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This is the deliverable D-JRP21-WP6.3 "Workshop 1 completed" of the OHEJP BIOPIGEE project produced in WP6 T3.

Summary Background Salmonella spp are a major cause of food-borne outbreaks in Europe. We investigated a large multi-country outbreak of Salmonella enterica serotype Enteritidis in the EU and European Economic Area (EEA). Methods A confirmed case was defined as a laboratory-confirmed infection with the outbreak strains of S Enteritidis based on whole-genome sequencing (WGS), occurring between May 1, 2015, and Oct 31, 2018. A probable case was defined as laboratory-confirmed infection with S Enteritidis with the multiple-locus variable-number tandem repeat analysis outbreak profile. Multi-country epidemiological, trace-back, trace-forward, and environmental investigations were done. We did a case-control study including confirmed and probable cases and controls randomly sampled from the population registry (frequency matched by age, sex, and postal code). Odds ratios (ORs) for exposure rates between cases and controls were calculated with unmatched univariable and multivariable logistic regression. Findings 18 EU and EEA countries reported 838 confirmed and 371 probable cases. 509 (42%) cases were reported in 2016, after which the number of cases steadily increased. The case-control study results showed that cases more often ate in food establishments than did controls (OR 3·4 [95% CI 1·6–7·3]), but no specific food item was identified. Recipe-based food trace-back investigations among cases who ate in food establishments identified eggs from Poland as the vehicle of infection in October, 2016. Phylogenetic analysis identified two strains of S Enteritidis in human cases that were subsequently identified in salmonella-positive eggs and primary production premises in Poland, confirming the source of the outbreak. After control measures were implemented, the number of cases decreased, but increased again in March, 2017, and the increase continued into 2018. Interpretation This outbreak highlights the public health value of multi-country sharing of epidemiological, trace-back, and microbiological data. The re-emergence of cases suggests that outbreak strains have continued to enter the food chain, although changes in strain population dynamics and fewer cases indicate that control measures had some effect. Routine use of WGS in salmonella surveillance and outbreak response promises to identify and stop outbreaks in the future. Funding European Centre for Disease Prevention and Control; Directorate General for Health and Food Safety, European Commission; and National Public Health and Food Safety Institutes of the authors' countries (see Acknowledgments for full list).

Introduction The article describes the occurrence and phylogenetic relationship of Salmonella isolates found in subcutaneous abscesses of leopard geckos. The aim of the study was to determine the cause of the abscesses and to characterise isolated Salmonella strains. Material and Methods Samples of abscesses from five animals and internal organs (lungs, liver, and gut) of three of them were tested for Salmonella according to the PN-EN ISO 6579:2002/A1:2007 standard. The antimicrobial resistance was evaluated by minimal inhibitory concentrations and the genetic similarity of the isolates was assessed with pulsed field gel electrophoresis (PFGE). Results In total, seventeen Salmonella isolates belonging to five different serovars were found to be susceptible to all tested antimicrobials except streptomycin. The serovars were S. Hadar, S. Fluntern, S. Tennessee, S. enterica subsp. salamae 55:k:z39, and S. Kentucky. Up to three serovars from different organs were isolated from the same individual. In two geckos, Salmonella were detected in the lungs. In three serovars, XbaI-PFGE typing revealed indistinguishable isolates from organs and abscesses. Conclusion Multiple Salmonella serovars might be involved in abscess formation and infections. The occurrence of the same PFGE profiles of the isolates may testify to the role of opportunistic organisms in causing infection.

This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr-positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre-enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr-1 or mcr-3 and mcr-4 or mcr-5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr-positive colistin-resistant E. coli or Salmonella spp. strains was 86% for CHROMID(R)Colistin R, 75% for CHROMagarTM COL-APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food-producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food-producing animals and food products in Europe.

Turtoises are a great puzzle when it comes to their bacterial flora, the composition and structure of which are still unknown in details. Its component which has been best described so far is Salmonella spp., presumably due to the threat of reptile-associated salmonellosis in humans. This investigation tried to assess and characterize intestinal bacterial flora of imported tortoises found dead during quarantine. Most of the animals carried various serovars of Salmonella showing no antimicrobial resistance. Presence of multiresistant Escherichia coli was possibly a result of industrial breeding and high usage of antimicrobials. Thirteen bacterial species or genera like Citrobacter spp., Morganella spp., Pseudomonas spp. were identified. Their commensal character is assumed, although pathogenic potential might be verified. The results indicate global tortoise trade as a source of common and exotic bacteria or antimicrobial resistance mechanisms in new geographical areas. These dangers indicate the need for a systematic survey of exotic pets and establishment of legal requirements for reptile health conditions on breeding, trade premises and in households with such pets.

Proposals to update the harmonised monitoring and reporting of antimicrobial resistance (AMR) from a public health perspective in Salmonella, Campylobacter coli, Campylobacter jejuni, Escherichia coli, Enterococcus faecalis, Enterococcus faecium and methicillin‐resistant Staphylococcus aureus (MRSA) from food‐producing animals and derived meat in the EU are presented in this report, accounting for recent trends in AMR, data collection needs and new scientific developments. Phenotypic monitoring of AMR in bacterial isolates, using microdilution methods for testing susceptibility and interpreting resistance using epidemiological cut‐off values is reinforced, including further characterisation of those isolates of E. coli and Salmonella showing resistance to extended‐spectrum cephalosporins and carbapenems, as well as the specific monitoring of ESBL/AmpC/carbapenemase‐producing E. coli. Combinations of bacterial species, food‐producing animals and meat, as well as antimicrobial panels have been reviewed and adapted, where deemed necessary. Considering differing sample sizes, numerical simulations have been performed to evaluate the related statistical power available for assessing occurrence and temporal trends in resistance, with a predetermined accuracy, to support the choice of harmonised sample size. Randomised sampling procedures, based on a generic proportionate stratified sampling process, have been reviewed and reinforced. Proposals to improve the harmonisation of monitoring of prevalence, genetic diversity and AMR in MRSA are presented. It is suggested to complement routine monitoring with specific cross‐sectional surveys on MRSA in pigs and on AMR in bacteria from seafood and the environment. Whole genome sequencing (WGS) of isolates obtained from the specific monitoring of ESBL/AmpC/carbapenemase‐producing E. coli is strongly advocated to be implemented, on a voluntary basis, over the validity period of the next legislation, with possible mandatory implementation by the end of the period; the gene sequences encoding for ESBL/AmpC/carbapenemases being reported to EFSA. Harmonised protocols for WGS analysis/interpretation and external quality assurance programmes are planned to be provided by the EU‐Reference Laboratory on AMR.

The aim of this study was to estimate virulence potential of Salmonella enterica strains colonizing the gut of free-living sand lizards (Lacerta agilis L.). The strains belonged to three Salmonella serovars: Abony, Schleissheim, and Telhashomer. Adhesion and invasion abilities of the strains were determined in quantitative assays using the gentamicin protection method. Induction of apoptosis was assessed using HeLa cell monolayers. PCR assays were used for detection of 26 virulence genes localised within mobile elements: pathogenicity islands, virulence plasmids, and prophage sequences. In vitro studies revealed that all strains had adhesion and invasion abilities to human epithelial cells. The isolates were cytotoxic and induced apoptosis of the cells. The serovars differed in the number of virulence-associated genes: up to 18 genes were present in Salmonella Schleissheim, 17 in Salmonella Abony, whereas as few as six genes were found in Salmonella Telhashomer. Generally, Salmonella Abony and Salmonella Schleissheim did not differ much in gene content connected with the presence SPI-1 to -5. All of the strains lacked genes localised within bacteriophages and plasmids. The presence of virulence-associated genes and in vitro pathogenicity assays suggest that Salmonella sp. strains originating from autochthonous, free-living lizards can potentially infect and cause disease in humans. Electronic supplementary material The online version of this article (10.1007/s10482-018-1079-8) contains supplementary material, which is available to authorized users.

Introduction: Data collection on the Salmonella occurrence is crucial in effective implementation of different actions or control programmes aiming to protect consumers’ health and to reduce the level of Salmonella prevalence in farm animals. The goal was to describe Salmonella serovar distribution along the food chain in Poland during 2010–2015 and to identify their epidemiological importance. Material and Methods: Slide agglutination according to White-Kauffmann-Le Minor scheme was used to identify Salmonella serovars of 6,928 isolates originating from animals, food, feeds, and fertilisers. Results: In total, 160 Salmonella serovars were identified. Differences in serovar distribution were observed depending on animal species. Among isolates from hens, S. Enteritidis and S. Infantis were the most prevalent. Serovar pattern in turkeys differed from those in hens, with S. Kentucky, S. Newport, S. Saintpaul being the most prevalent. Monophasic S. Typhimurium was predominant in pigs. Serovars found in food reflected those observed among livestock animals. Nine out of the ten most prevalent serovars in animals and humans were also found in organic fertilisers. Conclusion: Serotyping of large number of isolates from different sources is essential for insight on emerging serovars and trends of Salmonella occurrence. This may increase the value of epidemiological data and result in updating of Salmonella control programmes to target further epidemiologically important serovars in animals and better protection of consumers’ health.

There has been an increase in the number of reports on Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) isolated from animals and humans. Recent studies using whole genome sequencing (WGS) have provided evidence on the likely contribution of a unique conjugative megaplasmid (pESI; ~280 kb) to the dissemination of this serovar worldwide. In the present study, twenty-two unrelated Salmonella strains [S. Infantis (n = 20) and Salmonella 6,7:r:- (n = 2)] and their plasmids were investigated using next generation sequencing technologies (MiSeq and MinION) to unravel the significant expansion of this bacteria in Turkey. Multi-locus sequence typing, plasmid replicons, resistance gene contents as well as phylogenetic relations between strains were determined. According to the WGS data, all S. Infantis possessed the relevant megaplasmid backbone genes and belonged to sequence type 32 (ST32) with the exception of a single novel ST7091. Tetracycline and trimethoprim/sulfamethoxazole resistance were found to be widespread in S. Infantis strains and the resistant strains exclusively carried the tetA, sul1, sul2 and dfrA14 genes. One S. Infantis isolate was also a carrier of the plasmid-mediated ampC via blaCMY-2, gene. Moreover, full genomes of four S. Infantis isolates were reconstructed based on hybrid assembly. All four strains contained large plasmids (240–290 kb) similar to previously published megaplasmid (pESI) and accompanied by several small plasmids. The megaplasmid backbone contained a toxin-antitoxin system, two virulence cassettes and segments associated with heavy metals resistance, while variable regions possessed several antibiotic resistance genes flanked by mobile elements. This study indicated that pESI-like megaplasmid is widely disseminated within the tested S. Infantis strains of chicken meat, warranting further genomic studies on clinical strains from humans and animals to uncover the overall emergence and spread of this serovar.

The aim of the study was to estimate the herd-level, within-herd prevalence, the frequency of mixed infections and risk factors forL. intracellularis,B. hyodysenteriaeandSalmonellaspp. in selected farrow-to-finish Polish pig herds. A total of 254 pooled fecal samples were collected from 9 to 24 week-old pigs in 70 herds. Real time PCR for detection ofL. intracellularisandB. hyodysenteriaewas performed. ForSalmonellaspp. bacteriological examination was performed. The herd-level prevalences ofL. intracellularis, B. hyodysenteriaeandSalmonellaspp. among examined herds were 65.7%, 1.4% and 8.6%, respectively. The within-herd prevalences (in positive herds) forL. intracellularis,B. hyodysenteriaeandSalmonellaspp. were 51.5%, 75.0% and 30.4%, respectively. All herds with diarrhea observed during sampling were infected withL. intracellularisand 60% of herds with no diarrhea at the moment of sampling were infected withL. intracellularis(p=0.035). In herds with more than 200 sows the prevalence ofSalmonellaspp. was significantly higher compared to herds with less than 200 sows (p=0.027). In herds where all-in/all-out (AIAO) was respected, prevalence ofL. intracellulariswas significantly lower than in herds where this rule was not kept (p=0.024). Obtained results confirm thatL. intracellularisis the major cause of bacterial diarrhea in finishing pigs. The present study identified AIAO and herd size as a risk factor, at the herd level, forL. intracellularisandSalmonellaspp., respectively.

The background of quinolone resistance was characterized in ciprofloxacin-resistant commensal Escherichia coli selected out of 3,551 isolates from slaughtered animals in Poland between 2009 and 2012. Plasmid-mediated determinants were suspected in 6.2% of the study group, ranging from 1.1% in cattle to 9.7% in turkeys. Polymerase chain reaction and sequencing identified up to four quinolone resistance-determining substitutions in gyrA (Ser83, Asp87) and parC (Ala56, Ser80). Plasmid-mediated mechanisms were identified as qnrS1 (or qnrS3, n=70, including six isolates with chromosomal mutations), qnrB19 (or qnrB10, n=19), and qnrB17 (n=1). All tested isolates were negative for qnrA, qnrC, qnrD, qepA, and aac(6')-Ib-cr. Still, there were several E. coli suspected for both plasmid- and chromosome-mediated resistance with unrevealed genetic background of the phenomenon. Since all tested isolates showed diverse XbaI-PFGE profiles, chromosome-encoded quinolone resistance does not result from the spread of a single resistant clone, however, it is rather due to antimicrobial pressure leading to the selection of random gyr and par mutants. It also favors the selection and spread of plasmids carrying predominant qnr genes, since the same determinants were found in Salmonella, isolated from similar sources. The identification of carrier plasmids and mitigation of their spread might be essential for sustainable quinolone usage in animal husbandry and efficient protection of human health.

The aim of the study was to determine both environmental and infectious factors, influencing occurrence of bacterial enteropathogens in suckling piglets. For this purpose, a cross-sectional study in 70 Polish farrow-to-finish pig herds was performed. In each herd, presence of selected intestinal pathogens (Escherichia coli, Clostridium perfringens and Salmonella species) in faeces of suckling piglets were determined using bacteriological and PCR methods. Using logistic regression models, risk factors for enterotoxigenic E coli with fimbriae F4 (ETEC-F4) and C perfringens type A were established. Prevalence of bacterial enteropathogens in investigated herds was as follows (on herd and sample level): ETEC-F4 30.0 per cent and 9.3 per cent, respectively, C perfringens type A 91.4 per cent and 58.3 per cent, C perfringens type C 1.4 per cent and 0.3 per cent, Salmonella species 2.9 per cent and 0.5 per cent. The occurrence of ETEC-F4 was associated with presence of diarrhoea in piglets and vulval discharge in sows. Vulval discharge in sows, not slatted floor in farrowing pen and lack of coccidiosis prevention were determined as risk factors for C perfringens type A. The results obtained in the study revealed the high prevalence of enteropathogenic bacteria in pig herds and indicate management and organisation of production as factors having the greatest impact on the incidence of bacterial enteropathogens in piglets before weaning.

In the 1990s, [i]Salmonella enterica[/i] serovar (S.) Mbandaka occurred in feed and poultry in Poland. In the following years, the serovar also gained epidemiological importance in other EU countries. The objectives of current study were to evaluate the genetic relationship of contemporary S. Mbandaka with isolates originating from the beginning of the epidemics, and to assess the contribution of poultry as the source of infections in humans. Seventy S. Mbandaka isolated mainly in 2009 – 2010 from humans, poultry, food, and feed were typed with API ID32 [sup]®[/sup], MIC, plasmid profiling, PFGE, and MLST. PCR and sequencing were used to identify plasmid mediated quinolone and cephalosporin resistance mechanisms. Six biochemical profiles were identified and 59 of S. Mbandaka proved to be susceptible to the applied antimicrobials. Eight strains carried plasmids and a few of them were positive for [i]bla[/i][sub]CMY-2[/sub] and [i]qnr[/i]S1 genes. Two clusters of 15 [i]XbaI[/i]-PFGE profiles with similarity of 77.5% were found. The first cluster, gathered 7 profiles involving historical isolates and several contemporary non-human S. Mbandaka. The predominant profile in the second cluster consisted of 28 human and 1 broiler isolate. MLST analysis showed sequence type ST413 occurring among all tested isolates. The identification of close genetic relationships between S. Mbandaka of human and poultry origin indicates animals as a primal human infection route. Despite [i]Salmonella [/i]control programmes, the S. Mbandaka ST413 clone has been circulating for several years in Poland. [i]Salmonella[/i] control polices in food production chain should be continuously updated to target serovars of major epidemiological importance. Resistance noted in S. Mbandaka to such antimicrobials as fluoroquinolones and cephalosporins may hinder public health.

The epidemiological role of monophasic Salmonella enterica subsp. enterica serovar Typhimurium tends to increase, indicating pandemic spread. The aim of the present study was to confirm the occurrence of this serological variant in Poland and to report the first cases in Belarus and Ukraine. Genetic similarity of monophasic isolates with Salmonella Typhimurium already present in these countries was assessed. Serotyping, duplex-polymerase chain reaction (PCR) assay, antibiotic resistance and pulsed-field gel electrophoresis (PFGE) profiling have been used to meet the study objectives. Monophasic Salmonella Typhimurium was found at low frequency in various sources along the food chain, including feed, animals, meat, and sewage sludge. The first isolates date back to 2008. The clones observed in other European countries were found, along with a number of new, unrelated genetic lineages appearing locally in three countries. Monophasic Salmonella Typhimurium is claimed to replace and discontinue the domination of pentaresistant Salmonella Typhimurium. Pigs and pork are assumed to be the main vectors of monophasic Salmonella Typhimurium, but their relevance for public health is limited.

The aim of the study was the assessment of microbiological quality of compound feed used in Poland in 2007-2010. The examinations were done at all veterinary diagnostic laboratories operating in the frame of official laboratory system. The occurrence of Salmonella sp. and counts of Enterobacteriaceae family, mesophilic aerobic bacteria, total microorganisms, and fungi were assessed. Assays were done following Polish, European, and international standards. Percentage of contamination of compound feed for poultry, pigs, and cattle by Salmonella sp. ranged from 0% to 3.5%. The highest contamination level by Enterobacteriaceae bacteria were detected in wet petfood. No more than 106 cfu/g of aerobic bacteria and no more than 105 cfu/g of fungi were detected in the feed. The results of the study revealed that the microbiological quality of compound feed used in Poland in 2007-2010 was better than the quality of the feed used in 2003-2006.

The first CTX-M-producing Salmonella was described in primary animal production in Poland, due to the antimicrobial resistance monitoring and control program introduced in turkeys. It was associated with the outbreak of multiresistant Salmonella Kentucky in non-diseased turkeys, foods and food production environment, but found also in municipal sewage sludge. The emergence along the food chain of clonally related strains resistant to critically important antimicrobial agents, including cephalosporins, quinolones, sulfonamides, aminoglycosides, phenicols, and tetracycline, which are used against foodborne pathogens, poses a serious public health threat.

Objectives This study was initiated to collect retrospective information on the occurrence of plasmid-mediated quinolone resistance (PMQR) in Salmonella enterica and Escherichia coli isolates in Europe and to identify the responsible genes. Methods Databases of national reference laboratories containing MIC values for Salmonella and E. coli isolated between 1994 and 2009 in animals, humans, food and the environment from 13 European countries were screened for isolates exhibiting a defined quinolone resistance phenotype, i.e. reduced susceptibility to fluoroquinolones and nalidixic acid. PCR and sequence analysis were performed to identify the responsible PMQR genes. Results Screening of databases of 13 European countries resulted in a selection of 1215 Salmonella and 333 E. coli isolates. PMQR genes were identified in 59% of the Salmonella isolates and 15% of the E. coli isolates selected. In Salmonella, qnrS1 (n¿=¿125) and variants of qnrB (n¿=¿138) were frequently identified, whereas qnrA1 (n¿=¿3) and aac(6')-1b-cr (n¿=¿3) were rarely found. qnrD was detected in 22 Salmonella isolates obtained from humans and animals. In E. coli, qnrS1 was identified in 19 isolates and qnrB19 was found in one isolate. No qnrC or qepA genes were detected in either Salmonella or E. coli. Conclusions This study shows the occurrence and dissemination of PMQR genes in Salmonella and E. coli in Europe with a defined quinolone resistance phenotype. We also report the first detection of qnrD in Salmonella collected in Europe.

Many Member States of the European Union (EU) currently monitor antimicrobial resistance in zoonotic agents, including Salmonella and Campylobacter. According to Directive 2003/99/EC, Member States shall ensure that the monitoring provides comparable data on the occurrence of antimicrobial resistance. The European Commission asked the European Food Safety Authority to prepare detailed specifications for harmonised schemes for monitoring antimicrobial resistance. The objective of these specifications is to lay down provisions for a monitoring and reporting scheme for Salmonella in fowl ( Gallus gallus ), turkeys and pigs, and for Campylobacter jejuni and Campylobacter coli in broiler chickens. The current specifications are considered to be a first step towards a gradual implementation of comprehensive antimicrobial resistance monitoring at the EU level. These specifications propose to test a common set of antimicrobial agents against available cut-off values and a specified concentration range to determine the susceptibility of Salmonella and Campylobacter. Using isolates collected through programmes in which the sampling frame covers all epidemiological units of the national production, the target number of Salmonella isolates to be included in the antimicrobial resistance monitoring per Member State per year is 170 for each study population (i.e., laying hens, broilers, turkeys and slaughter pigs). The target number of Campylobacter isolates to be included in the antimicrobial resistance monitoring per Member State per year is 170 for each study population (i.e., broilers). The results of the antimicrobial resistance monitoring are assessed and reported in the yearly national report on trends and sources of zoonoses, zoonotic agents and antimicrobial resistance.

SUMMARYFifty-sevenSalmonellaTyphimurium strains isolated from poultry, swine and animal feed in Poland during the years 1979–1998 and 2000–2002 were analysed with conventional and molecular techniques. Antimicrobial resistance as well as multiresistance was found, respectively, in 80·1% and 56·1% of the isolates and most frequently among isolates from 2000–2002. Of several phage types noted, DT104 was prevalent among poultry, swine and feed isolates. DT104, U302 and non-typable strains had a multiple resistant profile (ACSSuT) due to the presence of class I integrons. Pulse-field gel electrophoresis ofXbaI andBlnI digest showed high genomic similarity between the strains and confirmed clonal spread ofS. Typhimurium infections. Plasmid profiling allowed further differentiation of the strains. We have, therefore, confirmed the appearance ofS. Typhimurium DT104 showing genome integrated integron-mediated antimicrobial resistance in Poland. These findings are significant for public and animal health risks and document the dissemination of DT104 epidemic strains into new geographical regions.

The study was focused on characterisation of quinolone resistance mechanisms in Salmonella isolated from animals, food, and feed between 2008 and 2011. Testing of Minimal Inhibitory Concentrations revealed 6.4% of 2680 isolates conferring ciprofloxacin resistance. Simultaneously 37.7% and 40.8% were accounted for, respectively, nalidixic acid and ciprofloxacin Non Wild-Type populations. Amplification and sequencing of quinolone resistance determining region of topoisomerases genes in 44 isolates identified multiple amino-acid substitutions in gyrA at positions Ser83 (N=22; → Leu, → Phe, → Tyr), Asp87 (N=22; → Asn, → Gly, → Tyr) and parC (Thr57Ser, N=23; Ala141Ser, N=1). No relevant mutations were identified in gyrB and parE. Twelve patterns combining one or two substitutions were related to neither serovar nor ciprofloxacin MIC. In 92 isolates suspected for plasmid mediated quinolone resistance two qnr alleles were found: qnrS1 (or qnrS3; N=50) and qnrB19 (or qnrB10; N=24). Additionally, two isolates with chromosomally encoded mechanisms carried qnrS1 and qnrS2. All tested isolates were negative for qnrA, qnrC, qnrD, qepA, aac(6')-Ib-cr. Both chromosomal and plasmid mediated quinolone resistance determinants were found in several Salmonella serovars and Pulsed Field Gel Electrophoresis was used to assess phylogenetic similarity of selected isolates (N=82). Salmonella Newport was found to accumulate quinolone resistance determinants and the serovar was spreading clonally with either variable gyrA mutations, qnrS1/S3, or qnrB10/B19. Alternatively, various determinants are dispersed among related S. Enteritidis isolates. Antimicrobial selection pressure, multiple resistance determinants and scenarios for their acquisition and spread make extremely difficult to combat quinolone resistance.

While the spread of Salmonella enterica serotype Kentucky resistant to ciprofloxacin across Africa and the Middle-East has been described recently, the presence of this strain in humans, food, various animal species (livestock, pets, and wildlife) and in environment is suspected in other countries of different continents. Here, we report results of an in-depth molecular epidemiological study on a global human and non-human collection of S. Kentucky (n = 70). We performed XbaI-pulsed field gel electrophoresis and multilocus sequence typing, assessed mutations in the quinolone resistance-determining regions, detected β-lactam resistance mechanisms, and screened the presence of the Salmonella genomic island 1 (SGI1). In this study, we highlight the rapid and extensive worldwide dissemination of the ciprofloxacin-resistant S. Kentucky ST198-X1-SGI1 strain since the mid-2000s in an increasingly large number of contaminated sources, including the environment. This strain has accumulated an increasing number of chromosomal and plasmid resistance determinants and has been identified in the Indian subcontinent, Southeast Asia and Europe since 2010. The second substitution at position 87 in GyrA (replacing the amino acid Asp) appeared helpful for epidemiological studies to track the origin of contamination. This global study provides evidence leading to the conclusion that high-level resistance to ciprofloxacin in S. Kentucky is a simple microbiological trait that facilitates the identification of the epidemic clone of interest, ST198-X1-SGI1. Taking this into account is essential in order to detect and monitor it easily and to take rapid measures in livestock to ensure control of this infection.

The purpose of the study was to define genetic diversity of reptilian Salmonella enterica serovar (S.) Kentucky isolates and their epidemiological relations to the ones from poultry, food, and environmental origin in Poland. Between 2010 and 2012 twenty-four S. Kentucky isolates derived from snakes (N = 8), geckos (N = 7), chameleons (N = 4), agamas (N = 1), lizard (N = 1), and environmental swabs taken from reptile exhibition (N = 3) were identified. They were characterized with antimicrobial minimal inhibitory concentration testing, XbaI–PFGE and MLST typing. The profiles compared to S. Kentucky available in BioNumerics local laboratory database (N = 40) showed 67.3% of relatedness among reptile isolates. Three genetic lineages were defined. The first lineage gathered 20 reptile isolates with 83.4% of similarity and wild-type MICs for all antimicrobials tested but streptomycin in single case. The remaining three reptilian and one post-exhibition environment S. Kentucky isolates were clustered (87.2%) with isolates originating from poultry, mainly turkey, food, and environment and presented variable non-wild type MICs to numerous antimicrobials. The third S. Kentucky lineage was composed of two isolates from feed (96.3%). The results suggest diverse sources and independent routes of infection. Most of the isolates belonged to reptile-associated clones spread both horizontally and vertically. Simultaneously, PFGE profiles and MLST type indistinguishable from the ones observed in poultry point out carnivore reptiles as possible vector of infection with multidrug and high-level ciprofloxacin resistant (MIC ≥ 8 mg/L) S. Kentucky. Public awareness and education are required to prevent potential reptile-associated S. Kentucky infections in humans.

Recently, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has gained some importance in the epidemiology of salmonellosis in Poland. Since biotyping, resistance typing, and plasmid profiling were insufficient for strain differentiation, genome macrorestriction by means of pulse-field gel electrophoresis (PFGE) was applied and proved to be the method of choice in S. Mbandaka epidemiological studies. XbaI and BcuI macrorestriction produced 15 and 14 pulse-field profiles (PFP), respectively, but in the case of each enzyme one profile was prevalent. When macrorestriction profiles were combined, a total 24 patterns were found. Based on the similarity of the profiles, four clonal lineages were identified. One clonal lineage contained the majority of poultry, feed and human isolates. Poultry was concluded to be an important source of S. Mbandaka for humans in Poland. Complementary use of various typing techniques improved efficacy of epidemiological studies giving possibility to subdivide S. Mbandaka into 35 types and the index of discrimination reached 0.947.

Free-living snakes may carry a broad range of pathogens that differ considerably by geographical locations and source species. The biological role of wildlife snakes for transmission of bacteria and helminths is hardly explored. The purpose of the study was a snapshot on the zoonotic agents load in free-living snakes found dead in a landscape park in central Poland. A total of 16 dead free-living snakes identified as European grass snake (Natrix natrix, N = 15) and a smooth snake (Coronella austriaca, N = 1) were tested. Abdominal organs were used for bacteriological testing, whereas, parasites were searched for in the remaining carcasses. Fourteen (87.5 %) individuals were positive for Salmonella spp., whereas, trematodes were found in 13 out of 16 samples (81.3 %). A total of 33 isolates representing 11 Salmonella serovars or antigenic forms were observed with Salmonella enterica subsp. diarizonae (IIIb) 38:r:z being the most frequent. The trematodes were recognized as Alaria alata mesocercariae (Distomum musculorum suis, DMS). Additionally, unidentified nematodes were detected in four samples. No Yersinia spp. and Mycobacterium spp. were found. The results indicate huge burden of Salmonella spp. and muscle parasites carriage by snakes present in the natural environment and indicate possible zoonotic and epidemiological impact. It justifies our concept of free-living snakes as possible source and vector of pathogens, especially for omnivores and scavengers foraging on snake corpses. Finding of rare and diverse Salmonella serovars and unidentified nematodes proves the need for in-depth studies and systematic approach to reveal the role of free-living reptiles in epidemiology of infectious agents.