Magdalena Kizerwetter-Świda

Phenotypic characteristics and virulence genotypes of Trueperella (Arcanobacterium) pyogenes strains isolated from European bison (Bison bonasus)

Trueperella (Arcanobacterium) pyogenes is an opportunistic animal pathogen, which in European bison is associated with different suppurative infections mainly of the urogenital tract. Little is known about the virulence of this bacterium and about the pathogenesis of infections. The main objective of this study was to determine phenotypic properties and virulence genotypes of the twenty-five T. pyogenes strains isolated from lesions in various tissues of free-living European bison. Classical bacteriological methods were used for phenotypic characterization. Genes encoding seven known and putative virulence factors of T. pyogenes were detected by PCR technique. Analysis of 16S rDNA partial sequences was performed to establish phylogenetic relationships of the isolated strains. All isolates showed typical morphological features of T. pyogenes and variable biochemical activity. Most of them displayed a strong positive effect in synergistic CAMP test. For all isolates the 16S rRNA gene partial sequence was identical to that of the T. pyogenes reference strain. All isolates carried the plo and fimA genes, while the nanH, nanP, cbpA, fimC and fimG genes were present in 40, 44, 12, 88 and 24% of the isolates, respectively. The T. pyogenes strains isolated from European bison represented various phenotypes and virulence genotypes, but there was no association between the investigated properties of the bacteria and the type of anatomopathological lesions from which they were isolated. These results indicate that the studied virulence factors of T. pyogenes are not significant determinants of the localization and type of infection caused by this bacterium.

Molecular characterization of Staphylococcus pseudintermedius strains isolated from clinical samples of animal origin

The aim of this study was to determine the species distribution among 44 randomly selected clinical isolates (30 mecA-positive and 14 mecA-negative) of animal origin previously identified as Staphylococcus intermedius by phenotypic tests and species-specific PCR amplification of the 16S rRNA gene. For this purpose, we used a multiplex PCR for the detection of the nuc gene and restriction fragment length polymorphism analysis of pta gene amplified by PCR. Both methods allow discrimination of Staphylococcus pseudintermedius from the other closely related members of the S. intermedius group and other coagulase-positive staphylococci isolated from animals. Genetic diversity of S. pseudintermedius strains was analyzed by staphylococcal protein A-encoding gene (spa) typing. Multiplex PCR method was used to identify staphylococcal cassette chromosome mec (SCCmec) type in mecA-positive strains. All isolates previously identified as S. intermedius were shown to belong to S. pseudintermedius. According to PCR-based SCCmec typing, SCCmecIII was the most prevalent type (n = 23), and solely seven isolates were designated as non-typeable. Furthermore, the assessment of spa-typing results revealed that the majority of all strains (n = 27) harbored spa type t02, and 17 strains were classified as non-typeable.

Characterization of Rhodococcus equi isolates from submaxillary lymph nodes of wild boars (Sus scrofa), red deer (Cervus elaphus) and roe deer (Capreolus capreolus)

Rhodococcus equi is a soil saprophyte and an opportunistic pathogen causing infections in animals, and rarely in humans. The presence of R. equi in tissues and faeces of some wild animal species was demonstrated previously. In this study we characterized R. equi isolates from submaxillary lymph nodes of free-living wild boars (n=23), red deer (n=2) and roe deer (n=2). This is the first description of R. equi strains isolated from tissues of the Cervidae. All isolates were initially recognized as R. equi based on the phenotypic properties. Their identification was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, detection of the choE gene and by sequence analysis of the 16S rRNA and rpoB genes. The presence of three plasmidic genes (traA, vapA and vapB) associated with R. equi virulence was investigated by PCR. In 16 wild boar isolates the traA and vapB genes were detected and they were located on virulence plasmids type 5, 7 or 11. The isolates from cervids and the remaining wild boar isolates were classified as avirulent based on a genotype traA(-)/vapA(-)B(-). In summary, these results confirm that wild boars can be a source of intermediately virulent R. equi strains, and indicate that red deer and roe deer can be a reservoir of avirulent R. equi strains.

Multidrug resistance in Escherichia coli strains isolated from infections in dogs and cats in Poland (2007-2013).

The antimicrobial susceptibility of Escherichia coli isolates associated with various types of infections in dogs and cats was determined. The studied isolates were most frequently susceptible to fluoroquinolones and the extended-spectrum cephalosporins (ESCs), antimicrobials commonly used in treatment of infections in companion animals. However, an increase in the percentage of strains resistant to β-lactam antibiotics including ESCs was noted between January 2007 and December 2013. The frequency of multidrug-resistant (MDR) E. coli isolation (66.8% of isolates) is alarming. Moreover, the statistically significant increase of the percentage of MDR isolates was observed during the study period. No difference in the prevalence of multidrug resistance was found between bacteria causing intestinal and extraintestinal infections and between canine and feline isolates. Nonhemolytic E. coli isolates were MDR more often than hemolytic ones. Our study showed the companion animals in Poland as an important reservoir of MDR bacteria. These results indicate that continuous monitoring of canine and feline E. coli antimicrobial susceptibility is required. Furthermore, introduction and application of recommendations for appropriate use of antimicrobials in small animal practice should be essential to minimize the emergence of multidrug resistance among E. coli in companion animals.

PFGE and AFLP genotyping of Staphylococcus aureus subsp. anaerobius isolated from goats with Morel’s disease

Staphylococcus aureus subsp. anaerobius is the etiological agent of the Morel’s disease in sheep and goats. The disease presents with subcutaneous abscesses, located mainly in the superficial lymph nodes. Forty-one isolates of S. aureus subsp. anaerobius were collected from two outbreaks of the Morel’s disease in Poland in years 2006–2008. Analysis of DNA SmaI digests by PFGE showed that 35 of 41 isolates belonged to the same PFGE type, identical to the type strain of S. aureus subsp. anaerobius ATCC 35844, confirming high level of clonality of the species. The DNA patterns of the remaining identical 6 isolates, different from the reference strain only by two bands, were found closely related. Genotyping performed with AFLP technique revealed two clonal groups including 16 and 25 isolates, respectively. The study indicated that AFLP technique might be a better discriminatory tool for genetic analysis of S. aureus subsp. anaerobius isolates, when compared to PFGE.

Characterisation of Staphylococcus aureus isolated from meat processing plants – a preliminary study

Abstract Introduction: Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) belonging to the clonal complex 398 (CC398) emerged recently in livestock as a new type of MRSA, which may cause zoonotic infections. This study presents data on the characterisation of S. aureus isolated from the meat processing plants. Material and Methods: S. aureus was isolated from 90 samples collected in the raw meat warehouse, from devices and surfaces of meat processing plants, and from finished meat products. The isolates were subjected to molecular analysis in order to investigate the presence of enterotoxin genes, the mecA gene, and to verify whether they belong to the clonal complex 398. The genetic relatedness of the isolates was determined using pulsed-field electrophoresis. Likewise, antimicrobial susceptibility was tested. Results: From 21 S. aureus strains isolated, five belonged to the CC398, two of which were recognised as MRSA and three as methicillin-sensitive Staphylococcus aureus (MSSA). The most prevalent enterotoxin genes were seg and sei. Two MRSA CC398 isolates, three MSSA CC398, and one MSSA were classified as multidrug-resistant. Conclusion: The first isolation of MSSA CC398 from beef in Poland indicates contamination of beef by strains belonging to this clonal complex. The occurrence of multidrug-resistant enterotoxigenic S. aureus isolates in the finished meat products constitutes a potential risk for the consumers.

Resistance of canine methicillin-resistant Staphylococcus pseudintermedius strains to pradofloxacin

We investigated in vitro activity of a novel veterinary fluoroquinolone, pradofloxacin, against methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates and compared with other fluoroquinolones. A total of 38 MRSP isolates were subjected to agar disk diffusion tests for sensitivity to pradofloxacin, orbifloxacin, marbofloxacin, enrofloxacin, and ciprofloxacin. The minimal inhibitory concentration (MIC) values of pradofloxacin, ciprofloxacin, and enrofloxacin were determined. Mutations in the genes encoding DNA gyrase subunit A (GyrA) and topoisomerase IV (GrlA) proteins associated with fluoroquinolone resistance were studied by an analysis of partial sequences of the genes encoding these proteins. Two MRSP isolates were susceptible in disk diffusion and microdilution test to all fluoroquinolones tested, including pradofloxacin. Based on the results of the disk diffusion testing, 33 of 38 isolates showed resistance to pradofloxacin and 3 were intermediate, whereas, by pradofloxacin MIC testing, 35 isolates were classified as resistant and 1 as intermediate. Single alterations in GyrA and GrlA proteins were observed in the 35 resistant isolates and the 1 intermediate isolate (MIC results). These same 36 isolates were also resistant to the other tested fluoroquinolones. The results of the current study showed that MRSP isolates are usually resistant to all fluoroquinolones, including pradofloxacin. Therefore, in routine susceptibility testing to pradofloxacin by disk diffusion, the results should be carefully interpreted for MRSP isolates, especially those resistant to other fluoroquinolones and, in questionable cases, the pradofloxacin MIC should be determined to confirm the susceptibility testing results.

Changes in the population structure of canine methicillin-resistant Staphylococcus pseudintermedius in Poland

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is being reported with an increasing frequency in small animal veterinary practice. The molecular typing of MRSP isolates revealed that the dominating European multidrug-resistant lineage is the sequence type 71 (ST71), associated with staphylococcal chromosomal cassette SCCmec type II-III. However, the recent reports indicated the emergence of other clones. The study aimed to determine the genetic properties of MRSP isolates obtained from dogs in Poland over a ten-year period. A total of 42 clinical MRSP isolates were subjected to multilocus-sequence typing (MLST) and SCCmec typing. MLST typing of 42 MRSP isolates yielded six STs belonging to two major clonal complexes (CCs): CC71 and CC551, associated with SCCmec element II-III and V, respectively. CC71 comprising ST71 and its newly described single locus variant (SLV) ST680. The second dominating CC551was represented by ST551 and newly described SLV ST771. The other, ST258 and ST85 were detected in single MRSP isolates. This is the first report concerning MLST typing of MRSP isolates in Poland. The results confirmed the domination of ST71 among MRSP until 2015, and the emergence of ST551 in 2015. Furthermore, in 2016 ST551 was identified in the majority of the strains, indicating the changes in the population structure of MRSP in Poland. Polish clinical MRSP isolates showed a shift in the population structure during the period of 2007 and 2016. The dominating MRSP lineage until 2015 was multidrug-resistant ST71-SCCmecII-III. The other lineage ST551-SCCmecV emerged in Poland since 2015, and in 2016 was found in the majority of MRSP isolates.