Search

Sequencing can provide genomic characterisation of a specific organism, as well as of a whole environmental or clinical sample. High Throughput Sequencing (HTS) makes it possible to generate an enormous amount of genomic data at gradually decreasing costs and almost in real-time. HTS is used, among others, in medicine, veterinary medicine, microbiology, virology and epidemiology. The paper presents practical aspects of the HTS technology. It describes generations of sequencing, which vary in throughput, read length, accuracy and costs ̶ and thus are used for different applications. The stages of HTS, as well as their purposes and pitfalls, are presented: extraction of the genetic material, library preparation, sequencing and data processing. For success of the whole process, all stages need to follow strict quality control measurements. Choosing the right sequencing platform, proper sample and library preparation procedures, as well as adequate bioinformatic tools are crucial for high quality results.

To understand the dynamics of antimicrobial resistance (AMR), in a One-Health perspective, surveillance play an important role. Monitoring systems already exist in the human health and livestock sectors, but there are no environmental monitoring programs. Therefore there is an urgent need to initiate environmental AMR monitoring programs nationally and globally, which will complement existing systems in different sectors. However, environmental programs should not only identify anthropogenic influences and levels of AMR, but they should also allow for identification of transmissions to and from human and animal populations. In the current review we therefore propose using antimicrobial resistant Escherichia coli as indicators for monitoring occurrence and levels of AMR in the environment, including wildlife.

Introduction: Factors other than PD-L1 (Programmed Death Ligand 1) are being sought as predictors for cancer immuno- or chemoimmunotherapy in ongoing studies and long-term observations. Despite high PD-L1 expression on tumor cells, some patients do not benefit from immunotherapy, while others, without the expression of this molecule, respond to immunotherapy. Attention has been paid to the composition of the gut microbiome as a potential predictive factor for immunotherapy effectiveness. Materials and Methods: Our study enrolled 47 Caucasian patients with stage IIIB or IV non-small cell lung cancer (NSCLC). They were eligible for treatment with first- or second-line immunotherapy or chemoimmunotherapy. We collected stool samples before the administration of immunotherapy. We performed next-generation sequencing (NGS) on DNA isolated from the stool sample and analyzed bacterial V3 and V4 of the 16S rRNA gene. Results: We found that bacteria from the families Barnesiellaceae, Ruminococcaceae, Tannerellaceae, and Clostridiaceae could modulate immunotherapy effectiveness. A high abundance of Bacteroidaaceae, Barnesiellaceae, and Tannerellaceae could extend progression-free survival (PFS). Moreover, the risk of death was significantly higher in patients with a high content of Ruminococcaceae family (HR = 6.3, 95% CI: 2.6 to 15.3, p < 0.0001) and in patients with a low abundance of Clostridia UCG-014 (HR = 3.8, 95% CI: 1.5 to 9.8, p = 0.005) regardless of the immunotherapy line. Conclusions: The Clostridia class in gut microbiota could affect the effectiveness of immunotherapy, as well as the length of survival of NSCLC patients who received this method of treatment.

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

Control and eradication of Salmonella serotypes of public health relevance (particularly S. Enteritidis and S. Typhimurium, including the monophasic variant) in poultry flocks is, despite its importance, a difficult objective to achieve due to the complex epidemiology of this zoonotic pathogen. The data gathered through the national control programs implemented in the European Union (EU) countries provide an excellent opportunity to identify factors associated with the isolation of this bacterium that may explain the main limitations preventing its eradication/favouring its reoccurrence in poultry flocks. In order to gather data about Salmonella control and management programs, data on the Salmonella National Control Programs (SNCP) in laying hens from all partners/countries involved in WP2 of the ADONIS project has been requested. This deliverable presents the full analysis conducted on the data from the Spanish SNCP facilitated by the Ministry of Agriculture, Fisheries and Food (Animal Health Department), which are the only complete database that has become available so far, though plans exist to extend the application of the developed analytic framework to other databases that may be accessed before the end of the project (options are France, United Kingdom and the Netherlands). The ultimate objective of the analysis obtained through the official surveillance activities conducted in the frame of the SNCP is to identify factors that may be associated with an increased probability of detection of Salmonella in poultry flocks, which may be indicative of a true higher risk of infection, or an increased sensitivity of the samplings conducted. This may help explain the temporal trends on the prevalence of Salmonella spp. in poultry observed in the EU in the last decade, and also help to assess the performance of the current monitoring system.

Anti-PD-1 or PD-L1 immunotherapy in some patients with non-small cell lung cancer (NSCLC) may not be effective, despite the high percentage of cancer cells with PD-L1 expression (≥ 50%). TMB (tumor mutation burden), smoking status and low intestinal microbiome diversity may be associated with lack of efficacy of immune checkpoints inhibitors treatment in NSCLC patients. The case presented here concerns a non-smoking female patient with lung adenocarcinoma, in whom, despite the high percentage of PD-L1 positive tumor cells (50%), pembrolizumab therapy was ineffective. Next generation sequencing (NGS) was performed using the FOCUS panel allowing the analysis of 52 genes whose damage is associated with various types of solid tumors, including lung cancer. Benign genetic changes clinically irrelevant for patients with non-small cell lung cancer have been observed. In the meantime, profiling of the patient’s intestinal microbiome was performed, due to the fact that the composition of the intestinal microbiome may be a decisive factor in the lack of response to immunotherapy in patients with high PD-L1 expression and no driver mutations. Low diversity of bacteria in the intestines, with a noticeable dysbiosis (dysbacteriosis), was observed. The presence of bacteria Akkermansia, Enterococcaceae, Bifidobacteriaceae or Coriobacteriaceae , especially the presence of Akkermansia mucinifila seems to be a favourable factor of the possibility of obtaining response to immunotherapy and prolongation of progression-free survival (PFS). In the intestinal microbiome of the presented case, no bacteria from the Verrucomicrobia phylum , to which A. mucinifila belongs , were found. In addition, only 0.011% of Enterococcaceae were found. Studies on the intestinal microbiome in cancer patients receiving immunotherapy appear to be necessary to correctly understand the effect of microbiome composition on the effectiveness of this treatment method.

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.

Bacteria of the Bacillus cereus group are Gram-positive rods and are widespread in nature, but little information is currently available about their presence in reptiles. Here, we report draft genome sequences of six Bacillus isolates belonging to three species, namely, Bacillus cereus , Bacillus paranthracis , and Bacillus toyonensis , isolated from pet reptiles in Poland.

Introduction The Shewanella putrefaciens group are ubiquitous microorganisms recently isolated from different freshwater fish species and causing serious health disorders. The purpose of the study was to characterise isolates of the S. putrefaciens group with special emphasis on elucidating serological diversity and determining putative virulence factors. Material and Methods Isolates collected from freshwater fish (n = 44) and reference strains were used. The identification of bacteria was carried out using biochemical kits and 16S rRNA sequencing. Polyclonal antibodies were prepared against the S. putrefaciens group. The bacterium’s susceptibility to antimicrobial agents, its enzymatic properties, and its adhesion ability to fish cell lines were also tested. Finally, selected isolates were used in challenge experiments in common carp and rainbow trout. Results Excluding six isolates undeterminable for species, the bacteria were classified to three species: S. putrefaciens, S. xiamenensis, and S. oneidensis, and showed some phenotypic diversity. Fourteen serological variants of the S. putrefaciens group were determined with the newly developed serotyping scheme. Conclusion Serodiversity may play an important role in the virulence of particular isolates. Further, S. putrefaciens group members adhere to epithelial cells and produce enzymes which may contribute to their virulence. Challenge tests confirmed the pathogenicity of the S. putrefaciens group for fish.

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.

Colistin is considered as a highest priority critically importantantimicrobial in human medicine. To evaluate the potential contribution offood-producing animals to colistin resistance in humans, WP1 of theOHEJP-IMPART project aimed to standardize a methodology to screenacquired-colistin resistance in most frequent commensal and zoonoticEnterobacteriaceae as E. coli and Salmonella and evaluate thismethodology in a multicentre approach.

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 resistance of bacteria to antimicrobial substances is one of the most serious epidemiological problems present on a global scale. The widespread use of same classes of antimicrobials in human and veterinary medicine, often without laboratory confirmation of the efficacy of active compounds used, contributes to the selection of resistant bacteria in humans and animals, and their spread in nature. The increasing resistance of pathogenic bacteria leads to serious consequences for both human and animal health. However, the resistance of commensal bacteria is equally important as they constitute a reservoir and vector of resistance determinants in the environment. Exposure to antimicrobials belonging to different classes can lead to cross-resistance and the selection of genes that may spread horizontally on mobile genetic elements. The emergence of plasmid-encoded resistance to critically important antibiotics for human medicine e.g. carbapenems or polymyxins is alarming. On the example of antibiotics classified as critically important for human medicine, it is possible to discuss almost all bacterial mechanisms of antimicrobial resistance. For effective combat against the growing antibiotic resistance of bacteria, it is necessary to know the mechanisms of resistance and the methods of their acquisition by bacteria. The aim of the paper is to review the ways that critically important antimicrobials act on bacterial cells and present complex mechanisms that are responsible for resistance to these substances as well as genes conferring for resistance. 1. Introduction. 2. Antimicrobials that cause loss of cell wall integrity: β-lactams, glycopeptides and phosphonic acid derivatives. 2.1. Mechanisms of antimicrobial action. 2.2. Mechanisms of resistance. 3. Antimicrobials affecting the cell membrane: polymyxins and lipopeptides. 3.1. Mechanisms of antimicrobial action. 3.2. Mechanisms of resistance. 4. Antimicrobial substances that inhibit the synthesis of nucleic acids: quinolones and ansamycins. 4.1. Mechanisms of antimicrobial action. 4.2. Mechanisms of resistance. 5. Antimicrobial substances inhibiting protein synthesis: macrolides, ketolides, aminoglycosides, glycylcyclines, oxazolidinones. 5.1. Mechanisms of antimicrobial action. 5.2. Mechanisms of resistance. 6. Summary

High throughput sequencing (HTS) creates an opportunity for comprehensive genomic studies. It can be applied in veterinary science, bacteriology and virology, diagnostics of animal diseases, food safety, examinations of the composition of environmental samples, and even in veterinary vaccinology. Thus HTS a wide-ranging method that can be applied in different areas of the One Health approach. In particular, the whole genome sequencing (WGS) of bacteria is routinely used in food hygiene and outbreak investigations for phylogenetic analysis of pathogenic bacteria isolated from various sources across timeline, molecular characterisation of bacteria, plasmids, antibiotic resistance and identification of virulence factors. Metagenomics can be used to characterize the composition of microbiota in environmental samples. It makes it possible to obtain a taxonomic identification of bacteria, fungi or plants present in a metasample. It can also be used for the monitoring and epidemiological tracing of viruses, such as SARS-CoV-2. The transcriptomic approach makes it possible to study the expression of genes associated with various infections and diseases. HTS is a highly versatile method, but the selection of the proper application is crucial to obtain expected outcomes. The paper presents some HTS approaches and examples of research in veterinary science.

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.

Salmonella enterica subspecies enterica serotype Choleraesuis is a swine adapted serovar. S. Choleraesuis variant Kunzendorf is responsible for the majority of outbreaks among pigs. S. Choleraesuis is rare in Europe, although there have been serious outbreaks in pigs including two outbreaks in Denmark in 1999-2000 and 2012-2013. Here, we elucidate the epidemiology, possible transmission routes and sources, and clonality of European S. Choleraesuis isolates including the Danish outbreak isolates. A total of 102 S. Choleraesuis isolates from different European countries and the United States, covering available isolates from the last two decades were selected for whole genome sequencing. We applied a temporally structured sequence analysis within a Bayesian framework to reconstruct a temporal and spatial phylogenetic tree. MLST type, resistance genes, plasmid replicons, and accessory genes were identified using bioinformatics tools. Fifty-eight isolates including 11 out of 12 strains from wild boars were pan-susceptible. The remaining isolates carried multiple resistance genes. Eleven different plasmid replicons in eight plasmids were determined among the isolates. Accessory genes were associated to the identified resistance genes and plasmids. The European S. Choleraesuis was estimated to have emerged in ∼ 1837 (95% credible interval, 1733-1983) with the mutation rate of 1.02 SNPs/genome/year. The isolates were clustered according to countries and neighbor countries. There were transmission events between strains from the United States and European countries. Wild boar and pig isolates were genetically linked suggesting cross-border transmission and transmission due to a wildlife reservoir. The phylogenetic tree shows that multiple introductions were responsible for the outbreak of 2012-2013 in Denmark, and suggests that poorly disinfected vehicles crossing the border into Denmark were potentially the source of the outbreak. Low levels of single nucleotide polymorphisms (SNPs) differences (0-4 SNPs) can be observed between clonal strains isolated from different organs of the same animal. Proper disinfection of livestock vehicles and improved quality control of livestock feed could help to prevent future spread of S. Choleraesuis or other more serious infectious diseases such as African swine fever (ASF) in the European pig production system.

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.

Different Shewanella species are isolated both from healthy and from diseased fish. To date, contemporary methods do not provide sufficient insight to determine species and detail differentiation between tested strains. Bacteria isolated from cultured (n = 33), wild (n = 12) and ornamental (n = 6) fish, as well as several reference strains, were tested by 16S rRNA gene sequencing, ERIC-PCR and pulsed-field gel electrophoresis (PFGE) assays. Our study indicates that isolates collected from freshwater fish were genetically diverse. Based on 16S rRNA gene sequences, bacteria were clustered into groups S. putrefaciens, S. xiamenensis and S. oneidensis. Some isolates were classified only to genus Shewanella; thus, 16S rRNA gene analyses were not enough to determine the species. ERIC-PCR revealed 49 different genotype profiles indicating that the method might be useful for differentiation of Shewanella isolates irrespectively to species identification, contrary to PFGE which is not suitable for Shewanella typing.

A total of 502 Campylobacter jejuni isolates from poultry in 12 different European countries (10 of them the largest poultry production countries in Europe) were whole genome sequenced to examine the genomic diversity of fluoroquinolone resistant (FQ‐R) and susceptible (FQ‐S) C. jejuni across the poultry producing European countries and to determine whether the emergence of fluoroquinolone resistance among C. jejuni is related to the transmission through countries or to the selection through fluoroquinolone use in the individual countries. A high genomic diversity was observed. The isolates clustered in four main clusters. All trees revealed that the isolates were clustered according to the presence/absence of the gyrA mutations causing fluoroquinolone resistance and ST‐types. The cgMLST trees of only FQ‐R and FQ‐S isolates showed that isolates from the same country of origin were distributed into multiple clusters similarly to the trees combining FQ‐R and FQ‐S isolates. The different phylogenetic methods, ranging from single nucleotide polymorphisms analysis to gene‐by‐gene approaches such as rMLST, cgMLST, wgMLST and core genome tree, provided concordant results, but it is not known which is the most accurate method for identifying the country of origin of the isolates. Allele frequency analysis of isolates under this study and a selection of previously published C. jejuni genomes in ENA showed association of geographical origin of poultry C. jejuni populations between Romania‐Poland, Italy‐Germany‐England, Portugal‐The Netherlands and USA‐Luxemburg. Allele frequency and phylogenetic analysis indicated that the isolates from Finland were genetically different from C. jejuni populations from other European countries included in this study. Trade pattern and antimicrobial use in livestock were not significantly associated with allele frequency or populations of C. jejuni, but data available to investigate these associations were limited.

The ENGAGE project (http://www.engage-europe.eu/) was a collaboration between eight institutions across Europe. The aim was to boost the scientific cooperation to use whole genome sequencing (WGS) analysis in food safety and public health protection. ENGAGE focused on Escherichia coli (commensal E. coli) and different Salmonella spp. serotypes. A total of 3,360 genomes, 778 and 2,582 of E. coli and Salmonella, respectively, were produced. These genomes were stored and shared among partners in a temporary repository to be submitted to the European Nucleotide Archive by the end of the project. Generated genomes were used for benchmarking exercises to assess the possibility of replacing conventional typing with WGS for outbreak investigation. For the analysed strains, the benchmarking exercises showed that SPAdes assembly performed better than Velvet and that, by using different bioinformatics tools, WGS Salmonella serotyping and antimicrobial resistance genes detection, were largely in concordance with phenotypic data. Discrepancies were related to sequence quality and phenotype misclassification rather than to limitations of the bioinformatics tools. All partners were able to infer the expected phylogeny for the Salmonella and Campylobacter isolates in benchmarking exercises. Two WGS proficiency tests (assessing different genomic quality markers) were conducted among partners with satisfactory results. Guidelines including available bioinformatics tools and standard operating procedures (wet and dry lab) were prepared and posted online. Workshops, training courses and twinning programmes were conducted. The training focused on online, Galaxy‐based, and command line bioinformatics tools. To reach out beyond ENGAGE, an e‐learning course (17 videos) was developed and made available online. Several proof of concept projects were run and some outcomes published, e.g. the discovery of colistin resistance gene, mcr‐5. Overall, the project showed that laboratories without previous WGS experience need a period of time to implement and perform WGS for foodborne pathogens routine analysis. All developed material will remain available on the ENGAGE website.

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.

Multidrug resistant Salmonella Kentucky strains have been isolated from turkeys in Poland since 2009. Multiple mutations within chromosomal genes gyrA and parC were responsible for high-level ciprofloxacin resistance. One of the isolates was extended spectrum β-lactamase- (ESBL) positive: the strain 1643/2010 carried a conjugative 167,779 bps plasmid of IncA/C family. The sequence analysis revealed that it carried a bla CTX-M-25 gene and an integron with another β-lactamase encoding gene— bla OXA-21. This is the first known report of a CTX-M-25 encoding gene both in Poland and in Salmonella Kentucky world-wide, as well as in the IncA/C plasmid. Analysis of the integron showed a novel arrangement of gene cassettes— aacA4 , aacC-A1 and bla OXA-21 where the latter might result from an intergeneric gene transfer. The study confirmed Salmonella Kentucky population isolated in Poland belongs to global epidemics of high level fluoroquinolone resistant clone ST198 that can carry rare β-lactamase genes.

Antimicrobial resistance was tested in Escherichia coli isolated from feces (n = 660) of red deer, roe deer, fallow deer, European bison, and wild boar shot in regional forests in Poland during two winter hunting seasons. Indicator E. coli (n = 542) was resistant against 11 of 14 tested compounds, mostly sulfamethoxazole, streptomycin, ampicillin, trimethoprim, and tetracycline (1.3-6.6% range). No significant differences were observed between boar and ruminant isolates. Most of deer and bison isolates showed no resistance. Selective screening of wildlife samples revealed 1.7% prevalence of cephalosporin-resistant E. coli found mostly in wild boars. They produced extended-spectrum beta-lactamases (bla

Invasiveness of alien turtles results from their impact on the functioning of the local ecosystem. It is due to predation on or competing with resident species, but also transfer of new and unknown pathogenic bacteria, viruses, parasites, or fungi. Salmonella is the most often reported microorganism, both in free-living and captive turtles. Zoonotic aspect of Salmonella spp. carriage has led to the definition of RAS (Reptile Associated Salmonellosis) acquired from domestic pet reptiles. Mycobacterium spp., Leptospira spp. and aquatic bacteria are also found in turtles. Additionally, nematode transmissions from invasive turtles to the autochthonic ones have been described. Alien turtles were less affected by parasitic invasion than animals living in a native location, but the infestation of alien parasites in native turtle species was usually more severe. Reports on viral or fungal infections in turtles are scarce. The identified knowledge gaps justify the need for research which will provide basic and systematic data on microbial threats related to alien and invasive turtles present in the natural environment of Poland. It will also give more insight in the scope and the impact of the problem on epidemiology and public health. 1. Introduction. 2. Invasive turtle species. 3. Bacteria. 3.1. Salmonella spp. 3.1.1. Reptile Associated Salmonellosis (RAS). 3.2. Mycobacteria. 3.3. Other bacteria. 4. Parasitofauna of turtles. 4.1. Invasive turtles as a source of helminth invasion of European pond turtle. 4.2. Occurrence and invasiveness of alien parasites in native and alien turtles. 5. Viral infections. 6. Mycotic infection. 7. Conclusions 1. Wstęp. 2. Inwazyjne gatunki żółwi. 3. Bakterie. 3.1. Salmonella spp. 3.1.1. Reptile Associated Salmonellosis (RAS). 3.2. Prątki. 3.3. Inne bakterie. 4. Parazytofauna żółwi. 4.1. Żółwie inwazyjne źródłem zarażenia helmintami żółwia błotnego. 4.2. Występowanie i patogenność obcych pasożytów u natywnych i obcych gatunków żółwi. 5. Infekcje wirusowe. 6. Infekcje grzybicze. 7. Podsumowanie

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.