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We characterized eight mcr-5 -positive Salmonella enterica subsp. enterica serovar Typhimurium sequence type 34 (ST34) isolates obtained from pigs and meat in Germany. Five plasmid types were identified harboring mcr-5 on Tn 6452 or putative mobile insertion cassettes. The mobility of mcr-5 was confirmed by integration of Tn 6452 into the bacterial chromosomes of two strains and the detection of conjugative mcr-5 plasmids. The association with mobile genetic elements might further enhance mcr-5 distribution., (Copyright © 2019 American Society for Microbiology.)

We investigated 543 Listeria monocytogenes isolates from food having a temporal and spatial distribution compatible with that of the invasive listeriosis outbreak occurring 2012-2016 in southern Germany. Using forensic microbiology, we identified several products from 1 manufacturer contaminated with the outbreak genotype. Continuous molecular surveillance of food isolates could prevent such outbreaks.

In order to reduce the prevalence of the Salmonella enterica serovars Typhimurium and Enteritidis as a main causative agent of human salmonellosis originating from poultry flocks and products, the EU regulations 2160/2003 and 2073/2005 and the German Hühner-Salmonellen-Verordnung were established ten years ago. A literature review shows that this aim could be reached to a large extend in many areas of the food production chain, e.g. in breeding and husbandry facilities in most EU member states including Germany. Nevertheless some exceptions exist, and there are other S. enterica serovars which have a human pathogenic potential comparable to S. Typhimurium and S. Enteritidis. Furthermore recent publications show, that especially processes in transport and slaughter of poultry can prevent successful husbandry sanitation measures. Especially in these areas a reasonable potential for hygiene improvements still exists. Based on the prevalence data obtained between 1996 and 2011 this review summarizes recent knowledge concerning possible risks of Salmonella cross contamination and suggests potential starting points for their mitigation.

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- is a monophasic variant of Salmonella Typhimurium. In this study, a total of 651 human and 107 food and environmental isolates of serovar 4,[5],12:i:- recovered from 2007 through 2011 in Switzerland were characterized by antibiotic resistance profiles and pulsed-field gel electrophoresis (PFGE). In addition, a selection of isolates belonging to the most frequent PFGE patterns was further subjected to multilocus variable-number tandem-repeat analysis (MLVA) and phage typing. Over the years 2007-2011, the reports of salmonellosis caused by Salmonella enterica serovar 4,[5],12:i:- significantly increased. A high prevalence of multidrug-resistant isolates, mainly showing an ampicillin-streptomycin-sulfonamide-tetracycline resistance pattern (ASSuT), was observed. In addition, four extended spectrum beta lactamase (ESBL) (CTX-M-55)-producing isolates were found. XbaI PFGE analysis of all isolates revealed over 150 different pulsotypes, and generally showed a considerable diversity within the monophasic isolates. Nevertheless, among these we identified seven dominant profiles, which encompassed 66% of all isolates tested. The PFGE type STYMXB.0131 dominated among human as well as food isolates. Multilocus variable-number tandem-repeat analysis profile 3-12-10-0-0211, which, in many cases, coincided with PFGE type STYMXB.0131 and phage type DT193 were the most prevalent types found for the isolates further characterized by these typing methods. Our data provide strong evidence for a spread of two specific Salmonella serovar 4,[5],12:i:- clones (PFGE pattern STYMXB.0131, resistance type ASSuT) and (PFGE pattern STYMXB.0131, resistance type SSuT). In contrast to the human isolates, the pork/poultry isolates expressed predominantly the SSuT resistance type.

Salmonella enterica serovar Infantis (Salmonella Infantis) is consistently isolated from broiler chickens, pigs, and humans worldwide. This study investigated 93 epidemiologically unrelated Salmonella Infantis strains isolated in Germany between 2005 and 2008 in respect to their transmission along the food chain. Various phenotypic and genotypic methods were applied, and the pathogenicity and resistance gene repertoire was determined. Phenotypically, 66% of the strains were susceptible to all 17 antimicrobials tested, while the others were almost all multidrug-resistant (two or more antimicrobial resistances), with different resistance profiles and preferentially isolated from broiler chickens. A number of phage types (PTs) were shared by strains from pigs, broiler chickens, and humans (predominated by PT 29). One, PT 1, was only detected in strains from pigs/pork and humans. Pulsed-field gel electrophoresis (PFGE) subdivided strains in seven different clusters, named A-G, consisting of 35 various XbaI profiles with coefficient of similarity values of 0.73-0.97. The majority of XbaI profiles were assigned to clusters A and C, and two predominant XbaI profiles were common in strains isolated from all sources investigated. Multi-locus sequence typing (MLST) analysis of selected strains representing the seven PFGE clusters revealed that they all belonged to ST32. The pathogenicity gene repertoire of 37 representative Salmonella Infantis strains analyzed by microarray was also identical. The resistance gene repertoire correlated perfectly with the phenotypic antimicrobial resistance profiles, and multidrug-resistant strains were associated with class 1 integrons. Overall, this study showed that two major closely related genotypes of Salmonella Infantis can transmit in Germany to humans through contaminated broiler meat or pork, and consequently presents a hazard for human health.

Pork contributes significantly to the public health disease burden caused by Salmonella infections. During the slaughter process pig carcasses can become contaminated with Salmonella. Contamination at the slaughter-line is initiated by pigs carrying Salmonella on their skin or in their faeces. Another contamination route could be resident flora present on the slaughter equipment. To unravel the contribution of these two potential sources of Salmonella a quantitative study was conducted. Process equipment (belly openers and carcass splitters), faeces and carcasses (skin and cutting surfaces) along the slaughter-line were sampled at 11 sampling days spanning a period of 4 months. Most samples taken directly after killing were positive for Salmonella. On 96.6% of the skin samples Salmonella was identified, whereas a lower number of animals tested positive in their rectum (62.5%). The prevalence of Salmonella clearly declined on the carcasses at the re-work station, either on the cut section or on the skin of the carcass or both (35.9%). Throughout the sampling period of the slaughter-line the total number of Salmonella per animal was almost 2 log lower at the re-work station in comparison to directly after slaughter. Seven different serovars were identified during the study with S. Derby (41%) and S. Typhimurium (29%) as the most prominent types. A recurring S. Rissen contamination of one of the carcass splitters indicated the presence of an endemic 'house flora' in the slaughterhouse studied. On many instances several serotypes per individual sample were found. The enumeration of Salmonella and the genotyping data gave unique insight in the dynamics of transmission of this pathogen in a slaughter-line. The data of the presented study support the hypothesis that resident flora on slaughter equipment was a relevant source for contamination of pork., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Salmonella enterica serovar Derby (S. Derby) is one of the most prevalent serovars in pigs in Europe and in the U.S. and ranks among the 10 most frequently isolated serovars in humans. Therefore, a set of 82 epidemiologically unrelated S. Derby strains isolated between 2006 and 2008 from pigs, pork and humans in Germany was selected and investigated in respect to the transmission of clonal groups of the serovar along the food chain. Various phenotypic and genotypic methods were applied and the pathogenicity and resistance gene repertoire was determined. Phenotypically 72% of the strains were susceptible to all 17 antimicrobials tested while the others were monoresistant to tetracycline or multi-resistant with different resistance profiles. Four major clonal groups were identified based on PFGE, sequence data of the virulence genes sopA, sopB and sopD, VNTR-locus STTR5 and MLST revealing also the new sequence type ST774. Thirty different PFGE profiles were detected resulting in four clusters representing the four groups. The pathogenicity gene repertoire of 32 representative S. Derby strains analyzed by microarray showed six types with differences in the Salmonella pathogenicity islands, pathogenicity genes on smaller islets or prophages and fimbriae coding genes. The pathogenicity gene repertoire of the predominant types PAT DE1 and DE2 were most similar to the ones of S. Paratyphi B (dT+, O5-) and to a minor degree to S. Infantis and S. Virchow PATs. Overall this study showed that in Germany currently one major S. Derby clone is frequently isolated from pigs and humans. Contaminated pork was identified as one vehicle and consequently is a risk for human health. To prevent this serovar from entering the food chain, control measurements should be applied at the farm level., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A robust duplex 5' nuclease (TaqMan) real-time PCR was developed and in-house validated for the specific detection of Salmonella enterica subspecies enterica serovar Enteritidis in whole chicken carcass rinses and consumption eggs. The assay uses specifically designed primers and a TaqMan probe to target the Prot6e gene located on the S. Enteritidis specific 60-kb virulence plasmid. As an internal amplification control to monitor Salmonella DNA in the sample, a second primer/TaqMan probe set detects simultaneously the Salmonella specific invA gene. The assay identified correctly 95% of the 79 Salmonella Enteritidis strains tested comprising 19 different phage types. None of the 119 non-Enteritidis strains comprising 54 serovars was positive for the Prot6e gene. The assay detection probability was for 10(2) or more genome equivalents 100% and for 10 equivalents 83%. A pre-PCR sample preparation protocol including a pre-enrichment step in buffered peptone water, followed by DNA extraction was applied on low levels of artificially contaminated whole chicken carcass rinses and eggs from hens as well as 25 potentially naturally contaminated chickens. The detection limit was less than three CFU per 50 ml carcass rinse or 10 ml egg. The sensitivity and specificity compared to the traditional culture-based detection method and serotyping were both 100%. Twenty-five potentially naturally contaminated chickens were compared by the real-time PCR and the traditional cultural isolation method resulting in four Salmonella positive samples of which two were positive for the Prot6e gene and serotyped as S. Enteritidis. We show also that Salmonella isolates which have a rough lipopolysaccharide structure could be assigned to the serovar Enteritidis by the real-time PCR. This methodology can contribute to meet the need of fast identification and detection methods for use in monitoring and control measures programmes.

In a previous study, we reported the performance of a PCR assay amplifying 285-bp of the invA gene of Salmonella spp. through an international ring-trial involving four participating laboratories [Int. J. Food Microbiol. 89 (2003) 241]. Based on the validated set of primers and recent advancements in PCR technology, we have designed two specific PCR assays for detecting Salmonella spp. We have compared PCR-enzyme-linked immunosorbent assay (PCR-ELISA) and LightCycler real-time PCR assay (LC-PCR) with the standard ISO 6579 bacteriological reference method. The two PCR tests incorporated an internal amplification control (IAC) co-amplified with the invA gene of Salmonella to monitor potential PCR inhibitors and ensure successful amplification. The selectivity study involved 84 Salmonella and 44 non-Salmonella strains and the samples tested were represented by 60 artificially-contaminated samples of fish, minced beef and raw milk, and 92 naturally-contaminated milk and meat samples. When using either PCR-ELISA or LC-PCR assays, only Salmonella strains were detected. PCR-ELISA and LC-PCR assays gave with pure Salmonella cultures the same detection limit level of 10(3)CFU/ml, which corresponds respectively to 50 and 10 cells per PCR tube. Data on artificially contaminated samples indicated that both PCR methods were able to detect after enrichment less than five Salmonella cells in 25 g of food, giving 100% concordance with the ISO 6579 reference method. The results on naturally contaminated samples demonstrated that despite certain inhibition problems, LC-PCR and PCR-ELISA assays were highly specific and sensitive, and provide a powerful tool for detection of Salmonella in food samples.

As part of a standardization project, an interlaboratory trial including 15 laboratories from 13 European countries was conducted to evaluate the performance of a noproprietary polymerase chain reaction (PCR)-based method for the detection of Salmonella on artificially contaminated chicken rinse and pig swab samples. The 3 levels were 1-10, 10-100, and 100-1000 colony-forming units (CFU)/100 mL. Sample preparations, including inoculation and pre-enrichment in buffered peptone water (BPW), were performed centrally in a German laboratory; the pre-PCR sample preparation (by a resin-based method) and PCR assay (gel electrophoresis detection) were performed by the receiving laboratories. Aliquots of BPW enrichment cultures were sent to the participants, who analyzed them using a thermal lysis procedure followed by a validated Salmonella-specific PCR assay. The results were reported as negative or positive. Outlier results caused, for example, by gross departures from the experimental protocol, were omitted from the analysis. For both the chicken rinse and the pig swab samples, the diagnostic sensitivity was 100%, with 100% accordance (repeatability) and concordance (reproducibility). The diagnostic specificity was 80.1% (with 85.7% accordance and 67.5% concordance) for chicken rinse, and 91.7% (with 100% accordance and 83.3% concordance) for pig swab. Thus, the interlaboratory variation due to personnel, reagents, thermal cyclers, etc., did not affect the performance of the method, which will be proposed as part of a developing international PCR standard.