Prevalence of Enterococcus spp. and the Whole-Genome Characteristics of Enterococcus faecium and Enterococcus faecalis Strains Isolated from Free-Living Birds in Poland.

Enterococci as opportunistic bacteria are important for human health. Due to the prevalence and ease of acquisition and transfer of their genes, they are an excellent indicator of environmental contamination and the spread of antimicrobial resistance. The aim of the study was to assess the prevalence of Enterococcus spp. in wild birds in Poland, determination of antimicrobial susceptibility and WGS analysis of Enterococcus (E.) faecium and E. faecalis. For this purpose, 138 samples from various species of free-living birds were tested, with 66.7% positive results. Fourteen species were detected, with E. faecalis being the most common, followed by E. casseliflavus and E. hirae. In antimicrobial susceptibility testing, 10.0% of E. faecalis and 50.0% of E. faecium showed resistance to one antimicrobial agent, in addition the MDR phenotype which was found in one E. faecium. The most common resistance phenotype included tetracycline and quinupristin/dalfopristin. The WGS analysis confirmed the significant advantage of the virulence gene diversity of E. faecalis strains over E. faecium. In addition, plasmid replicons were found in 42.0% of E. faecalis and 80.0% of E. faecium. The obtained results confirm free-living birds can be a reservoir of Enterococcus spp. with a considerable zoonotic potential.

The first description of the complete genome sequence of multidrug-resistant Salmonella enterica serovar monophasic Typhimurium (1,4,[5],12:i:-) isolate with the mcr-1.1 gene on IncHI2 found in pig in Poland.

Monophasic Salmonella Typhimurium (1,4,[5],12:i:-) is one of the leading Salmonella serovars causing human salmonellosis in Europe. It has been observed in Poland since 2008. This serovar is considered the one with the highest rate of mcr prevalence. This report presents a sequence characteristic of the multidrug-resistant (MDR) monophasic S. Typhimurium isolated from a pig faecal sample with the confirmed presence of the mcr-1.1 gene. The genome was assembled into the complete chromosome and 4 plasmids: IncHI2 (232 119 bp), IncFIB/IncFIC (133 901 bp), ColRNAI (6659 bp), and Col8282 (4066bp). The strain identified as ST34 carried multiple antimicrobial resistance genes located both on chromosome (tet(B)) and plasmids: mcr-1.1 and blaTEM-1B on ST4-IncHI2, and mef(B), blaTEM-1B, aadA1, qacL, dfrA12, aadA2, cmlA1, sul3, tet(M) on IncFIB/FIC. The mcr-1.1 gene was previously identified in E. coli deriving mainly from poultry, but this is the first case of the occurrence of mcr-positive Salmonella in Poland. The obtained results of analysis of the genome content draw attention to the problem of multidrug-resistant pathogens, especially in the context of resistance to colistin which is a last-resort antimicrobial.

Draft genome sequences data of four Salmonella enterica subsp. enterica serovar Dublin archival strains originating from animals in Poland, 1956 - 1957.

Salmonella enterica subsp. enterica serovar Dublin (S. Dublin) is a zoonotic pathogen causing infections in animals, especially in cattle. In this study, we report draft genome sequences of four S. Dublin isolated between 1956 and 1957 from cattle and fox in Poland. Whole genome sequencing was performed on the Illumina platform and the data is available at National Center for Biotechnology Information under the BioProject accession number PRJNA865912. In order to better understand the genetic basis of epidemiology of S. Dublin infection, the obtained sequences were analyzed using the tools which are available at Center of Genomic Epidemiology (https://www.genomicepidemiology.org/) including core genome multilocus sequence typing (cgMLST) and core genome single nucleotide polymorphisms (cgSNPs).

Salmonella in Captive Reptiles and Their Environment-Can We Tame the Dragon?

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.

Genetic Relationship of Salmonella Isolates Found in Subcutaneous Abscesses in Leopard Geckos (Eublepharis Macularius).

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.

Antimicrobial Resistance in Escherichia coli Isolated from Wild Animals in Poland.

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 (blaCTX-M-1, blaCTX-M-15) and plasmid-mediated AmpC-type cephalosporinase (blaCMY-2). The majority of the isolates originated from boars shot in a narrow time frame and space; therefore, common antimicrobial selection pressure in the environment was assumed. Three E. coli isolates carried plasmid-mediated quinolone resistance genes (qnrS1/S3). No transferable colistin resistance mechanisms were found in two resistant E. coli. Transferability of resistance was proved in a single pAmpC-positive isolate carrying IncI1-alpha 95 kb plasmid. No cephalosporin-resistant E. coli harbored pathogenicity markers; therefore, they might be considered a vector of resistance determinants, but not a pathogen themselves.

Distribution of Salmonella Serovars along the Food Chain in Poland, 2010-2015.

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.

Fifteen years of successful spread of Salmonella enterica serovar Mbandaka clone ST413 in Poland and its public health consequences.

In the 1990s, Salmonella enterica 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 (®), 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 blaCMY-2 and qnrS1 genes. Two clusters of 15 XbaI-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 Salmonella control programmes, the S. Mbandaka ST413 clone has been circulating for several years in Poland. Salmonella 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.

Mechanisms of cephalosporin resistance in indicator Escherichia coli isolated from food animals.

Resistance to ß-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 ß-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.