Real-Time Fluorescence PCR Assays for Detection and Characterization of Heat-Labile I and Heat-Stable I Enterotoxin Genes from Enterotoxigenic \u3ci\u3eEscherichia coli\u3c/i\u3e

To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC

Emergence and clonal dissemination of<i> Salmonella enterica</i> serovar Enteritidis causing salmonellosis in Mauritius

Introduction: For decades, Salmonella enterica serovar Enteritidis has been among the most prevalent serovars reported worldwide. However, it was rarely encountered in Mauritius until 2007; since then the number of non-typhoidal Salmonella serogroup O:9 (including serovar Enteritidis) increased. A study was conducted to investigate the genetic relatedness between S. Enteritidis isolates recovered in Mauritius from food and clinical specimens (stool, blood, and exudate). Methodology: Forty-seven isolates of S. Enteritidis obtained in 2009 from human stools, blood cultures and exudates, and from food specimens were characterized by antimicrobial susceptibility testing and Multiple-Locus Variable-number tandem repeat Analysis (MLVA). Results: With the exception of a single isolate which demonstrated intermediate susceptibility to streptomycin, all isolates were pansusceptible to the 14 antimicrobials tested. Thirty seven out of the 47 isolates (78.7%) exhibited an indistinguishable MLVA profile which included isolates from ready-to-eat food products, chicken, and human clinical isolates from stool, blood and exudate. Conclusions: The presence of highly related strains in both humans and raw chicken, and the failure to isolate the serovar from other foods, suggests that poultry is the main reservoir of S. Enteritidis in Mauritius and that the majority of human cases are associated with chicken consumption which originated from one major producer. Stool isolates were indistinguishable or closely related to blood and exudate isolates, indicating that, besides gastroenteritis, the same strain caused invasive infections. Control of S.Enteritidis by poultry breeders would lower the financial burden associated with morbidity in humans caused by this organism in Mauritius.</jats:p

Characterisation of ropy slime-producing Lactobacillus sakei using repetitive element sequence -based PCR

http://www.elsevier.nl/locate/01681605Eighteen earlier well characterised Lactobacillus sake strains representing different slime production capabilities in vacuum-packaged meat products were analysed using repetitive element sequence-based PCR (rep-PCR). The single primers BOXA1R and RW3A and the primer pair REP1R-Dt & REP2R-Dt were evaluated for their applicability in L. sake genotyping. The five different patterns produced by RW3A were least revealing, with the discriminatory power equalling to ribotyping. BOXA1R and REP-primer pair both produced six different banding patterns and the combination of these results yielded seven different rep-types. Rep-PCR was concluded to have approximately the same discriminatory power as randomly amplified polymorphic DNA (RAPD) analysis, but it was inferior to pulsed-field gel electrophoresis (PFGE). How-ever, if the results of rep-PCR and RAPD were combined, the discrimination was comparable PFGE, with the exception that within Ribogroup I the non-slime-producing strains were not distinguished from weak slime producers. It was concluded that the combination of the two PCR-based typing techniques, rep-PCR and RAPD, would be a valuable tool in large scale contamination studies at the meat processing plants, since results can be obtained rapidly and fewer isolates need further analysis by PFGE

Safety evaluation of sous vide-processed products with respect to nonproteolytic Clostridium botulinum by use of challenge studies and predictive microbiological models.

Sixteen different types of sous vide-processed products were evaluated for safety with respect to nonproteolytic group II Clostridium botulinum by using challenge tests with low (2.0-log-CFU/kg) and high (5.3-log-CFU/kg) inocula and two currently available predictive microbiological models, Food MicroModel (FMM) and Pathogen Modeling Program (PMP). After thermal processing, the products were stored at 4 and 8°C and examined for the presence of botulinal spores and neurotoxin on the sell-by date and 7 days after the sell-by date. Most of the thermal processes were found to be inadequate for eliminating spores, even in low-inoculum samples. Only 2 of the 16 products were found to be negative for botulinal spores and neurotoxin at both sampling times. Two products at the high inoculum level showed toxigenesis during storage at 8°C, one of them at the sell-by date. The predictions generated by both the FMM thermal death model and the FMM and PMP growth models were found to be inconsistent with the observed results in a majority of the challenges. The inaccurate predictions were caused by the limited number and range of the controlling factors in the models. Based on this study, it was concluded that the safety of sous vide products needs to be carefully evaluated product by product. Time-temperature combinations used in thermal treatments should be reevaluated to increase the efficiency of processing, and the use of additional antibotulinal hurdles, such as biopreservatives, should be assessed

Characterization of Salmonella enterica serovar Enteritidis isolates recovered from blood and stool specimens in Thailand

<p>Abstract</p> <p>Background</p> <p>Bacteremia due to <it>Salmonella</it> spp. is a life-threatening condition and is commonly associated with immune compromise. A 2009 observational study estimated risk factors for the ten most common non-typhoidal <it>Salmonella</it> (NTS) serovars isolated from Thai patients between 2002–2007. In this study, 60.8% of <it>Salmonella enterica</it> serovar Enteritidis isolates (n = 1517) were recovered from blood specimens and infection with <it>Salmonella</it> serovar Enteritidis was a statistically significant risk factor for bacteremia when compared to other NTS serovars. Based on this information, we characterized a subset of isolates collected in 2008 to determine if specific clones were recovered from blood or stool specimens at a higher rate. Twenty blood isolates and 20 stool isolates were selected for antimicrobial resistance testing (MIC), phage typing, PFGE, and MLVA.</p> <p>Result</p> <p>Eight antibiogrammes, seven MLVA types, 14 <it>Xba</it>I/<it>Bln</it>I PFGE pattern combinations, and 11 phage types were observed indicating considerable diversity among the 40 isolates characterized. Composite analysis based on PFGE and MLVA data revealed 22 genotypes. Seven of the genotypes containing two or more isolates were from both stool and blood specimens originating from various months and zones. Additionally, those genotypes were all further discriminated by phage type and/or antibiogramme. Ninety percent of the isolates were ciprofloxacin resistant.</p> <p>Conclusions</p> <p>The increased percentage of bloodstream infections as described in the 2009 observational study could not be attributed to a single clone. Future efforts should focus on assessing the immune status of bacteriaemic patients and identifying prevention and control measures, including attribution studies characterizing non-clinical (animal, food, and environmental) isolates.</p

Comparative analysis of subtyping methods against a whole-genome-sequencing standard for Salmonella enterica serotype Enteritidis.

A retrospective investigation was performed to evaluate whole-genome sequencing as a benchmark for comparing molecular subtyping methods for Salmonella enterica serotype Enteritidis and survey the population structure of commonly encountered S. enterica serotype Enteritidis outbreak isolates in the United States. A total of 52 S. enterica serotype Enteritidis isolates representing 16 major outbreaks and three sporadic cases collected between 2001 and 2012 were sequenced and subjected to subtyping by four different methods: (i) whole-genome single-nucleotide-polymorphism typing (WGST), (ii) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), (iii) clustered regularly interspaced short palindromic repeats combined with multi-virulence-locus sequence typing (CRISPR-MVLST), and (iv) pulsed-field gel electrophoresis (PFGE). WGST resolved all outbreak clusters and provided useful robust phylogenetic inference results with high epidemiological correlation. While both MLVA and CRISPR-MVLST yielded higher discriminatory power than PFGE, MLVA outperformed the other methods in delineating outbreak clusters whereas CRISPR-MVLST showed the potential to trace major lineages and ecological origins of S. enterica serotype Enteritidis. Our results suggested that whole-genome sequencing makes a viable platform for the evaluation and benchmarking of molecular subtyping methods

PulseNet International: vision for the implementation of whole genome sequencing (WGS) for global food-borne disease surveillance

FWD-NEXT Expert Panel - Portugal/INSA - Vítor Borges (Instituto Nacional de Saúde Doutor Ricardo Jorge, Lisbon, Portugal)PulseNet International is a global network dedicated to laboratory-based surveillance for food-borne diseases. The network comprises the national and regional laboratory networks of Africa, Asia Pacific, Canada, Europe, Latin America and the Caribbean, the Middle East, and the United States. The PulseNet International vision is the standardised use of whole genome sequencing (WGS) to identify and subtype food-borne bacterial pathogens worldwide, replacing traditional methods to strengthen preparedness and response, reduce global social and economic disease burden, and save lives. To meet the needs of real-time surveillance, the PulseNet International network will standardise subtyping via WGS using whole genome multilocus sequence typing (wgMLST), which delivers sufficiently high resolution and epidemiological concordance, plus unambiguous nomenclature for the purposes of surveillance. Standardised protocols, validation studies, quality control programmes, database and nomenclature development, and training should support the implementation and decentralisation of WGS. Ideally, WGS data collected for surveillance purposes should be publicly available, in real time where possible, respecting data protection policies. WGS data are suitable for surveillance and outbreak purposes and for answering scientific questions pertaining to source attribution, antimicrobial resistance, transmission patterns, and virulence, which will further enable the protection and improvement of public health with respect to food-borne disease.info:eu-repo/semantics/publishedVersio

Multiple-locus variable-number tandem repeat analysis for strain discrimination of non-O157 Shiga toxin-producing \u3ci\u3eEscherichia coli\u3c/i\u3e

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens of growing concern worldwide that have been associated with several recent multistate and multinational outbreaks of foodborne illness. Rapid and sensitive molecular-based bacterial strain discrimination methods are critical for timely outbreak identification and contaminated food source traceback. One such method, multiple-locus variable-number tandemrepeat analysis(MLVA), is being used with increasing frequency in foodborne illness outbreak investigations to augment the current gold standard bacterial subtyping technique, pulsed-field gel electrophoresis (PFGE). The objective of this studywas to develop a MLVA assay for intra- and inter-serogroup discrimination of six major non-O157 STEC serogroups—O26, O111, O103, O121, O45, and O145—and performa preliminary internal validation of the method on a limited number of clinical isolates. The resultant MLVA scheme consists of ten variable number tandem repeat (VNTR) loci amplified in three multiplex PCR reactions. Sixty-five unique MLVA types were obtained among 84 clinical non-O157 STEC strains comprised of geographically diverse sporadic and outbreak related isolates. Compared to PFGE, the developed MLVA scheme allowed similar discrimination among serogroups O26, O111, O103, and O121 but not among O145 and O45. To more fully compare the discriminatory power of this preliminary MLVA method to PFGE and to determine its epidemiological congruence, a thorough internal and external validation needs to be performed on a carefully selected large panel of strains, including multiple isolates from single outbreaks