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Due to its high case fatality rate, foodborne listeriosis is considered a major public health concern worldwide. We describe one of the largest listeriosis outbreaks in Germany with 83 cases of inv...

We compared the consistency, accuracy and reproducibility of next-generation short read sequencing between ten laboratories involved in food safety (research institutes, state laboratories, universities and companies) from Germany and Austria. Participants were asked to sequence six DNA samples of three bacterial species (Campylobacter jejuni, Listeria monocytogenes and Salmonella enterica) in duplicate, according to their routine in-house sequencing protocol. Four different types of Illumina sequencing platforms (MiSeq, NextSeq, iSeq, NovaSeq) and one Ion Torrent sequencing instrument (S5) were involved in the study. Sequence quality parameters were determined for all data sets and centrally compared between laboratories. SNP and cgMLST calling were performed to assess the reproducibility of sequence data collected for individual samples. Overall, we found Illumina short read data to be more accurate (higher base calling accuracy, fewer miss-assemblies) and consistent (little variability between independent sequencing runs within a laboratory) than Ion Torrent sequence data, with little variation between the different Illumina instruments. Two laboratories with Illumina instruments submitted sequence data with lower quality, probably due to the use of a library preparation kit, which shows difficulty in sequencing low GC genome regions. Differences in data quality were more evident after assembling short reads into genome assemblies, with Ion Torrent assemblies featuring a great number of allele differences to Illumina assemblies. Clonality of samples was confirmed through SNP calling, which proved to be a more suitable method for an integrated data analysis of Illumina and Ion Torrent data sets in this study.

Foodborne infection with Listeria causes potentially life-threatening disease listeriosis. Listeria monocytogenes is widely recognized as the only species of public health concern, and the closely related species Listeria innocua is commonly used by the food industry as an indicator to identify environmental conditions that allow for presence, growth, and persistence of Listeria spp. in general. In our study, we analyze the occurrence of Listeria spp. in a farm-to-fork approach in a poultry production chain in Egypt and identify bacterial entry gates and transmission systems. Prevalence of Listeria innocua at the three production stages (farm, slaughterhouse, food products) ranged from 11% to 28%. The pathogenic species Listeria monocytogenes was not detected, and Listeria innocua strains under study did not show genetic virulence determinants. However, the close genetic relatedness of Listeria innocua isolates (maximum 63 SNP differences) indicated cross-contamination between all stages from farm to final food product. Based on these results, chicken can be seen as a natural source of Listeria. Last but not least, sanitary measures during production should be reassessed to prevent bacterial contamination from entering the food chain and to consequently prevent human listeriosis infections. For this purpose, surveillance must not be restricted to pathogenic species.

Emerging infectious diseases 23(10), 1680-1683 (2017). doi:10.3201/eid2310.161623, Published by CDC, Atlanta, Ga.

An outbreak with a remarkable Listeria monocytogenes clone causing 163 cases of non-invasive listeriosis occurred in Germany in 2015. Core genome multi locus sequence typing grouped non-invasive outbreak isolates and isolates obtained from related food samples into a single cluster, but clearly separated genetically close isolates obtained from invasive listeriosis cases. A comparative genomic approach identified a premature stop codon in the chiB gene, encoding one of the two L. monocytogenes chitinases, which clustered with disease outcome. Correction of this premature stop codon in one representative gastroenteritis outbreak isolate restored chitinase production, but effects in infection experiments were not found. While the exact role of chitinases in virulence of L. monocytogenes is still not fully understood, our results now clearly show that ChiB-derived activity is not required to establish L. monocytogenes gastroenteritis in humans. This limits a possible role of ChiB in human listeriosis to later steps of the infection.

Foodborne pathogens such as L. monocytogenes can persist in food production environments for a long time, causing perennial outbreaks. Hence, bacterial pathogens are able to survive cleaning and disinfection procedures. Accordingly, they may be repeatedly exposed to sublethal concentrations of disinfectants, which might result in bacterial adaptation to these biocides. Furthermore, antibiotic coresistance and cross-resistance are known to evolve under biocide selection pressure in vitro. Hence, antimicrobial tolerance seems to play a crucial role in the resilience and persistence of foodborne pathogens in the food chain and might reduce therapeutic options in infectious diseases., Contamination of food during processing is recognized as a main transmission route of Listeria monocytogenes. To prevent microbial contamination, biocides are widely applied as disinfectants in food processing plants. However, there are concerns about the development of antimicrobial resistance in foodborne pathogens due to widespread biocide usage. In our study, 93 L. monocytogenes isolates from German food production facilities were (i) tested for biocide and antibiotic susceptibility using broth microdilution assays, (ii) analyzed for links between reduced biocide susceptibility and antibiotic resistance, and (iii) characterized by whole-genome sequencing, including the detection of genes coding for biocide tolerance, antibiotic resistance, and other virulence factors. Fifteen L. monocytogenes isolates were tolerant to benzalkonium chloride (BAC), and genes conferring BAC tolerance were found in 13 of them. Antibiotic resistance was not associated with biocide tolerance. BAC-tolerant isolates were assigned to 6 multilocus sequence type (MLST) clonal complexes, and most of them harbored internalin A pseudogenes with premature stop codons or deletions (n = 9). Our study demonstrated a high genetic diversity among the investigated isolates including genotypes that are frequently involved in human infections. Although in vitro adaptation studies to biocides have raised concerns about increasing cross-resistance to antibiotics, our results do not provide evidence for this phenomenon in field isolates. IMPORTANCE Foodborne pathogens such as L. monocytogenes can persist in food production environments for a long time, causing perennial outbreaks. Hence, bacterial pathogens are able to survive cleaning and disinfection procedures. Accordingly, they may be repeatedly exposed to sublethal concentrations of disinfectants, which might result in bacterial adaptation to these biocides. Furthermore, antibiotic coresistance and cross-resistance are known to evolve under biocide selection pressure in vitro. Hence, antimicrobial tolerance seems to play a crucial role in the resilience and persistence of foodborne pathogens in the food chain and might reduce therapeutic options in infectious diseases.

Background: The objective of this study was to evaluate the susceptibility of 259 Listeria monocytogenes strains isolated from food and food-processing environments and patient samples in Germany to 14 antibiotics widely used in veterinary and human medicine. L. monocytogenes strains were isolated mainly from milk and milk products and classified according to their molecular serotypes IIa (n = 112), IIb (n = 41), IIc (n = 36), IVa (n = 1), IVb (n = 66), and IVb-v1 (n = 3). Methods: Susceptibility tests were performed by using the automated 96-well based microdilution system Micronaut-S. Ampicillin, benzylpenicillin, ceftriaxone, ciprofloxacin, daptomycin, erythromcyin, gentamicin, linezolid, meropenem, rifampicin, tetracycline, tigecycline, trimethoprim/sulfamethoxazole and vancomycin were tested in at least five different concentrations. Results: Among the 259 strains under study, 145 strains revealed multidrug-resistance (resistance to ≥3 antibiotics) and predominantly belonged to serotype IV (59%). Strains were mainly resistant to daptomycin, tigecycline, tetracycline, ciprofloxacin, ceftriaxone, trimethropim/sulfamethoxazole and gentamicin. Conclusions: Antibiotic resistance in general and multidrug-resistance in particular were more prevalent in L. monocytogenes strains isolated in Germany compared to similar reference stocks from other European countries and the USA but similar to stocks from China. Keywords: Antibiotic resistance, Antimicrobials, Germany, Listeria monocytogenes, Susceptibility testing

Enteropathogenic Escherichia coli (EPEC) is recognized as an important intestinal pathogen that frequently causes acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probiotic E. coli strain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. While both in vitro and in vivo studies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including pathogenic E. coli , the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenic E. coli (aEPEC) with respect to single infection steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of the inhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression.

Biofilms develop successively on devices of milk production without sufficient cleaning and originate from the microbial community of raw milk. The established biofilm matrices enable incorporation of pathogens like Listeria monocytogenes, which can cause a continuous contamination of food processing plants. L. monocytogenes is frequently found in raw milk and non-pasteurized raw milk products and as part of a biofilm community in milk meters and bulk milk tanks. The aim of this study was to analyze whether different L. monocytogenes strains are interacting with the microbial community of raw milk in terms of biofilm formation in the same manner, and to identify at which stage of biofilm formation a selected L. monocytogenes strain settles best. Bacterial community structure and composition of biofilms were analyzed by a cloning and sequencing approach and terminal restriction fragment length polymorphism analysis (T-RFLP) based on the bacterial 16S rRNA gene. The chemical composition of biofilms was analyzed by Fourier transform infrared spectroscopy (FTIR), while settled L. monocytogenes cells were quantified by fluorescence in situ hybridization (FISH). Addition of individual L. monocytogenes strains to raw milk caused significant shifts in the biofilm biomass, in the chemical as well as in the bacterial community composition. Biofilm formation and attachment of L. monocytogenes cells were not serotype but strain specific. However, the added L. monocytogenes strains were not abundant since mainly members of the genera Citrobacter and Lactococcus dominated the bacterial biofilm community. Overall, added L. monocytogenes strains led to a highly competitive interaction with the raw milk community and triggered alterations in biofilm formation.

In the past, Listeria monocytogenes has been isolated from game feces and meat. However, less information is available on the occurrence of L. monocytogenes in other specimens originating from game animals. Hence, the aim of this study was to get an overview of the occurrence and distribution of L. monocytogenes in game animals by characterization of isolates from different matrices. For that purpose, samples were collected from red deer (Cervus elaphus), wild boars (Sus scrofa), and feed during the hunting season 2011-2012 in three different regions of Germany and Austria. Six samples from each animal were examined: tonsils, content of the rumen or the stomach, liver, intestinal lymph nodes, cecum content, and feces. Nineteen of 45 red deer and 12 of 49 wild boars were found to be positive for L. monocytogenes as well as 4 of 22 pooled feed samples. L. monocytogenes was isolated most frequently from the rumen of red deer (14 of 19) and the tonsils of wild boars (7 of 12). Serotypes 1/2a, 1/2b, 4a, and 4b were detected in samples of game animals and feed, and serotypes 1/2a and 4b were the most prevalent serotypes. The presence of L. monocytogenes serotype 4a had not yet been described in red deer. This might be due to the fact that it was only isolated from the content of rumen and that no other study has yet examined ruminal content. Pulsed-field gel electrophoresis showed a wide variety of strains. Some strains occurred in both species and feed samples, but one strain was dominant in one region. The results show that red deer and wild boars can be carriers of L. monocytogenes in different matrices, although the feces samples can be negative.

Nonpathogenic, intestinal Escherichia coli (commensal E. coli ) supports the physiological intestinal balance of the host, whereas pathogenic E. coli with typical virulence factor gene profiles can cause severe outbreaks of diarrhea. In many reports, E. coli isolates from diarrheic animals were classified as putative pathogens. Here we describe a broad variety of virulence gene-positive E. coli isolates from swine with no clinical signs of intestinal disease. The isolation of E. coli from 34 pigs from the same population and the testing of 331 isolates for genes encoding heat-stable enterotoxins I and II, heat-labile enterotoxin I, Shiga toxin 2e, and F4, F5, F6, F18, and F41 fimbriae revealed that 68.6% of the isolates were positive for at least one virulence gene, with a total of 24 different virulence factor gene profiles, implying high rates of horizontal gene transfer in this E. coli population. Additionally, we traced the occurrence of hemolytic E. coli over a period of 1 year in this same pig population. Hemolytic isolates were differentiated into seven clones; only three were found to harbor virulence genes. Hemolytic E. coli isolates without virulence genes or with only the fedA gene were found to be nontypeable by slide agglutination tests with OK antisera intended for screening live cultures against common pathogenic E. coli serogroups. The results appear to indicate that virulence gene-carrying E. coli strains are a normal part of intestinal bacterial populations and that high numbers of E. coli cells harboring virulence genes and/or with hemolytic activity do not necessarily correlate with disease.

In vitro studies on the pathogenesis in swine have been hampered by the lack of relevant porcine cell lines. Since many bacterial infections are swine-specific, studies on pathogenic mechanisms require appropriate cell lines of porcine origin. We have characterized the permanent porcine intestinal epithelial cell line, IPEC-J2, using a variety of methods in order to assess the usefulness of this cell line as an in vitro infection model. Electron microscopic analyses and histochemical staining revealed the cells to be enterocyte-like with microvilli, tight junctions and glycocalyx-bound mucin. The functional integrity of monolayers was determined by transepithelial electrical resistance (TEER) measurements. Both commensal bacteria and important bacterial pathogens were chosen for study based on their principally different infection mechanisms: obligate extracellular Escherichia coli, facultative intracellular Salmonella and obligate intracellular Chlamydia. We determined the colonization and proliferation of the bacteria on and within the host cells and monitored the host cell response. We verified the expression of mRNAs encoding the cytokines IL-1alpha, -6, -7, -8, -18, TNF-alpha and GM-CSF, but not TGF-beta or MCP-1. IL-8 protein expression was enhanced by Salmonella invasion. We conclude that the IPEC-J2 cell line provides a relevant in vitro model system for porcine intestinal pathogen-host cell interactions.

Background: The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known. Methodology/Principal Findings: In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra-and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN. Conclusions: We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion.

Aims: To verify the presence of Escherichia coli Nissle 1917 as a natural isolate in swine and to characterize in vitro probiotic properties as well as in vivo persistence in a feeding experiment. Methods and Results: During studies on the intestinal microflora of pigs, we isolated E. coli Nissle 1917 sporadically from a pig population over a period of 1 year. The identity of the isolates as E. coli Nissle 1917 was verified by serotyping, Nissle-specific PCR, macrorestriction analysis (pulsed field gel electrophoresis) and the determination of in vitro probiotic properties in invasion and adhesion assays using a porcine intestinal epithelial cell line. Both the E. coli isolates and the E. coli Nissle 1917 strain showed strong reductions in adhesion of porcine enteropathogenic E. coli and invasion of Salmonella typhimurium with epithelial cells in vitro, with a probiotic effect. Screening of five epidemiologically unlinked swine farms and two wild boar groups showed one farm positive for E. coli Nissle 1917. A feeding experiment with four piglets showed viable E. coli Nissle 1917 in the intestine of three animals. Conclusions: The results of this study suggest that the E. coli Nissle 1917 strain is already partially established in swine herds, but the colonization of individual animals is variable. Significance and Impact of the Study: We report natural, long-term colonization and transmission of the probiotic E. coli Nissle 1917 strain in a swine herd, characterized individual persistence and colonization properties in swine and established an in vitro porcine intestinal epithelial cell model of probiotic action. The results of this study would have implications in the use of this strain as a probiotic in swine and contribute to a better understanding of the individual nature of intestinal bacterial persistence and establishment.