Search

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 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.

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.

Reptiles are considered a reservoir of a variety of Salmonella (S.) serovars. Nevertheless, due to a lack of large-scale research, the importance of Reptilia as a Salmonella vector still remains not completely recognized. A total of 731 samples collected from reptiles and their environment were tested. The aim of the study was to assess the prevalence of Salmonella in exotic reptiles kept in Poland and to confirm Salmonella contamination of the environment after reptile exhibitions. The study included Salmonella isolation and identification, followed by epidemiological analysis of the antimicrobial resistance of the isolates. Implementation of a pathway additional to the standard Salmonella isolation protocol led to a 21% increase in the Salmonella serovars detection rate. The study showed a high occurrence of Salmonella, being the highest at 92.2% in snakes, followed by lizards (83.7%) and turtles (60.0%). The pathogen was also found in 81.2% of swabs taken from table and floor surfaces after reptile exhibitions and in two out of three egg samples. A total of 918 Salmonella strains belonging to 207 serovars and serological variants were obtained. We have noted the serovars considered important with respect to public health, i.e., S. Enteritidis, S. Typhimurium, and S. Kentucky. The study proves that exotic reptiles in Poland are a relevant reservoir of Salmonella.

Transmission of pathogenic and resistant bacteria from wildlife to the bacterial gene pool in nature affects the ecosystem. Hence, we studied intestine content of five wild rodent species: the yellow-necked wood mouse (Apodemus flavicollis, n = 121), striped field mouse (Apodemus agrarius, n = 75), common vole (Microtus arvalis, n = 37), bank vole (Myodes glareolus, n = 3), and house mouse (Mus musculus, n = 1) to assess their potential role as an antimicrobial resistance (AMR) and Salmonella vector. The methods adopted from official AMR monitoring of slaughtered animals were applied and supplemented with colistin resistance screening. Whole-genome sequencing of obtained bacteria elucidated their epidemiological relationships and zoonotic potential. The study revealed no indications of public health relevance of wild rodents from the sampled area in Salmonella spread and their limited role in AMR dissemination. Of 263 recovered E. coli, the vast majority was pan-susceptible, and as few as 5 E. coli showed any resistance. In four colistin-resistant strains neither the known mcr genes nor known mutations in pmr genes were found. One of these strains was tetracycline-resistant due to tet(B). High diversity of virulence factors (n = 43) noted in tested strains including ibeA, cdtB, air, eilA, astA, vat, pic reported in clinically relevant types of enteric E. coli indicate that rodents may be involved in the ecological cycle of these bacteria. Most of the strains represented unique sequence types and ST10805, ST10806, ST10810, ST10824 were revealed for the first time, showing genomic heterogeneity of the strains. The study broadened the knowledge on phylogenetic diversity and structure of the E. coli population in wild rodents.

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

Antimicrobial resistance (AMR) in bacteria is a complex subject, why one need to look at this phenomenon from a wider and holistic perspective. The extensive use of the same antimicrobial classes in human and veterinary medicine as well as horticulture is one of the main drivers for the AMR selection. Here, we applied shotgun metagenomics to investigate the AMR epidemiology in several animal species including farm animals, which are often exposed to antimicrobial treatment opposed to an unique set of wild animals that seems not to be subjected to antimicrobial pressure. The comparison of the domestic and wild animals allowed to investigate the possible anthropogenic impact on AMR spread. Inclusion of animals with different feeding behaviors (carnivores, omnivores) enabled to further assess which AMR genes that thrives within the food chain. We tested fecal samples not only of intensively produced chickens, turkeys, and pigs, but also of wild animals such as wild boars, red foxes, and rodents. A multi-directional approach mapping obtained sequences to several databases provided insight into the occurrence of the different AMR genes. The method applied enabled also analysis of other factors that may influence AMR of intestinal microbiome such as diet. Our findings confirmed higher levels of AMR in farm animals than in wildlife. The results also revealed the potential of wildlife in the AMR dissemination. Particularly in red foxes, we found evidence of several AMR genes conferring resistance to critically important antimicrobials like quinolones and cephalosporins. In contrast, the lowest abundance of AMR was observed in rodents originating from natural environment with presumed limited exposure to antimicrobials. Shotgun metagenomics enabled us to demonstrate that discrepancies between AMR profiles found in the intestinal microbiome of various animals probably resulted from the different antimicrobial exposure, habitats, and behavior of the tested animal species.

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.

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.

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.

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 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.

The prevalence of Escherichia coli with putative extended-spectrum cephalosporin resistance was assessed in cattle, pigs, broilers, layers, and turkey slaughtered in Poland. The occurrence of random E. coli isolates recovered from MacConkey agar plates with non–wild-type minimal inhibitory concentrations for cefotaxime and ceftazidime reached 0.6% in layers, 2.3% in turkey, and 4.7% in broilers, whereas all cattle and pigs isolates fell into the wild-type subpopulation. The use of MacConkey agar supplemented with cefotaxime (2 mg/L) increased the recovery of resistant strains up to 33.3% of samples from pigs, 42.3% from layers, 48.0% from turkey, and 54.5% from broilers. Still, no cephalosporin-resistant E. coli was found in cattle. E-test identified extended-spectrum beta-lactamase (ESBL) and ampC-type resistance phenotypes in 15 and 33 strains, respectively. Molecular characterization identified CTX-M-1 gene in 13 ESBL strains, 5 of which possessed also TEM-1b. One strain harbored SHV-12 gene. CMY-2 was found in all of 20 tested ampC-type cephalosporinase-positive strains either alone (n = 14) or in combination with mutations in ampC promoter region (n = 6). CTX-M-1 and CMY-2 genes were noted also in five strains from laying hens and broilers originated from Belgium and Germany. Nosocomial infections in Poland are caused by E. coli carrying other determinants than those found in our study. Thus, our results indicate that animals colonized with cephalosporin-resistant strains might not be the major source of human infections in Poland. However, the contribution to community-acquired infections by spread of resistant clones or resistance genes may not be excluded.

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.

Resistance to β-lactams is considered one of the major global problems and recently it became the most frequently studied topic in the area of antimicrobial resistance. The study was focused on phenotypic and genetic characterisation of commensal Escherichia coli (E. coli), including those producing cephalosporinases, isolated from gut flora of healthy slaughter animals. E. coli were cultured simultaneously on MacConkey agar (MCA) and cefotaxime supplemented MCA. The isolates were confirmed with ONPG and indol tube tests as well as PCR targeting uspA gene. Microbroth dilution method was applied for determination of Minimal Inhibitory Concentrations and interpreted according to EUCAST epidemiological cut-off values. Cephalosporin resistance phenotypes were defined by E-tests (BioMerieux) and relevant gene amplicons from selected strains were sequenced. A total of 298 E. coli isolates with cephalosporin resistance (ESC) found in 99 ones, were obtained from 318 cloacal or rectal swabs deriving from broilers, layers, turkeys, pigs and cattle. Both extended spectrum β-lactamase (ESBL) and ampC-cephalosporinase resistance phenotypes were noted in all tested animal species but cattle. At least one of the analysed genes was identified in 90 out of 99 cephalosporin-resistant isolates: blaTEM (n=44), blaCMY (n=38), blaCTX-M (n=33) and blaSHV (n=12). None of the phenotypes was identified in nine isolates. Sequencing of PCR products showed occurrence of ESBL-genes: blaCTX-M-1/-61, blaSHV-12, blaTEM-1,-52/-92,-135 and ampC-gene blaCMY-2. They were located on numerous and diverse plasmids and resistance transferability was proved by electroporation of blaSHV-12 and blaCTX-M-1/-61 located on X1 plasmids. Detection of cephalosporin resistant E. coli confirms the existence of resistance genes reservoir in farm animals and their possible spread (i.e. via IncX1 plasmids) to other bacteria including human and animal pathogens. The identified genetic background indicates on ecological aspects of selection and dissemination of cephalosporin resistance in E. coli isolated from food-producing animals rather than its potential role for public health threats.

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.

Monitoring of antimicrobial resistance in commensal Escherichia coli (N = 3430) isolated from slaughtered broilers, laying hens, turkeys, swine, and cattle in Poland has been run between 2009 and 2012. Based on minimal inhibitory concentration (MIC) microbiological resistance to each of 14 tested antimicrobials was found reaching the highest values for tetracycline (43.3%), ampicillin (42.3%), and ciprofloxacin (39.0%) whereas the lowest for colistin (0.9%), cephalosporins (3.6 ÷ 3.8%), and florfenicol (3.8%). The highest prevalence of resistance was noted in broiler and turkey isolates, whereas it was rare in cattle. That finding along with resistance patterns specific to isolation source might reflect antimicrobial consumption, usage preferences or management practices in specific animals. Regression analysis has identified changes in prevalence of microbiological resistance and shifts of MIC values. Critically important fluoroquinolone resistance was worrisome in poultry isolates, but did not change over the study period. The difference (4.7%) between resistance to ciprofloxacin and nalidixic acid indicated the scale of plasmid-mediated quinolone resistance. Cephalosporin resistance were found in less than 3.8% of the isolates but an increasing trends were observed in poultry and MIC shift in the ones from cattle. Gentamycin resistance was also increasing in E. coli of turkey and cattle origin although prevalence of streptomycin resistance in laying hens decreased considerably. Simultaneously, decreasing MIC for phenicols observed in cattle and layers isolates as well as tetracycline values in E. coli from laying hens prove that antimicrobial resistance is multivariable phenomenon not only directly related to antimicrobial usage. Further studies should elucidate the scope of commensal E. coli as reservoirs of resistance genes, their spread and possible threats for human and animal health.

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.

Background The project "Antibiotic resistance in bacteria of animal origin – II" (ARBAO-II) was funded by the European Union (FAIR5-QLK2-2002-01146) for the period 2003–2005, with the aim to establish a continuous monitoring of antimicrobial susceptibility among veterinary laboratories in European countries based on validated and harmonised methodologies. Available summary data of the susceptibility testing of the bacterial pathogens from the different laboratories were collected. Method Antimicrobial susceptibility data for several bovine pathogens were obtained over a three year period (2002–2004). Each year the participating laboratories were requested to fill in excel-file templates with national summary data on the occurrence of antimicrobial resistance from different bacterial species. A proficiency test (EQAS – external quality assurance system) for antimicrobial susceptibility testing was conducted each year to test the accuracy of antimicrobial susceptibility testing in the participating laboratories. The data from this testing demonstrated that for the species included in the EQAS the results are comparable between countries. Results Data from 25,241 isolates were collected from 13 European countries. For Staphylococcus aureus from bovine mastitis major differences were apparent in the occurrence of resistance between countries and between the different antimicrobial agents tested. The highest frequency of resistance was observed for penicillin. For Mannheimia haemolytica resistance to ampicillin, tetracycline and trimethoprim/sulphonamide were observed in France, the Netherlands and Portugal. All isolates of Pasteurella multocida isolated in Finland and most of those from Denmark, England (and Wales), Italy and Sweden were susceptible to the majority of the antimicrobials. Streptococcus dysgalactiae and Streptococcus uberis isolates from Sweden were fully susceptible. For the other countries some resistance was observed to tetracycline, gentamicin and erythromycin. More resistance and variation of the resistance levels between countries were observed for Escherichia coli compared to the other bacterial species investigated. Conclusion In general, isolates from Denmark, England (and Wales), the Netherlands, Norway, Sweden and Switzerland showed low frequencies of resistance, whereas many isolates from Belgium, France, Italy, Latvia and Spain were resistant to most antimicrobials tested. In the future, data on the prevalence of resistance should be used to develop guidelines for appropriate antimicrobial use in veterinary medicine.