In vitro activity of selected antimicrobials against methicillin-resistant Staphylococcus pseudintermedius of canine origin in Poland.

BACKGROUND: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an important veterinary pathogen. In general, only a few antimicrobials show in vitro activity against MRSP isolates. OBJECTIVES: The objective of this study was to determine the in vitro activity of selected antimicrobials, including last-choice drugs, against clinical MRSP isolates of canine origin. The activity of 10 selected agents was evaluated against 41 clinical MRSP isolates. METHODS: The disk diffusion method and minimal inhibitory concentration values were used for antimicrobial susceptibility testing (AST). The guidelines for staphylococci of canine or human origin were employed for the interpretation of the results. RESULTS: Among the examined MRSP isolates, resistance to enrofloxacin and clindamycin was the most prevalent (n = 40; 97.6%). Resistance to doxycycline and gentamicin was observed in 83.0% (n = 34) and 68.3% (n = 28) of the isolates, respectively. Single isolates were resistant to chloramphenicol (n = 5; 12.2%) and rifampicin (n = 3; 7.3%), whereas all showed susceptibility to amikacin, vancomycin, mupirocin and linezolid. Predominantly, the results of AST obtained by both methods were consistent. Some discrepancies were observed for gentamicin; however, clinical breakpoints for staphylococci of human origin were used. CONCLUSIONS: Amikacin and chloramphenicol constitute potential treatment options in infections caused by MRSP and may be included in extended susceptibility testing in our geographical region. The determination of clinical breakpoints for some antimicrobials not incorporated in the recommendations should be a high priority in the veterinary diagnostics.

A Fatal A/H5N1 Avian Influenza Virus Infection in a Cat in Poland.

A European Shorthair male cat, neutered, approximately 6 years of age, was presented to the veterinary clinic due to apathy and anorexia. The cat lived mostly outdoors and was fed raw chicken meat. After 3 days of diagnostic procedures and symptomatic treatment, respiratory distress and neurological signs developed and progressed into epileptic seizures, followed by respiratory and cardiac arrest within the next 3 days. Post-mortem examination revealed necrotic lesions in the liver, lungs, and intestines. Notably, the brain displayed perivascular infiltration of lymphocytes and histiocytes. Few foci of neuronal necrosis in the brain were also confirmed. Microscopic examination of the remaining internal organs was unremarkable. The A/H5N1 virus infection was confirmed using a one-step real-time reverse transcription polymerase chain reaction (RT-qPCR). The disease caused severe neurological and respiratory signs, evidence of consolidations and the presence of numerous B lines, which were detected on lung ultrasound examination; the postmortem findings and detection of A/H5N1 viral RNA in multiple tissues indicated a generalized A/H5N1 virus infection. Moreover, a multidrug-resistant strain of Enterococcus faecium was isolated in pure culture from several internal organs. The source of infection could be exposure to infected birds or their excrements, as well as contaminated raw poultry meat but, in this case, the source of infection could not be identified.

Is Vitamin D3 a Worthy Supplement Protecting against Secondary Infections in Dogs with Atopic Dermatitis?

Canine atopic dermatitis (CAD) is a common, chronic, inflammatory skin disease in dogs worldwide. This disease often predisposes for secondary organisms overgrowth and skin infections with pathogens, such as Staphylococcus pseudintermedius and Malassezia pachydermatis. Unfortunately, the causes of this disease in both humans and animals are not fully understood; therefore, the only possible option is a lifelong, symptomatic treatment. The management of CAD is mainly based on limiting contact with allergens and antipruritic therapy, most often with glucocorticoids and antihistamines. A serious problem in this situation is the fact, that long-term administration of glucocorticoids leads to side effects like polyuria, alopecia, increased susceptibility to infection, muscle atrophy, and many others. For this reason, great emphasis is placed on the development of replacement and supportive therapies. It is a well-documented fact that reduced concentrations of serum vitamin D3 contribute to the severity of atopic dermatitis symptoms in humans. Moreover, unlike the most commonly used therapeutic methods, of which the main goal is to ameliorate inflammation and pruritus, namely the symptoms of AD, vitamin D3 supplementation affects some underlying factors of this disease. Therefore, in this review, we summarize the current state of knowledge regarding the role of vitamin D3 in CAD, its protective effect against secondary bacterial and fungal infections, and the potential of its supplementation in dogs.

Tetracycline, Macrolide and Lincosamide Resistance in Streptococcus canis Strains from Companion Animals and Its Genetic Determinants.

Growing antimicrobial resistance (AMR) in companion-animal pathogens, including Streptococcus canis (S. canis), is a significant concern for pet treatment as well for public health. Despite the importance of S. canis in veterinary and human medicine, studies concerning the AMR of this bacterium are still scarce. A total of 65 S. canis strains, isolated from dogs and cats, were assessed to test for susceptibility to six clinically relevant antimicrobials via a microdilution method. The prevalence of the selected acquired-resistance genes was also investigated via PCR. High MIC50 and MIC90 values (≥128 µg/mL) were noted for tetracycline, erythromycin and clindamycin. Only a few strains were resistant to the tested beta-lactams (6.2%). Tetracycline resistance was found in 66.2% of the strains. Resistance to erythromycin and clindamycin (ML resistance) was found in 55.4% of the strains. Strains with a phenotype showing concurrent resistance to tetracycline and ML were predominant (53.8%). AMR in the tested S. canis strains was associated with a variety of acquired and potentially transferable genes. Tetracycline resistance was conferred by tet(O) (40.0%), tet(M) (9.2%), and tet(T) (1.5%), which is reported for the first time in S. canis. In most cases, the tet(M) gene was detected in relation to the conjugative transposon Tn916. The MLSB phenotype was confirmed in the strains harboring erm(B) (43.1%) and erm(TR) (7.7%). To conclude, a high rate of S. canis strains occurring in dogs and cats displayed resistance to antimicrobials important for treatment; moreover, they are a potential reservoirs of various resistance determinants. Therefore, AMR in these pathogens should be continuously monitored, especially regarding the One Health concept.

Prevalence and Genetic Diversity of Trueperella pyogenes Isolated from Infections in European Bison (Bison bonasus).

Trueperella pyogenes is a Gram-positive bacterium causing purulent infections in many animal species, including the European bison. However, the data about the virulence and genetic relationships of T. pyogenes strains isolated from these wild ruminants are strongly limited. The aim of this study was to investigate the prevalence of T. pyogenes infections in the European bison, and to evaluate the genetic diversity of isolates from these animals. In the time span of 10 years, 328 European bison from 16 different locations were examined. The standard bacteriological methods were used for T. pyogenes isolation and identification from clinical specimens obtained from urogenital tract infections and abscesses of different locations. The presence of genes encoding known virulence factors was investigated by PCR, and the genetic diversity of T. pyogenes strains was examined with the RAPD-PCR method. The prevalence of T. pyogenes infections was 14.6%, and the pathogen was isolated from both female (47.9% of isolates) and male (52.1% of isolates) European bison. It should be highlighted that a considerable number of strains were isolated from the prepuce and penis infections. Therefore, the role of T. pyogenes in the pathogenesis of balanoposthitis should be seriously perceived. A total of 39 T. pyogenes strains were subjected to genetic characterization. All studied strains carried the plo gene, while the nanH (25.6%), nanP (23.1%), cbpA (7.7%), fimA (97.4%), fimC (69.2%), fimE (92.3%) and fimG (15.4%) genes were present with a variable frequency among the tested strains. The virulence genotype plo/fimA/fimC/fimE was dominant. RAPD-PCR typing showed a high level of genetic diversity among European bison T. pyogenes strains, and a total of 31 different RAPD profiles were distinguished. In a few cases, the same RAPD profile was found in strains obtained from animals living in the same area. This study provided the first data about the prevalence and genetic relationships of T. pyogenes in the Polish population of European bison. However, further epidemiological investigations are needed to understand the routes of transmission and dissemination of the pathogen in these wild animals.

The Prevalence of Histopathological Features of Pneumonia in Goats with Symptomatic Caprine Arthritis-Encephalitis.

Chronic interstitial pneumonia (CIP) is a main pathology of sheep infected with small ruminant lentivirus (SRLV). Caprine arthritis-encephalitis (CAE) is caused by the same pathogen; however, the presence of CIP has been only occasionally reported in SRLV-infected goats. We carried out a cross-sectional study to determine the prevalence of histopathological lesions indicative of CIP in goats with symptomatic CAE, and to investigate whether CIP was associated with a higher prevalence of other types of pneumonia (purulent bronchopneumonia, fibrinous pleuropneumonia) or bacterial infections. Lung specimens and bronchial swabs were collected for histopathological and bacteriological examination, respectively, from 116 goats from a CAE-affected herd. All goats were euthanized due to severe clinical signs of CAE. The goats were seropositive for SRLV infection in two different ELISAs and the presence of SRLV antigen in the lung tissue was confirmed by immunohistochemistry. Histopathologically, pneumonia of any type was confirmed in 82 goats (70.7%) and CIP was present in 67 goats (57.8%). In most goats, the severity of the histopathological features of pneumonia was mild. Bacteria were detected in bronchial swabs from 73 goats (62.9%). CIP proved to be significantly positively linked to the occurrence of purulent bronchopneumonia (p < 0.001), fibrinous pleuropneumonia (p = 0.001), and of the infection of lungs with bacteria capable of causing pneumonia (p = 0.050). The causal character of these associations should be considered and warrants further investigation.

Focused Cardiac Ultrasound Examination as a Tool for Diagnosis of Infective Endocarditis and Myocarditis in Dogs and Cats.

Symptoms of infective endocarditis (IE) and myocarditis are usually nonspecific and include fever, apathy, and loss of appetite. This condition can lead to severe heart failure with ascites or/and fluid in the thoracic cavity or/and in the pericardial sac. We describe infective endocarditis and myocarditis in 3 dogs and 4 cats. In all animals, the initial diagnosis was performed on the basis of a focused cardiac ultrasound examination performed by a general practitioner after a training in this technique. The initial findings were confirmed by a board-certified specialist in veterinary cardiology. Post mortem positive microbiological results from valves were obtained in 4 of 7 patients. Methicillin-resistant Staphylococcus aureus was confirmed in 2 cases and Staphylococcus epidermidis was confirmed in 2 cases, one of which included Enterococcus sp. coinfection. Histopathological examination confirmed initial diagnosis in 5 of 7 animals. In the remaining 2 patients, the time elapsed from the onset of clinical symptoms to death was about 1 month and no active inflammation but massive fibrosis was found microscopically. This is, to our best knowledge, the first report of IE and myocarditis diagnosed in small animals using focused cardiac ultrasound examination. Therefore, we conclude that common usage of this technique by trained general veterinarians may increase the rate of diagnosed patients with these conditions.

Molecular characterization of high-level mupirocin resistance in methicillin-resistant staphylococci isolated from companion animals.

High-level mupirocin resistance (HLMR) is determined by the plasmid-located ileS2 gene flanked by two copies of the insertion sequence 257 (IS257). The molecular epidemiology of high-level mupirocin-resistant isolates could be assessed by the determination of their IS257-ileS2 spacer regions conformation. In this study, 188 isolates of methicillin-resistant staphylococci were subjected to the detection of HLMR, and analysis of the conformation of the IS257-ileS2 spacer regions. Mupirocin resistance was detected in five (2,6%) isolates, among which two were recognized as Staphylococcus pseudintermedius, two as Staphylococcus haemolyticus, and one as Staphylococcus aureus. High-level mupirocin resistance was revealed by the agar disk diffusion method, and MIC values, and was confirmed by the detection of the ileS2 gene. The conformations of the IS257-ileS2 spacer regions were homologous in two S. haemolyticus strains tested. The remaining three isolates showed diverse IS257-ileS2 conformations. The results of this study indicate that HLMR occasionally occurs in staphylococci isolated from companion animals. The heterogeneity and the homogeneity of the IS257-ileS2 spacer regions confirm that the ileS2 gene spread among staphylococci of animal origin by the transfer of different as well as the same plasmids. Surveillance of the occurrence of mupirocin resistance and molecular characterization of resistant isolates are strongly recommended due to the possibility of plasmid-located resistance gene transfer between staphylococci.

Pigeons as Carriers of Clinically Relevant Multidrug-Resistant Pathogens-A Clinical Case Report and Literature Review.

Pigeons are widespread bird species in urban regions (Columba livia forma urbana) and may carry pathogens with zoonotic potential. In recent years, more and more data indicate that these zoonotic pathogens are multidrug resistant. Our results confirmed that global trend. Three different multidrug-resistant pathogens were isolated from an oral cavity of a racing pigeon with lesions typical for pigeon pox virus infection. Staphylococcus aureus was recognized as methicillin resistant, thus resistant to all beta-lactams. Additionally, it was also resistant to many other classes of antibiotics, namely: aminoglycosides, tetracyclines, phenicols, lincosamides, and macrolides. Escherichia coli showed resistance to all antimicrobials tested, and it was classified as intermediate to amikacin. Moreover, Candida albicans resistant to clotrimazole, natamycin, flucytosine, and amphotericin and intermediate to ketoconazole, nystatin, and econazole was also isolated. This raises the question how pigeons acquire such highly resistant strains. Therefore, more data are needed concerning the resistance to antibiotics in strains from domestic and wild pigeons in Poland. Until the problem is fully understood, it will be challenging to implement adequate planning of any control measures and check their effectiveness.

Trueperella pyogenes Isolates from Livestock and European Bison (Bison bonasus) as a Reservoir of Tetracycline Resistance Determinants.

Determinants of tetracycline resistance in Trueperella pyogenes are still poorly known. In this study, resistance to tetracycline was investigated in 114 T. pyogenes isolates from livestock and European bison. Tetracycline minimum inhibitory concentration (MIC) was evaluated by a microdilution method, and tetracycline resistance genes were detected by PCR. To determine variants of tetW and their linkage with mobile elements, sequencing analysis was performed. Among the studied isolates, 43.0% were tetracycline resistant (MIC ≥ 8 µg/mL). The highest MIC90 of tetracycline (32 µg/mL) was noted in bovine and European bison isolates. The most prevalent determinant of tetracycline resistance was tetW (in 40.4% of isolates), while tetA(33) was detected only in 8.8% of isolates. Four variants of tetW (tetW-1, tetW-2, tetW-3, tetW-4) were recognized. The tetW-3 variant was the most frequent and was linked to the ATE-1 transposon. The tetW-2 variant, found in a swine isolate, was not previously reported in T. pyogenes. This is the first report on determinants of tetracycline resistance in T. pyogenes isolates from European bison. These findings highlight that wild animals, including wild ruminants not treated with antimicrobials, can be a reservoir of tetracycline-resistant bacteria carrying resistance determinants, which may be easily spread among pathogenic and environmental microorganisms.

New Determinants of Aminoglycoside Resistance and Their Association with the Class 1 Integron Gene Cassettes in Trueperella pyogenes.

Trueperella pyogenes is an important opportunistic animal pathogen. Different antimicrobials, including aminoglycosides, are used to treat T. pyogenes infections. The aim of the present study was to evaluate aminoglycoside susceptibility and to detect aminoglycoside resistance determinants in 86 T. pyogenes isolates of different origin. Minimum inhibitory concentration of gentamicin, streptomycin, and kanamycin was determined using a standard broth microdilution method. Genetic elements associated with aminoglycoside resistance were investigated by PCR and DNA sequencing. All studied isolates were susceptible to gentamicin, but 32.6% and 11.6% of them were classified as resistant to streptomycin and kanamycin, respectively. A total of 30 (34.9%) isolates contained class 1 integrons. Class 1 integron gene cassettes carrying aminoglycoside resistance genes, aadA11 and aadA9, were found in seven and two isolates, respectively. Additionally, the aadA9 gene found in six isolates was not associated with mobile genetic elements. Moreover, other, not carried by gene cassettes, aminoglycoside resistance genes, strA-strB and aph(3')-IIIa, were also detected. Most importantly, this is the first description of all reported genes in T. pyogenes. Nevertheless, the relevance of the resistance phenotype to genotype was not perfectly matched in 14 isolates. Therefore, further investigations are needed to fully explain aminoglycoside resistance mechanisms in T. pyogenes.

Prevalence, antibiotic susceptibility and virulence factors of Enterococcus species in racing pigeons (Columba livia f. domestica).

BACKGROUND: This study was aimed to investigate the intestinal microbiota in racing pigeons with regard to Enterococcus species distribution, virulence factors and antibiotic susceptibility. Three methods (API, Multiplex sodA-PCR, 16S rRNA sequencing) were compared for Enterococcus species identification. Cloacal samples from 179 apparently healthy pigeons of 13 different flocks were tested. RESULTS: Multiplex sodA-PCR and 16S rRNA gene sequencing showed almost perfect agreement in Enterococcus species identification. Isolates were identified as Enterococcus columbae (34.5%), Enterococcus hirae (20.7%), Enterococcus faecalis (11.7%), Enterococcus faecium (11.7%), Enterococcus gallinarum (9%), Enterococcus mundtii (4.8%), Enterococcus casseliflavus (3.4%), Enterococcus cecorum (2.1%), Enterococcus durans (2.1%). More Enterococcus species were found after the race season than before. The study showed differences between Enterococcus species in relation to 68.8% (22/32) biochemical parameters. Six out of seven virulence genes were detected: gelE (43.5%), asa1 (42.1%), efaA (30.3%), ace (30.3%), cylA (27.6%), and esp (9%). None of the isolates harboured hyl gene. Overall 15.2% of Enterococcus isolates produced gelatinase, but 66.7% gelE genes were silent. Enterococcus faecalis showed the most often efaA, ace and gelatinase activity than other enterococcal species. Nearly all isolates (93.1%) were resistant to at least one antibiotic. The most frequent resistance was to enrofloxacin (80%), doxycycline with teicoplanin (73.1%), erythromycin (49.7%). The study revealed significant differences between some enterococcal species in the antibiotic susceptibility to different antibiotics. Enterococcus columbae and E. cecorum showed significantly more frequent resistance to chloramphenicol than other enterococci. The presence of VRE (19.3%), HLGR (2.8%) and no LRE were found. Overall 30.3% of isolates were positive for vancomycin resistance genes, where vanC1 (E. gallinarum), vanC2-C3 (E. hirae, E. casseliflavus), vanB (E. columbae) predominated. CONCLUSIONS: We conclude, that intestinal microbiota in racing pigeons is composed by 9 different Enterococcus species. Given that racing pigeons are kept in close contact with humans and backyard animals, combined with their long-distance flight abilities, they can serve as potential source of virulent and antibiotic resistant Enterococcus spp. in the environment.

High-level mupirocin resistance in methicillin-resistant staphylococci isolated from dogs and cats.

BACKGROUND: Mupirocin is one of the few antimicrobials active against methicillin-resistant Staphylococcus aureus (MRSA), and is frequently used for the eradication of MRSA nasal colonisation in humans. Initially, mupirocin resistance was recognised in human S. aureus, including MRSA isolates, then also among coagulase-negative staphylococci (CoNS). Nowadays, mupirocin resistance is occasionally observed in canine staphylococci, along with Staphylococcus pseudintermedius (MRSP) strains, as well as CoNS, which usually show methicillin resistance. In the current study, high-level mupirocin resistance in methicillin-resistant staphylococci isolated from diseased dogs and cats was investigated. RESULTS: Among 140 methicillin-resistant staphylococci isolates from dogs and cats, three showed high-level mupirocin resistance in a screening test using the agar disk diffusion method. One was recognised as methicillin-resistant S. aureus, one as methicillin-resistant S. pseudintermedius, and one as methicillin-resistant Staphylococcus haemolyticus. S. pseudintermedius and S. aureus were isolated from dogs, S. haemolyticus was obtained from a cat. All isolates showed high-level mupirocin resistance, confirmed by minimum inhibitory concentration (MIC) values of above 1024 µg/ml and the presence of the plasmid-located gene ileS2. This is the first report on the detection of high-level mupirocin resistance (HLMR) in S. haemolyticus of feline origin. CONCLUSIONS: This study revealed the occurrence of HLMR in three Staphylococcus isolates obtained from companion animals in Poland. The results of this study indicate that the monitoring of mupirocin resistance in staphylococci of animal origin, especially in methicillin-resistant isolates, is strongly recommended.

Pathogenicity and Virulence of Trueperella pyogenes: A Review.

Bacteria from the species Trueperella pyogenes are a part of the biota of skin and mucous membranes of the upper respiratory, gastrointestinal, or urogenital tracts of animals, but also, opportunistic pathogens. T. pyogenes causes a variety of purulent infections, such as metritis, mastitis, pneumonia, and abscesses, which, in livestock breeding, generate significant economic losses. Although this species has been known for a long time, many questions concerning the mechanisms of infection pathogenesis, as well as reservoirs and routes of transmission of bacteria, remain poorly understood. Pyolysin is a major known virulence factor of T. pyogenes that belongs to the family of cholesterol-dependent cytolysins. Its cytolytic activity is associated with transmembrane pore formation. Other putative virulence factors, including neuraminidases, extracellular matrix-binding proteins, fimbriae, and biofilm formation ability, contribute to the adhesion and colonization of the host tissues. However, data about the pathogen-host interactions that may be involved in the development of T. pyogenes infection are still limited. The aim of this review is to present the current knowledge about the pathogenic potential and virulence of T. pyogenes.

Characterization of pathogenic Enterococcus cecorum from different poultry groups: Broiler chickens, layers, turkeys, and waterfowl.

Enterococcus cecorum (EC) is known as a commensal in the intestines of mammals and birds. However, it has been described as an emerging pathogen in poultry industry worldwide. The aim of this study was to analyze and compare EC isolated from clinical material collected from poultry groups with different production purposes. The genetic diversity among pathogenic EC in relation to each specific poultry type was examined. In total, 148 isolates from independent infection outbreaks (2011-2016) were used: 76 broiler chickens (CB), 37 broiler breeders (BB), 23 layers (CL), 7 waterfowl (W) and 5 turkey (T) flocks (1 isolate/1 flock). We provided age ranges at diagnosis of EC-infection for 5 poultry groups. Isolates obtained from CB were significantly more frequently retrieved from bone marrow, joints, spine, and contrary to BB, CL less frequently retrieved from respiratory system. The study showed differences between EC of various poultry types in relation to 10/32 (31.3%) biochemical parameters. EC isolates from CB were significantly more often positive for ßGAL, ßNAG, MLZ, and less often positive for PAL and ßMAN than isolates from other poultry types. However, BB and W isolates showed higher ability to metabolise mannitol than CB, CL, and T. CB isolates showed lower ability to survive at 60°C. Only chicken EC-isolates harbored virulence genes: CB (8.1%) > BB (3.4%) > CL (2%). No specific pulsotype of EC was associated with a specific poultry. One or several various (up to 6) genetic types of EC may be involved in outbreaks in CB flocks within one year in one region. Outbreaks reported in following years in the same region were usually caused by a distinct set of EC-genetic types. PFGE results indicated at the genetic heterogeneity among pathogenic isolates involved in outbreaks in relation to each poultry type. To our best knowledge, this is the first study which provides a comparison between clinical EC from 5 poultry groups. The study provides a new insight into EC as pathogen of different bird species. The obtained data may be useful in further studies on EC-infections more focused on a specific type of poultry.

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.

Distribution of antimicrobial resistance determinants, virulence-associated factors and clustered regularly interspaced palindromic repeats loci in isolates of Enterococcus faecalis from various settings and genetic lineages.

Enterococcus faecalis represents an important factor of hospital-associated infections (HAIs). The knowledge on its evolution from a commensal to an opportunistic pathogen is still limited; thus, we performed a study to characterise distribution of factors that may contribute to this adaptation. Using a collection obtained from various settings (hospitalised patients, community carriers, animals, fresh food, sewage, water), we investigated differences in antimicrobial susceptibility, distribution of antimicrobial resistance genes, virulence-associated determinants and phenotypes, and CRISPR loci in the context of the clonal relatedness of isolates. Bayesian Analysis of Population Structure revealed the presence of three major groups; two subgroups comprised almost exclusively HAI isolates, belonging to previously proposed enterococcal high-risk clonal complexes (HiRECCs) 6 and 28. Isolates of these two subgroups were significantly enriched in antimicrobial resistance genes, presumably produced a polysaccharide capsule and often carried the aggregation substance asa1; distribution of other virulence-associated genes, such as esp and cyl, formation of a biofilm and gelatinase production were more variable. Moreover, both subgroups showed a low prevalence of CRISPR-Cas 1 and 3 and presence of small CRISPR2 variants. Our study confirms the importance of HiRECCs in the population of E. faecalis and their confinement to the hospital settings.

Molecular characterization of Rhodococcus equi isolates from horses in Poland: pVapA characteristics and plasmid new variant, 85-kb type V.

BACKGROUND: Rhodococcus equi is one of the most significant bacterial pathogens affecting foals up to 6 months of age worldwide. Rhodococcosis is present in Poland however information about molecular characterization of R. equi isolates is scarce. This study describes molecular characterization of Rhodococcus equi infection on 13 horse breeding farms in Poland between 2001 and 2012. Samples were collected by tracheobronchial aspiration from pneumonic foals or during necropsy. The R. equi isolates were genotyped by plasmid profiling and pulsed-field gel electrophoresis. RESULTS: Totally, 58 R. equi isolates were investigated. One isolate lost its plasmid. Among the 57 VapA-positive isolates, 48 contained 85-kb type I plasmid (82.8%), 8 contained 87-kb type I plasmid (13.8%). One isolate (1.7%) had a unique restriction cleavage pattern and the 2nd fragment of EcoRI digests of this plasmid DNA was about 2600 bases smaller than that of the 85 kb type I. This new plasmid variant was designated as the "85-kb type V". Among the 58 isolates typeable with VspI-PFGE, ten PFGE clusters were detected. The majority of foals were infected mostly with isolates of low genetic diversity. CONCLUSIONS: Most of clinical isolates of R. equi from foals in Poland contain pVapA 85-kb type I and 87-kb type I similarly to the other European countries and the United States. However, the new variant of pVapA 85-kb type V was identified. The chromosomal variability was detected among some of the investigated isolates and the presence of farm-specific isolates might be possible.

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.

Phenotypic and genotypic characterization of Enterococcus cecorum strains associated with infections in poultry.

BACKGROUND: From the beginning of the 21(st) century Enterococcus cecorum has emerged as a significant health problem for poultry raised under intensive production systems. To obtain new insights into this bacterial species, we investigated 82 clinical isolates originating from different poultry flocks in Poland between 2011 and 2014. RESULTS: Phenotypically, isolates from clinical cases showed ability to growth at low temperatures (4 °C, 10 °C), and differences in growth at 45 °C (74.4 %). Survival at high temperatures (60 °C, 70 °C) was observed for 15, 30 min. More than half of strains survived at 60 °C even after prolonged incubation (1 h), but none survived after 1 h at 70 °C. Total growth inhibition was observed on agar supplemented with tergitol or potassium tellurite. Relatively high number of isolates gave positive reactions for ß-galactosidase (ßGAL 80 %), Voges Proskauer test (60 %), less for ß-mannosidase (17 %), glycogen and mannitol (12 %). The metabolic fingerprinting for E. cecorum obtained in Biolog system revealed ability to metabolise 22 carbon sources. Only 27/82 strains contained ≥ 1 virulence genes of tested 7, however 2.4 % isolates carried 6. Increased antimicrobial resistance was observed to enrofloxacin (87 %), teicoplanin (85 %), doxycycline (83 %), erythromycin (46 %). Most strains (75/82) showed multidrug resistance. The single isolate was resistant to vancomycin (VRE) and high level gentamicin (HLGR). Linezolid resistance among clinical isolates was not found. PFGE revealed diversity of E. cecorum from cases. It could be assumed that transmission of pathogenic strains between flocks regardless of type of production or geographical region may be possible. CONCLUSIONS: Clinical infections in poultry caused by E. cecorum may indicated on new properties of this bacterial species, previously known as a commensal. Despite many common phenotypic features, differences were found among clinical isolates. Several, widely distributed pathogenic E. cecorum strains seemed to be responsible for infection cases found in different poultry types.