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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.

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.

Introduction: The aim of the study was to optimise and compare two multiplex PCR assays for the detection of Listeria spp. and Listeria monocytogenes in biological samples including the liver, brain, and blood. Material and Methods: Three strains of L. monocytogenes and single strains of each of the species: L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used. Additionally, five other species of bacterium were used to evaluate the specificity of the tests. Results: Specific amplification products were obtained for both multiplex PCR assays, which confirmed the tested strains as Listeria spp. and L. monocytogenes, respectively. Isolates of other species did not yield PCR products. Conclusion: Both multiplex PCR assays proved to be significantly sensitive and highly-specific methods for the detection of Listeria strains.

The accurate identification of Taylorella equigenitalis strains is essential to improve worldwide prevention and control strategies for contagious equine metritis (CEM). This study compared 367 worldwide equine strains using multilocus sequence typing according to the geographical origin, isolation year and equine breed. The strains were divided into 49 sequence types (STs), including 10 described for the first time. Three major and three minor clonal complexes (CCs), and 11 singletons, were identified. The genetic heterogeneity was low (0.13 STs/strain) despite the wide diversity of geographical origins (n = 16), isolation years (1977 to 2018) and equine breeds (n = 18). It was highest outside Europe and in the 1977-1997 period; current major STs and CCs already existed before 1998. Previous data associated the major CC1 with the first CEM outbreaks in 1977-1978 in the United Kingdom, Australia and the United States, and revealed its circulation in France. Our study confirms its circulation in France over a longer period of time (1992-2018) and its distribution in Spain and Germany but not throughout Europe. In addition to CC1, relationships between non-European and European countries were observed only through ST4, ST17 and ST30. Within Europe, several STs emerged with cross-border circulation, in particular ST16 and ST46 from the major complexes CC2 and CC8. These results constitute a baseline for monitoring the spread of CEM outbreaks. A retrospective analysis of a higher number of strains isolated worldwide between 1977 and the early 2000s would be helpful to obtain an exhaustive picture of the original CEM situation.

The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes. Five strains of L. monocytogenes and single strains of each of the species: L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of the tests. QuantiTect SYBR Green PCR and QuantiTect Probe PCR kits were selected for the study. In the first stage of the study, SYBR Green I real-time PCRs were performed under several methods, the first one allowing detection of the 23S rDNA gene and the remainder based on the amplification of the hlyA gene. In the next part, three varied in method TaqMan probe-based real-time PCRs allowing confirmation of strains belonging to Listeria spp. and L. monocytogenes were conducted. The observation of amplification curves in real-time PCR methods enabled the detection of both genes, and these methods demonstrated a significant sensitivity and high specificity. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes, which confirmed the tested strains as Listeria spp. and L. monocytogenes respectively. Isolates of other microbial species did not yield real-time PCR products.

The aim of study was the preliminary evaluation of the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum ß-lactamases (ESBL) - producing Escherichia coli in 650 milk and inflammatory secretions from cows with clinical or subclinical mastitis. One millilitre of the sample was added to Mueller-Hinton broth supplemented with 6.5% NaCl, Tryptone Soya Broth with cefoxitin and aztreonam, and then to MRSA ID agar. Presumptive MRSA colonies were analysed for the presence of mecA gene. Parallel to MRSA identification, the samples were incubated in buffered peptone water, lauryl tryptose broth and McConkey agar supplemented with cefotaxim for ESBL-producing E. coli isolation. These bacteria were identified using API Rapid 32 E and the ability of ESBL production was initially established using disc test D68C and confirmed by MIC technique using Sensititre ESBL plates. The primers (blaCTX, blaTEM, blaSHV, and blaCMY-2-group) for the detection of some of the genes encoding ESBL production were used. The 45 strains of S. aureus with mecA gene and 41 strains of E. coli with blaTEM gene were detected.

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

The paper concerns molecular study on pathogenicity markers of fourteen Y. enterocolitica O:9 isolated from pigs in which initially positive serological reactions for brucellosis were observed (n = 41), healthy pigs, which were brucellosis-negative (n = 258), and wild boars serologically negative for brucellosis (n = 209). PCR identification proved that all isolates were ail, ystA- and myfA-positive. The plasmid encoding yadA marker was detected in nine isolates that originated from pigs serologically positive or negative for brucellosis, and from one isolate of wild boar origin. Furthermore, none of the examined isolates was ystB-positive. Results of the investigations indicate that the Y. enterocolitica O:9 isolates from pigs or wild boars, regardless of whether they were serologically positive or negative for brucellosis, may also be potentially pathogenic for humans, due to the presence of chromosomal and plasmid encoded molecular markers.

Systemic mycobacteriosis was diagnosed in a group of ornamental fish. Although a large number of acid-fast bacterial rods were identified in the kidneys, liver, and muscles of each fish, no granulomas were observed in internal organs. Mycobacterium peregrinum was identified using the GenoType Mycobacterium CM assay. This study illustrates a considerable risk of atypical mycobacteriosis in humans.

Bovine tuberculosis is an infectious disease that occurs in many species of both domestic and wild animals, as well as those held in captivity. The etiological factor is the acid resistant bacillus (Mycobacterium bovis or Mycobacterium caprae), which is characterized by the major pathogenicity among mycobacteria belonging to the Mycobacterium tuberculosis complex. The material from 8 antelopes from the zoo, suspected for tuberculosis were examined, and M. bovis strains were isolated from 6 of them. The spoligotyping method showing spoligo pattern 676763777777600. In Poland, this spoligotype has not been observed so far.

The paper presents the results of bacteriological and molecular investigations on the presence of Y. enterocolitica O:9 in the head, mammary and genital lymph nodes, spleen, liver, and uterus samples originating from 58 cows slaughtered due to the positive results of serological examinations for brucellosis. All samples were cultured for Brucella and Y. enterocolitica and examined in multiplex PCR assay (mPCR), in order to identify the universal 16S rRNA Brucella sp. marker and amplify the perosamine synthetase (per) gene, specific for Y. enterocolitica O:9 only. Out of 58 examined animals, in 23 cases the presence of Y. enterocolitica was demonstrated. Typical Yersinia-suspected colonies were seen after 24-48 h incubation on Cefsulodin-Irgasan- Novobiocin (CIN) Yersinia selective solid medium. The mPCR analysis confirmed the presence of predicted amplicon of 312 bp, typical for Y. enterocolitica O:9 in 20 out of 58 lymph node samples tested and similarly, as in bacteriological examination, other samples were negative. The presence of the 16S rRNA gene of Brucella, generating the amplicon of 905 bp, was not observed in any of the samples tested.

The aim of the study was to perform a molecular investigations for the presence of pathogenicity genotypic markers of Y. enterocolitica O:9 isolated from cattle, in which initially positive serological reactions for brucellosis were observed. Almost all isolates were ail-, ystA- and myfA-positive (n=19). On the other hand, one isolate, which harboured plasmid encoding gene yadA was ail- , ystA- and myfA-negative. The plasmid encoding yadA marker was present in half of the isolates tested. None of the examined isolates was ystB-positive. The results of the investigations revealed that the Y. enterocolitica O:9 isolates, related to false positive serological results for brucellosis, may be also potentially pathogenic for humans, due to the presence of chromosomal and plasmidencoded molecular markers.

Since 2009, Poland has had a TB-free status, although over the last seven years 12-34 cases of bovine TB have been recorded annually. In 2009-2012 the largest number of cattle infected with Mycobacterium bovis and Mycobacterium caprae were culled in Masovian Voivodeship. Likewise, the largest number of sources of this zoonosis were recorded in that voivodeship. The vicinity of farms where bTB was found indicated that it could have been transmitted between their herds. The aim of this study was to characterise the molecular patterns of bovine bacillus strains isolated from cattle in Masovian Voivodeship and the molecular relationships between them. The material for microbiological examination came from 38 cattle (Bos taurus) located in 7 counties of Masovian Voivodeship. These 38 strains of MTBC were further identified as M. bovis (24 isolates; 63%) and M. caprae (14 isolates; 37%). A two-step genotyping analysis of the 38 MTBC strains identified 24 molecular patterns, closely related phylogenetically, which were assigned to 8 clusters of 2-6 strains. Sources of transmission were identified in 8 out of 13 herds examined in the 7 counties of Masovian Voivodeship. The results of the genotyping analysis excluded the possibility of TB transmission between different herds in Masovian Voivodeship. It was proved, however, that TB had been transmitted between animals bred on one of the farms..

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.

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.

A total of 42 Brucella strains were isolated from animals in Poland in years 2003-2012. Most of them (N=37) originated from wild animals, 3 from cattle, 1 from pig and 1 from sheep. The strains were characterised using both bacteriological and molecular (Bruce-ladder and MLVA) methods. The examinations revealed that all strains from wild boars, hares, cattle and pigs (N=41) had the same phenotypic characteristics and were classified as B. suis biovar 2. The remaining strain, isolated from sheep, was classified as B. ovis. The molecular examination showed that all B. suis biovar 2 strains, except one, had the same molecular profile as reference strain B. suis bv2 Thomsen. Different from the others strain originated from boars imported to Poland and its VNTR profile was typical for Iberian strains.

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.