antonio; Lucas López, Rosario

    2016-10-01

    Bacteriophages have attracted great attention for application in food biopreservation. Lytic bacteriophages specific for human pathogenic bacteria can be isolated from natural sources such as animal feces or industrial wastes where the target bacteria inhabit. Lytic bacteriophages have been tested in different food systems for inactivation of main food-borne pathogens including Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, Salmonella enterica, Shigella spp., Campylobacter jejuni and Cronobacter sakazkii, and also for control of spoilage bacteria. Application of lytic bacteriophages could selectively control host populations of concern without interfering with the remaining food microbiota. Bacteriophages could also be applied for inactivation of bacteria attached to food contact surfaces or grown as biofilms. Bacteriophages may receive a generally recognized as safe status based on their lack of toxicity and other detrimental effects to human health. Phage preparations specific for L. monocytogenes, E. coli O157:H7 and S. enterica serotypes have been commercialized and approved for application in foods or as part of surface decontamination protocols. Phage endolysins have a broader host specificity compared to lytic bacteriophages. Cloned endolysins could be used as natural preservatives, singly or in combination with other antimicrobials such as bacteriocins.

  20. Erwinia gerundensis sp. nov., a cosmopolitan epiphyte originally isolated from pome fruit trees.

    PubMed

    Rezzonico, Fabio; Smits, Theo H M; Born, Yannick; Blom, Jochen; Frey, Jürg E; Goesmann, Alexander; Cleenwerck, Ilse; de Vos, Paul; Bonaterra, Anna; Duffy, Brion; Montesinos, Emilio

    2016-01-26

    A survey to obtain potential antagonists of pome fruit tree diseases yielded two yellow epiphytic bacterial isolates morphologically similar to Pantoea agglomerans, but showing no biocontrol activity. Whole-Cell MALDI-TOF mass spectrometry and analysis of 16S rRNA and gyrB gene sequences suggested the possibility of a new species with a phylogenetic position in either Pantoea or Erwinia. Multilocus sequence analysis (MLSA) placed the two strains in the genus Erwinia and supported their classification as a novel species. The strains showed general phenotypic characteristics typical of this genus and results of DNA-DNA hybridizations confirmed that they represent a single novel species. Both strains showed a DNA G+C content, as determined by HPLC, of 54.5 mol% and could be discriminated from phylogenetically related species of the genus Erwinia by their ability to utilize potassium gluconate, potassium 2-ketogluconate, D-maltose, D-melibiose and D-raffinose. Whole-genome sequencing of strain EM595T revealed the presence of a chromosomal carotenoid biosynthesis gene cluster similar to those found in Cronobacter and Pantoea spp. that explains the pigmentation of the strain, which is atypical for the genus Erwinia. Additional strains belonging to the same species were recovered from different plant hosts in three different continents, revealing the cosmopolitan nature of this epiphyte. The name Erwinia gerundensis sp. nov. is proposed, with EM595T (= LMG 28990T; = CCOS 903T) as the designated type strain.

  1. The 216-bp marB gene of the marRAB operon in Escherichia coli encodes a periplasmic protein which reduces the transcription rate of marA.

    PubMed

    Vinué, Laura; McMurry, Laura M; Levy, Stuart B

    2013-08-01

    The marRAB operon is conserved in seven genera of enteric bacteria (Escherichia, Shigella, Klebsiella, Enterobacter, Salmonella, Cronobacter, and Citrobacter). MarA is a transcriptional regulator affecting many genes involved in resistance to stresses, and MarR is an autorepressor of the operon, but a role for the marB gene has been unclear. A recent work reported that deletion of marB causes resistance to certain stresses and increases the amount of marA transcript. We show here that the small (216 bp) marB gene encodes a protein, not an sRNA, because two different stop codons within the predicted open reading frame of marB prevented plasmid-borne marB from complementing ΔmarB::Kan. The ΔmarB::Kan mutation did not increase the stability of the marA transcript, suggesting that MarB does not destabilize the marA transcript but rather reduces its rate of transcription. Placing the putative signal sequence of MarB upstream of signal-sequence-less alkaline phosphatase guided the phosphatase to its normal periplasmic location. We conclude that MarB is a small periplasmic protein that represses the marRAB promoter by an indirect mechanism, possibly involving a signal to one of the cytoplasmic regulators of that promoter.

  2. Novel Approaches to Improve the Intrinsic Microbiological Safety of Powdered Infant Milk Formula

    PubMed Central

    Kent, Robert M.; Fitzgerald, Gerald F.; Hill, Colin; Stanton, Catherine; Ross, R. Paul

    2015-01-01

    Human milk is recognised as the best form of nutrition for infants. However; in instances where breast-feeding is not possible, unsuitable or inadequate, infant milk formulae are used as breast milk substitutes. These formulae are designed to provide infants with optimum nutrition for normal growth and development and are available in either powdered or liquid forms. Powdered infant formula is widely used for convenience and economic reasons. However; current manufacturing processes are not capable of producing a sterile powdered infant formula. Due to their immature immune systems and permeable gastro-intestinal tracts, infants can be more susceptible to infection via foodborne pathogenic bacteria than other age-groups. Consumption of powdered infant formula contaminated by pathogenic microbes can be a cause of serious illness. In this review paper, we discuss the current manufacturing practices present in the infant formula industry, the pathogens of greatest concern, Cronobacter and Salmonella and methods of improving the intrinsic safety of powdered infant formula via the addition of antimicrobials such as: bioactive peptides; organic acids; probiotics and prebiotics. PMID:25685987

  3. A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells.

    PubMed

    Soejima, Takashi; Xiao, Jin-Zhong; Abe, Fumiaki

    2016-06-23

    Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 10(0) cfu/ml for the test sample compared with a detection limit of 1.6 × 10(3) cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research.

  4. The two peptide lantibiotic lacticin 3147 acts synergistically with polymyxin to inhibit Gram negative bacteria

    PubMed Central

    2013-01-01

    Background The emergence of bacterial drug resistance encourages the re-evaluation of the potential of existing antimicrobials. Lantibiotics are post-translationally modified, ribosomally synthesised antimicrobial peptides with a broad spectrum antimicrobial activity. Here, we focussed on expanding the potential of lacticin 3147, one of the most studied lantibiotics and one which possesses potent activity against a wide range of Gram positive species including many nosocomial pathogens. More specifically, our aim was to investigate if lacticin 3147 activity could be enhanced when combined with a range of different clinical antibiotics. Results Initial screening revealed that polymyxin B and polymyxin E (colistin) exhibited synergistic activity with lacticin 3147. Checkerboard assays were performed against a number of strains, including both Gram positive and Gram negative species. The resultant fractional inhibitory concentration (FIC) index values established that, while partial synergy was detected against Gram positive targets, synergy was obvious against Gram negative species, including Cronobacter and E. coli. Conclusions Combining lacticin 3147 with low levels of a polymyxin could provide a means of broadening target specificity of the lantibiotic, while also reducing polymyxin use due to the lower concentrations required as a result of synergy. PMID:24069959

  5. Diversity, antimicrobial and antioxidant activities of culturable bacterial endophyte communities in Aloe vera.

    PubMed

    Akinsanya, Mushafau Adewale; Goh, Joo Kheng; Lim, Siew Ping; Ting, Adeline Su Yien

    2015-12-01

    Twenty-nine culturable bacterial endophytes were isolated from surface-sterilized tissues (root, stem and leaf) of Aloe vera and molecularly characterized to 13 genera: Pseudomonas, Bacillus, Enterobacter, Pantoea, Chryseobacterium, Sphingobacterium, Aeromonas, Providencia, Cedecea, Klebsiella, Cronobacter, Macrococcus and Shigella. The dominant genera include Bacillus (20.7%), Pseudomonas (20.7%) and Enterobacter (13.8%). The crude and ethyl acetate fractions of the metabolites of six isolates, species of Pseudomonas, Bacillus, Chryseobacterium and Shigella, have broad spectral antimicrobial activities against pathogenic Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Salmonella Typhimurium, Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli, Streptococcus pyogenes and Candida albicans, with inhibition zones ranging from 6.0 ± 0.57 to 16.6 ± 0.57 mm. In addition, 80% of the bacterial endophytes produced 1,1-diphenyl-2-picrylhydrazyl (DPPH) with scavenging properties of over 75% when their crude metabolites were compared with ascorbic acid (92%). In conclusion, this study revealed for the first time the endophytic bacteria communities from A. vera (Pseudomonas hibiscicola, Macrococcus caseolyticus, Enterobacter ludwigii, Bacillus anthracis) that produce bioactive compounds with high DPPH scavenging properties (75-88%) and (Bacillus tequilensis, Pseudomonas entomophila, Chryseobacterium indologenes, Bacillus aerophilus) that produce bioactive compounds with antimicrobial activities against bacterial pathogens. Hence, we suggest further investigation and characterization of their bioactive compounds.

  6. Detoxification of mercury pollutant leached from spent fluorescent lamps using bacterial strains.

    PubMed

    Al-Ghouti, Mohammad A; Abuqaoud, Reem H; Abu-Dieyeh, Mohammed H

    2016-03-01

    The spent fluorescent lamps (SFLs) are being classified as a hazardous waste due to having mercury as one of its main components. Mercury is considered the second most toxic heavy metal (arsenic is the first) with harmful effects on animal nervous system as it causes different neurological disorders. In this research, the mercury from phosphor powder was leached, then bioremediated using bacterial strains isolated from Qatari environment. Leaching of mercury was carried out with nitric and hydrochloric acid solutions using two approaches: leaching at ambient conditions and microwave-assisted leaching. The results obtained from this research showed that microwave-assisted leaching method was significantly better in leaching mercury than the acid leaching where the mercury leaching efficiency reached 76.4%. For mercury bio-uptake, twenty bacterial strains (previously isolated and purified from petroleum oil contaminated soils) were sub-cultured on Luria Bertani (LB) plates with mercury chloride to check the bacterial tolerance to mercury. Seven of these twenty strains showed a degree of tolerance to mercury. The bio-uptake capacities of the promising strains were investigated using the mercury leached from the fluorescent lamps. Three of the strains (Enterobacter helveticus, Citrobacter amalonaticus, and Cronobacter muytjensii) showed bio-uptake efficiency ranged from 28.8% to 63.6%.

  7. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis.

    PubMed

    Santiago-Felipe, S; Tortajada-Genaro, L A; Puchades, R; Maquieira, A

    2014-02-06

    Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR-ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA-ELISA combination is proposed for amplification at a low, constant temperature (40°C) in a short time (40 min), for the hybridisation of labelled products to specific 5'-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA-ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA-ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings.

  8. Low Diversity Bacterial Community and the Trapping Activity of Metabolites from Cultivable Bacteria Species in the Female Reproductive System of the Oriental Fruit Fly, Bactrocera dorsalis Hendel (Diptera: Tephritidae)

    PubMed Central

    Shi, Zhanghong; Wang, Lili; Zhang, Hongyu

    2012-01-01

    Our goal was to identify the bacteria inhabiting the reproductive system of the female oriental fruit fly, Bactrocera dorsalis (Hendel), and evaluate the chemotaxis of B. dorsalis to the metabolites produced by the bacteria. Based on 16S rRNA-based polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), 18 operational taxonomic units (OTUs) were assigned to the five bacterial classes Betaproteobacteria, Alphaproteobacteria, Gammaproteobacteria, Bacilli and Actinobacteria. Nine OTUs were assigned to Gammaproteobacteria, which was the most highly represented class. Enterobacteriaceae constituted the dominant family, and within this family, three genera and five species were identified, including Enterobacter sakazakii, Klebsiella oxytoca, Klebsiella pneumoniae, Raoultella terrigena and Enterobacter amnigenus. In this set, the first two species were the dominant components, and the latter three species were the minor ones. Finally, we found that the metabolites produced by R. terrigena, K. oxytoca and K. pneumoniae were attractive to the B. dorsalis adults, and in field studies, B. dorsalis adults were most attracted to K. oxytoca. Collectively, our results suggest that the female reproductive system plays an important role in the transfer of enterobacteria from the gut to fruit. Our data may prompt the development of a female-targeted population control strategy for this fly. PMID:22754363

  9. Multiplexed bead-based mesofluidic system for detection of food-borne pathogenic bacteria.

    PubMed

    Jin, Sheng-Quan; Yin, Bin-Cheng; Ye, Bang-Ce

    2009-11-01

    In the present study, a simple and rapid multiplexed bead-based mesofluidic system (BMS) was developed for simultaneous detection of food-borne pathogenic bacteria, including Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella, Enterobacter sakazakii, Shigella, Escherichia coli O157:H7, and Campylobacter jejuni. This system is based on utilization of isothiocyanate-modified microbeads that are 250 mum in diameter, which were immobilized with specific amino-modified oligonucleotide probes and placed in polydimethylsiloxane microchannels. PCR products from the pathogens studied were pumped into microchannels to hybridize with the oligonucleotide-modified beads, and hybridization signals were detected using a conventional microarray scanner. The short sequences of nucleic acids (21 bases) and PCR products characteristic of bacterial pathogens could be detected at concentrations of 1 pM and 10 nM, respectively. The detection procedure could be performed in less than 30 min with high sensitivity and specificity. The assay was simple and fast, and the limits of quantification were in the range from 500 to 6,000 CFU/ml for the bacterial species studied. The feasibility of identification of food-borne bacteria was investigated with samples contaminated with bacteria, including milk, egg, and meat samples. The results demonstrated that the BMS method can be used for effective detection of multiple pathogens in different foodstuffs.

  10. Production of Cellulose and Curli Fimbriae by Members of the Family Enterobacteriaceae Isolated from the Human Gastrointestinal Tract

    PubMed Central

    Zogaj, Xhavit; Bokranz, Werner; Nimtz, Manfred; Römling, Ute

    2003-01-01

    Citrobacter spp., Enterobacter spp., and Klebsiella spp. isolated from the human gut were investigated for the biosynthesis of cellulose and curli fimbriae (csg). While Citrobacter spp. produced curli fimbriae and cellulose and Enterobacter spp. produced cellulose with various temperature-regulatory programs, Klebsiella spp. did not show pronounced expression of those extracellular matrix components. Investigation of multicellular behavior in two Citrobacter species and Enterobacter sakazakii showed an extracellular matrix, cell clumping, pellicle formation, and biofilm formation associated with the expression of cellulose and curli fimbriae. In those three strains, the csgD-csgBA region and the cellulose synthase gene bcsA were conserved. PCR screening for the presence of csgD, csgA and bcsA revealed that besides Klebsiella pneumoniae and Klebsiella oxytoca, all species investigated harbored the genetic information for expression of curli fimbriae and cellulose. Since Citrobacter spp., Enterobacter spp., and Klebsiella spp. are frequently found to cause biofilm-related infections such as catheter-associated urinary tract infections, the human gut could serve as a reservoir for dissemination of biofilm-forming isolates. PMID:12819107

  11. Enterobacter turicensis sp. nov. and Enterobacter helveticus sp. nov., isolated from fruit powder.

    PubMed

    Stephan, Roger; Van Trappen, Stefanie; Cleenwerck, Ilse; Vancanneyt, Marc; De Vos, Paul; Lehner, Angelika

    2007-04-01

    Four Gram-negative, facultatively anaerobic, non-spore-forming isolates of coccoid rods were obtained from fruit powder and investigated in a polyphasic taxonomic study. Comparative 16S rRNA gene sequence analysis allocated the isolates to the family Enterobacteriaceae. Their phylogenetic position within the family Enterobacteriaceae was confirmed by rpoB sequence analysis and as the highest rpoB sequence similarities were obtained with Enterobacter radicincitans, Enterobacter cowanii and Enterobacter sakazakii, the isolates clearly belong to the genus Enterobacter. Biochemical data revealed that the isolates can be separated into two distinct groups that represent two novel species, as confirmed by DNA-DNA hybridizations. The two novel species can be differentiated from their nearest neighbours by the following characteristics: the utilization of sucrose, D-sorbitol, putrescine and mucate, the hydrolysis of aesculin and a negative result in the Voges-Proskauer reaction. It is therefore proposed that these novel isolates are classified as Enterobacter turicensis sp. nov. (type strain 508/05(T)=LMG 23730(T)=DSM 18397(T)) and Enterobacter helveticus sp. nov. (type strain 513/05(T)=LMG 23732(T)=DSM 18396(T)).

  12. Enterobacter pulveris sp. nov., isolated from fruit powder, infant formula and an infant formula production environment.

    PubMed

    Stephan, Roger; Van Trappen, Stefanie; Cleenwerck, Ilse; Iversen, Carol; Joosten, Han; De Vos, Paul; Lehner, Angelika

    2008-01-01

    Six Gram-negative, facultatively anaerobic, non-spore-forming, coccoid rod-shaped isolates were obtained from fruit powder (n=3), infant formula (n=2) and an infant formula production environment (n=1) and investigated in a polyphasic taxonomic study. Comparative 16S rRNA gene sequence analysis combined with rpoB gene sequence analysis allocated the isolates to the family Enterobacteriaceae. The highest rpoB gene sequence similarities (91.2-95.8%) were obtained with Enterobacter helveticus, Enterobacter radicincitans, Enterobacter turicensis and Enterobacter sakazakii and the phylogenetic branch formed by these species was supported by a high bootstrap value. Biochemical data revealed that the isolates could be differentiated from their nearest neighbours by their ability to utilize melibiose, sucrose, D-arabitol, mucate and 1-O-methyl-alpha-galactopyranoside and their negative reactions for D-sorbitol utilization and the Voges-Proskauer test. On the basis of the phylogenetic analyses, DNA-DNA hybridization data, and unique physiological and biochemical characteristics, it is proposed that the isolates represent a novel species of the genus Enterobacter, Enterobacter pulveris sp. nov. The type strain is 601/05(T) (=LMG 24057(T)=DSM 19144(T)).

  13. Biological and quantitative-SAR evaluations, and docking studies of (E)-N -benzylidenebenzohydrazide analogues as potential antibacterial agents

    PubMed Central

    Alam, Mohammad Sayed; Jebin, Sefat; Rahman, M. Mostafizur; Bari, Md. Latiful; Lee, Dong-Ung

    2016-01-01

    A series of 15 (E)-N'-benzylidenebenzohydrazide analogues were evaluated for their antimicrobial activities against eleven pathogenic and food-borne microbes, namely, S. aureus (G+), L. monocytogenes (G+), B. subtilis (G+), K. pneumonia (G¯), C. sakazakii (G¯), C. freundii (G¯), S. enterica (G¯), S. enteritidis (G¯), E. coli (G¯), Y. pestis (G¯), and P. aeruginosa (G¯). Most of the compounds exhibited selective activity against some Gram-negative bacterial strains. Of the compounds tested (3a-o), 3b and 3g were most active against C. freundii (MIC = ~19 µg mL-1). Whereas, compounds 3d, 3i, 3k and 3n exhibited MIC values ranging from 37.5 to 75 μg mL-1 against C. freundii, and compounds 3e, 3l and 3n had MIC values of ~75 μg mL-1 against K. pneumonia. Quantitative structure-antibacterial activity relationships were studied using physicochemical parameters and a good correlation was found between calculated octanol-water partition coefficients (clogP; a lipophilic parameter) and antibacterial activities. In silico screening was also performed by docking high (3b and 3g) and low (3n) activity compounds on the active site of E. coli FabH receptor, which is an important therapeutic target. The findings of these in silico screening studies provide a theoretical basis for the design and synthesis of novel benzylidenebenzohydrazide analogues that inhibit bacterial FabH. PMID:27540348

  14. Study of the micro-organisms associated with the fermented bread (khamir) produced from sorghum in Gizan region, Saudi Arabia.

    PubMed

    Gassem, M A

    1999-02-01

    Traditional bread (khamir) was made from sorghum flour of two local varieties, Bayadh and Hamra. The bread was prepared by mixing the sorghum flour with water and spices (onion, garlic, lemon juice and fenugreek) in a 1:0.8 (w/w) ratio and fermented for 24 h at 30 degrees C. Two other fermentations were carried out using an inoculum from the previous fermentation. The micro-organisms were isolated from different plates and identified using different characterization systems. Both total bacterial populations and lactic acid bacteria increased with fermentation time and reached the highest number at 16 h (first fermentation) and at 8 h (second and third fermentation). The content of lactic acid was increased with time to reach 1.2%, but the increase was higher for the second and third fermentations (1.6% each). The pH dropped with time from 6.77 to 4.35 in the first fermentation and from 6.65 to 4.18, and 6.57-3.93, in the second and third fermentations, respectively. The microorganisms, which were isolated and characterized during the 24 h fermentation, included: bacteria (Pediococcus pentosaceus, Lactobacillus brevis, Lact. lactis subsp. lactis, Lact. cellobiosus, Klebsiella oxytoca, Kl. pneumoniae, Enterobacter aerogenes, Ent. sakazakii, Serratia marcescens and Ser. odourifera), moulds (Penicillium sp., Rhizopus sp., Aspergillus niger, Alternaria sp., Fusarium sp. and Mucor sp.) and yeasts (Candida parapsilosis, C. orvegnsis and Rhodotorula glutinis).

  15. Inhibitory effect of commercial green tea and rosemary leaf powders on the growth of foodborne pathogens in laboratory media and oriental-style rice cakes.

    PubMed

    Lee, Sun-Young; Gwon, So-Young; Kim, Seung-Ju; Moon, Bo Kyung

    2009-05-01

    The antimicrobial effects of green tea and rosemary added to foods as antagonists to foodborne pathogens were determined in laboratory media and oriental-style rice cakes. The growth of each pathogen (Bacillus cereus, Salmonella Typhimurium, Enterobacter sakazakii, Escherichia coli O157:H7, Staphylococcus aureus, and Listeria monocytogenes) in tryptic soy broth or rice cake with or without addition of green tea or rosemary leaf powders before autoclaving or cooking, respectively, was investigated after inoculation. The addition of 1% green tea or rosemary produced similar results for inhibiting the growth of pathogens in tryptic soy broth. However, green tea was more effective than rosemary for inhibiting the growth of L. monocytogenes. Both botanicals had inhibitory effects against all pathogens tested in this study. Green tea was particularly effective against B. cereus, S. aureus, and L. monocytogenes, and rosemary was strongly inhibitory against B. cereus and S. aureus. The addition of 1 or 3% green tea or rosemary to rice cakes did not significantly reduce total aerobic counts; however, levels of B. cereus and S. aureus were significantly reduced in rice cakes stored for 3 days at room temperature (22 degrees C). The order of antimicrobial activities against B. cereus in rice cake was 1% rosemary < 1% green tea < 3% rosemary = 3% green tea. These results indicate that the use of natural plant materials such as green tea and rosemary could improve the microbial quality of foods in addition to their functional properties.

  16. Phytochemical and Cytogenetic Characterization of Centaurea solstitialis L. (Asteraceae) from Croatia.

    PubMed

    Carev, Ivana; Ruščić, Mirko; Skočibušić, Mirjana; Maravić, Ana; Siljak-Yakovlev, Sonja; Politeo, Olivera

    2017-02-01

    The cytogenetic characterization of Centaurea solstitialis L. (Asteraceae) showed a chromosome number of 2n = 16. Karyotype is composed by four pairs of metacentric, two pairs of submetacentric and two pairs of subtelocentric chromosomes. Physical mapping of two rDNA probes revealed two loci of 35S and one locus of 5S rRNA genes. Chromomycin fluorochrome banding revealed that all rDNA loci were GC rich. The genome size (2C-value) of 1.95 pg classes this species in the group of very small genomes. Chemical composition of C. solstitialis volatile oil (VO) from Croatia, studied with gas chromatography-mass spectrometry showed dominant components as it follows: hexadecanoic acid, α-linolenic acid, germacrene D and heptacosane. Antioxidant capacity, measured by ferric reducing power assay and 2,2-diphenyl-1-picrylhydrazyl methods, as well as inhibition of acetyl- and butyrylcholinesterase of VO was lower comparing to a standard solutions. Volatile oil tested with disc diffusion method showed good inhibitory potential against Pseudomonas aeruginosa, Escherichia coli and all tested fungi: Candida albicans, Penicillium funiculosum and Aspergillus fumigatus. The microdilution method showed best activity against Chronobacter sakazakii and A. fumigatus.

  17. [Aerobic bacterial flora from the digestive tract of the common vampire bat, Desmodus rotundus (Chiroptera: Phyllostomidae)].

    PubMed

    Chaverri, Gloriana

    2006-09-01

    This study addresses the composition of microbial flora in the vampire bat (Desmodus rotundus) primarily because all available data are outdated, and because of the economical significance of this bat species. Twenty-one bats were collected and their aerobic bacteria documented separately for stomach and intestine. Bacteria were identified through the Analytical Profile Index (API), and results analyzed with the APILAB software. A total of thirty bacterial species were isolated from sixteen females and five males. The most common species were Escherichia coli and Staphylococcus aureus, although other bacteria, such as Acinetobacterjohnsonii, Enterobacter sakazakii, Staphylococcus chromogenes, S. hyicus and S. xylosus were also common. The number of species found in the stomach and intestine was significantly different, and the intestine presented a higher diversity compared to the stomach. This has previously been found in other mammals and it is attributed to a reduction of acidity. Most of the species found in this study are considered normal components of the digestive tract of mammals, although other bacteria common in the skin of mammals and from aquatic environments were found. Bacteria from the skin may invade the vampire's stomach and/or intestine when the bat has contact with its prey, and may suggest that the vampire's feeding habit facilitates the invasion of other microbes not common in its digestive tract. The fact that bacteria from aquatic environments were also found suggests that D. rotundus, as previously found by other researchers, drinks free water when available, and water may be another source of microbial invasion.

  18. Roles of nitric oxide and intestinal microbiota in the pathogenesis of necrotizing enterocolitis.

    PubMed

    Grishin, Anatoly; Bowling, Jordan; Bell, Brandon; Wang, Jin; Ford, Henri R

    2016-01-01

    Necrotizing enterocolitis remains one of the most vexing problems in the neonatal intensive care unit. Risk factors for NEC include prematurity, formula feeding, and inappropriate microbial colonization of the GI tract. The pathogenesis of NEC is believed to involve weakening of the intestinal barrier by perinatal insults, translocation of luminal bacteria across the weakened barrier, an exuberant inflammatory response, and exacerbation of the barrier damage by inflammatory factors, leading to a vicious cycle of inflammation-inflicted epithelial damage. Nitric oxide (NO), produced by inducible NO synthase (iNOS) and reactive NO oxidation intermediates play a prominent role in the intestinal barrier damage by inducing enterocyte apoptosis and inhibiting the epithelial restitution processes, namely enterocyte proliferation and migration. The factors that govern iNOS upregulation in the intestine are not well understood, which hampers efforts in developing NO/iNOS-targeted therapies. Similarly, efforts to identify bacteria or bacterial colonization patterns associated with NEC have met with limited success, because the same bacterial species can be found in NEC and in non-NEC subjects. However, microbiome studies have identified the three important characteristics of early bacterial populations of the GI tract: high diversity, low complexity, and fluidity. Whether NEC is caused by specific bacteria remains a matter of debate, but data from hospital outbreaks of NEC strongly argue in favor of the infectious nature of this disease. Studies in Cronobacter muytjensii have established that the ability to induce NEC is the property of specific strains rather than the species as a whole. Progress in our understanding of the roles of bacteria in NEC will require microbiological experiments and genome-wide analysis of virulence factors.

  19. Fabrication of Polymerase Chain Reaction Plastic Lab-on-a-Chip Device for Rapid Molecular Diagnoses

    PubMed Central

    2016-01-01

    Purpose: We aim to fabricate a thermoplastic poly(methylmethacrylate) (PMMA) Lab-on-a-Chip device to perform continuous- flow polymerase chain reactions (PCRs) for rapid molecular detection of foodborne pathogen bacteria. Methods: A miniaturized plastic device was fabricated by utilizing PMMA substrates mediated by poly(dimethylsiloxane) interfacial coating, enabling bonding under mild conditions, and thus avoiding the deformation or collapse of microchannels. Surface characterizations were carried out and bond strength was measured. The feasibility of the Lab-on-a-Chip device for performing on-chip PCR utilizing a lab-made, portable dual heater was evaluated. The results were compared with those obtained using a commercially available thermal cycler. Results: A PMMA Lab-on-a-Chip device was designed and fabricated for conducting PCR using foodborne pathogens as sample targets. A robust bond was established between the PMMA substrates, which is essential for performing miniaturized PCR on plastic. The feasibility of on-chip PCR was evaluated using Escherichia coli O157:H7 and Cronobacter condimenti, two worldwide foodborne pathogens, and the target amplicons were successfully amplified within 25 minutes. Conclusions: In this study, we present a novel design of a low-cost and high-throughput thermoplastic PMMA Lab-on-a-Chip device for conducting microscale PCR, and we enable rapid molecular diagnoses of two important foodborne pathogens in minute resolution using this device. In this regard, the introduced highly portable system design has the potential to enable PCR investigations of many diseases quickly and accurately. PMID:27230459

  20. Isolation and characterization of Enterobacteriaceae species infesting post-harvest strawberries and their biological control using bacteriophages.

    PubMed

    Kurtböke, D Ipek; Palk, A; Marker, A; Neuman, C; Moss, L; Streeter, K; Katouli, M

    2016-10-01

    Strawberry is a significantly consumed fruit worldwide, mostly without being subjected to disinfection processes. During the harvest and transfer from farm to consumers as well as where organic farming practises have been employed, the surface of the fruit may become contaminated by pathogenic bacteria. Post-harvest strawberry fruits in punnets available for public consumption were thus screened for the presence of enteric bacteria in the Sunshine Coast region of Queensland, Australia. Some of the tested samples (13 %) were found to carry such bacteria and even in greater numbers if organic amendments were used (69 %). The bacteria were found to belong in the genera of Escherichia, Enterobacter, Raoultella, Klebsiella, Pantoea, Shigella, Citrobacter and Cronobacter within the family Enterobacteriaceae. Some of the isolates were found to adhere to Caco-2 cells representing human gut epithelium as well as carrying virulence and toxin genes. Resistance mostly against sulphafurazole, cefoxitin, ampicillin and nitrofurantoin was found among 14 different antimicrobial agents tested including 100 % resistance to cefoxitin and ampicillin in the genus Pantoea. In the second phase of the study, bacteriophages were isolated against the isolates and were subsequently applied to post-harvest fruits. A significant (P ≤ 0.001) reduction in the number of enteric bacteria was observed when a high-titre polyvalent bacteriophage suspension (×10(12) PFU/mL) was applied to the fruit surface. Bacteriophages also decreased the adhesion of the Escherichia coli isolates to Caco-2 cells. Findings might indicate that biological control using bacteriophages might be of significant value for the industry targeting to reduce pathogenic loads of bacteria on the fruit.

  1. Characterization of Five Podoviridae Phages Infecting Citrobacter freundii.

    PubMed

    Hamdi, Sana; Rousseau, Geneviève M; Labrie, Simon J; Kourda, Rim S; Tremblay, Denise M; Moineau, Sylvain; Slama, Karim B

    2016-01-01

    Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus.

  2. Characterization of Beta-lactamases in Faecal Enterobacteriaceae Recovered from Healthy Humans in Spain: Focusing on AmpC Polymorphisms.

    PubMed

    Porres-Osante, Nerea; Sáenz, Yolanda; Somalo, Sergio; Torres, Carmen

    2015-07-01

    The intestinal tract is a huge reservoir of Enterobacteriaceae, some of which are opportunist pathogens. Several genera of these bacteria harbour intrinsic antibiotic resistance genes, such as ampC genes in species of Citrobacter, Enterobacter or Escherichia genera. In this work, beta-lactamases and other resistance mechanisms have been characterized in Enterobacteriaceae isolates recovered from healthy human faecal samples, focusing on the ampC beta-lactamase genes. Fifty human faecal samples were obtained, and 70 Enterobacteriaceae bacteria were isolated: 44 Escherichia coli, 4 Citrobacter braakii, 9 Citrobacter freundii, 8 Enterobacter cloacae, 1 Proteus mirabilis, 1 Proteus vulgaris, 1 Klebsiella oxytoca, 1 Serratia sp. and 1 Cronobacter sp. A high percentage of resistance to ampicillin was detected (57%), observing the AmpC phenotype in 22 isolates (31%) and the ESBL phenotype in 3 isolates. AmpC molecular characterization showed high diversity into bla CMY and bla ACT genes from Citrobacter and Enterobacter species, respectively, and the pulsed-field gel electrophoresis (PFGE) analysis demonstrated low clonality among them. The prevalence of people colonized by strains carrying plasmid-mediated ampC genes obtained in this study was 2%. The unique plasmid-mediated bla AmpC identified in this study was the bla CMY-2 gene, detected in an E. coli isolate ascribed to the sequence type ST405 which belonged to phylogenetic group D. The hybridization and conjugation experiments demonstrated that the ISEcp1-bla CMY-2-blc structure was carried by a ~78-kb self-transferable IncK plasmid. This study shows a high polymorphism among beta-lactamase genes in Enterobacteriaceae from healthy people microbiota. Extensive AmpC-carrier studies would provide important information and could allow the anticipation of future global health problems.

  3. Characterization of Five Podoviridae Phages Infecting Citrobacter freundii

    PubMed Central

    Hamdi, Sana; Rousseau, Geneviève M.; Labrie, Simon J.; Kourda, Rim S.; Tremblay, Denise M.; Moineau, Sylvain; Slama, Karim B.

    2016-01-01

    Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus. PMID:27446058

  4. Model approach to estimate the probability of accepting a lot of heterogeneously contaminated powdered food using different sampling strategies.

    PubMed

    Valero, Antonio; Pasquali, Frédérique; De Cesare, Alessandra; Manfreda, Gerardo

    2014-08-01

    Current sampling plans assume a random distribution of microorganisms in food. However, food-borne pathogens are estimated to be heterogeneously distributed in powdered foods. This spatial distribution together with very low level of contaminations raises concern of the efficiency of current sampling plans for the detection of food-borne pathogens like Cronobacter and Salmonella in powdered foods such as powdered infant formula or powdered eggs. An alternative approach based on a Poisson distribution of the contaminated part of the lot (Habraken approach) was used in order to evaluate the probability of falsely accepting a contaminated lot of powdered food when different sampling strategies were simulated considering variables such as lot size, sample size, microbial concentration in the contaminated part of the lot and proportion of contaminated lot. The simulated results suggest that a sample size of 100g or more corresponds to the lower number of samples to be tested in comparison with sample sizes of 10 or 1g. Moreover, the number of samples to be tested greatly decrease if the microbial concentration is 1CFU/g instead of 0.1CFU/g or if the proportion of contamination is 0.05 instead of 0.01. Mean contaminations higher than 1CFU/g or proportions higher than 0.05 did not impact on the number of samples. The Habraken approach represents a useful tool for risk management in order to design a fit-for-purpose sampling plan for the detection of low levels of food-borne pathogens in heterogeneously contaminated powdered food. However, it must be outlined that although effective in detecting pathogens, these sampling plans are difficult to be applied since the huge number of samples that needs to be tested. Sampling does not seem an effective measure to control pathogens in powdered food.

  5. [Outbreaks of Salmonella in infants associated with powdered infant formula].

    PubMed

    Toyofuku, Hajime; Kubota, Kunihiro; Morikawa, Kaoru

    2006-01-01

    Historically, outbreaks associated with Salmonella-contaminated milk products were recognized as early as the 1950's in the United Kingdom and Bulgaria. In the 1960's and 1970's there were also a number of outbreaks related to Salmonella in various powdered milk products. As a result, Salmonella criterion was included in the Codex Code of hygienic practice for foods for infants and children. Between 1985 and 2005 at least 6 outbreaks of salmonellosis, involving as many as 250 infants, have been associated with powdered infant formula (PIF). In 2005, in France, an outbreak affecting more than 100 infants was associated with PIF contaminated with Salmonella Agona. These reported outbreaks indicated that problems persisted. Experts from two FAO/WHO Expert Consultations, held in 2004 and 2006, concluded that intrinsic contamination of PIF with Enterobacter sakazakii and Salmonella has been a cause of infection and illness in infants, including severe disease which can lead to serious developmental sequelae and death. Most of the Salmonella outbreaks associated with PIF involved unusual Salmonella serotypes, which likely aided in the recognition of these outbreaks. In many regions of the world where Salmonella serotyping is not routinely performed, identification of geographically or temporarily diffused outbreaks could be difficult. It is therefore important to use the appropriate methodology to detect unusual strains of Salmonella that cause illnesses in infants, such as the lactose-positive strain, and to perform serotyping and/or pulsed-field gel electrophoresis (PFGE) genotyping for rapid identification of Salmonella outbreaks and to establish linkages between the illness and implicated food.

  6. Automated species and strain identification of bacteria in complex matrices using FTIR spectroscopy

    NASA Astrophysics Data System (ADS)

    Puzey, K. A.; Gardner, P. J.; Petrova, V. K.; Donnelly, C. W.; Petrucci, G. A.

    2008-04-01

    Fourier Transform Infrared (FTIR) spectroscopy provides a highly selective and reproducible means for the chemically-based discrimination of intact microbial cells which make the method valuable for large-scale screening of foods. The goals of the present study were to assess the effect of chemical interferents, such as food matrices, different sanitizing compounds and growth media, on the ability of the method to accurately identify and classify L. innocua, L. welshimeri, E. coli, S. cholerasuis, S. subterranea, E. sakazakii, and E. aerogenes. Moreover, the potential of FTIR spectroscopy for discrimination of L. innocua and L. welshimeri of different genotypes and the effect of growth phase on identification accuracy of L. innocua and L. welshimeri were tested. FTIR spectra were collected using two different sample presentation techniques - transmission and attenuated total reflection (ATR), and then analyzed using multivariate discriminant analysis based on the first derivative of the FTIR spectra with the unknown spectra assigned to the species group with the shortest Mahalanobis distance. The results of the study demonstrated 100% correct identification and differentiation of all bacterial strains used in this study in the presence of chemical interferents or food matrices, better than 99% identification rate in presence of media matrices, and 100% correct detection for specific bacteria in mixed flora species. Additionally, FTIR spectroscopy proved to be 100% accurate when differentiating between genotypes of L. innocua and L. welshimeri, with the classification accuracy unaffected by the growth stage. These results suggest that FTIR spectroscopy can be used as a valuable tool for identifying pathogenic bacteria in food and environmental samples.

  7. Occurrence of yeasts, enterococci and other enteric bacteria in subgingival biofilm of HIV-positive patients with chronic gingivitis and necrotizing periodontitis.

    PubMed

    Gaetti-Jardim Júnior, Elerson; Nakano, Viviane; Wahasugui, Thais C; Cabral, Fátima C; Gamba, Rosa; Avila-Campos, Mario Julio

    2008-04-01

    The purpose of this study was to determine the prevalence of enteric bacteria and yeasts in biofilm of 80 HIV-positive patients with plaque-associated gingivitis or necrotizing periodontitis. Patients were subjected to extra, intra oral and radiographic examinations. The oral hygiene, bleeding on probing, gingival conditions, and attachment loss were evaluated. Clinical specimens were collected from gingival crevices or periodontal pockets, transferred to VMGA III, diluted and transferred to Sabouraud Dextrose agar with 100 μg/ml of chloramphenicol, peptone water, EVA broth, EMB agar, SS agar, Bile esculin agar and Brilliant green agar. Isolation of yeasts was carried out at room temperature, for 3-7 days; and for the isolation of enteric microorganisms plates were incubated at 37°C, for 24-48 h. The yeasts identification was performed according to the carbon and nitrogen assimilation, fermentation of carbohydrates and germ tube formation. Bacteria were identified according to their colonial and cellular morphologies and biochemical tests. Yeasts were identified as Candida albicans and its occurrence was more common in patients with CD4+ below 200/mm(3) and was affected by the extension of periodontal involvement (P = 0.0345). Enteric bacteria recovered from clinical specimens were identified as Enterobacter sakazakii, Enterobacter cloacae, Serratia liquefaciens, Klebsiella oxytoca and Enterococcus sp. Enterobacteriaceae and enterococci were detected in 32.5% of clinical samples from patients with necrotizing periodontitis. In conclusion, non-oral pathogenic bacteria and C. albicans were more prevalent in periodontal sites of HIV-positive patients with necrotizing periodontitis and chronic gingivitis.

  8. A modified molecular beacons-based multiplex real-time PCR assay for simultaneous detection of eight foodborne pathogens in a single reaction and its application.

    PubMed

    Hu, Qinghua; Lyu, Dongyue; Shi, Xiaolu; Jiang, Yixiang; Lin, Yiman; Li, Yinghui; Qiu, Yaqun; He, Lianhua; Zhang, Ran; Li, Qingge

    2014-03-01

    Foodborne disease outbreaks are often caused by one of the major pathogens. Early identification of the causal pathogen is crucial for disease control and prevention. We describe a real-time polymerase chain reaction (rtPCR) assay that can identify, in a single reaction, up to eight common foodborne bacterial pathogens, including Salmonella enterica subsp. enterica, Listeria monocytogenes, Escherichia coli O157, Vibrio parahaemolyticus, V. vulnificus, Campylobacter jejuni, Enterobacter sakazakii, and Shigella spp. This multiplex rtPCR assay takes advantage of modified molecular beacons and the multicolor combinational probe coding strategy to discriminate each pathogen and the homo-tag assisted non-dimer (HAND) system to prevent dimer formation. The detection limits of the assay ranged from 1.3×10(3) colony-forming units (CFU)/g stool (L. monocytogenes) to 1.6×10(4) CFU/g stool (Shigella spp.). The target genes were 100% specific as assessed on 986 reference strains covering 41 species since no cross-reactions were observed. The assay was applied to the detection of foodborne pathogens in 11,167 clinical samples and the results were compared with culture methods for further validation. The sensitivity and specificity of the rtPCR were 100% and 99%, respectively. When performed in a 96-well rtPCR system, more than 90 samples could be analyzed within 3 h. Given the high accuracy, sensitivity, specificity, and short turn-around time, the established assay could be used for the rapid and reliable identification of the causative pathogens responsible for a certain foodborne disease outbreak and rapid screening of these major foodborne pathogens in laboratory-based surveillance of outpatient clinical samples or even food samples.

  9. Occurrence of yeasts, enterococci and other enteric bacteria in subgingival biofilm of HIV-positive patients with chronic gingivitis and necrotizing periodontitis

    PubMed Central

    Gaetti-Jardim Júnior, Elerson; Nakano, Viviane; Wahasugui, Thais C.; Cabral, Fátima C.; Gamba, Rosa; Avila-Campos, Mario Julio

    2008-01-01

    The purpose of this study was to determine the prevalence of enteric bacteria and yeasts in biofilm of 80 HIV-positive patients with plaque-associated gingivitis or necrotizing periodontitis. Patients were subjected to extra, intra oral and radiographic examinations. The oral hygiene, bleeding on probing, gingival conditions, and attachment loss were evaluated. Clinical specimens were collected from gingival crevices or periodontal pockets, transferred to VMGA III, diluted and transferred to Sabouraud Dextrose agar with 100 μg/ml of chloramphenicol, peptone water, EVA broth, EMB agar, SS agar, Bile esculin agar and Brilliant green agar. Isolation of yeasts was carried out at room temperature, for 3-7 days; and for the isolation of enteric microorganisms plates were incubated at 37°C, for 24-48 h. The yeasts identification was performed according to the carbon and nitrogen assimilation, fermentation of carbohydrates and germ tube formation. Bacteria were identified according to their colonial and cellular morphologies and biochemical tests. Yeasts were identified as Candida albicans and its occurrence was more common in patients with CD4+ below 200/mm3 and was affected by the extension of periodontal involvement (P = 0.0345). Enteric bacteria recovered from clinical specimens were identified as Enterobacter sakazakii, Enterobacter cloacae, Serratia liquefaciens, Klebsiella oxytoca and Enterococcus sp. Enterobacteriaceae and enterococci were detected in 32.5% of clinical samples from patients with necrotizing periodontitis. In conclusion, non-oral pathogenic bacteria and C. albicans were more prevalent in periodontal sites of HIV-positive patients with necrotizing periodontitis and chronic gingivitis. PMID:24031212

  10. In vitro probiotic characteristics of Lactobacillus plantarum ZDY 2013 and its modulatory effect on gut microbiota of mice.

    PubMed

    Huang, Renhui; Tao, Xueying; Wan, Cuixiang; Li, Shengjie; Xu, Hengyi; Xu, Feng; Shah, Nagendra P; Wei, Hua

    2015-09-01

    Lactobacillus plantarum ZDY 2013, a novel strain isolated from Chinese traditional fermented acid beans, was systematically evaluated for its survival capacity under stress conditions (pH, bile salt, simulated gastrointestinal tract, and antibiotics), production of exopolysaccharide and antagonism against 8 pathogens. Its effect on mice gut microbiota was also investigated by quantitative PCR and PCR-denaturing gradient gel electrophoresis. The results showed that ZDY 2013 can grow at pH 3.5 and survive at pH 2.0 for 6 h and at 0.45% bile salt for 3 h. The exopolysaccharide yield was up to 204±7.68 mg/L. The survival rate of ZDY 2013 in a simulated gastrointestinal tract was as high as 65.84%. Antagonism test with a supernatant of ZDY 2013 showed maximum halo of 28 mm against Listeria monocytogenes. The inhibition order was as follows: Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Shigella sonnei, Enterobacter sakazakii, and Staphylococcus aureus. Lactobacillus plantarum ZDY 2013 was sensitive to some antibiotics (e.g., macrolide, sulfonamides, aminoglycoside, tetracyclines and β-lactams), whereas it was resistant to glycopeptides, quinolones, and cephalosporins antibiotics. Denaturing gradient gel electrophoresis profile demonstrated that ZDY 2013 administration altered the composition of the microbiota at various intestinal loci of the mice. Moreover, the quantitative PCR test showed that the administration of ZDY 2013 enhanced the populations of Bifidobacterium and Lactobacillus in either the colon or cecum, and reduced the potential enteropathogenic bacteria (e.g., Enterococcus, Enterobacterium, and Clostridium perfringens). Lactobacillus plantarum ZDY 2013 exhibited high resistance against low pH, bile salt, and gastrointestinal fluid, and possessed antibacterial and gut microbiota modulation properties with a potential application in the development of dairy food and nutraceuticals.

  11. Quantification of hurdles: predicting the combination of effects -- Interaction vs. non-interaction.

    PubMed

    Bidlas, Eva; Lambert, Ronald J W

    2008-11-30

    Combination of disparate as well as related antimicrobial effects constitutes the concept of hurdle technology. Quantification of combined effects, including claims of synergy, can be accomplished using surface response modelling, as is frequently done and reported. The Gamma hypothesis, however, states that the relative effects of different antimicrobial factors combine independently. Studies performed using time to detection have shown that the Gamma hypothesis is an adequate foundation for the analysis of multi-factor environmental stresses placed on microorganisms, including pH, weak acids and temperature. Data from the combined action of Na acetate and pH on Aeromonas hydrophila, Na acetate/pH , K sorbate/pH and combined Na acetate/K sorbate at pH 6.5, 6.0 and 5.5 on Escherichia coli and the combined action of Na acetate/pH and temperature on Enterobacter sakazakii were examined using nominal logistic modelling, response surface modelling (RS) and by using a Gamma model. The Gamma model can be used in a predictive manner unlike the RS models and the parameters of the RS models can be approximated from the fit of the Gamma model to the observed data. The expansion of the Gamma model explains the occurrence of the statistically significant cross terms of the RS polynomials. The emphasis within the literature of seeking interactions or synergies between environmental factors should be replaced with one emphasising the falsification of the Gamma approach. This can be done by examining the relative ratios of the gamma factors when in combination, but this also requires the use of appropriate functions to do this.

  12. Mangrovibacter phragmitis sp. nov., an endophyte isolated from the roots of Phragmites karka.

    PubMed

    Behera, Pratiksha; Ramana, V Venkata; Maharana, Bhagirathi; Joseph, Neetha; Vaishampayan, Parag; Singh, Nitin; Shouche, Yogesh; Bhadury, Punyasloke; Mishra, Samir R; Raina, Vishakha; Suar, Mrutyunjay; Pattnaik, Ajit K; Rastogi, Gurdeep

    2017-01-12

    A facultative anaerobic, Gram-stain-negative, rod-shaped, nitrogen fixing, endophytic bacterial strain MP23T was isolated from the roots of Phragmites karka growing in Chilika Lagoon, Odisha, India. Strain MP23T was slightly halophilic and optimal NaCl concentration and temperature for growth was 1% and 30°C, respectively. On the basis of 16S rRNA gene sequence similarities, strain MP23T was affiliated to family Enterobacteriaceae and most closely related to Mangrovibacter yixingensis KCTC 42181T and Mangrovibacter plantisponsor DSM 19579T by 99.71%, followed by Salmonella enterica subsp. salamae DSM 9220T (97.22%), Cronobacter condimenti LMG 26250T (97.14%), and Salmonella enterica subsp. diarizone DSM 14847T (97%). Sequence analysis of 16S rRNA, hsp60, gyrB, and rpoB genes showed that strain MP23T formed a phylogenetic cluster with Mangrovibacter yixingensis KCTC 42181T and Mangrovibacter plantisponsor DSM 19579T indicating that it belong to genus Mangrovibacter. The major cellular fatty acids were C16:0, C18:1ω6c and/or C18:1ω7c, C16:1ω6c and/or C16:1ω7c, C14:0, C14:03OH and/or C16:1isoI, and C17:0cyclo. Polar lipid pattern of strain MP23T consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine. The DNA G+C content was 50.3 mol%. Based on experimental DNA-DNA hybridization values and average nucleotide identity derived from in silico comparison of whole genome sequences, strain MP23T could be distinguished from its closest neighbours. We therefore conclude that strain MP23T represents a novel species of the genus Mangrovibacter for which the name Mangrovibacter phragmitis sp. nov. is proposed. The type strain is MP23T (= DSM 100250 T = KCTC 42580 T).

  13. Evaluation of probiotic properties of Lactobacillus plantarum WLPL04 isolated from human breast milk.

    PubMed

    Jiang, Meiling; Zhang, Fen; Wan, Cuixiang; Xiong, Yonghua; Shah, Nagendra P; Wei, Hua; Tao, Xueying

    2016-03-01

    Lactobacillus plantarum WLPL04, a specific strain isolated from human breast milk, was investigated for its survival capacity (acid and bile salt tolerance, survival in simulated gastrointestinal tract, inhibition of pathogens, antibiotic susceptibility, yield of exopolysaccharides) and probiotic properties (antiadhesion of pathogens, protection from harmful effect of sodium dodecyl sulfate, and antiinflammatory stress on Caco-2 cells). The results showed that Lb. plantarum WLPL04 had broad-spectrum activity against gram-positive strains (Listeria monocytogenes CMCC54007, Bacillus cereus ATCC14579, and Staphylococcus aureus CMCC26003) and gram-negative strains (Pseudomonas aeruginosa MCC10104, Shigella sonnei ATCC25931, Enterobacter sakazakii ATCC29544, Salmonella typhimurium ATCC13311, and Escherichia coli O157:H7). Antibiotic susceptibility tests showed that Lb. plantarum WLPL04 was susceptible to 8 of 14 antibiotics (e.g., erythromycin and nitrofurantoin) and resistant to 6 of 14 antibiotics (e.g., kanamycin and bacitracin). Lactobacillus plantarum WLPL04 was able to survive at pH 2.5 for 3h and at 0.45% bile salt for 12h, suggesting that it can survive well in the gastrointestinal tract. In addition, the exopolysaccharide yield of Lb. plantarum WLPL04 reached 426.73 ± 65.56 mg/L at 24h. With strategies of competition, inhibition, and displacement, Lb. plantarum WLPL04 reduced the adhesion of E. coli O157:H7 (35.51%), Sal. typhimurium ATCC 13311 (8.10%), and Staph. aureus CMCC 26003 (40.30%) on Caco-2 cells by competition, and subsequently by 59.80, 62.50, and 42.60%, respectively, for the 3 pathogens through inhibition, and by 75.23, 39.97, and 52.88%, respectively, through displacement. Lactobacillus plantarum WLPL04 attenuated the acute stress induced by sodium dodecyl sulfate on Caco-2 cells and significantly inhibited the expression of inflammatory cytokines (IL-6, IL-8 and tumor necrosis factor-α) on Caco-2 cells but increased IL-10 expression in vitro

  14. New trends in emerging pathogens.

    PubMed

    Skovgaard, Niels

    2007-12-15

    The emergence of pathogens is the result of a number of impact in all parts of the food chain. The emerging technologies in food production explain how new pathogens can establish themselves in the food chain and compromise food safety. The impact of the food technology is analysed for several bacteria, such as Yersinia, Campylobacter, Arcobacter, Helicobacter pullorum, Enterobacter sakazakii, Mycobacterium avium spp. paratuberculosis, prions related to vCJD and others. The importance of the ability of many microbes to form VBNC forms is elaborated on. Research on culture independent methods may address this outstanding issue to the better understanding of emerging pathogens. The "demerging" of pathogens also occur, and examples of this are explained. The reaction of bacteria to stresses and sublethal treatments, and how exposure to one stress factor can confer resistance to other stresses, literally speaking causing contagious resistance, are explained. The implication of this e.g. in modern approaches of food preservation, such as Minimally processed Foods, is considerable. Intestinal colonization of EHEC may be regulated by Quorum sensing, and this ability of microbes plays an important role in the colonization of microbes in food and on food processing equipment, an important factor in the emergence of pathogens. The emergence of Saccharomyces cerevisiae, as an opportunistic human pathogen, used for centuries for food and production of alcoholic beverages, calls for research in molecular tools to distinguish between probiotic and clinical strains. Cyclospora cayetanensis and Norovirus outbreaks can no longer be designated as emerging pathogens, they share however one characteristic in the epidemiology of emerging nature, the importance of the hygiene in the primary production stage, including supply of potable water, and the application of GMP and the HACCP principles in the beginning of the food chain. Hepatitis E virus is a potential emerging food borne

  15. Confirmation of E. coli among other thermotolerant coliform bacteria in paper mill effluents, wood chips screening rejects and paper sludges.

    PubMed

    Beauchamp, Chantal J; Simao-Beaunoir, Anne-Marie; Beaulieu, Carole; Chalifour, François-Philippe

    2006-07-01

    Paper sludges are solid wastes material generated from the paper production, which have been characterized for their chemical contents. Some are rich in wood fiber and are a good carbon source, for example the primary and de-inking paper sludges. Others are made rich in nitrogen and phosphorus by pressing the activated sludge, resulting from the biological water treatments, with the primary sludge, yielding the combined paper sludge. Still, in the absence of sanitary effluents very few studies have addressed the characterization of their coliform microflora. Therefore, this study investigated the thermotolerant coliform population of one paper mill effluent and two paper mill sludges and wood chips screening rejects using chromogenic media. For the first series of analyses, the medium used was Colilert broth and positive tubes were selected to isolate bacteria in pure culture on MacConkey agar. In a second series of analyses, double selective media, based on ss-galactosidase and ss-glucuronidase activities, were used to isolate bacteria. First, the presence of thermotolerant coliforms was detected in low numbers in most water effluents, but showed that the entrance of the thermotolerant coliforms was early in the industrial process. Also, large numbers of thermotolerant coliforms, i.e., 7,000,000 MPN/g sludge (dry weight; d.w.), were found in combined sludges. From this first series of isolations, bacteria were purified on MacConkey medium and identified as Citrobacter freundii, Enterobacter sp, E. sakazakii, E. cloacae, Escherichia coli, K. pneumoniae, K. pneumoniae subsp. rhinoscleromatis, K. pneumoniae subsp. ozaenae, K. pneumoniae subsp. pneumoniae, Pantoea sp, Raoultella terrigena, R. planticola. Second, the presence of thermotolerant coliforms was measured at more than 3,700-6,000 MPN/g (d.w) sludge, whereas E. coli was detected from 730 to more than 3,300 MPN/g (d.w.) sludge. The presence of thermotolerant coliform bacteria and E. coli was sometimes detected