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Sample records for cronobacter sakazakii baa-894

  1. 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. PMID:26481623

  2. 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. PMID:26384977

  3. 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.

  4. 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

  5. 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

  6. Complete genome sequence and phenotype microarray analysis of Cronobacter sakazakii SP291: a persistent isolate cultured from a powdered infant formula production facility

    PubMed Central

    Yan, Qiongqiong; Power, Karen A.; Cooney, Shane; Fox, Edward; Gopinath, Gopal R.; Grim, Christopher J.; Tall, Ben D.; McCusker, Matthew P.; Fanning, Séamus

    2013-01-01

    Outbreaks of human infection linked to the powdered infant formula (PIF) food chain and associated with the bacterium Cronobacter, are of concern to public health. These bacteria are regarded as opportunistic pathogens linked to life-threatening infections predominantly in neonates, with an under developed immune system. Monitoring the microbiological ecology of PIF production sites is an important step in attempting to limit the risk of contamination in the finished food product. Cronobacter species, like other microorganisms can adapt to the production environment. These organisms are known for their desiccation tolerance, a phenotype that can aid their survival in the production site and PIF itself. In evaluating the genome data currently available for Cronobacter species, no sequence information has been published describing a Cronobacter sakazakii isolate found to persist in a PIF production facility. Here we report on the complete genome sequence of one such isolate, Cronobacter sakazakii SP291 along with its phenotypic characteristics. The genome of C. sakazakii SP291 consists of a 4.3-Mb chromosome (56.9% GC) and three plasmids, denoted as pSP291-1, [118.1-kb (57.2% GC)], pSP291-2, [52.1-kb (49.2% GC)], and pSP291-3, [4.4-kb (54.0% GC)]. When C. sakazakii SP291 was compared to the reference C. sakazakii ATCC BAA-894, which is also of PIF origin, the annotated genome data identified two interesting functional categories, comprising of genes related to the bacterial stress response and resistance to antimicrobial and toxic compounds. Using a phenotypic microarray (PM), we provided a full metabolic profile comparing C. sakazakii SP291 and the previously sequenced C. sakazakii ATCC BAA-894. These data extend our understanding of the genome of this important neonatal pathogen and provides further insights into the genotypes associated with features that can contribute to its persistence in the PIF environment. PMID:24032028

  7. Sialic acid utilization by Cronobacter sakazakii

    PubMed Central

    2013-01-01

    Background The Cronobacter genus is composed of seven species, and can cause infections in all age groups. Of particular concern is C. sakazakii, as this species is strongly associated with severe and often fatal cases of necrotizing enterocolitis and meningitis in neonates and infants. Whole genome sequencing has revealed that the nanAKT gene cluster required for the utilisation of exogenous sialic acid is unique to the C. sakazakii species (ESA_03609–13). Sialic acid is found in breast milk, infant formula, intestinal mucin, and gangliosides in the brain, hence its metabolism by C. sakazakii is of particular interest. Therefore its metabolism could be an important virulence factor. To date, no laboratory studies demonstrating the growth of C. sakazakii on sialic acid have been published nor have there been reports of sialidase activity. The phylogenetic analysis of the nan genes is of interest to determine whether the genes have been acquired by horizontal gene transfer. Results Phylogenetic analysis of 19 Cronobacter strains from 7 recognised species revealed the nanAKTR genes formed a unique cluster, separate from other Enterobacteriaceae such as E. coli K1 and Citrobacter koseri, which are also associated with neonatal meningitis. The gene organisation was similar to Edwardsiella tarda in that nanE gene (N-acetylmannosamine-6-phosphate-2epimerase) was not located within the nanATK cluster. Laboratory studies confirmed that only C. sakazakii, and not the other six Cronobacter species, was able to use sialic acid as a carbon source for growth. Although the ganglioside GM1 was also used as carbon source, no candidate sialidase genes were found in the genome, instead the substrate degradation is probably due to β–galactosidase activity. Conclusions Given the relatively recent evolution of both C. sakazakii (15–23 million years ago) and sialic acid synthesis in vertebrates, sialic acid utilization may be an example of co-evolution by one species of the

  8. 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.

  9. 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.

  10. Acid stress management by Cronobacter sakazakii.

    PubMed

    Alvarez-Ordóñez, Avelino; Cummins, Conor; Deasy, Thérèse; Clifford, Tanya; Begley, Máire; Hill, Colin

    2014-05-16

    Cronobacter sakazakii is a foodborne pathogenic microorganism associated with sporadic cases of neonatal meningitis, necrotising enterocolitis, septicaemia, bloody diarrhoea and brain abscesses acquired through the consumption of contaminated powdered infant formula (PIF). This study aimed to investigate the growth of C. sakazakii DPC6529, a particularly stress tolerant clinical isolate, in acidified laboratory media and PIF. The possibility of a stationary-phase acid tolerance response (ATR) was also investigated. C. sakazakii DPC6529 grew in LB broth acidified to pH4.2 with hydrochloric acid (HCl) and was capable of relatively fast growth in PIF acidified to pH5.0 with HCl, representing the stomach pH reported for newborns and infants. Moreover, bacterial growth in LB broth supplemented with 1% (w/v) glucose gave rise to a stationary-phase ATR which resulted in enhanced survival against a subsequent acid challenge at pH3.0. A transposon mutagenesis approach was used to shed light on some of the molecular mechanisms involved in the response C. sakazakii DPC6529 to normally lethal acid exposures. The data suggests that repairing damage in proteins and nucleic acids, posttranscriptional modification of tRNA molecules and maintenance of the integrity of the cellular envelope are key processes in the defence against acid stress. Clones carrying transposon insertions in genes encoding the envelope stress response regulators CpxR and OmpR were identified as acid-sensitive mutants. Further analyses of the ompR defective mutant and its complemented counterpart evidenced that OmpR is a key player in the response of C. sakazakii to acid stress, although it was not essential to mount an active stationary-phase ATR, at least under the tested conditions. The ability of C. sakazakii DPC6529 to grow in acid environments and to develop an adaptive stationary-phase ATR may allow for its survival or even proliferation within the infant gastrointestinal tract after consumption of

  11. 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

  12. Insights into virulence factors determining the pathogenicity of Cronobacter sakazakii.

    PubMed

    Singh, Niharika; Goel, Gunjan; Raghav, Mamta

    2015-01-01

    Cronobacter sakazakii is an opportunistic pathogen associated with outbreaks of life-threatening necrotizing enterocolitis, meningitis and sepsis in neonates and infants. The pathogen possesses an array of virulence factors which aid in tissue adhesion, invasion and host cell injury. Although the identification and validation of C. sakazakii virulence factors has been hindered by availability of suitable neonatal animal model, various studies has reported outer membrane protein A (ompA) as a potential virulence marker. Various other plasmid associated genes such as filamentous hemagglutinin (fhaBC), Cronobacter plasminogen activator (cpa) and genes responsible for iron acquisition (eitCBAD and iucABD/iutA) have been reported in different strains of C. sakazakii. Besides these proposed virulence factors, several biophysical growth factors such as formation of biofilms and resistance to various environmental stresses also contributes to the pathogenic potential of this pathogen. This review provides an update on virulence determinants associated with the pathogenesis of C. sakazakii. The potential reservoirs of the pathogen, mode of transmission and epidemiology are also discussed. PMID:25950947

  13. 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. PMID:25062020

  14. 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.

  15. 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.

  16. Immunoliposome-based immunomagnetic concentration and separation assay for rapid detection of Cronobacter sakazakii.

    PubMed

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

    2016-03-15

    This study aimed to develop an immunoliposome-based immunomagnetic concentration and separation assay for the rapid detection of Cronobacter sakazakii (C. sakazakii), an acute opportunistic foodborne pathogenic bacterium, in both pure culture and infant formula. To develop the assay, magnetic nanoparticles (diameter 30 nm) were coated with immunoglobulin G (IgG), specifically anti-C. sakazakii IgG, and applied for the sensitive and efficient detection of C. sakazakii using immunoliposomes. The binding efficiency of anti-C. sakazakii IgG to the magnetic nanoparticles was 86.23 ± 0.59%. The assay developed in this study detected as few as 3.3 × 10(3) CFUmL(-1) of C. sakazakii in pure culture within 2h 30 min; in comparison, an indirect non-competitive enzyme-linked immunosorbent assay was able to detect 6.2 × 10(5) CFUmL(-1) of C. sakazakii in pure culture after 17 h. The developed assay did not show any cross-reactivity with other Cronobacter spp. or pathogens belonging to other genera. In addition, the method was able to detect 10(3) CFUmL(-1) of C. sakazakii in infant formula without any pre-incubation. These results confirm that the immunoliposome-based immunomagnetic concentration and separation assay may facilitate highly sensitive, efficient, and rapid detection of C. sakazakii. PMID:26547009

  17. 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.

  18. 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...

  19. 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.

  20. 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

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

    PubMed

    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

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

    PubMed

    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; 10(7) 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

  3. Efficacy of the thin agar layer method for the recovery of stressed Cronobacter spp. (Enterobacter sakazakii).

    PubMed

    Osaili, Tareq M; Al-Nabulsi, Anas A; Shaker, Reyad R; Al-Holy, Murad M; Al-Haddaq, Mohammed S; Olaimat, Amin N; Ayyash, Mutamed M; Al Ta'ani, Mahmoud K; Forsythe, Stephen J

    2010-10-01

    Cronobacter spp. (Enterobacter sakazakii) are emerging opportunistic pathogens for all age groups, and are of particular concern when it comes to infants. Prior to contaminating food, the organism may be exposed to a variety of stresses, leading to a generation of sublethally injured cells that may not be detected by selective media unless a protracted recovery period is included in the isolation procedure. This study evaluated the efficacy of the thin agar layer (TAL) method for the recovery of Cronobacter cells that had been exposed to various stress conditions. Five strains of C. sakazakii and C. muytjensii were exposed to starvation, heat, cold, acid, alkaline, chlorine, or ethanol, with or without further exposure to desiccation stress. The recovery of the stressed cells was determined on tryptone soy agar (TSA; nonselective control medium), violet red bile glucose agar (VRBGA; selective agar), Druggan-Forsythe-Iversen (DFI; selective agar), and TAL media (viz., VRBGA overlaid with TSA, and DFI overlaid with TSA). Regardless of stress type, there were no significant differences among the recoveries of stressed desiccated Cronobacter spp. cultures on TSA, DFI+TSA, and VRBGA+TSA, but there was significantly less recovery on VRBGA. The recovery of prestressed desiccated Cronobacter spp. on DFI+TSA was similar to that on TSA, whereas the recovery on VRBGA+TSA was lower. DFI+TSA performed better than VRBGA+TSA did in differentiating Cronobacter spp. within mixed bacterial cultures. The results of this study suggest the use of the TAL method DFI+TSA as an improved method for the direct recovery of stressed Cronobacter spp.

  4. 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.

  5. 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.

  6. An in silico analysis of osmotolerance in the emerging gastrointestinal pathogen Cronobacter sakazakii.

    PubMed

    Feeney, Audrey; Sleator, Roy D

    2011-01-01

    Up to 80% of infants infected with Cronobacter sakazakii die within days of birth, making this emerging gastrointestinal pathogen a serious cause for concern. The mode of transmission most often associated with C. sakazakii infection is powdered infant formula (PIF), which typically has a water activity (aw) of ca 0.2--inhospitable to most bacterial pathogens. In the current study a comparative genomic approach was used to investigate the distinctive ability of this pathogen to survive and persist in such low aW conditions. A comprehensive review of the mechanisms involved in bacterial osmoadaptation was followed by an exhaustive homology transfer based approach to identify putative osmotolerance loci in the C. sakazakii genome. In total 53 osmotoleance loci were identified, including both hyper- and hypo-osmotic stress response systems, helping to construct a concise overview of the C. sakazakii osmotolerance response. Interestingly, while C. sakazakii contains homologues of all the principal osmotolerance loci of Escherichia coli; a key difference is that C. sakazakii contains multiple copies of certain osmotolerance loci; including seven copies of the E. coli proP homologue and two copies of OpuC--a multi component carnitine uptake system associated with Listeria monocytogenes and which has also been found to transport other compatible solutes such as glycine betaine, proline, ectoine and choline. Furthermore, the osmotic stress response of C. sakazakii appears to be regulated at the transcriptional, translational and post-translational levels, with RpoS most likely functioning as the global transcriptional regulator of the osmotolerance response. PMID:21918371

  7. Impact of environmental stress desiccation, acidity, alkalinity, heat or cold on antibiotic susceptibility of Cronobacter sakazakii.

    PubMed

    Al-Nabulsi, Anas A; Osaili, Tareq M; Elabedeen, Noor A Zain; Jaradat, Ziad W; Shaker, Reyad R; Kheirallah, Khalid A; Tarazi, Yaser H; Holley, Richard A

    2011-03-30

    Cronobacter sakazakii is an emerging foodborne pathogen that has been implicated in severe forms of meningitis, septicemia or necrotizing colitis in pre-term neonates. Although illness outbreaks (primarily associated with powdered infant formula, PIF) caused by this pathogen are rare, the case-fatality rate may reach 50%. Successful treatment of C. sakazakii infection is reliant upon clinical use of antibiotics (AB) such as ampicillin. Recent reports showed increased resistance of C. sakazakii to broad-spectrum antibiotics. The objective of this study was to evaluate the effect of extreme pH (3.5 for 30 min or 11.25 for 5 min), cold (4°C for 24h), heat (55°C for 5 min), and desiccation (cells were dried at 40°C for 2h and held at 21°C for 4 d) stresses on susceptibility of five isolated strains of C. sakazakii to streptomycin, gentamicin, kanamycin, neomycin, tetracycline, doxycycline, tilmicosin, florfenicol, ampicillin, amoxicillin, vancomycin, ciprofloxacin and enrofloxacin. All unstressed strains of C. sakazakii were sensitive to streptomycin, gentamycin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and amoxicillin, but were moderately resistant or resistant to the rest. Exposing cells to alkaline or acidic stress did not change their sensitivity toward streptomycin, gentamycin, kanamycin or ciprofloxacin, but their resistance toward the other AB was increased. Cells stressed by desiccation showed increased sensitivity toward streptomycin, gentamicin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and doxycycline, but showed resistance toward the others. Cold-stressed cells were more sensitive to streptomycin, gentamicin, kanamycin, and ciprofloxacin compared with heat-stressed cells, but both heat and cold-stressed cells showed increased resistance toward all the other AB. Results obtained will help in understanding the effect of environmental stresses during processing on C. sakazakii susceptibility to AB. PMID:21402424

  8. Antimicrobial activity of syringic acid against Cronobacter sakazakii and its effect on cell membrane.

    PubMed

    Shi, Chao; Sun, Yi; Zheng, Zhiwei; Zhang, Xiaorong; Song, Kaikuo; Jia, Zhenyu; Chen, Yifei; Yang, Miaochun; Liu, Xin; Dong, Rui; Xia, Xiaodong

    2016-04-15

    Syringic acid (SA) has been reported to exhibit antibacterial ability against various microorganisms, but little work has been done on its effect on Cronobacter sakazakii. In this study, minimum inhibitory concentrations (MICs) of SA against various C. sakazakii strains were determined. Moreover, changes in intracellular ATP concentration, intracellular pH (pHin), membrane potential and membrane integrity were measured to evaluate the influence of SA on cell membrane. Finally, field emission scanning electron microscope (FESEM) was used to assess the morphological changes of bacterial cells caused by SA. It was shown that the MICs of SA against all tested C. sakazakii strains were 5mg/mL. SA retarded bacterial growth, and caused cell membrane dysfunction, which was evidenced by intracellular ATP concentration decrease, pHin reduction, cell membrane hyperpolarization and changes in cellular morphology. These findings indicated that SA has potential to be developed as a natural preservative to control C. sakazakii in foods associated with this pathogen and prevent related infections.

  9. Screening of genes involved in interactions with intestinal epithelial cells in Cronobacter sakazakii.

    PubMed

    Du, Xin-Jun; Zhang, Xia; Li, Ping; Xue, Rui; Wang, Shuo

    2016-12-01

    Cronobacter sakazakii possesses a significant ability to adhere to and invade epithelial cells in its host. However, the molecular mechanisms underlying this process are poorly understood. In the current study, the adhesive and invasive capabilities of 56 C. sakazakii strains against human epithelial cells were evaluated, and one of them was selected for construction of a mutant library using the Tn5 transposon. In a systematic analysis of the adhesive and invasive capabilities of 1084 mutants, 10 mutants that showed more than a 50 % reduction in adhesion or invasion were obtained. Tail-PCR was used to sequence the flanking regions of the inserted transposon and 8 different genes (in 10 different mutants) were identified that encoded an exonuclease subunit, a sugar transporter, a transcriptional regulator, two flagellar biosynthesis proteins, and three hypothetical proteins. Raman spectroscopy was used to analyze variations in the biochemical components of the mutants, and the results showed that there were fewer amide III proteins, protein -CH deformations, nucleic acids and tyrosines and more phenylalanine, carotenes, and fatty acids in the mutants than in the wild type strain. Real-time PCR was used to further confirm the involvement of the genes in the adhesive and invasive abilities of C. sakazakii, and the results indicated that the expression levels of the 8 identified genes were upregulated 1.2- to 11.2-fold. The results of this study provide us with insight into the mechanism by which C. sakazakii infects host cells at molecular level. PMID:27637944

  10. 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.

  11. Conditioned medium from Bifidobacteria infantis protects against Cronobacter sakazakii-induced intestinal inflammation in newborn mice.

    PubMed

    Weng, Meiqian; Ganguli, Kriston; Zhu, Weishu; Shi, Hai Ning; Walker, W Allan

    2014-05-01

    Necrotizing enterocolitis (NEC) is associated with a high morbidity and mortality in very low birth weight infants. Several hypotheses regarding the pathogenesis of NEC have been proposed but to date no effective treatment is available. Previous studies suggest that probiotic supplementation is protective. We recently reported that probiotic (Bifidobacterium infantis) conditioned medium (PCM) has an anti-inflammatory effect in cultured fetal human intestinal cells (H4) and fetal intestine explants. In this study, we tested in vivo whether PCM protects neonatal mice from developing intestinal inflammation induced by exposure to Cronobacter sakazakii (C. sakazakii), an opportunistic pathogen associated with NEC. We found that infected neonatal mice had a significantly lower body weight than control groups. Infection led to ileal tissue damage including villous rupture, disruption of epithelial cell alignment, intestinal inflammation, apoptotic cell loss, and decreased mucus production. Pretreatment with PCM prevented infection caused decrease in body weight, attenuated enterocyte apoptotic cell death, mitigated reduced mucin production, and maintained ileal structure. Infected ileum expressed reduced levels of IκBα, which could be restored upon pretreatment with PCM. We also observed a nuclear translocation of NF-κB p65 in H4 cells exposed to C. sakazakii, which was prevented in PCM-pretreated cells. Finally, treatment of neonatal mice with PCM prior to infection sustained the capacity of ileal epithelial proliferation. This study suggests that an active component(s) released into the culture medium by B. infantis may prevent ileal damage by a pathogen linked to NEC.

  12. 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

  13. A Novel Bacteriophage Targeting Cronobacter sakazakii Is a Potential Biocontrol Agent in Foods

    PubMed Central

    Lee, Ju-Hoon; Bai, Jaewoo; Shin, Hakdong; Kim, Yeran; Park, Bookyung; Heu, Sunggi

    2015-01-01

    Cronobacter sakazakii is an important pathogen that causes high mortality in infants. Due to its occasional antibiotic resistance, a bacteriophage approach might be an alternative effective method for the control of this pathogen. To develop a novel biocontrol agent using bacteriophages, the C. sakazakii-infecting phage CR5 was newly isolated and characterized. Interestingly, this phage exhibited efficient and relatively durable host lysis activity. In addition, a specific gene knockout study and subsequent complementation experiment revealed that this phage infected the host strain using the bacterial flagella. The complete genome sequence analysis of phage CR5 showed that its genome contains 223,989 bp of DNA, including 231 predicted open reading frames (ORFs), and it has a G+C content of 50.06%. The annotated ORFs were classified into six functional groups (structure, packaging, host lysis, DNA manipulation, transcription, and additional functions); no gene was found to be related to virulence or toxin or lysogen formation, but >80% of the predicted ORFs are unknown. In addition, a phage proteomic analysis using SDS-PAGE and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) revealed that seven phage structural proteins are indeed present, supporting the ORF predictions. To verify the potential of this phage as a biocontrol agent against C. sakazakii, it was added to infant formula milk contaminated with a C. sakazakii clinical isolate or food isolate, revealing complete growth inhibition of the isolates by the addition of phage CR5 when the multiplicity of infection (MOI) was 105. PMID:26497465

  14. 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.

  15. Genetic Characterization of Cronobacter sakazakii Recovered from the Environmental Surveillance Samples During a Sporadic Case Investigation of Foodborne Illness.

    PubMed

    Sulaiman, Irshad M; Jacobs, Emily; Segars, Katharine; Simpson, Steven; Kerdahi, Khalil

    2016-08-01

    Cronobacter sakazakii is an opportunistic human-pathogenic bacterium known to cause acute meningitis and necrotizing enterocolitis in neonates and immunocompromised individuals. This human-pathogenic microorganism has been isolated from a variety of food and environmental samples, and has been also linked to foodborne outbreaks associated with powdered infant formula (PIF). The U.S. Food and Drug Administration have a policy of zero tolerance of these organisms in PIF. Thus, this agency utilizes the presence of these microorganisms as one of the criteria in implementing regulatory actions and assessing adulteration of food products of public health importance. In this study, we recovered two isolates of Cronobacter from the 91 environmental swab samples during an investigation of sporadic case of foodborne illness following conventional microbiological protocols. The isolated typical colonies were identified using VITEK2 and real-time PCR protocols. The recovered Cronobacter isolates were then characterized for species identification by sequencing the 16S rRNA locus. Further, multilocus sequence typing (MLST) was accomplished characterizing seven known C. sakazakii-specific MLST loci (atpD, fusA, glnS, gltB, gyrB, infB, and pps). Results of this study confirmed all of the recovered Cronobacter isolates from the environmental swab samples to be C. sakazakii. The MLST profile matched with the published profile of the complex 31 of C. sakazakii. Thus, rRNA and 7-loci MLST-based sequencing protocols are robust techniques for rapid detection and differentiation of Cronobacter species, and these molecular diagnostic tools can be used in implementing successful surveillance program and in the control and prevention of foodborne illness.

  16. Genetic Characterization of Cronobacter sakazakii Recovered from the Environmental Surveillance Samples During a Sporadic Case Investigation of Foodborne Illness.

    PubMed

    Sulaiman, Irshad M; Jacobs, Emily; Segars, Katharine; Simpson, Steven; Kerdahi, Khalil

    2016-08-01

    Cronobacter sakazakii is an opportunistic human-pathogenic bacterium known to cause acute meningitis and necrotizing enterocolitis in neonates and immunocompromised individuals. This human-pathogenic microorganism has been isolated from a variety of food and environmental samples, and has been also linked to foodborne outbreaks associated with powdered infant formula (PIF). The U.S. Food and Drug Administration have a policy of zero tolerance of these organisms in PIF. Thus, this agency utilizes the presence of these microorganisms as one of the criteria in implementing regulatory actions and assessing adulteration of food products of public health importance. In this study, we recovered two isolates of Cronobacter from the 91 environmental swab samples during an investigation of sporadic case of foodborne illness following conventional microbiological protocols. The isolated typical colonies were identified using VITEK2 and real-time PCR protocols. The recovered Cronobacter isolates were then characterized for species identification by sequencing the 16S rRNA locus. Further, multilocus sequence typing (MLST) was accomplished characterizing seven known C. sakazakii-specific MLST loci (atpD, fusA, glnS, gltB, gyrB, infB, and pps). Results of this study confirmed all of the recovered Cronobacter isolates from the environmental swab samples to be C. sakazakii. The MLST profile matched with the published profile of the complex 31 of C. sakazakii. Thus, rRNA and 7-loci MLST-based sequencing protocols are robust techniques for rapid detection and differentiation of Cronobacter species, and these molecular diagnostic tools can be used in implementing successful surveillance program and in the control and prevention of foodborne illness. PMID:27155844

  17. 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

  18. Plasmid-encoded MCP is involved in virulence, motility, and biofilm formation of Cronobacter sakazakii ATCC 29544.

    PubMed

    Choi, Younho; Kim, Seongok; Hwang, Hyelyeon; Kim, Kwang-Pyo; Kang, Dong-Hyun; Ryu, Sangryeol

    2015-01-01

    The aim of this study was to elucidate the function of the plasmid-borne mcp (methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles in Cronobacter sakazakii ATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, in C. sakazakii ATCC 29544. An in silico analysis of pCSA2 revealed that it included six putative open reading frames, and one of them was mcp. The mcp mutant was defective for invasion into and adhesion to epithelial cells, and the virulence of the mcp mutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility of C. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functional mcp gene. Furthermore, a lack of the mcp gene also impaired the ability of C. sakazakii to form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence of C. sakazakii ATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in the C. sakazakii sequence type 8 (ST8) lineage.

  19. 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.

  20. 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.

  1. Effect of ethanol shock pretreatment on the tolerance of Cronobacter sakazakii BCRC 13988 exposed to subsequent lethal stresses.

    PubMed

    Huang, Yi-Ting; Cheng, Kuan-Chen; Yu, Roch-Chui; Chou, Cheng-Chun

    2013-02-01

    Cronobacter spp., formerly Enterobacter sakazakii, are human pathogens. They are the etiological agent of life-threating bacterial infections in infants. In this study, the survival behavior of C. sakazakii Bioresources Collection and Research Center (BCRC) 13988 in the presence of various ethanol concentrations was first examined. Besides, the test organism was subjected to treatment with 5% ethanol for 60 min (ethanol shock). The effect of ethanol shock on the resistance of C. sakazakii to lethal stresses, including high ethanol concentration (15%), low temperature (4°C and -20°C), high temperature (51°C) and high acidity (pH 3.3), was investigated. Results revealed that 4-5% ethanol is the maximum concentration that will allow C. sakazakii BCRC 13988 to grow in trypticase soy broth (TSB). Etthanol at 6% and 7% or more, respectively, exerted bacteriostatic and bactericidal effects, respectively, on the test organism. Although ethanol shock did not affect the resistance of C. sakazakii to a refrigerated temperature (4°C), the ethanol-shocked C. sakazakii survived better under other lethal stress conditions. After 50 min of exposure to 15% ethanol, the ethanol-shocked C. sakazakii showed a survival percentage of 16.11%, which was a 6400-fold increase over the control cells (<0.01%). On the other hand, the ethanol-shocked C. sakazakii exhibited a 45-fold higher survival after 120-min exposure to 51°C. At the end of the 7-day storage at -20°C, the ethanol-shocked cells exhibited a survival percentage of 0.25% which was significantly (p<0.05) higher than that (less than 0.01%) of the control. Additionally, the ethanol-shocked cells showed a survival percentage of 13.80% compared to only 1.60% noted with the control cells after exposure to pH 3.3 for 60 min.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26048942

  3. 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. PMID:26048942

  4. 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

  5. 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...

  6. 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.

  7. The Membrane Proteins Involved in Virulence of Cronobacter sakazakii Virulent G362 and Attenuated L3101 Isolates

    PubMed Central

    Ye, YingWang; Gao, Jina; Jiao, Rui; Li, Hui; Wu, Qingping; Zhang, Jumei; Zhong, Xian

    2015-01-01

    Cronobacter sakazakii is an opportunistic foodborne pathogen and the virulence differences were previously documented. However, information about membranous proteins involved in virulence differences was not available. In this study, virulent characterization such as biofilm formation and flagella motility between virulent C. sakazakii isolate G362 and attenuated L3101 were determined. Then, two-dimensional gel electrophoresis (2-DE) technology was used to preliminarily reveal differential expression of membranous proteins between G362 and L3101. On the mass spectrometry (MS) analysis and MASCOT research results, fourteen proteins with differential expression were successfully identified. At the threshold of twofold changes, five out of eight membranous proteins were up-regulated in G362. Using RT-PCR, the expression abundance of the protein (enzV, ompX, lptE, pstB, and OsmY) genes at mRNA levels was consistent with the results by 2-DE method. The findings presented here provided novel information and valuable knowledge for revealing pathogenic mechanism of C. sakazakii. PMID:26617581

  8. Reservoir and routes of transmission of Enterobacter sakazakii (Cronobacter spp.) in a milk powder-producing plant.

    PubMed

    Jacobs, C; Braun, P; Hammer, P

    2011-08-01

    Several outbreaks of Cronobacter spp. (Enterobacter sakazakii) have been described as food-borne illness in neonates and infants. Powdered infant formula has been identified as a source of infection, especially in hospital nurseries, where a bulk of formula nutrient is prepared for the whole day and instructions for preparation are not always followed correctly. Neonates who are underweight or immunosuppressed are especially at risk for an E. sakazakii infection. Considering that milk powder is the main ingredient of powdered infant formula, we analyzed the incidence and distribution of E. sakazakii in a milk powder-producing plant. We looked specifically at the spray-drying towers and roller dryers. Selected isolates from samples taken from the environment and final product were typed by pulsed-field gel electrophoresis to investigate the epidemiology of the organism within the production area of the plant. Seven pulsed-field gel electrophoresis types were detected in the spray-drying area, which presumably entered the plant through an aperture for process air and an improperly controlled roller shutter. Furthermore, textile filters for exhaust air of both the spray-drying towers were identified as internal reservoirs of the pathogen. For economic reasons, powder from the textile filters is reintroduced into the product flow; this can contaminate the final product. For the production of milk powder to be used as an ingredient of powdered infant formula, it was suggested to terminate the process of reintroducing the filtered powder into the product flow. A second transmission route was identified in the roller dryer section of the factory. It could be shown that contaminated milk concentrate could pass the process unheated, thus leading to a contamination of the product with E. sakazakii.

  9. 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

  10. 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. PMID:27148223

  11. 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

  12. Rapid Detection and Simultaneous Genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in Powdered Infant Formula Using Real-time PCR and High Resolution Melting (HRM) Analysis

    PubMed Central

    Cai, Xian-Quan; Yu, Hai-Qiong; Ruan, Zhou-Xi; Yang, Lei-Liang; Bai, Jian-Shan; Qiu, De-Yi; Jian, Zhi-Hua; Xiao, Yi-Qian; Yang, Jie-Yang; Le, Thanh Hoa; Zhu, Xing-Quan

    2013-01-01

    Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 102 CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula. PMID:23825624

  13. 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

  14. 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. PMID:27163653

  15. Comparison of desiccation tolerance among Listeria monocytogenes, Escherichia coli O157:H7, Salmonella enterica, and Cronobacter sakazakii in powdered infant formula.

    PubMed

    Koseki, Shigenobu; Nakamura, Nobutaka; Shiina, Takeo

    2015-01-01

    Bacterial pathogens such as Listeria monocytogenes, Escherichia coli O157:H7, Salmonella enterica, and Cronobacter sakazakii have demonstrated long-term survival in/on dry or low-water activity (aw) foods. However, there have been few comparative studies on the desiccation tolerance among these bacterial pathogens separately in a same food matrix. In the present study, the survival kinetics of the four bacterial pathogens separately inoculated onto powdered infant formula as a model low-aw food was compared during storage at 5, 22, and 35°C. No significant differences in the survival kinetics between E. coli O157:H7 and L. monocytogenes were observed. Salmonella showed significantly higher desiccation tolerance than these pathogens, and C. sakazakii demonstrated significantly higher desiccation tolerance than all other three bacteria studied. Thus, the desiccation tolerance was represented as C. sakazakii > Salmonella > E. coli O157:H7 = L. monocytogenes. The survival kinetics of each bacterium was mathematically analyzed, and the observed kinetics was successfully described using the Weibull model. To evaluate the variability of the inactivation kinetics of the tested bacterial pathogens, the Monte Carlo simulation was performed using assumed probability distribution of the estimated fitted parameters. The simulation results showed that the storage temperature significantly influenced survival of each bacterium under the dry environment, where the bacterial inactivation became faster with increasing storage temperature. Furthermore, the fitted rate and shape parameters of the Weibull model were successfully modelled as a function of temperature. The numerical simulation of the bacterial inactivation was realized using the functions of the parameters under arbitrary fluctuating temperature conditions. PMID:25581184

  16. Comparison of desiccation tolerance among Listeria monocytogenes, Escherichia coli O157:H7, Salmonella enterica, and Cronobacter sakazakii in powdered infant formula.

    PubMed

    Koseki, Shigenobu; Nakamura, Nobutaka; Shiina, Takeo

    2015-01-01

    Bacterial pathogens such as Listeria monocytogenes, Escherichia coli O157:H7, Salmonella enterica, and Cronobacter sakazakii have demonstrated long-term survival in/on dry or low-water activity (aw) foods. However, there have been few comparative studies on the desiccation tolerance among these bacterial pathogens separately in a same food matrix. In the present study, the survival kinetics of the four bacterial pathogens separately inoculated onto powdered infant formula as a model low-aw food was compared during storage at 5, 22, and 35°C. No significant differences in the survival kinetics between E. coli O157:H7 and L. monocytogenes were observed. Salmonella showed significantly higher desiccation tolerance than these pathogens, and C. sakazakii demonstrated significantly higher desiccation tolerance than all other three bacteria studied. Thus, the desiccation tolerance was represented as C. sakazakii > Salmonella > E. coli O157:H7 = L. monocytogenes. The survival kinetics of each bacterium was mathematically analyzed, and the observed kinetics was successfully described using the Weibull model. To evaluate the variability of the inactivation kinetics of the tested bacterial pathogens, the Monte Carlo simulation was performed using assumed probability distribution of the estimated fitted parameters. The simulation results showed that the storage temperature significantly influenced survival of each bacterium under the dry environment, where the bacterial inactivation became faster with increasing storage temperature. Furthermore, the fitted rate and shape parameters of the Weibull model were successfully modelled as a function of temperature. The numerical simulation of the bacterial inactivation was realized using the functions of the parameters under arbitrary fluctuating temperature conditions.

  17. 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...

  18. New decontamination method based on caprylic acid in combination with citric acid or vanillin for eliminating Cronobacter sakazakii and Salmonella enterica serovar Typhimurium in reconstituted infant formula.

    PubMed

    Choi, M J; Kim, S A; Lee, N Y; Rhee, M S

    2013-09-16

    The antimicrobial effects of natural compounds (caprylic acid, CA; citric acid, CTA; and vanillin, VNL) on the inactivation of Cronobacter sakazakii and Salmonella enterica serovar Typhimurium were examined in reconstituted infant formula. The samples were treated with: 1) CA, CTA, or VNL alone (0, 10, 20, 30, 40, 60, and 80 mM); 2) a combination of CA (10 and 20 mM) and CTA (15 and 30 mM); and 3) a combination of CA (10 and 20 mM) and VNL (15 and 30 mM), at mild feeding temperatures (40 °C and 45 °C), and the bacterial populations were assayed periodically (0, 5, 10, and 30 min). For both bacteria, the combined treatments had marked synergistic antimicrobial effects compared with the sum of the effects of each individual treatment. For example, there was no noticeable reduction (P > 0.05) in the population of C. sakazakii following an individual treatment with 20 mM CA or 30 mM CTA for 5 min at 40 °C, whereas the population was reduced to undetectable levels (reduction > 7.3 log CFU/ml) following treatment with a combination of CA and CTA (20 CA+30 CTA for 5 min at 40 °C). As the temperature increased, the bactericidal effect was stronger at all time points with a synergistic effect. In a validation assay using a low level inoculum (approximately 10³ CFU/ml) of desiccation-stressed bacteria in certain conditions, the combined treatments (e.g., CA 10 mM+CTA 30 mM for 5 min at 45 °C for C. sakazakii, and CA 10mM+VNL 15 mM for 10 min at 45 °C for S. Typhimurium) completely destroyed the bacteria with no recovery of cell viability. Disintegration of the membrane and changes in the cell structure or morphology, such as plasmolysis and membrane disruption, were detected by flow cytometry and electron microscopy, respectively. These methods use antimicrobials that could be applied as food additives in infant formula, which may help to eliminate bacteria. PMID:24042002

  19. Effect of heat shock on the fatty acid and protein profiles of Cronobacter sakazakii BCRC 13988 as well as its growth and survival in the presence of various carbon, nitrogen sources and disinfectants.

    PubMed

    Li, Po-Ting; Hsiao, Wan-Ling; Yu, Roch-Chui; Chou, Cheng-Chun

    2013-12-01

    In the present study, Cronobacter sakazakii, a foodborne pathogen, was first subjected to heat shock at 47 °C for 15 min. Effect of heat shock on the fatty acid and protein profiles, carbon and nitrogen source requirements as well as the susceptibilities of C. sakazakii to Clidox-S, a chlorine-containing disinfectant and Quatricide, a quaternary ammonium compound were investigated. Results revealed that heat shock increased the proportion of myristic acid (14:0), palmitic acid (16:0) and the ratio of saturated fatty acid to unsaturated fatty acid, while reducing the proportion of palmitoleic acid (16:1) and cis-vacceric acid (18:1). In addition, eleven proteins showed enhanced expression, while one protein showed decreased expression in the heat-shocked compared to the non-heat-shocked cells. Non-heat-shocked cells in the medium supplemented with beef extract exhibited the highest maximum population. On the contrary, the highest maximum population of heat-shocked C. sakazakii was noted in the medium having either tryptone or yeast extract as the nitrogen source. Among the various carbon sources examined, the growth of the test organism, regardless of heat shock, was greatest in the medium having glucose as the carbon source. Furthermore, heat shock enhanced the resistance of C. sakazakii to Clidox-S or Quatricide.

  20. 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

  1. 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

    Feng, Shaolong; Eucker, Tyson P; Holly, Mayumi K; Konkel, Michael E; Lu, Xiaonan; Wang, Shuo

    2014-02-01

    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

  2. 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.

  3. Analysis of a consensus fragment in ERIC-PCR fingerprinting of Enterobacter sakazakii.

    PubMed

    Ye, Yingwang; Wu, Qingping; Yao, Lin; Dong, Xiaohui; Wu, Kui; Zhang, Jumei

    2009-06-30

    Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR was employed to generate ERIC-PCR fingerprints of 23 strains of E. sakazakii. A consensus fragment was observed in all 23 strains and was purified, cloned and sequenced from type strain ATCC29544. A comparison of the nucleotide acid with other sequences available in the GenBank revealed 98% of identity with E. sakazakii ATCC BAA-894 and 71%-75% identity with oligopeptidase gene or protease II gene of some species from the Enterobacteriaceae family. The consensus fragment from ATCC29544 was used to synthesize probes using DIG-labeling by PCR for dot hybridization. The consensus fragment was found to be highly conserved. Diversity analysis based on the consensus fragment sequencing showed a high heterogeneity between E. sakazakii strains and other related strains. In addition, 24 E. sakazakii strains could be distributed into two groups, while E. sakazakii ATCC51329 formed a separate branch. Genetic diversity and potential taxonomic complexity were evident within the E. sakazakii strains.

  4. 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.

  5. Molecular Characterization of Cronobacter Lipopolysaccharide O-Antigen Gene Clusters and Development of Serotype-Specific PCR Assays ▿

    PubMed Central

    Jarvis, K. G.; Grim, C. J.; Franco, A. A.; Gopinath, G.; Sathyamoorthy, V.; Hu, L.; Sadowski, J. A.; Lee, C. S.; Tall, B. D.

    2011-01-01

    Cronobacter (formerly Enterobacter sakazakii) is a recently defined genus consisting of six species, C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, and Cronobacter genomospecies 1. In this study, MboII restriction fragment length polymorphism (RFLP) patterns of O-antigen gene clusters, located between galF and gnd, were used to identify serotypes in Cronobacter spp. Seven O-antigen RFLP clusters were generated, including three C. sakazakii clusters, previously identified as serotypes O1, O2, and O3. The O-antigen regions of six strains with unique RFLP patterns, including two C. sakazakii strains, two C. malonaticus strains, one C. turicensis strain, and one C. muytjensii strain, revealed three O-antigen gene clusters shared among Cronobacter species. PCR assays were developed, targeting the wzx O-antigen polymerase gene, and used to screen 231 Cronobacter strains to determine the frequency of these newly identified serotypes. PMID:21531829

  6. 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

  7. Characterization of Cronobacter spp. isolated from food of plant origin and environmental samples collected from farms and from supermarkets in the Czech Republic.

    PubMed

    Vojkovska, Hana; Karpiskova, Renata; Orieskova, Maria; Drahovska, Hana

    2016-01-18

    The Cronobacter genus (previously known as Enterobacter sakazakii) comprises seven species (Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti)which cause serious infections in neonates and immunocompromised people.Most of the documented outbreaks of these bacteria have been associated with consumption of contaminated powdered infant formula. The plant environment is considered to be the natural habitat of these bacteria. Therefore, a total number of 563 samples of vegetables, fruit, water and environmental swabs were collected from local farms and supermarkets in the Czech Republic and investigated for the presence of Cronobacter spp. The obtained 45 isolates (8.0%) were further characterized by phenotyping (antimicrobial resistance, capsule and pigment production) and genotyping (fusA sequencing,MLST, PCR-serotyping) methods. Most of the Cronobacter isolates (42.2%) were identified as C. sakazakii, followed by C. turicensis (31.1%), C. dublinensis (22.2%), C. malonaticus (2.2%) and C. universalis (2.2%). The 25 identified sequence types, out of which 17 were unique for only one strain, indicated a high diversity of strains. C. sakazakii sequence type 4 (ST 4), which has been associated with many cases of meningitis, was isolated only in one case. A strong association of C. turicensis and C. dublinensis with the plant environment can be deduced from our results.

  8. Characterization of Cronobacter spp. isolated from food of plant origin and environmental samples collected from farms and from supermarkets in the Czech Republic.

    PubMed

    Vojkovska, Hana; Karpiskova, Renata; Orieskova, Maria; Drahovska, Hana

    2016-01-18

    The Cronobacter genus (previously known as Enterobacter sakazakii) comprises seven species (Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti)which cause serious infections in neonates and immunocompromised people.Most of the documented outbreaks of these bacteria have been associated with consumption of contaminated powdered infant formula. The plant environment is considered to be the natural habitat of these bacteria. Therefore, a total number of 563 samples of vegetables, fruit, water and environmental swabs were collected from local farms and supermarkets in the Czech Republic and investigated for the presence of Cronobacter spp. The obtained 45 isolates (8.0%) were further characterized by phenotyping (antimicrobial resistance, capsule and pigment production) and genotyping (fusA sequencing,MLST, PCR-serotyping) methods. Most of the Cronobacter isolates (42.2%) were identified as C. sakazakii, followed by C. turicensis (31.1%), C. dublinensis (22.2%), C. malonaticus (2.2%) and C. universalis (2.2%). The 25 identified sequence types, out of which 17 were unique for only one strain, indicated a high diversity of strains. C. sakazakii sequence type 4 (ST 4), which has been associated with many cases of meningitis, was isolated only in one case. A strong association of C. turicensis and C. dublinensis with the plant environment can be deduced from our results. PMID:26513253

  9. 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%.

  10. Characterization of Cronobacter recovered from dried milk and related products

    PubMed Central

    2009-01-01

    Background Cronobacter is a recently proposed genus consisting of six genomospecies that encompass the organisms previously identified as Enterobacter sakazakii. Cronobacter are opportunistic pathogens and are known to cause serious infections in infants, particularly neonates. High case fatality rates have been associated with infections and acute sequelae can occur in survivors with severe ramifications on neurological development. Infant formula has been identified as one route of transmission for infection in infants. However, the primary reservoirs for subsequent contamination of foods with Cronobacter remain undefined due to the ubiquitous nature of these organisms. More recently, infections in adults have been reported, especially amongst the elderly and patients who are immunocompromised. To help prevent the transmission of infection, it is important to identify the main food sources for Cronobacter. The aim of this study was to identify and characterize Cronobacter isolated from dried-milk and related products available in an Egyptian food market. Results In total sixteen Cronobacter strains were isolated from 152 dairy-based products. These were identified and characterized using pheno- and genotyping experiments. Real-time PCR confirmed the detection of Cronobacter. Following antibiotic susceptibility tests, 3 strains showed resistance to trimethoprim and/or neomycin. Phenotype profiles were generated based on key biochemical distinguishing tests. Pulsed-field gel electrophoresis (PFGE) identified 8 PFGE types amongst the collection of strains. Repetitive sequence based PCR (rep-PCR) analysis identified 3 rep-PCR types amongst the collection of strains. Sequencing of the recN gene was used to differentiate among the recently described species of Cronobacter. Conclusion This study identified the presence of Cronobacter in dried milk and related products sourced from the Nile-Delta region of Egypt. Although the majority of the strains were susceptible to

  11. Development of a Custom-Designed, Pan Genomic DNA Microarray to Characterize Strain-Level Diversity among Cronobacter spp.

    PubMed Central

    Tall, Ben Davies; Gangiredla, Jayanthi; Gopinath, Gopal R.; Yan, Qiongqiong; Chase, Hannah R.; Lee, Boram; Hwang, Seongeun; Trach, Larisa; Park, Eunbi; Yoo, YeonJoo; Chung, TaeJung; Jackson, Scott A.; Patel, Isha R.; Sathyamoorthy, Venugopal; Pava-Ripoll, Monica; Kotewicz, Michael L.; Carter, Laurenda; Iversen, Carol; Pagotto, Franco; Stephan, Roger; Lehner, Angelika; Fanning, Séamus; Grim, Christopher J.

    2015-01-01

    Cronobacter species cause infections in all age groups; however neonates are at highest risk and remain the most susceptible age group for life-threatening invasive disease. The genus contains seven species:Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite an abundance of published genomes of these species, genomics-based epidemiology of the genus is not well established. The gene content of a diverse group of 126 unique Cronobacter and taxonomically related isolates was determined using a pan genomic-based DNA microarray as a genotyping tool and as a means to identify outbreak isolates for food safety, environmental, and clinical surveillance purposes. The microarray constitutes 19,287 independent genes representing 15 Cronobacter genomes and 18 plasmids and 2,371 virulence factor genes of phylogenetically related Gram-negative bacteria. The Cronobacter microarray was able to distinguish the seven Cronobacter species from one another and from non-Cronobacter species; and within each species, strains grouped into distinct clusters based on their genomic diversity. These results also support the phylogenic divergence of the genus and clearly highlight the genomic diversity among each member of the genus. The current study establishes a powerful platform for further genomics research of this diverse genus, an important prerequisite toward the development of future countermeasures against this foodborne pathogen in the food safety and clinical arenas. PMID:25984509

  12. Cronobacter spp.--opportunistic food-borne pathogens. A review of their virulence and environmental-adaptive traits.

    PubMed

    Jaradat, Ziad W; Al Mousa, Waseem; Elbetieha, Ahmed; Al Nabulsi, Anas; Tall, Ben D

    2014-08-01

    The genus Cronobacter consists of a diverse group of Gram-negative bacilli and comprises seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti. Cronobacter are regarded as opportunistic pathogens, and have been implicated in newborn and infant infections, causing meningitis, necrotizing enterocolitis and bacteraemia or sepsis. Cronobacter virulence is believed to be due to multiple factors. Some strains were found to produce diarrhoea or cause significant fluid accumulation in suckling mice. Two iron acquisition systems (eitCBAD and iucABCD/iutA), Cronobacter plasminogen activator gene (cpa), a 17 kb type VI secretion system (T6SS), and a 27 kb filamentous haemagglutinin gene (fhaBC) and associated putative adhesins locus are harboured on a family of RepFIB-related plasmids (pESA3 and pCTU1), suggesting that these are common virulence plasmids; 98% of 229 tested Cronobacter strains possessed these plasmids. Even though pESA3 and pCTU1 share a common backbone composed of the repA gene and eitCBAD and iucABCD/iutA gene clusters, 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. Other factors were observed, in that Cronobacter form biofilms, and show unusual resistance to heat, dry and acid stress growth conditions. The outer-membrane protein A is probably one of the best-characterized virulence markers of Cronobacter. Furthermore, it was reported that Cronobacter employ phosphatidylinositide 3-kinase/Akt signalling, which activates protein kinase C-α and impairs the host cell's mitogen-activated protein kinase pathway, in order to invade cells. Cronobacter can also use immature dendritic cells and macrophages to escape the immune response. This review addresses the various virulence and environmental-adaptive characteristics

  13. Culture media for the isolation of Cronobacter spp.

    PubMed

    Druggan, Patrick; Iversen, Carol

    2009-12-31

    Enterobacter sakazakii is a member of the Enterobacteriaceae and has been implicated in causing necrotising enterocolitis, as well as bacteraemia and meningitis in infants. In some cases, the infection has been linked to ingestion of infant formula milk (IFM) that has not been terminally sterilised. The nomenclature of E. sakazakii has been clarified and it has now been accepted as a group of six species comprising a novel genus, Cronobacter. Outbreaks in neonatal intensive care units resulting in relatively high case fatality rates and the recognition of IFM as a significant route of infection prompted the development of culture-based detection methods. Development of enrichment broths specific for Cronobacter spp., coupled to the use of fluorogenic and chromogenic substrates in culture media has significantly improved the sensitivity and specificity of methods. This review presents the history and rationale behind the currently available methods, and gives an overview of the principles involved in designing these microbiological media. PMID:19896231

  14. Identification of Natural Animicrobial Substances in Red Muscadine Juice against Cranonbacter sakazakii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Muscadine grape (Vitis rotundifolia Michx.) juice with natural organic, phenolic acids and polyphenol compounds identified in red muscadine juice (‘Noble’) were tested against Cronobacter sakazakii. Commercial baby juices with high polyphenol content (176.7~347.7 mg/mL), showed poor antimicrobial a...

  15. Identification of Natural Antimicrobial Substances in Red Muscadine Juice against Enterobacter sakazakii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red muscadine (Vitis rotundifolia Michx.) juices with natural organic, phenolic acids and polyphenol compounds were tested against Cronobacter sakazakii. The concentration of total phenolic compounds of commercial baby juices ranged from 176.7 to 347.7 mg/mL. Commercial baby juices showed poor antim...

  16. Occurrence and Characterization of Cronobacter spp. in Dehydrated Rice Powder from Chinese Supermarket.

    PubMed

    Huang, Yan; Pang, Yiheng; Wang, Hong; Tang, Zhengzhu; Zhou, Yan; Zhang, Weiyu; Li, Xiugui; Tan, Dongmei; Li, Jian; Lin, Ying; Liu, Xiaoling; Huang, Weiyi; Shi, Yunliang

    2015-01-01

    Cronobacter spp. are emerging food-borne pathogens and have been identified as causative agents of meningitis and necrotizing enterocolitis in infants. Dehydrated rice is popular with a wide range of people and it is frequently used as a substitute for infant milk powder to baby older than four months. The occurrence of Cronobacter spp. was investigated in 1,012 samples of dehydrated rice powder collected from 14 manufacturers in China during 2010 to 2012. The isolates were identified using fusA allele sequencing and subtyped using pulsed-field gel electrophoresis. Seventy-six samples (7.5%) contained Cronobacter spp. The prevalence among manufacturers ranged from 0-28.8%. The 76 isolates included 4 species [Cronobacter sakazakii (52 isolates) Cronobacter malonaticus (14 isolates), Cronobacter dublinensis (7 isolates), and Cronobacter muytjensii (3 isolates)]. Twenty-three unique fusA alleles and sixty-six PFGE-patterns were detected. All isolated strains were observed to be sensitive or to show intermediate susceptibility to eight tested antimicrobial agents. The study revealed serious contamination of dehydrated rice powder by Cronobacter spp., with prevalence varying among manufacturers in China. Identified Cronobacter species, fusA alleles, and subtypes were diverse.

  17. Occurrence and Characterization of Cronobacter spp. in Dehydrated Rice Powder from Chinese Supermarket

    PubMed Central

    Huang, Yan; Pang, Yiheng; Wang, Hong; Tang, Zhengzhu; Zhou, Yan; Zhang, Weiyu; Li, Xiugui; Tan, Dongmei; Li, Jian; Lin, Ying; Liu, Xiaoling; Huang, Weiyi; Shi, Yunliang

    2015-01-01

    Cronobacter spp. are emerging food-borne pathogens and have been identified as causative agents of meningitis and necrotizing enterocolitis in infants. Dehydrated rice is popular with a wide range of people and it is frequently used as a substitute for infant milk powder to baby older than four months. The occurrence of Cronobacter spp. was investigated in 1,012 samples of dehydrated rice powder collected from 14 manufacturers in China during 2010 to 2012. The isolates were identified using fusA allele sequencing and subtyped using pulsed-field gel electrophoresis. Seventy-six samples (7.5%) contained Cronobacter spp. The prevalence among manufacturers ranged from 0-28.8%. The 76 isolates included 4 species [Cronobacter sakazakii (52 isolates) Cronobacter malonaticus (14 isolates), Cronobacter dublinensis (7 isolates), and Cronobacter muytjensii (3 isolates)]. Twenty-three unique fusA alleles and sixty-six PFGE-patterns were detected. All isolated strains were observed to be sensitive or to show intermediate susceptibility to eight tested antimicrobial agents. The study revealed serious contamination of dehydrated rice powder by Cronobacter spp., with prevalence varying among manufacturers in China. Identified Cronobacter species, fusA alleles, and subtypes were diverse. PMID:26132635

  18. Desiccation resistance and persistence of Cronobacter species in infant formula.

    PubMed

    Osaili, T; Forsythe, S

    2009-12-31

    Cronobacter is a newly described genus which includes opportunistic pathogens formerly known as 'Enterobacter sakazakii'. These organisms have been isolated from a wide variety of sources, including powdered infant formula (PIF). This review focuses on the desiccation survival of Cronobacter, and its relevance to vehicles of infection. Due to its probable natural habitat of plant material, the organism has an array of survival mechanisms which includes resistance to desiccation and osmotic stresses. The organism can survive for long periods of time (>2years) in the desiccated state, and can be recovered from a large number of powdered foods in addition to powdered infant formula. On reconstitution, the organism may rapidly multiply and present a risk to immunocompromised infants. It is expected that an improved understanding of the nature of Cronobacter persistence may aid in further improved control measures and eliminate the bacterium from the critical food production environments.

  19. The Genotypic Characterization of Cronobacter spp. Isolated in China

    PubMed Central

    Cui, Jinghua; Du, Xiaoli; Liu, Hui; Hu, Guangchun; Lv, Guoping; Xu, Baohong; Yang, Xiaorong; Li, Wei; Cui, Zhigang

    2014-01-01

    Cronobacter spp. (Enterobacter sakazakii) is an important pathogen contaminating powdered infant formula (PIF). To describe the genotypic diversity of Cronobacter isolated in China, we identified the isolates using fusA allele sequencing, and subtyped all of the isolates using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multiple-locus variable-number tandem-repeat analysis (MLVA). A total of 105 isolates were identified, which included C. sakazakii (58 isolates), C. malonaticus (30 isolates), C. dublinensis (11 isolates), C. turicensis (5 isolates), and C. muytjensii (1 isolate). These isolates were showed to have 85 PFGE-patterns, 71 sequence types (STs), and 55 MLVA-patterns. Comparisons among the three molecular subtyping methods revealed that the PFGE method was the most distinguishable tool in identifying clusters of Cronobacter spp. through DNA fingerprinting, and MLST method came second. However, ESTR-1, ESTR-2, ESTR-3, and ESTR-4 were not effective loci for subtyping Cronobacter spp. such that the MLVA method requires further improvement. PMID:25029018

  20. 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

  1. The speciation and genotyping of Cronobacter isolates from hospitalised patients.

    PubMed

    Alsonosi, A; Hariri, S; Kajsík, M; Oriešková, M; Hanulík, V; Röderová, M; Petrželová, J; Kollárová, H; Drahovská, H; Forsythe, S; Holý, O

    2015-10-01

    The World Health Organization (WHO) has recognised all Cronobacter species as human pathogens. Among premature neonates and immunocompromised infants, these infections can be life-threatening, with clinical presentations of septicaemia, meningitis and necrotising enterocolitis. The neurological sequelae can be permanent and the mortality rate as high as 40-80%. Despite the highlighted issues of neonatal infections, the majority of Cronobacter infections are in the elderly population suffering from serious underlying disease or malignancy and include wound and urinary tract infections, osteomyelitis, bacteraemia and septicaemia. However, no age profiling studies have speciated or genotyped the Cronobacter isolates. A clinical collection of 51 Cronobacter strains from two hospitals were speciated and genotyped using 7-loci multilocus sequence typing (MLST), rpoB gene sequence analysis, O-antigen typing and pulsed-field gel electrophoresis (PFGE). The isolates were predominated by C. sakazakii sequence type 4 (63%, 32/51) and C. malonaticus sequence type 7 (33%, 17/51). These had been isolated from throat and sputum samples of all age groups, as well as recal and faecal swabs. There was no apparent relatedness between the age of the patient and the Cronobacter species isolated. Despite the high clonality of Cronobacter, PFGE profiles differentiated strains across the sequence types into 15 pulsotypes. There was almost complete agreement between O-antigen typing and rpoB gene sequence analysis and MLST profiling. This study shows the value of applying MLST to bacterial population studies with strains from two patient cohorts, combined with PFGE for further discrimination of strains. PMID:26173692

  2. Evaluation of Cronobacter Growth and Phenotypic Variation Under Modified Culture Conditions.

    PubMed

    Segars, Katharine; Simpson, Steven; Kerdahi, Khalil; Sulaiman, Irshad M

    2016-02-01

    Cronobacter sakazakii is an opportunistic pathogen known to cause acute meningitis and necrotizing enterocolitis in neonates and immunocompromised individuals. It has been isolated from a wide range of food and environmental samples, and has been linked to outbreaks associated with powdered infant formula. This study was carried out to assess variations in growth conditions (temperature, pH, and sugar supplement) and to establish how these changes impact phenotypic characteristics for successful recovery and identification of Cronobacter, particularly for routine surveillance purposes. A total of six Cronobacter isolates were tested to evaluate the above growth conditions, including three ATCC Cronobacter reference and three environmental isolates obtained from regulatory sample screening. Although only slight changes in colony-forming units were observed across the pH range and the sugars tested, the morphology was significantly impacted by changes in these growth factors. Incubation between 30 and 50 °C resulted in growth after 24 h, and the growth was slower at ambient temperature and colony formation was most robust at 30 °C. Results of this study suggest that 30 °C may be suitable for recovery of some Cronobacter strains, and minor variations in growth conditions can alter colony morphology and appearance. Expression of unique biological characteristics based on phenotypic observations may be beneficial for differentiating various Cronobacter strains.

  3. Strategies for the Identification and Tracking of Cronobacter Species: An Opportunistic Pathogen of Concern to Neonatal Health

    PubMed Central

    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. PMID:26000266

  4. Cronobacter Illness and Infant Formula

    MedlinePlus

    ... many germs. Breastfeeding helps prevent many kinds of sicknesses among infants. Wash your hands carefully with soap ... do: Breastfeed. Breastfeeding helps prevent many kinds of sicknesses among infants. Almost no cases of Cronobacter sickness ...

  5. 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. PMID:23043820

  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. Prevalence and Characterization of Cronobacter spp. from Various Foods, Medicinal Plants, and Environmental Samples.

    PubMed

    Singh, Niharika; Goel, Gunjan; Raghav, Mamta

    2015-07-01

    Dairy or non-dairy based products were explored to determine the prevalence, molecular characterization, and antibiotic susceptibility of Cronobacter spp. The isolation was done as per ISO 22964:2006 on chromogenic media followed by further confirmation by biochemical- and 16S rRNA-based identification. From 219 samples, the chromogenic agar assay and biochemical tests yielded presumptive 45 isolates. Among them, only 36 isolates showed 282 bp band amplified from ITS-G gene confirming as Cronobacter sakazakii. The Cronobacter spp. prevalence was highest in herbs and spices (34 %) while environmental samples had contamination rates of 23 % indicating plants as a possible reservoir of this pathogen. All the isolates were resistant to β-lactam derivatives (68 %), macrolides (88.6 %), and aminoglycosides (79.9 %) but susceptible to phenicoles (31.6 %) and tetracyclines (15 %) derivatives. The results emphasize the screening of plant materials before their incorporation in food matrices. PMID:25855303

  9. Insights into the Emergent Bacterial Pathogen Cronobacter spp., Generated by Multilocus Sequence Typing and Analysis

    PubMed Central

    Joseph, Susan; Forsythe, Stephen J.

    2012-01-01

    Cronobacter spp. (previously known as Enterobacter sakazakii) is a bacterial pathogen affecting all age groups, with particularly severe clinical complications in neonates and infants. One recognized route of infection being the consumption of contaminated infant formula. As a recently recognized bacterial pathogen of considerable importance and regulatory control, appropriate detection, and identification schemes are required. The application of multilocus sequence typing (MLST) and analysis (MLSA) of the seven alleles atpD, fusA, glnS, gltB, gyrB, infB, and ppsA (concatenated length 3036 base pairs) has led to considerable advances in our understanding of the genus. This approach is supported by both the reliability of DNA sequencing over subjective phenotyping and the establishment of a MLST database which has open access and is also curated; http://www.pubMLST.org/cronobacter. MLST has been used to describe the diversity of the newly recognized genus, instrumental in the formal recognition of new Cronobacter species (C. universalis and C. condimenti) and revealed the high clonality of strains and the association of clonal complex 4 with neonatal meningitis cases. Clearly the MLST approach has considerable benefits over the use of non-DNA sequence based methods of analysis for newly emergent bacterial pathogens. The application of MLST and MLSA has dramatically enabled us to better understand this opportunistic bacterium which can cause irreparable damage to a newborn baby’s brain, and has contributed to improved control measures to protect neonatal health. PMID:23189075

  10. Characterization of a single-chain variable fragment specific to Cronobacter spp. from hybridoma based on outer membrane protein A.

    PubMed

    Chen, Qiming; Tao, Tingting; Bie, Xiaomei; Lu, Fengxia; Li, Yuanhong; Lu, Zhaoxin

    2016-10-01

    Monoclonal antibody and polyclonal antibody specific to Cronobacter spp. had been reported in previous studies. However, the preparation of single-chain variable fragment (scFv) was faster and convenient. Hence, the aim of this study was to construct a scFv using outer membrane protein A (OmpA) of C. sakazakii as antigen. The protein sequences of OmpA of Cronobacter spp. were analyzed first. The results showed protein OmpA with length of 347 amino acids was conserved in Cronobacter genus (94.83%-100% of protein identity) and was greater than that observed for the other genera tested (8.28-91.64% of protein identity). Then, purified protein OmpA expressed in E. coli was used to prepare hybridoma and to construct scFv further. The scFv was named scFvH81 and analyzed by bioinformatics. The model of scFvH81 built by homologous modeling had a good quality (residues in disallowed regions: 3%) and showed that scFvH81 had a standard pocket-like site. Purified scFvH81 was prepared by denaturation and renaturation of inclusion body and it showed a good specificity and its affinity of Ka=2.39×10(6)M(-1). Therefore, it could be used in the detection and the pathogenesis study of Cronobacter spp. PMID:27498230

  11. Growth of Cronobacter spp. under dynamic temperature conditions occurring during cooling of reconstituted powdered infant formula.

    PubMed

    Kandhai, M C; Breeuwer, P; Gorris, L G M; Zwietering, M H; Reij, M W

    2009-12-01

    Reconstituted infant formulae are excellent growth media for Cronobacter spp. (formerly Enterobacter sakazakii) and other microorganisms that may be present in such products. Immediate consumption or rapid cooling and storage at a low temperature are therefore recommended as control measures to prevent microbial growth. Placing a container filled with reconstituted liquid formula in the refrigerator, however, does not mean that the temperature of the liquid is directly the same as the set-point of the refrigerator. This study describes the temperature profiles and methods to predict lag time and possible growth of Cronobacter spp. during the cooling process in three types of containers. The overall heat transfer coefficients (alpha) were determined and were shown to have a very large variability in both household refrigerators and an air-ventilated refrigerator equipped with a fan. A mathematical model was built to predict the growth of Cronobacter spp. under dynamic temperature conditions using three models for the lag time. The various estimations for the lag time had a remarkably strong impact on the predicted growth. The assumption of a constant k-value (k = lag time x specific growth rate = lambda x micro = 2.88) fitted the experimental data best. Predictions taking into account the large variability in heat transfer showed that proliferation of Cronobacter spp. during cooling may be prevented by limiting the volume to be cooled to portion size only, or by reconstituting at temperatures of 25 degrees C or lower. The model may also be used to predict growth in other situations where dynamic temperature conditions exist.

  12. Fully Closed Genome Sequences of Five Type Strains of the Genus Cronobacter and One Cronobacter sakazakii Strain.

    PubMed

    Moine, Deborah; Kassam, Mohamed; Baert, Leen; Tang, Yanjie; Barretto, Caroline; Ngom Bru, Catherine; Klijn, Adrianne; Descombes, Patrick

    2016-01-01

    Cronobacteris associated with infant infections and the consumption of reconstituted infant formula. Here we sequenced and closed six genomes ofC. condimenti(T),C. muytjensii(T),C. universalis(T),C. malonaticus(T),C. dublinensis(T), andC. sakazakiithat can be used as reference genomes in single nucleotide polymorphism (SNP)-based next-generation sequencing (NGS) analysis for source tracking investigations. PMID:27013041

  13. 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.

  14. 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.

  15. Comparative Genotypic and Phenotypic Analysis of Cronobacter Species Cultured from Four Powdered Infant Formula Production Facilities: Indication of Pathoadaptation along the Food Chain

    PubMed Central

    Yan, Qiongqiong; Wang, Juan; Gangiredla, Jayanthi; Cao, Yu; Martins, Marta; Gopinath, Gopal R.; Stephan, Roger; Lampel, Keith; Tall, Ben D.

    2015-01-01

    Cronobacter species are opportunistic pathogens commonly found in the environment. Among the seven Cronobacter species, Cronobacter sakazakii sequence type 4 (ST-4) is predominantly associated with recorded cases of infantile meningitis. This study reports on a 26-month powdered infant formula (PIF) surveillance program in four production facilities located in distinct geographic regions. The objective was to identify the ST(s) in PIF production environments and to investigate the phenotypic features that support their survival. Of all 168 Cronobacter isolates, 133 were recovered from a PIF production environment, 31 were of clinical origin, and 4 were laboratory type strains. Sequence type 1 (n = 84 isolates; 63.9%) was the dominant type in PIF production environments. The majority of these isolates clustered with an indistinguishable pulsotype and persisted for at least an 18-month period. Moreover, DNA microarray results identified two phylogenetic lineages among ST-4 strains tested. Thereafter, the ST-1 and -4 isolates were phenotypically compared. Differences were noted based on the phenotypes expressed by these isolates. The ST-1 PIF isolates produced stronger biofilms at both 28°C and 37°C, while the ST-4 clinical isolates exhibited greater swimming activity and increased binding to Congo red dye. Given the fact that PIF is a low-moisture environment and that the clinical environment provides for an interaction between the pathogen and its host, these differences may be consistent with a form of pathoadaptation. These findings help to extend our current understanding of the epidemiology and ecology of Cronobacter species in PIF production environments. PMID:25911470

  16. Comparative Genotypic and Phenotypic Analysis of Cronobacter Species Cultured from Four Powdered Infant Formula Production Facilities: Indication of Pathoadaptation along the Food Chain.

    PubMed

    Yan, Qiongqiong; Wang, Juan; Gangiredla, Jayanthi; Cao, Yu; Martins, Marta; Gopinath, Gopal R; Stephan, Roger; Lampel, Keith; Tall, Ben D; Fanning, Séamus

    2015-07-01

    Cronobacter species are opportunistic pathogens commonly found in the environment. Among the seven Cronobacter species, Cronobacter sakazakii sequence type 4 (ST-4) is predominantly associated with recorded cases of infantile meningitis. This study reports on a 26-month powdered infant formula (PIF) surveillance program in four production facilities located in distinct geographic regions. The objective was to identify the ST(s) in PIF production environments and to investigate the phenotypic features that support their survival. Of all 168 Cronobacter isolates, 133 were recovered from a PIF production environment, 31 were of clinical origin, and 4 were laboratory type strains. Sequence type 1 (n = 84 isolates; 63.9%) was the dominant type in PIF production environments. The majority of these isolates clustered with an indistinguishable pulsotype and persisted for at least an 18-month period. Moreover, DNA microarray results identified two phylogenetic lineages among ST-4 strains tested. Thereafter, the ST-1 and -4 isolates were phenotypically compared. Differences were noted based on the phenotypes expressed by these isolates. The ST-1 PIF isolates produced stronger biofilms at both 28°C and 37°C, while the ST-4 clinical isolates exhibited greater swimming activity and increased binding to Congo red dye. Given the fact that PIF is a low-moisture environment and that the clinical environment provides for an interaction between the pathogen and its host, these differences may be consistent with a form of pathoadaptation. These findings help to extend our current understanding of the epidemiology and ecology of Cronobacter species in PIF production environments.

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

    PubMed

    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) × 10(3) to (2.1 ± 0.01) × 10(5) 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 (10(8) 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

  18. 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

  19. 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. PMID:17600483

  20. Prevalence and relative risk of Cronobacter spp., Salmonella spp., and Listeria monocytogenes associated with the body surfaces and guts of individual filth flies.

    PubMed

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

    2012-11-01

    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.

  1. 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

  2. Multiplex PCR Assay Targeting a Diguanylate Cyclase-Encoding Gene, cgcA, To Differentiate Species within the Genus Cronobacter

    PubMed Central

    Carter, L.; Lindsey, L. A.; Grim, C. J.; Sathyamoorthy, V.; Jarvis, K. G.; Gopinath, G.; Lee, C.; Sadowski, J. A.; Trach, L.; Pava-Ripoll, M.; McCardell, B. A.; Tall, B. D.

    2013-01-01

    In a comparison to the widely used Cronobacter rpoB PCR assay, a highly specific multiplexed PCR assay based on cgcA, a diguanylate cyclase gene, that identified all of the targeted six species among 305 Cronobacter isolates was designed. This assay will be a valuable tool for identifying suspected Cronobacter isolates from food-borne investigations. PMID:23144142

  3. 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.

  4. 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. PMID:27116956

  5. Cronobacter Species Contamination of Powdered Infant Formula and the Implications for Neonatal Health

    PubMed Central

    Kalyantanda, Gautam; Shumyak, Lyudmila; Archibald, Lennox Kenneth

    2015-01-01

    Cronobacter is a class of Enterobacteriaceae that cause infections in neonates, especially those born prematurely. Over 90% of these infections have been linked epidemiologically to powdered infant formula (PIF). Contamination of PIF can occur at manufacture, reconstitution, or storage of reconstituted product. Intrinsic properties that enable Cronobacter to cause disease include resistance to heat, ultraviolet radiation, oxygen radicals, stomach acids, and pasteurization; an ability to utilize sialic acid (a nutrition additive to PIF that facilitates the organism’s growth and survival), and an exceptional affinity for biofilms in enteral feeding tubes. As part of ongoing endeavors to reduce the incidence of neonatal PIF-associated Cronobacter infections, the World Health Organization and the US Food and Drug Administration have established guidelines for PIF production, preparation for infant feeding, and storage of reconstituted product. PMID:26191519

  6. 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.

  7. 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.

  8. 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.

  9. Inhibition of growth of Enterobacter sakazakii in reconstituted infant formula by the lactoperoxidase system.

    PubMed

    Gurtler, Joshua B; Beuchat, Larry R

    2007-09-01

    Neonatal bacteremia and meningitis caused by the opportunistic pathogen Enterobacter sakazakii have been associated with the consumption of reconstituted powdered infant formula. Lactoperoxidase (LPO), present in mammalian milk, is known to inhibit the growth of enteric pathogens. We undertook a study to determine if the lactoperoxidase system (LPOS) will inhibit the growth of E. sakazakii in a milk-based powdered infant formula reconstituted with water. Initially at 0.04 CFU/ml, E. sakazakii grew to 2.40 to 2.74 log CFU/ml in reconstituted infant formula held at 30 or 37 degrees C for 8 h and to 0.6 log CFU/ ml in formula held for 12 h at 21 degrees C. The pathogen was not detected (less than 1 CFU/227 ml) by enrichment of formula treated with 10 to 30 microg/ml LPO and stored for 24 h at 37 degrees C or 30 microg/ml LPO and stored for 24 h at 30 degrees C. Populations of E. sakazakii, initially at 4.40 log CFU/ml of reconstituted infant formula containing 5 microg/ml LPO, did not increase significantly (P > 0.05) for up to 12 h at 21 and 30 degrees C. Populations either decreased significantly or were unchanged in formula supplemented with 10 microg/ml LPO and stored at 21, 30, or 37 degrees C for up to 24, 8, and 8 h, respectively. Results indicate that LPOS can be used to control the growth of E. sakazakii in reconstituted infant formula, thereby potentially reducing the risk of neonatal infections resulting from consumption of formula that may be contaminated with the pathogen.

  10. Survival of Enterobacter sakazakii in powdered infant formula as affected by composition, water activity, and temperature.

    PubMed

    Gurtler, Joshua B; Beuchat, Larry R

    2007-07-01

    A study was done to determine survival characteristics of Enterobacter sakazakii in four milk-based and two soybean-based powdered infant formulas. A 10-strain mixture of E. sakazakii was inoculated into the six infant formulas at water activity (aw) 0.25 to 0.30, 0.31 to 0.33, and 0.43 to 0.50 to give low (0.80 log CFU/g) and high (4.66 to 4.86 log CFU/g) populations. At an initial population of 0.80 log CFU/g, E. sakazakii was detected by enrichment in six of six, four of six, and one of six formulas stored for 12 months at 4, 21, and 30 degrees C, respectively. In four of six formulas at aw values of 0.25 to 0.30, initially high populations decreased significantly (P < or = 0.05), although by less than 1 log CFU/g, within 6 months at 4 degrees C. Populations decreased significantly in all formulas in the aw range of 0.25 to 0.50 during storage for 1 month at 21 or 30 degrees C and again between 1 and 6 months in most formulas. Significant reductions occurred between 6 and 12 months in some formulas. At all storage temperatures, reductions in populations tended to be greater in formulas at aw 0.43 to 0.50 than in formulas at aw 0.25 to 0.30. The rate of inactivation of E. sakazakii in formulas was not markedly influenced by formula composition. Cells from mucoid and nonmucoid colonies formed by two strains on violet red bile glucose agar supplemented with pyruvate were inoculated into a milk-based powdered infant formula and a soybean-based powdered infant formula having a high aw range of 0.43 to 0.86 and stored at 4, 21, and 30 degrees C for up to 36 weeks. With few exceptions, populations of both strains decreased significantly in both formulas within 2 weeks at all temperatures; rates of death increased with increased aw and storage temperature. The presence of mucoidal extracellular materials on the surface of E. sakazakii cells was not associated with protection against death. This study shows that the retention of viability of E. sakazakii in powdered

  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. PMID:25846909

  12. 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.

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

    PubMed Central

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

    2015-01-01

    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 23827T, 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 104 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. PMID:26060602

  14. 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.

  15. Enterobacter sakazakii in dried infant formulas and milk kitchens of maternity wards in São Paulo, Brazil.

    PubMed

    Palcich, Gabriela; Gillio, Cintia de Moraes; Aragon-Alegro, Lina Casale; Pagotto, Franco J; Farber, Jeffrey M; Landgraf, Mariza; Destro, Maria Teresa

    2009-01-01

    This study was the first conducted in Brazil to evaluate the presence of Enterobacter sakazakii in milk-based powdered infant formula manufactured for infants 0 to 6 months of age and to examine the conditions of formula preparation and service in three hospitals in São Paulo State, Brazil. Samples of dried and rehydrated infant formula, environments of milk kitchens, water, bottles and nipples, utensils, and hands of personnel were analyzed, and E. sakazakii and Enterobacteriaceae populations were determined. All samples of powdered infant formula purchased at retail contained E. sakazakii at <0.3 [corrected] most probable number (MPN)/100 g. In hospital samples, E. sakazakii was found in one unopened formula can (0.3 MPN/100 g) and in the residue from one nursing bottle from hospital A. All other cans of formula from the same lot bought at a retail store contained E. sakazakii at <0.3 [corrected] MPN/100 g. The pathogen also was found in one cleaning sponge from hospital B. Enterobacteriaceae populations ranged from 10(1) to 10(5) CFU/g in cleaning aids and <5 CFU/g in all formula types (dry or rehydrated), except for the sample that contained E. sakazakii, which also was contaminated with Enterobacteriaceae at 5 CFU/g. E. sakazakii isolates were not genetically related. In an experiment in which rehydrated formula was used as the growth medium, the temperature was that of the neonatal intensive care unit (25 degrees C), and the incubation time was the average time that formula is left at room temperature while feeding the babies (up to 4 h), a 2-log increase in levels of E. sakazakii was found in the formula. Visual inspection of the facilities revealed that the hygienic conditions in the milk kitchens needed improvement. The length of time that formula is left at room temperature in the different hospitals while the babies in the neonatal intensive care unit are being fed (up to 4 h) may allow for the multiplication of E. sakazakii and thus may lead to an

  16. Effects of extended dry storage of powdered infant milk formula on susceptibility of Enterobacter sakazakii to hot water and ionizing radiation.

    PubMed

    Osaili, Tareq M; Al-Nabulsi, Anas A; Shaker, Reyad R; Ayyash, Mutamed M; Olaimat, Amin N; Al-Hasan, Ashraf S Abu; Kadora, Khaled M; Holley, Richard A

    2008-05-01

    Infant milk formula has been identified as a potential source of Enterobacter sakazakii, which has been implicated in neonatal meningitis and necrotizing enterocolitis. This study was undertaken to determine whether the length of E. sakazakii storage in powdered infant milk formula (PIMF) affected the ability of the pathogen to survive subsequent reconstitution of the powder with hot water or treatment with gamma radiation. Five E. sakazakii strains were mixed individually with PIMF and kept for up to 12 months at 25 degrees C. After storage PIMF was reconstituted with water at 60 to 100 degrees C or was exposed to < or = 5 kGy of gamma radiation. Without any treatment secondary to drying, E. sakazakii counts decreased < 1 log/g after 1 month but decreased about 4 log/g during storage for 8 to 12 months. Dry storage decreased thermal resistance but increased resistance of E. sakazakii to ionizing radiation in PIMF. Reconstitution of contaminated powder with water at 70 degrees C after 1 month of dry storage reduced E. sakazakii viability slightly, > 2 log/g, and after powder was stored for 12 months all E. sakazakii strains were eliminated. In contrast, desiccation substantially increased the resistance of E. sakazakii strains to ionizing radiation. Although the D-value for E. sakazakii IMF1 following overnight storage in PIMF was 0.98 kGy, > 4 kGy was required to kill 1.5 log/g of the same strain that had survived 12 months in dry PIMF. Results suggested that low-dose irradiation will more effectively eliminate E. sakazakii from PIMF if the treatment is applied shortly after PIMF manufacture.

  17. 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.

  18. 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

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

    PubMed

    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

  20. Performance of Media for Recovering Stressed Cells of Enterobacter sakazakii as Determined Using Spiral Plating and Ecometric Techniques

    PubMed Central

    Gurtler, J. B.; Beuchat, L. R.

    2005-01-01

    A study was done to determine the performance of differential, selective media for supporting resuscitation and colony development by stressed cells of Enterobacter sakazakii. Cells of four strains of E. sakazakii isolated from powdered infant formula were exposed to five stress conditions: heat (55°C for 5 min), freezing (−20°C for 24 h, thawed, frozen again at −20°C for 2 h, thawed), acidic pH (3.54), alkaline pH (11.25), and desiccation in powdered infant formula (water activity, 0.25; 21°C for 31 days). Control and stressed cells were spiral plated on tryptic soy agar supplemented with 0.1% pyruvate (TSAP, a nonselective control medium); Leuschner, Baird, Donald, and Cox (LBDC) agar (a differential, nonselective medium); Oh and Kang agar (OK); fecal coliform agar (FCA); Druggan-Forsythe-Iversen (DFI) medium; violet red bile glucose (VRBG) agar; and Enterobacteriaceae enrichment (EE) agar. With the exception of desiccation-stressed cells, suspensions of stressed cells were also plated on these media and on R&F Enterobacter sakazakii chromogenic plating (RF) medium using the ecometric technique. The order of performance of media for recovering control and heat-, freeze-, acid-, and alkaline-stressed cells by spiral plating was TSAP > LBDC > FCA > OK, VRBG > DFI > EE; the general order for recovering desiccated cells was TSAP, LBDC, FCA, OK > DFI, VRBG, EE. Using the ecometric technique, the general order of growth indices of stressed cells was TSAP, LBDC > FCA > RF, VRBG, OK > DFI, EE. The results indicate that differential, selective media vary greatly in their abilities to support resuscitation and colony formation by stressed cells of E. sakazakii. The orders of performance of media for recovering stressed cells were similar using spiral plating and ecometric techniques, but results from spiral plating should be considered more conclusive. PMID:16332738

  1. A comparative study between overlay method and selective-differential media for recovery of stressed Enterobacter sakazakii cells from infant formula.

    PubMed

    Al-Holy, Murad A; Lin, Mengshi; Al-Qadiri, Hamzah M; Rasco, Barbara A

    2008-02-01

    This study compares the performance of different selective-differential media with the overlay method for recovery of stressed cells of Enterobacter sakazakii from infant formula milk (IFM). Five different selective-differential media were used in this study: OK medium, violet red bile agar (VRBA), Druggan-Forsythe-Iversen agar (DFI), Enterobacteriaceae enrichment (EE) agar, and fecal coliform agar (FCA). Tryptic soy agar supplemented with 0.1% sodium pyruvate (TSAP) was used as a control. The overlay method involved applying a thin layer (8ml) of each of the selective media onto TSAP after spreading a sample onto TSAP. Reconstituted IFM was inoculated by ca 1x10(7)CFU/ml of a mixture of four strains of E. sakazakii and subjected to different stress conditions: heat (55 degrees C for 10min), a freeze-thaw cycle (-20 degrees C for 24h, thawed at room temperature, frozen again at -20 degrees C, and thawed), acidic pH (pH 3.56 for 15min), alkaline pH (pH 11.04 for 15min), and desiccation (E. sakazakii was inoculated onto powdered IFM at a level of ca 1x10(6)CFU/g, held at 21 degrees C, water activity of the inoculated product was 0.29 and examined at 0, 15, and 30d). No major differences were noticed between the control (TSAP) and the overlay methods. However, the overlay method recovered significantly higher numbers of stressed E. sakazakii cells compared to selective-differential media. Also, the selective-differential media exhibited some variability in terms of their capabilities to recover stressed cells of E. sakazakii. Among all the examined selective-differential media, DFI performed better for recovering stressed E. sakazakii cells. This study suggests that the overlay method may serve as a potential alternative to direct selective plating for best recovery of E. sakazakii from IFM.

  2. Development of Multiple-Locus Variable-Number Tandem-Repeat Analysis for the Molecular Subtyping of Enterobacter sakazakii▿

    PubMed Central

    Mullane, N. R.; Ryan, M.; Iversen, C.; Murphy, M.; O'Gaora, P.; Quinn, T.; Whyte, P.; Wall, P. G.; Fanning, S.

    2008-01-01

    The genomic content of Enterobacter sakazakii strain ATCC BAA-894 was analyzed for variable-number tandem repeats (VNTRs). In this study we report the development of a multiple-locus VNTR analysis (MLVA) strategy for the subtyping of E. sakazakii. The method is based on a GeneScan analysis of four VNTR loci labeled with multiple fluorescent dyes. This approach was applied to a collection of 112 isolates representing all 16 of the currently defined E. sakazakii biogroups. MLVA successfully discriminated among these isolates and compared favorably with pulsed-field gel electrophoresis. The method was relatively fast and easy to perform. The potential value of MLVA as an epidemiological tool is discussed. PMID:18083860

  3. A comparison of polymerase chain reaction and international organization for standardization methods for determination of Enterobacter sakazakii contamination of infant formulas from Chinese mainland markets.

    PubMed

    Ye, Yingwang; Wu, Qingping; Yao, Lin; Dong, Xiaohui; Wu, Kui; Zhang, Jumei

    2009-12-01

    Enterobacter sakazakii is an emerging foodborne pathogen associated with meningitis, necrotizing enterocolitis, and sepsis in infants. One of the main transmission vehicles is the commercially available infant formulas. To provide efficient options and direction for detecting E. sakazakii in infant formulas, evaluation of different polymerase chain reaction (PCR) assays targeting the 16S-23S rDNA internal transcribed spacer (ITS), the ompA gene, and the alpha-1,4-glucosidase gene (gluA) of this organism, were compared to the International Organization for Standardization (ISO) method for detecting E. sakazakii in the 243 commercial infant formula samples. Twelve samples were found to be positive for E. sakazakii by all the PCR assays used, followed by sequencing of PCR products. Ten samples were found to be positive by the ISO method, and all 10 gave positive signals for all the PCR amplifications. In contrast, four false-positive results were generated by single-PCR of the ITS region and one false-positive result targeting the ompA gene, while two false-negative results occurred with the ISO method. Combined with selective enrichment step(s), duplex-PCR targeting ITS and ompA and targeting ompA and gluA genes or single-PCR of the gluA gene can be used to test for contamination by E. sakazakii in infant formulas before they enter the market. PCR techniques will be helpful for routine monitoring and risk assessment for large-scale screenings.

  4. Comparison of three chromogenic media and evaluation of two molecular-based identification systems for the detection of Enterobacter sakazakii from environmental samples from infant formulae factories.

    PubMed

    Derzelle, Sylviane; Dilasser, Françoise; Maladen, Véronique; Soudrie, Nicole; Leclercq, Alexandre; Lombard, Bertrand; Lafarge, Veŕonique

    2007-07-01

    Enterobacter sakazakii is an occasional contaminant of powdered infant formula that can cause rare but severe foodborne infections in infants. To determine optimal methods for the detection and identification of E. sakazakii, 38 naturally contaminated samples from infant formulae factories were analyzed by two PCR-based methods and by a method (TS 22964/RM 210) developed by the International Organization for Standardization and the International Dairy Federation (ISO-IDF) using three different commercial chromogenic agars. The ISO-IDF method includes two enrichment steps, plating of the second enrichment broth on E. sakazakii isolation agar (a chromogenic selective agar), picking of five typical colonies for transfer onto tryptone soy agar, and subsequent confirmation of yellow-pigmented colonies by biochemical characterization. Twenty-two of the 38 samples were positive by the culture method. E. sakazakii isolation agar (ESIA; AES Laboratoires), COMPASS agar (Biokar Diagnostics), and Druggan-Forsythe-Iversen agar (Oxoid) compared favorably with violet red bile glucose agar (VRBG, a selective medium for Enterobacteriaceae), with positive predictive values of 86.96, 88, and 74.07%, respectively, in contrast to 47.83% for VRBG. One additional positive sample was detected using the nonpatented real-time PCR method evaluated, and those results were in 97.3% concordance with the ISO-IDF results. Some discrepancies between the results of the DuPont Qualicon BAX system and those of the ISO-IDF method could be explained by heterogeneity of contamination and sampling. Thus, both PCR-based systems were suitable for detecting and specifically identifying E. sakazakii within 1 to 2 days, and COMPASS agar and ESIA could be used interchangeably as a first-step medium to isolate presumptive E. sakazakii colonies.

  5. A comparison of polymerase chain reaction and international organization for standardization methods for determination of Enterobacter sakazakii contamination of infant formulas from Chinese mainland markets.

    PubMed

    Ye, Yingwang; Wu, Qingping; Yao, Lin; Dong, Xiaohui; Wu, Kui; Zhang, Jumei

    2009-12-01

    Enterobacter sakazakii is an emerging foodborne pathogen associated with meningitis, necrotizing enterocolitis, and sepsis in infants. One of the main transmission vehicles is the commercially available infant formulas. To provide efficient options and direction for detecting E. sakazakii in infant formulas, evaluation of different polymerase chain reaction (PCR) assays targeting the 16S-23S rDNA internal transcribed spacer (ITS), the ompA gene, and the alpha-1,4-glucosidase gene (gluA) of this organism, were compared to the International Organization for Standardization (ISO) method for detecting E. sakazakii in the 243 commercial infant formula samples. Twelve samples were found to be positive for E. sakazakii by all the PCR assays used, followed by sequencing of PCR products. Ten samples were found to be positive by the ISO method, and all 10 gave positive signals for all the PCR amplifications. In contrast, four false-positive results were generated by single-PCR of the ITS region and one false-positive result targeting the ompA gene, while two false-negative results occurred with the ISO method. Combined with selective enrichment step(s), duplex-PCR targeting ITS and ompA and targeting ompA and gluA genes or single-PCR of the gluA gene can be used to test for contamination by E. sakazakii in infant formulas before they enter the market. PCR techniques will be helpful for routine monitoring and risk assessment for large-scale screenings. PMID:19743923

  6. 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.

  7. Evaluation of a revised U.S. Food and Drug Administration method for the detection of Cronobacter in powdered infant formula: a collaborative study.

    PubMed

    Chen, Yi; Noe, Kathy E; Thompson, Sandra; Elems, Carol A; Brown, Eric W; Lampel, Keith A; Hammack, Thomas S

    2012-06-01

    A revised U.S. Food and Drug Administration (FDA) method for the isolation and detection of Cronobacter from powdered infant formula was recently developed, which combines real-time PCR, chromogenic agars, and RAPID ID 32E biochemical tests. This method provides an expedient analysis within 24 to 48 h. A collaborative validation study involving four different laboratories was conducted to compare the revised FDA method with the reference FDA method using casein- and soy-based powdered infant formula inoculated with different Cronobacter strains. Valid results from 216 test portions and controls from collaborating laboratories were obtained and showed that the revised FDA method performed significantly better than the reference FDA method. Newly revised PCR protocols and VITEK 2 were also evaluated to be integrated into the complete detection procedure.

  8. A Cronobacter turicensis O1 Antigen-Specific Monoclonal Antibody Inhibits Bacterial Motility and Entry into Epithelial Cells

    PubMed Central

    Lehner, Angelika; Dietrich, Richard; Kleinsteuber, Ina; Canals, Rocío; Zurfluh, Katrin; Weiner, Kerstin; Märtlbauer, Erwin

    2014-01-01

    Cronobacter turicensis is an opportunistic foodborne pathogen that can cause a rare but sometimes lethal infection in neonates. Little is known about the virulence mechanisms and intracellular lifestyle of this pathogen. In this study, we developed an IgG monoclonal antibody (MAb; MAb 2G4) that specifically recognizes the O1 antigen of C. turicensis cells. The antilipopolysaccharide antibody bound predominantly monovalently to the O antigen and reduced bacterial growth without causing cell agglutination. Furthermore, binding of the antibody to the O1 antigen of C. turicensis cells caused a significant reduction of the membrane potential which is required to energize flagellar rotation, accompanied by a decreased flagellum-based motility. These results indicate that binding of IgG to the O antigen of C. turicensis causes a direct antimicrobial effect. In addition, this feature of the antibody enabled new insight into the pathogenicity of C. turicensis. In a tissue culture infection model, pretreatment of C. turicensis with MAb 2G4 showed no difference in adhesion to human epithelial cells, whereas invasion of bacteria into Caco-2 cells was significantly inhibited. PMID:25534937

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

    PubMed Central

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

    2016-01-01

    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 23827T 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. PMID:26725701

  10. Prediction of a Required Log Reduction with Probability for Enterobacter sakazakii during High-Pressure Processing, Using a Survival/Death Interface Model▿

    PubMed Central

    Koseki, Shige; Matsubara, Maki; Yamamoto, Kazutaka

    2009-01-01

    A probabilistic model for predicting Enterobacter sakazakii inactivation in trypticase soy broth (TSB) and infant formula (IF) by high-pressure processing was developed. The modeling procedure is based on a previous model (S. Koseki and K. Yamamoto, Int. J. Food Microbiol. 116:136-143, 2007) that describes the probability of death of bacteria. The model developed in this study consists of a total of 300 combinations of pressure (400, 450, 500, 550, or 600 MPa), pressure-holding time (1, 3, 5, 10, or 20 min), temperature (25 or 40°C), inoculum level (3, 5, or 7 log10 CFU/ml), and medium (TSB or IF), with each combination tested in triplicate. For each replicate response of E. sakazakii, survival and death were scored with values of 0 and 1, respectively. Data were fitted to a logistic regression model in which the medium was treated as a dummy variable. The model predicted that the required pressure-holding times at 500 MPa for a 5-log reduction in IF with 90% achievement probability were 26.3 and 7.9 min at 25 and 40°C, respectively. The probabilities of achieving 5-log reductions in TSB and IF by treatment with 400 MPa at 25°C for 10 min were 92 and 3%, respectively. The model enabled the identification of a minimum processing condition for a required log reduction, regardless of the underlying inactivation kinetics pattern. Simultaneously, the probability of an inactivation effect under the predicted processing condition was also provided by taking into account the environmental factors mentioned above. PMID:19201951

  11. Prediction of a required log reduction with probability for Enterobacter sakazakii during high-pressure processing, using a survival/death interface model.

    PubMed

    Koseki, Shige; Matsubara, Maki; Yamamoto, Kazutaka

    2009-04-01

    A probabilistic model for predicting Enterobacter sakazakii inactivation in trypticase soy broth (TSB) and infant formula (IF) by high-pressure processing was developed. The modeling procedure is based on a previous model (S. Koseki and K. Yamamoto, Int. J. Food Microbiol. 116:136-143, 2007) that describes the probability of death of bacteria. The model developed in this study consists of a total of 300 combinations of pressure (400, 450, 500, 550, or 600 MPa), pressure-holding time (1, 3, 5, 10, or 20 min), temperature (25 or 40 degrees C), inoculum level (3, 5, or 7 log(10) CFU/ml), and medium (TSB or IF), with each combination tested in triplicate. For each replicate response of E. sakazakii, survival and death were scored with values of 0 and 1, respectively. Data were fitted to a logistic regression model in which the medium was treated as a dummy variable. The model predicted that the required pressure-holding times at 500 MPa for a 5-log reduction in IF with 90% achievement probability were 26.3 and 7.9 min at 25 and 40 degrees C, respectively. The probabilities of achieving 5-log reductions in TSB and IF by treatment with 400 MPa at 25 degrees C for 10 min were 92 and 3%, respectively. The model enabled the identification of a minimum processing condition for a required log reduction, regardless of the underlying inactivation kinetics pattern. Simultaneously, the probability of an inactivation effect under the predicted processing condition was also provided by taking into account the environmental factors mentioned above.

  12. Multiplex PCR and a chromogenic DNA macroarray for the detection of Listeria monocytogens, Staphylococcus aureus, Streptococcus agalactiae, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio parahaemolyticus, Salmonella spp. and Pseudomonas fluorescens in milk and meat samples.

    PubMed

    Chiang, Yu-Cheng; Tsen, Hau-Yang; Chen, Hsin-Yen; Chang, Yu-Hsin; Lin, Chien-Ku; Chen, Chih-Yuan; Pai, Wan-Yu

    2012-01-01

    Food products, such as milk and meat products including cheese, milk powder, fermented milk, sausage, etc. are susceptible to the contamination by pathogenic and deteriorative bacteria. These bacteria include Listeria monocytogens, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Salmonella spp., Vibrio parahaemolyticus, Streptococcus agalactiae and Pseudomonas fluorescens, etc. Traditional methods for the detection of these microorganisms are laborious and time consuming. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and PCR primers for the detection of aforementioned microorganisms. By using two sets of multiplex PCR, followed by a chromogenic macroarray system, these organisms in milk or other food products could be simultaneously detected. When the system was used for the inspection of milk or meat homogenate containing 10(0) target cells per milliliter or gram of the sample, all these bacterial species could be identified after an 8h pre-enrichment step. The system consisting of a multiplex PCR step followed by macroarray allowed us to detect multiple target bacterial species simultaneously without the use of agarose gel electrophoresis. Compared to the commonly used multiplex PCR method, this approach has the additional advantage of detecting more bacterial strains because some bacterial strains generate PCR products with the same molecular sizes which can be differentiated by macroarray but not by electrophoresis. PMID:22101309

  13. 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 Central

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

    2014-01-01

    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 601/05T = LMG 24057T = DSM 19144T) and Franconibacter helveticus comb. nov. (type strain 513/05T = LMG 23732T = DSM 18396T), respectively, and Enterobacter turicensis in the genus Siccibacter gen. nov. as Siccibacter turicensis comb. nov. (type strain 508/05T = LMG 23730T = DSM 18397T). PMID:25028159

  14. 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.

  15. 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

    2012-09-01

    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.

  16. 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

    2012-09-01

    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. PMID:22884171

  17. 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.

  18. 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). PMID:26893037

  19. 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).

  20. 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.

  1. Safety aspects in preparation and handling of infant food.

    PubMed

    Turck, Dominique

    2012-01-01

    Powdered infant formula (PIF) can become contaminated during production with harmful bacteria such as Cronobacter spp. (formerly Enterobacter sakazakii). Inadequate conditions of preparation and handling of PIF can therefore exacerbate the risk of severe infection, especially in preterm infants. The WHO emphasized three main interventions for preparation and handling of PIF: (1) to dilute the powdered milk in water at a temperature of at least 70°C to inactivate Cronobacter spp.; (2) to consume milk right after each preparation, and (3) to store reconstituted milk at <5°C. The European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and the French Food Safety Agency (Afssa) disagree with the heating process because of possible adverse effects on nutrients and the risk of severe burns. In home settings, PIF should be prepared fresh for each meal and be kept warm in bottle warmers or thermos bottles. In institutional settings, written guidelines for preparation and handling of PIF should be established. The use of sterile liquid formula is encouraged for healthy newborn infants in maternity wards; PIF should be prepared on a daily basis. Safe infant feeding involves the production of microbiologically cleaner infant formula by industry, and both education and support for the caregivers in preparing and handling the formula. PMID:22699771

  2. Safety aspects in preparation and handling of infant food.

    PubMed

    Turck, Dominique

    2012-01-01

    Powdered infant formula (PIF) can become contaminated during production with harmful bacteria such as Cronobacter spp. (formerly Enterobacter sakazakii). Inadequate conditions of preparation and handling of PIF can therefore exacerbate the risk of severe infection, especially in preterm infants. The WHO emphasized three main interventions for preparation and handling of PIF: (1) to dilute the powdered milk in water at a temperature of at least 70°C to inactivate Cronobacter spp.; (2) to consume milk right after each preparation, and (3) to store reconstituted milk at <5°C. The European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and the French Food Safety Agency (Afssa) disagree with the heating process because of possible adverse effects on nutrients and the risk of severe burns. In home settings, PIF should be prepared fresh for each meal and be kept warm in bottle warmers or thermos bottles. In institutional settings, written guidelines for preparation and handling of PIF should be established. The use of sterile liquid formula is encouraged for healthy newborn infants in maternity wards; PIF should be prepared on a daily basis. Safe infant feeding involves the production of microbiologically cleaner infant formula by industry, and both education and support for the caregivers in preparing and handling the formula.

  3. 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.

  4. P-glycoprotein induction by breast milk attenuates intestinal inflammation in experimental necrotizing enterocolitis

    PubMed Central

    Guner, Yigit S.; Franklin, Ashanti L.; Chokshi, Nikunj K.; Castle, Shannon L.; Pontarelli, Elizabeth; Wang, Jin; Wang, Larry; Prasadarao, Nemani V.; Upperman, Jeffrey S.; Grishin, Anatoly V.; Ford, Henri R.

    2014-01-01

    P-glycoprotein (Pgp), a product of the multi-drug resistance gene MDR1a, is a broad specificity efflux ATP cassette transmembrane transporter that is predominantly expressed in epithelial tissues. Because mdr1a−/− mice tend to develop spontaneous colitis in bacteria-dependent manner, Pgp is believed to have a role in protection of the intestinal epithelium from luminal bacteria. Here we demonstrate that levels of Pgp in the small intestine of newborn rodents dramatically increase during breastfeeding, but not during formula feeding (FF). In rats and mice, levels of intestinal Pgp peak on days 3–7 and 1–5 of breastfeeding, respectively. The mdr1a−/− neonatal mice subjected to FF, hypoxia, and hypothermia have significantly higher incidence and pathology, as well as significantly earlier onset of necrotizing enterocolitis (NEC) than congenic wild type mice. Breast-fed mdr1a−/− neonatal mice are also more susceptible to intestinal damage caused by the opportunistic pathogen Cronobacter sakazakii that has been associated with hospital outbreaks of NEC. Breast milk, but not formula, induces Pgp expression in enterocyte cell lines in a dose- and time-dependent manner. High levels of ectopically expressed Pgp protect epithelial cells in vitro from apoptosis induced by C. sakazakii. Taken together, these results show that breast milk-induced expression of Pgp may have a role in the protection of the neonatal intestinal epithelium from injury associated with nascent bacterial colonization. PMID:21788941

  5. 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.

  6. 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. PMID:26613918

  7. 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.

  8. 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...

  9. 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

  10. 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

  11. 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.

  12. Short communication: radio frequency dielectric heating of nonfat dry milk affects solubility and whey protein nitrogen index.

    PubMed

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

    2013-03-01

    The US infant formula market is estimated at over $3.5 billion, of which 75% are dairy-based formulas. Dried dairy powders pose a significant food safety risk, with Cronobacter sakazakii and Salmonella spp. being pathogens of particular concern. Radio frequency dielectric heating (RFDH) can provide rapid, uniform heat treatment of dry powders; thus, it potentially may be used as a postprocess lethality treatment for nonfat dry milk (NDM) or powdered infant formula. Because RFDH is a heat treatment, the functionality of the NDM may be altered and should be evaluated. High heat- and low heat-NDM were RFDH processed at temperatures ranging from 75 to 90°C for 5 to 125 min. Products were then assessed for whey protein nitrogen index (WPNI), solubility, and color. In low heat-NDM, RFDH decreased WPNI and solubility if the process was done at ≥ 80°C; however, in high heat-NDM, RFDH had a greater effect on solubility than WPNI and some color properties were altered. Further investigation of RFDH is merited to validate its application as a pathogen control process for NDM across processing parameters that result in acceptable functional properties for infant formula and other food products containing NDM.

  13. 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. PMID:26187843

  14. Extended-spectrum-β-lactamase-producing Enterobacteriaceae isolated from vegetables imported from the Dominican Republic, India, Thailand, and Vietnam.

    PubMed

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

    2015-05-01

    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.

  15. 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.

  16. 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

  17. Identification of bacterial endophytes associated with traditional medicinal plant Tridax procumbens Linn.

    PubMed Central

    Preveena, Jagadesan; Bhore, Subhash J.

    2013-01-01

    Background: In traditional medicine, Tridax procumbens Linn. is used in the treatment of injuries and wounds. The bacterial endophytes (BEs) of medicinal plants could produce medicinally important metabolites found in their hosts; and hence, the involvement of BEs in conferring wound healing properties to T. Procumbens cannot be ruled out. But, we do not know which types of BEs are associated with T. Procumbens. Objective: The objective of this study was to investigate the fast growing and cultivable BEs associated with T. procumbens. Materials and Methods: Leaves and stems of healthy T. Procumbens plants were collected and cultivable BEs were isolated from surface-sterilized leaf and stem tissue samples using Luria-Bertani (LB) agar (medium) at standard conditions. A polymerase chain reaction was employed to amplify 16S rRNA coding gene fragments from the isolates. Cultivable endophytic bacterial isolates (EBIs) were identified using 16S rRNA gene nucleotide sequence similarity based method of bacterial identification. Results: Altogether, 50 culturable EBIs were isolated. 16S rRNA gene nucleotide sequences analysis using the Basic Local Alignment Search Tool (BLAST) revealed identities of the EBIs. Analysis reveals that cultivable Bacillus spp., Cronobacter sakazakii, Enterobacter spp., Lysinibacillus sphaericus, Pantoea spp., Pseudomonas spp. and Terribacillus saccharophilus are associated with T. Procumbens. Conclusion: Based on the results, we conclude that 24 different types of culturable BEs are associated with traditionally used medicinal plant, T. Procumbens, and require further study. PMID:24501447

  18. In vitro and in vivo examination of anticolonization of pathogens by Lactobacillus paracasei FJ861111.1.

    PubMed

    Deng, Kan; Chen, Tingtao; Wu, Qinglong; Xin, Hongbo; Wei, Qiang; Hu, Ping; Wang, Xiaolei; Wang, Xin; Wei, Hua; Shah, Nagendra P

    2015-10-01

    Very limited information exists on the exclusion of pathogens by probiotics in the gut of the host challenged with pathogens. In this study, we tested probiotic characteristics in vitro and anticolonization ability of Lactobacillus paracasei FJ861111.1 in mice infected with selected pathogenic microorganisms. The in vitro results indicated that L. paracasei FJ861111.1 had a high survival in acidic conditions at pH 2.5 and bile salt concentration at 0.3%, and strong inhibition ability against common pathogens including Shigella dysenteriae, Staphylococcus aureus, Cronobacter sakazakii, Escherichia coli, and Candida albicans. The cell adhesion assays showed that L. paracasei FJ861111.1 exhibited strong adherence to HT-29 cells and excluded the adhesion of selected food-borne pathogens to HT-29 cells. The in vivo results showed that fermented milk with L. paracasei and viili (a Nordic yogurt product) significantly improved the population of total bacteria and of Lactobacillus in the feces of mice, and significantly inhibited the colonization of C. albicans to the intestines of mice post-C. albicans infection. Thus, it appears that this strain could be used as a probiotic organism for manufacturing functional fermented milk. PMID:26254535

  19. 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. PMID:25763038

  20. 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

  1. 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.

  2. 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.

  3. Bioengineered Nisin A Derivatives with Enhanced Activity against Both Gram Positive and Gram Negative Pathogens

    PubMed Central

    Field, Des; Begley, Maire; O’Connor, Paula M.; Daly, Karen M.; Hugenholtz, Floor; Cotter, Paul D.; Hill, Colin; Ross, R. Paul

    2012-01-01

    Nisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazakii. This is the first report of derivatives of nisin, or indeed any lantibiotic, with enhanced antimicrobial activity against both Gram positive and Gram negative bacteria. PMID:23056510

  4. 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.

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

    PubMed

    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

  6. 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

  7. 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. PMID:21902929

  8. Neonatal enteral feeding tubes as loci for colonisation by members of the Enterobacteriaceae

    PubMed Central

    2009-01-01

    Background The objective of this study was to determine whether neonatal nasogastric enteral feeding tubes are colonised by the opportunistic pathogen Cronobacter spp. (Enterobacter sakazakii) and other Enterobacteriaceae, and whether their presence was influenced by the feeding regime. Methods One hundred and twenty-nine tubes were collected from two neonatal intensive care units (NICU). A questionnaire on feeding regime was completed with each sample. Enterobacteriaceae present in the tubes were identified using conventional and molecular methods, and their antibiograms determined. Results The neonates were fed breast milk (16%), fortified breast milk (28%), ready to feed formula (20%), reconstituted powdered infant formula (PIF, 6%), or a mixture of these (21%). Eight percent of tubes were received from neonates who were 'nil by mouth'. Organisms were isolated from 76% of enteral feeding tubes as a biofilm (up to 107 cfu/tube from neonates fed fortified breast milk and reconstituted PIF) and in the residual lumen liquid (up to 107 Enterobacteriaceae cfu/ml, average volume 250 μl). The most common isolates were Enterobacter cancerogenus (41%), Serratia marcescens (36%), E. hormaechei (33%), Escherichia coli (29%), Klebsiella pneumoniae (25%), Raoultella terrigena (10%), and S. liquefaciens (12%). Other organisms isolated included C. sakazakii (2%),Yersinia enterocolitica (1%),Citrobacter freundii (1%), E. vulneris (1%), Pseudomonas fluorescens (1%), and P. luteola (1%). The enteral feeding tubes were in place between < 6 h (22%) to > 48 h (13%). All the S. marcescens isolates from the enteral feeding tubes were resistant to amoxicillin and co-amoxiclav. Of additional importance was that a quarter of E. hormaechei isolates were resistant to the 3rd generation cephalosporins ceftazidime and cefotaxime. During the period of the study, K. pneumoniae and S. marcescens caused infections in the two NICUs. Conclusion This study shows that neonatal enteral feeding tubes

  9. 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. PMID:26100713

  10. In vitro fermentation of fructooligosaccharides with human gut bacteria.

    PubMed

    Mao, Bingyong; Li, Dongyao; Zhao, Jianxin; Liu, Xiaoming; Gu, Zhennan; Chen, Yong Q; Zhang, Hao; Chen, Wei

    2015-03-01

    Fructooligosaccharides (FOS) are one of the most studied prebiotics, selectively stimulating the growth of health-promoting bacteria in the host. However, there is increasing evidence that commensal gut bacteria, such as Bacteroides fragilis, Clostridium butyricum, Enterobacter cloacae, and even the pathogenic Escherichia coli BEN2908, are also able to metabolize FOS in vitro, and in some cases, FOS displayed adverse effects. Therefore, it is necessary to identify FOS-metabolizing species that are present in the gut. Unlike previous studies focusing on individual strains, this study used the traditional culture method combined with an alignment search on the gut bacteria database established from the Human Microbiome Project (HMP). The alignment results showed that homologous proteins for FOS transporters and glycosidases were distributed in 237 of the 453 strains of gut bacteria. La506 msmK encoding the ATP-binding protein and Aec45 fosGH1 encoding glycoside hydrolase were most widely distributed, in 155 and 55 strains, respectively. Seven of eight strains with both transporters and glycosidases were proven to be capable of metabolizing FOS, while five strains without either transporters or glycosidases were not. Fifteen species isolated from human feces and 11 species from the alignment search were identified to be FOS-metabolizing, of which Cronobacter sakazakii, Marvinbryantia formatexigens, Ruminococcus gnavus, and Weissella paramesenteroides are reported here for the first time. Thus, alignment search combined with the culture method is an effective method for obtaining a global view of the FOS-metabolizing bacteria in the gut and will be helpful in further investigating the relationship between FOS and human gut bacteria.

  11. 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

  12. 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

  13. 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

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

    PubMed

    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.

  15. 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.

  16. 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.

  17. Prevalence of foodborne pathogens in retailed foods in Thailand.

    PubMed

    Ananchaipattana, Chiraporn; Hosotani, Yukie; Kawasaki, Susumu; Pongsawat, Sirikae; Latiful, Bari Md; Isobe, Seiichiro; Inatsu, Yasuhiro

    2012-09-01

    The consumption of foodborne pathogens contaminated in food is one of the major causes of diarrheal diseases in Thailand. The objective of this study was to evaluate the prevalence and types of contaminating bacteria in retailed foodstuffs in Thailand. Food from four categories (137 samples total), including meat (51 samples), vegetables (38 samples), fish or seafood (37 samples), and fermented food (11 samples), was purchased randomly from seven different open-markets and seven supermarkets in Thailand from August 2010 to March 2011. Seven types of major foodborne pathogens were identified using conventional culture methods. Approximately 80% of meat samples tested was contaminated with Salmonella spp. In contrast, the Salmonella spp. contamination rate of vegetable (5%) or fermented food (9%) samples was comparatively low. Six strains of Cronobacter sakazakii and two strains of Yersinia enterocolitica were also isolated. A substantially higher rate of contamination by Bacillus cereus was observed in fermented food (82%) than in samples of meat (2%) and fish or seafood (5%). Seven Listeria spp. isolates were obtained from meat and fish or seafood samples. Approximately 39% of samples tested were found to be contaminated with Staphylococcus spp. (54 isolates). The rate of bacterial contamination of meat did not depend on the type of market. However, the contamination rate of Staphylococcus spp. in vegetables was higher in open markets than in supermarkets, and the contamination rate of Salmonella spp. and Staphylococcus spp. in fish or seafood samples purchased in open markets was likewise higher than in those purchased in supermarkets. Therefore, improvement of hygienic practices throughout the food chain may be required to reduce the risk of food poisoning.

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

    PubMed

    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

  19. Comparison of Media for the Isolation of Enterobacter sakazakii▿

    PubMed Central

    Iversen, Carol; Forsythe, Stephen J.

    2007-01-01

    Enterobacter sakazakii is associated with neonatal infections and is occasionally present at low levels (<1 CFU/g) in powdered infant formula milk (IFM). It has been previously reported that some E. sakazakii strains do not grow in standard media for Enterobacteriaceae and coliform bacteria; therefore, a reliable method is needed for recovery of the organism. Three E. sakazakii enrichment broths—Enterobacteriaceae enrichment broth (EE), E. sakazakii selective broth (ESSB), and modified lauryl sulfate broth (mLST)—were compared with a novel broth designed for maximum recovery of E. sakazakii, E. sakazakii enrichment broth (ESE). One hundred seventy-seven strains (100%) grew in ESE, whereas between 2 and 6% of strains did not grow in EE, mLST, or ESSB. E. sakazakii possesses α-glucosidase activity, and a number of selective, chromogenic agars for E. sakazakii isolation based on this enzyme have been developed. E. sakazakii isolation agar produced fewer false-positive colonies than did Druggan-Forsythe-Iversen agar. However, the latter supported the growth of more E. sakazakii strains. It was also determined that 2% of E. sakazakii strains did not produce yellow pigmentation on tryptone soya agar at 25°C, a characteristic frequently cited in the identification of E. sakazakii. The recovery of desiccated E. sakazakii (0.2 to 2000 CFU/25 g) from powdered IFM in the presence of a competing flora was determined with various enrichment broths and differential selective media. Current media designed for the isolation and presumptive identification of E. sakazakii do not support the growth of all currently known E. sakazakii phenotypes; therefore, improvements in the proposed methods are desirable. PMID:17071794

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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...

  6. 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...

  7. 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

  8. The response of foodborne pathogens to osmotic and desiccation stresses in the food chain.

    PubMed

    Burgess, Catherine M; Gianotti, Andrea; Gruzdev, Nadia; Holah, John; Knøchel, Susanne; Lehner, Angelika; Margas, Edyta; Esser, Stephan Schmitz; Sela Saldinger, Shlomo; Tresse, Odile

    2016-03-16

    In combination with other strategies, hyperosmolarity and desiccation are frequently used by the food processing industry as a means to prevent bacterial proliferation, and particularly that of foodborne pathogens, in food products. However, it is increasingly observed that bacteria, including human pathogens, encode mechanisms to survive and withstand these stresses. This review provides an overview of the mechanisms employed by Salmonella spp., Shiga toxin producing E. coli, Cronobacter spp., Listeria monocytogenes and Campylobacter spp. to tolerate osmotic and desiccation stresses and identifies gaps in knowledge which need to be addressed to ensure the safety of low water activity and desiccated food products.

  9. 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

    2015-05-01

    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.

  10. 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

    2015-05-01

    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. PMID:25679309

  11. 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.

  12. 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

  13. Enterobacteriaceae in dehydrated powdered infant formula manufactured in Indonesia and Malaysia.

    PubMed

    Estuningsih, Sri; Kress, Claudia; Hassan, Abdulwahed A; Akineden, Omer; Schneider, Elisabeth; Usleber, Ewald

    2006-12-01

    To determine the occurrence of Salmonella and Shigella in infant formula from Southeast Asia, 74 packages of dehydrated powdered infant follow-on formula (recommended age, > 4 months) from five different manufacturers, four from Indonesia and one from Malaysia, were analyzed. None of the 25-g test portions yielded Salmonella or Shigella. However, further identification of colonies growing on selective media used for Salmonella and Shigella detection revealed the frequent occurrence of several other Enterobacteriaceae species. A total of 35 samples (47%) were positive for Enterobacteriaceae. Ten samples (13.5%) from two Indonesian manufacturers yielded Enterobacter sakazakii. Other Enterobacteriaceae isolated included Pantoea spp. (n = 12), Escherichia hermanii (n = 10), Enterobacter cloacae (n = 8), Klebsiella pneumoniae subsp. pneumoniae (n = 3), Citrobacter spp. (n = 2), Serratia spp. (n = 2), and Escherichia coli (n = 2). To our knowledge, this is the first report to describe the contamination of dehydrated powdered infant formula from Indonesia with E. sakazakii and several other Enterobacteriaceae that could be opportunistic pathogens. Improper preparation and conservation of these products could result in a health risk for infants in Indonesia.

  14. Microbial selenite reduction with organic carbon and electrode as sole electron donor by a bacterium isolated from domestic wastewater.

    PubMed

    Nguyen, Van Khanh; Park, Younghyun; Yu, Jaecheul; Lee, Taeho

    2016-07-01

    Selenium is said to be multifaceted element because it is essential at a low concentration but very toxic at an elevated level. For the purpose of screening a potential microorganism for selenite bioremediation, we isolated a bacterium, named strain THL1, which could perform both heterotrophic selenite reduction, using organic carbons such as acetate, lactate, propionate, and butyrate as electron donors under microaerobic condition, and electrotrophic selenite reduction, using an electrode polarized at -0.3V (vs. standard hydrogen electrode) as the sole electron donor under anaerobic condition. This bacterium determined to be a new strain of the genus Cronobacter, could remove selenite with an efficiency of up to 100%. This study is the first demonstration on a pure culture could take up electrons from an electrode to perform selenite reduction. The selenium nanoparticles produced by microbial selenite reduction might be considered for recovery and use in the nanotechnology industry. PMID:27099943

  15. Isothermal DNA amplification strategies for duplex microorganism detection.

    PubMed

    Santiago-Felipe, Sara; Tortajada-Genaro, Luis Antonio; Morais, Sergi; Puchades, Rosa; Maquieira, Ángel

    2015-05-01

    A valid solution for micro-analytical systems is the selection of a compatible amplification reaction with a simple, highly-integrated efficient design that allows the detection of multiple genomic targets. Two approaches under isothermal conditions are presented: recombinase polymerase amplification (RPA) and multiple displacement amplification (MDA). Both methods were applied to a duplex assay specific for Salmonella spp. and Cronobacter spp., with excellent amplification yields (0.2-8.6 · 10(8) fold). The proposed approaches were successfully compared to conventional PCR and tested for the milk sample analysis as a microarray format on a compact disc (support and driver). Satisfactory results were obtained in terms of resistance to inhibition, selectivity, sensitivity (10(1)-10(2)CFU/mL) and reproducibility (below 12.5%). The methods studied are efficient and cost-effective, with a high potential to automate microorganisms detection by integrated analytical systems working at a constant low temperature.

  16. 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. PMID:23141821

  17. 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.

  18. 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

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

    PubMed Central

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

    2013-01-01

    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. 1×109 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. PMID:24293788

  20. 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.

  1. FIRST REPORT OF METALLO-β-LACTAMASES PRODUCING Enterobacter spp. STRAINS FROM VENEZUELA

    PubMed Central

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

    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 bla VIM-2 in two E. cloacae and one Enterobacter sp., which also showed multiple mechanisms of resistance. Both E. cloacae showed bla TEM-1, but only one showed bla CTX-M-15 gene, while no bla SHV was detected. PMID:24553611

  2. 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. PMID:25148969

  3. 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.

  4. Bacterial pathogens recovered from vegetables irrigated by wastewater in Morocco.

    PubMed

    Ibenyassine, K; Mhand, R Ait; Karamoko, Y; Anajjar, B; Chouibani, M M; Ennaji, M

    2007-06-01

    The authors obtained 50 vegetable samples from various regions in Morocco and examined them to determine the microbiological quality of these products. Aerobic count, coliform, enterococci, and Staphylococcus aureus were evaluated. This analysis revealed high levels of enterococci, fecal coliforms, and total coliforms. No coagulase-positive Staphylococcus aureus was detected in any of the samples analyzed. Biochemical identification of Enterobacteriaceae showed the presence of Citrobacter freundii (28 percent), Enterobacter cloacae (27 percent), Escherichia coli (16 percent), Enterobacter sakazakii (12 percent), Klebsiella pneumoniae (17 percent), Serratia liquefaciens (11 percent), and Salmonella arizonae (0.7 percent). The results clearly demonstrate that vegetables irrigated with untreated wastewater have a high level of microbiological contamination. Consequently, these vegetables may be a threat for the Moroccan consumer and may be considered a serious risk to Moroccan public health.

  5. 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. PMID:24553611

  6. Killing activity of microwaves in milk.

    PubMed

    Kindle, G; Busse, A; Kampa, D; Meyer-König, U; Daschner, F D

    1996-08-01

    The killing activity of microwaves of 2450 MHz frequency and 600 W power on Pseudomonas aeruginosa, Escherichia coli, Enterobacter sakazakii, Klebsiella pneumoniae, Staphylococcus aureus, Candida albicans, Mycobacterium terrae and poliomyelitis vaccine-virus suspended in five infant formula preparations was investigated. The samples were brought to the boil (85-100 s depending on milk type). They had reached average temperatures of 82-93 degrees C at this point. Most of the vegetative organisms were killed. In those samples where growth was still detectable after microwave treatment, a significant reduction in viable micro-organisms (at least 5000-fold) was noted. We conclude that microwave beating to the boil is a convenient and fast method to reduce microbial contamination of infant feeds. However, care should be taken to ensure that milk is adequately cooled to the required temperature before it is fed to an infant. PMID:8864939

  7. Prevalence and characterization of foodborne pathogens from Australian dairy farm environments.

    PubMed

    McAuley, Catherine M; McMillan, Kate; Moore, Sean C; Fegan, Narelle; Fox, Edward M

    2014-12-01

    The ability of foodborne pathogens to gain entry into food supply systems remains an ongoing concern. In dairy products, raw milk acts as a major vehicle for this transfer; however, the sources of pathogenic bacteria that contaminate raw milk are often not clear, and environmental sources of contamination or the animals themselves may contribute to the transfer. This survey examined the occurrence of 9 foodborne pathogens in raw milk and environments of 7 dairy farms (3 bovine, 3 caprine, and 1 ovine farm) in summer and autumn, in Victoria, Australia. A total of 120 samples were taken from sampling points common to dairy farms, including pasture, soil, feed, water sources, animal feces, raw milk, and milk filters. The prevalence of the Bacillus cereus group, Campylobacter, Clostridium perfringens, Cronobacter, Shiga-toxigenic Escherichia coli, Listeria, Salmonella, coagulase-positive staphylococci (CPS), and Yersinia enterocolitica across the farms was investigated. The 2 most prevalent bacteria, which were detected on all farms, were the B. cereus group, isolated from 41% of samples, followed by Cl. perfringens, which was isolated from 38% of samples. The highest occurrence of any pathogen was the B. cereus group in soil, present in 93% of samples tested. Fecal samples showed the highest diversity of pathogens, containing 7 of the 9 pathogens tested. Salmonella was isolated from 1 bovine farm, although it was found in multiple samples on both visits. Out of the 14 occurrences where any pathogen was detected in milk filters, only 5 (36%) of the corresponding raw milk samples collected at the same time were positive for the same pathogen. All of the CPS were Staphylococcus aureus, and were found in raw milk or milk filter samples from 6 of the 7 farms, but not in other sample types. Pathogenic Listeria species were detected on 3 of the 7 farms, and included 4 L. ivanovii-positive samples, and 1 L. monocytogenes-positive water sample. Shiga-toxigenic Escherichia coli

  8. 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.

  9. 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

    2014-02-01

    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. PMID:24456598

  10. Bacteriological survey of ready-to-eat lettuce, fresh-cut fruit, and sprouts collected from the Swiss market.

    PubMed

    Althaus, D; Hofer, E; Corti, S; Julmi, A; Stephan, R

    2012-07-01

    The consumption of ready-to-eat fresh vegetables has increased significantly in the recent decades. So far, no data are available on the bacteriological burden and the prevalence of foodborne pathogens in ready-to-eat lettuce, fresh-cut fruit, and sprouts on the Swiss market. This study was based on investigations carried out during 2 months of the summer season in 2011. Samples of 142 salads, 64 fresh-cut fruit, and 27 sprouts were included in this study. Escherichia coli, an indicator microorganism for fecal contamination, was only found in 5 lettuce samples, with amounts ranging between 2 and 3 log CFU/g. No Salmonella spp. were detected from any of the 233 samples analyzed in this study, and a low occurrence was found for contamination with L. monocytogenes, Shiga toxin-producing E. coli, enteropathogenic E. coli, and Cronobacter. From the results of the present study, we conclude that even in a country where the use of chlorine solutions to sanitize fruits and vegetables in the fresh-cut industry is not allowed, it is possible to produce ready-to-eat lettuce, fresh-cut fruit, and sprouts with high microbiological standards. Strict maintenance of good practices of hygiene at preharvest, harvest, and postharvest levels is of central importance to ensure both public health protection and product quality.

  11. 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

  12. 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

    2014-02-01

    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.

  13. Behavior of Enterobacter pulveris in amorphous and crystalline powder matrices treated with supercritical carbon dioxide.

    PubMed

    Callanan, M; Paes, M; Iversen, C; Kleijn, R; Bravo Almeida, C; Peñaloza, W; Johnson, N; Vuataz, G; Michel, M

    2012-11-01

    The resistance of an Enterobacter pulveris strain to combined heat and supercritical carbon dioxide (scCO(2)) treatments in different powder matrices was examined. The strain proved resistant to scCO(2) treatment up to 50 MPa pressure at temperatures >73 °C for at least 20 min in a commercial infant formula. Water availability was shown to be important for the observed thermotolerance, because introduction of water in the scCO(2) gas flow during treatment resulted in a 1 log(10) cfu/g reduction of the initial inoculum. Interestingly, similar tolerance to heat and scCO(2) treatment was observed in a less complex matrix, a maltodextrin powder. In contrast, the bacterial strain proved sensitive to lower temperatures (55-65 °C) over shorter times (≤10 min) in a dextrose powder composed of crystalline particles. Therefore, the microorganism demonstrates heat sensitivity in the crystalline powder matrix closer to that of nonpowder liquid matrices. These data demonstrate the increased heat tolerance of the bacterium specifically in amorphous powders and indicate that this characteristic is not dependent on fat and other components commonly found in infant formula. The information is important in designing strategies to deal with contamination of powders with Enterobacteriacae, including pathogenic Cronobacter spp.

  14. Purification and Characterization of a Rabbit Serum Factor That Kills Listeria Species and Other Foodborne Bacterial Pathogens.

    PubMed

    Kothary, Mahendra H; Franco, Augusto A; Tall, Ben D; Gopinath, Gopal R; Datta, Atin R

    2016-08-01

    In an in-vitro assay, rabbit serum, but not human serum, killed Listeria monocytogenes, a foodborne pathogen. The aim of our study was to purify and partially characterize this killing factor. Listericidin was purified from rabbit serum by a single-step ion-exchange chromatography with DEAE-Sephadex A-50 and its antimicrobial activity was assessed by a microdilution method. Listericidin is a protein with a molecular weight of 9 kDa and an isoelectric point of 8.1. It kills L. monocytogenes at 4°C, 25°C, and 37°C, and its activity is resistant to heat (boiling) and acidic conditions (pH <2). Listericidin's activity is inhibited by sodium chloride and various growth media, is sensitive to proteolytic enzymes and is enhanced by calcium chloride, and is neutralized by monoclonal antibodies to human complement C3a. However, the listericidin reacts weakly with these antibodies in an ELISA. The first 33 N-terminal residues of listericidin (SVQLTEKRMDKVGQYTNKELRKXXEDGMRDNPM) have homology to various complement C3a components. Listericidin also kills other Listeria spp., Vibrio spp., Salmonella spp., Escherichia spp., Cronobacter spp., and Bacillus spp. The listericidin peptide purified in a single-step chromatography is pH and heat stable, and has a broad antimicrobial spectrum against major foodborne pathogens in addition to L. monocytogenes. PMID:27455064

  15. Behavior of Enterobacter pulveris in amorphous and crystalline powder matrices treated with supercritical carbon dioxide.

    PubMed

    Callanan, M; Paes, M; Iversen, C; Kleijn, R; Bravo Almeida, C; Peñaloza, W; Johnson, N; Vuataz, G; Michel, M

    2012-11-01

    The resistance of an Enterobacter pulveris strain to combined heat and supercritical carbon dioxide (scCO(2)) treatments in different powder matrices was examined. The strain proved resistant to scCO(2) treatment up to 50 MPa pressure at temperatures >73 °C for at least 20 min in a commercial infant formula. Water availability was shown to be important for the observed thermotolerance, because introduction of water in the scCO(2) gas flow during treatment resulted in a 1 log(10) cfu/g reduction of the initial inoculum. Interestingly, similar tolerance to heat and scCO(2) treatment was observed in a less complex matrix, a maltodextrin powder. In contrast, the bacterial strain proved sensitive to lower temperatures (55-65 °C) over shorter times (≤10 min) in a dextrose powder composed of crystalline particles. Therefore, the microorganism demonstrates heat sensitivity in the crystalline powder matrix closer to that of nonpowder liquid matrices. These data demonstrate the increased heat tolerance of the bacterium specifically in amorphous powders and indicate that this characteristic is not dependent on fat and other components commonly found in infant formula. The information is important in designing strategies to deal with contamination of powders with Enterobacteriacae, including pathogenic Cronobacter spp. PMID:22959935

  16. Evaluation of different buffered peptone water (BPW) based enrichment broths for detection of Gram-negative foodborne pathogens from various food matrices.

    PubMed

    Margot, H; Zwietering, M H; Joosten, H; O'Mahony, Emer; Stephan, R

    2015-12-01

    This study evaluated the effects of changing the composition of the pre-enrichment medium buffered peptone water (BPW) on the growth of stressed and unstressed Gram-negative foodborne pathogens in a one-broth enrichment strategy. BPW supplemented with an available iron source and sodium pyruvate, along with low levels of 8-hydroxyquinoline and sodium deoxycholate (BPW-S) improved the recovery of desiccated Cronobacter spp. from powdered infant formula. Growth of Salmonella and STEC was comparable in all BPW variants tested for different food matrices. In products with high levels of Gram-negative background flora (e.g. sprouts), the target organisms could not be reliably detected by PCR in any of the BPW variants tested unless the initial level exceeded 10(3) cfu/10 g of sprouts. Based on these results we suggest BPW-S for a one-broth enrichment strategy of stressed Gram-negative foodborne pathogens from dry products. However, a one-broth enrichment strategy based on BPW variants tested in this evaluation is not recommended for produce with a high level of Gram-negative background flora due to very high detection limits. PMID:26267889

  17. 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

    2016-01-01

    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. PMID:27334801

  18. Novel approaches to improve the intrinsic microbiological safety of powdered infant milk formula.

    PubMed

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

    2015-02-12

    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.

  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. Evaluation of different buffered peptone water (BPW) based enrichment broths for detection of Gram-negative foodborne pathogens from various food matrices.

    PubMed

    Margot, H; Zwietering, M H; Joosten, H; O'Mahony, Emer; Stephan, R

    2015-12-01

    This study evaluated the effects of changing the composition of the pre-enrichment medium buffered peptone water (BPW) on the growth of stressed and unstressed Gram-negative foodborne pathogens in a one-broth enrichment strategy. BPW supplemented with an available iron source and sodium pyruvate, along with low levels of 8-hydroxyquinoline and sodium deoxycholate (BPW-S) improved the recovery of desiccated Cronobacter spp. from powdered infant formula. Growth of Salmonella and STEC was comparable in all BPW variants tested for different food matrices. In products with high levels of Gram-negative background flora (e.g. sprouts), the target organisms could not be reliably detected by PCR in any of the BPW variants tested unless the initial level exceeded 10(3) cfu/10 g of sprouts. Based on these results we suggest BPW-S for a one-broth enrichment strategy of stressed Gram-negative foodborne pathogens from dry products. However, a one-broth enrichment strategy based on BPW variants tested in this evaluation is not recommended for produce with a high level of Gram-negative background flora due to very high detection limits.

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

    PubMed Central

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

    2016-01-01

    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 × 100 cfu/ml for the test sample compared with a detection limit of 1.6 × 103 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. PMID:27334801

  2. 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%.

  3. 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%. PMID:26725036

  4. 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. PMID:26454221

  5. 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

  6. Molecular epidemiology of the integron-located VEB-1 extended-spectrum beta-lactamase in nosocomial enterobacterial isolates in Bangkok, Thailand.

    PubMed

    Girlich, D; Poirel, L; Leelaporn, A; Karim, A; Tribuddharat, C; Fennewald, M; Nordmann, P

    2001-01-01

    Over a 21/2-month period in 1999, 37 ceftazidime-resistant nonrepetitive enterobacterial isolates were collected from 37 patients in a Bangkok hospital, Thailand. Eighty-one percent of these strains expressed a clavulanic acid-inhibited extended-cephalosporin resistance profile. An identical extended-spectrum beta-lactamase (ESBL), VEB-1, was found in 16 unrelated enterobacterial isolates (Escherichia coli, n = 10; Enterobacter cloacae, n = 2; Enterobacter sakazakii, n = 1; and Klebsiella pneumoniae, n = 3) and in two clonally related E. cloacae isolates. The bla(VEB-1) gene was located on mostly self-conjugative plasmids (ca. 24 to 200 kb) that conferred additional non-beta-lactam antibiotic resistance patterns. Additionally, the bla(VEB-1) gene cassette was part of class 1 integrons varying in size and structure. The bla(VEB-1)-containing integrons were mostly associated with bla(OXA-10)-like and arr-2-like gene cassettes, the latter conferring resistance to rifampin. These data indicated the spread of bla(VEB-1) in Bangkok due to frequent transfer of different plasmids and class 1 integrons and rarely to clonally related strains. Plasmid- and integron-mediated resistance to rifampin was also found in enterobacterial isolates.

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

    PubMed

    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.

  8. 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.

  9. [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.

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

    PubMed

    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

  11. 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

  12. Mitogenic response and probiotic characteristics of lactic acid bacteria isolated from indigenously pickled vegetables and fermented beverages.

    PubMed

    Kumar, Mukesh; Ghosh, Moushumi; Ganguli, Abhijit

    2012-02-01

    Lactic acid bacteria from indigenous pickled vegetables and fermented beverages (fermented rice and Madhuca longifolia flowers) were isolated and investigated for their functional characteristics in vitro as potential new probiotic strains. Four isolates (all Lactobacillus spp.) selected on the basis of high tolerance to bile (0.2%) were identified by standard and molecular methods (16S rDNA) as L. helveticus, L. casei, L. delbrueckii and L. bulgaricus from pickled vegetables and fermented beverages respectively. These selected strains had antibiotic resistance, tolerance to artificial gastric juice and phenol (0.4%), enzymatic profile, and antagonistic activity against enteric pathogens (Enterobacter sakazakii, Salmonella typhimurium, Shigella flexneri 2a, Listeria monocytogenes, Yersinia enterocolitica and Aeromonas hydrophila). All strains survived well in artificial gastric juice at low pH (3.0) values for 4 h, possessed bile salt hydrolase activity and were susceptible to most antibiotics including vancomycin. Additionally, the isolates exhibited high tolerance to phenol, high cell surface hydrophobicity (>60%) and induced proliferation of murine splenocytes. All the four strains of present study suppressed the Con A-stimulated proliferation of the mouse spleen cells, although L. casei had the strongest suppressive effect. The results of this study suggest a potential application of the strains (following human clinical trials), for developing probiotic foods.

  13. Safety assessment and probiotic evaluation of Enterococcus faecium YF5 isolated from sourdough.

    PubMed

    Tan, Qianglai; Xu, Hengyi; Aguilar, Zoraida P; Peng, Shanshan; Dong, Suqin; Wang, Baogui; Li, Ping; Chen, Tingtao; Xu, Feng; Wei, Hua

    2013-04-01

    Enterococcus faecium YF5, a strain previously isolated from sourdough, was assessed for safety and probiotic potential. Its virulence and antibiotic resistant phenotypes (cytolysin and gelatinase production, antibiotic susceptibility) and genes (cylA, gelE, ace, agg, esp, and vanA) were surveyed. Results indicated that the tested virulence determinants were nontoxic. In addition, E. faecium YF5 was sensitive to 3 antibiotics such as amoxicillin, vancomycin, and chloramphenicol. Furthermore, results of in vivo animal acute oral toxicity of E. faecium YF5 studies were similar to the control group that indicated no abnormalities. In addition, E. faecium YF5 stably survived in low pH, bile salts, gastric, and intestinal fluids in vitro. Moreover, E. faecium YF5 was found to adhere to human colon cancer cell line HT-29 at 3.39 (±0.67) × 10(5) CFU/mL. When cocultured with pathogenic organisms (Enterobacter sakazakii CMCC45402, Escherichia coli CMCC44102, enterohemorrhage Escherichia coli O157: H7 CMCC44828, Salmonella Typhimurium CMCC50071, Shigella flexneri 301, and Shigella sonnei ATCC 29930) and 2 gram-positive strains (Listeria monocytogenes CMCC54001 and Staphylococcus aureus CMCC 26003), it inhibited these foodborne pathogens with exception of S. aureus. Therefore, E. faecium YF5 can be regarded as a safe strain and it may be used as a probiotic preparation or for microecologics. PMID:23488799

  14. [Coliform bacteria in rinsed beer mugs--identification with the API 20 E system and resistance behavior].

    PubMed

    Schindler, P R; Metz, H

    1990-10-01

    653 11 beer mugs rinsed mechanically by conveyor belt dishwashing machines and 182 mugs cleaned in open vats were investigated for their content of faecal and total coliforms. Ten and more faecal coliform germs could be detected in 4.7% of the mechanically rinsed tankards and in 12.1% of those cleaned in open vats. Respectively, ten and more total coliforms were found in 30.3% and in 67.6%. Altogether 213 coliform strains as defined by the German drinking water regulation were characterised biochemically by the API 20 E-system and by their susceptibility to 15 antimicrobial agents. A total of 19 different species from eight genera could be differentiated, whereas 12 strains were unidentifiable by the applied system. Klebsiella oxytoca was the most frequently encountered germ, followed by Enterobacter cloacae, K. pneumoniae and Ent. sakazakii. 59 of the 213 strains were fully susceptible to all antibiotics used. 44 strains, however, revealed complete or moderate resistance against three and more agents. These results indicate that unhygienic dishwashing procedures may play a decisive role in the epidemiology of clinically relevant and multiresistant germs. PMID:2149435

  15. The ErbB4 Ligand Neuregulin-4 Protects against Experimental Necrotizing Enterocolitis

    PubMed Central

    McElroy, Steven J.; Castle, Shannon L.; Bernard, Jessica K.; Almohazey, Dana; Hunter, Catherine J.; Bell, Brandon A.; Al Alam, Denise; Wang, Larry; Ford, Henri R.; Frey, Mark R.

    2015-01-01

    Necrotizing enterocolitis (NEC) affects up to 10% of premature infants, has a mortality of 30%, and can leave surviving patients with significant morbidity. Neuregulin-4 (NRG4) is an ErbB4-specific ligand that promotes epithelial cell survival. Thus, this pathway could be protective in diseases such as NEC, in which epithelial cell death is a major pathologic feature. We sought to determine whether NRG4-ErbB4 signaling is protective in experimental NEC. NRG4 was used i) in the newborn rat formula feeding/hypoxia model; ii) in a recently developed model in which 14- to 16-day-old mice are injected with dithizone to induce Paneth cell loss, followed by Klebsiella pneumoniae infection to induce intestinal injury; and iii) in bacterially infected IEC-6 cells in vitro. NRG4 reduced NEC incidence and severity in the formula feed/hypoxia rat model. It also reduced Paneth cell ablation–induced NEC and prevented dithizone-induced Paneth cell loss in mice. In vitro, cultured ErbB4−/− ileal epithelial enteroids had reduced Paneth cell markers and were highly sensitive to inflammatory cytokines. Furthermore, NRG4 blocked, through a Src-dependent pathway, Cronobacter muytjensii–induced IEC-6 cell apoptosis. The potential clinical relevance of these findings was demonstrated by the observation that NRG4 and its receptor ErbB4 are present in human breast milk and developing human intestine, respectively. Thus, NRG4-ErbB4 signaling may be a novel pathway for therapeutic intervention or prevention in NEC. PMID:25216938

  16. 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.

  17. 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

  18. 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

  19. 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. PMID:27357225

  20. Longitudinal Analysis of the Premature Infant Intestinal Microbiome Prior to Necrotizing Enterocolitis: A Case-Control Study

    PubMed Central

    Zhou, Yanjiao; Shan, Gururaj; Sodergren, Erica; Weinstock, George; Walker, W. Allan; Gregory, Katherine E.

    2015-01-01

    Necrotizing enterocolitis (NEC) is an inflammatory disease of the newborn bowel, primarily affecting premature infants. Early intestinal colonization has been implicated in the pathogenesis of NEC. The objective of this prospective case-control study was to evaluate differences in the intestinal microbiota between infants who developed NEC and unaffected controls prior to disease onset. We conducted longitudinal analysis of the 16S rRNA genes of 312 samples obtained from 12 NEC cases and 26 age-matched controls with a median frequency of 7 samples per subject and median sampling interval of 3 days. We found that the microbiome undergoes dynamic development during the first two months of life with day of life being the major factor contributing to the colonization process. Depending on when the infant was diagnosed with NEC (i.e. early vs. late onset), the pattern of microbial progression was different for cases and controls. The difference in the microbiota was most overt in early onset NEC cases and controls. In proximity to NEC onset, the abundances of Clostridium sensu stricto from Clostridia class were significantly higher in early onset NEC subjects comparing to controls. In late onset NEC, Escherichia/Shigella among Gammaproteobacteria, showed an increasing pattern prior to disease onset, and was significantly higher in cases than controls six days before NEC onset. Cronobacter from Gammaproteobacteria was also significantly higher in late onset NEC cases than controls 1-3 days prior to NEC onset. Thus, the specific infectious agent associated with NEC may vary by the age of infant at disease onset. We found that intravenously administered antibiotics may have an impact on the microbial diversity present in fecal material. Longitudinal analysis at multiple time points was an important strategy utilized in this study, allowing us to appreciate the dynamics of the premature infant intestinal microbiome while approaching NEC at various points. PMID:25741698

  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 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. PMID:27446058

  3. Pathogen prevalence and influence of composted dairy manure application on antimicrobial resistance profiles of commensal soil bacteria.

    PubMed

    Edrington, Tom S; Fox, William E; Callaway, Todd R; Anderson, Robin C; Hoffman, Dennis W; Nisbet, David J

    2009-03-01

    Composting manure, if done properly, should kill pathogenic bacteria such as Salmonella and Escherichia coli O157:H7, providing for an environmentally safe product. Over a 3-year period, samples of composted dairy manure, representing 11 composting operations (two to six samples per producer; 100 total samples), were screened for Salmonella and E. coli O157:H7 and were all culture negative. Nonpathogenic bacteria were cultured from these compost samples that could theoretically facilitate the spread of antimicrobial resistance from the dairy to compost application sites. Therefore, we collected soil samples (three samples per plot; 10 plots/treatment; 90 total samples) from rangeland that received either composted dairy manure (CP), commercial fertilizer (F), or no treatment (control, CON). Two collections were made appoximately 2 and 7 months following treatment application. Soil samples were cultured for Pseudomonas and Enterobacter and confirmed isolates subjected to antimicrobial susceptibility testing. Three species of Enterobacter (cloacae, 27 isolates; aeroginosa, two isolates; sakazakii, one isolate) and two species of Pseudomonas (aeruginosa, 11 isolates; putida, seven isolates) were identified. Five Enterobacter isolates were resistant to ampicillin and one isolate was resistant to spectinomycin. All Pseudomonas isolates were resistant to ampicillin, ceftiofur, florfenicol, sulphachloropyridazine, sulphadimethoxine, and trimethoprim/sulfamethoxazole and most isolates were resistant to chlortetracycline and spectinomycin. Pseudomonas isolates were resistant to an average of 8.6, 7.9, and 8 antibiotics for CON, CP, and F treatments, respectively. No treatment differences were observed in antimicrobial resistance patterns in any of the soil isolates examined. Results reported herein support the use of composted dairy manure as an environmentally friendly soil amendment. PMID:19105635

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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