Publications by authors named "Przemysław Zięba"

26 Publications

  • Page 1 of 1

A rich mosaic of resistance in extended-spectrum β-lactamase-producing Escherichia coli isolated from red foxes (Vulpes vulpes) in Poland as a potential effect of increasing synanthropization.

Sci Total Environ 2021 Nov 20:151834. Epub 2021 Nov 20.

University of Life Sciences, Faculty of Veterinary Medicine, Department of Preclinical Veterinary Sciences, Sub-Department of Veterinary Microbiology, Akademicka 12, 20-033 Lublin, Poland. Electronic address:

In our research, we analyzed the resistance of cephalosporin-resistant E. coli strains to antimicrobial agents. The strains were collected during five years from wild animal species commonly inhabiting Poland. We have identified the type of β-lactamases produced and the multidrug-resistance profile. Most strains (73.8%) had genes encoding ESBL enzymes, mainly CTX-M-1 and TEM. Almost all AmpC-β-lactamase-producing isolates had the bla gene. Almost 70% of the strains tested showed a multi-drug resistance profile. The dominant phenotype was resistance to tetracycline (69.05%), and/or sulfamethoxazole (57.1%). We also found high resistance to quinolones: ciprofloxacin 35.7% and nalidixic acid 52.4%. The phenotypic resistance of the strains was in most cases confirmed by the presence of corresponding genes. Among strains, 26.2% were carriers of plasmid-mediated quinolone resistance genes (PMQR). MLST analysis revealed a large clonal variation of the strains, which was reflected in 28 different sequence types. More than half of the strains (54.7%) were classified into the following sequence complexes: 10, 23, 69, 101, 155, 156, 168, 354, 398, 446, and 648. Only one strain in the studied group was assigned to the ExPEC pathotype and represented sequence type 117. The results of our research have confirmed that isolates obtained from wild animals possess many resistance determinants and sequence types, which are also found in food-producing animals and humans. This reflects the doctrine of "One health", which clearly indicates that human health is inextricably linked with animal health as well as degree of environmental contamination. We conclude that the resistance and virulence profiles of strains isolated from wildlife animals may be a resultant of various sources encountered by animals, creating a rich and varied mosaic of genes, which is very often unpredictable and not reflected in the correlation between the sequence type and the gene profile of resistance or virulence observed in epidemic clones.
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http://dx.doi.org/10.1016/j.scitotenv.2021.151834DOI Listing
November 2021

Virulence and Antimicrobial Resistance Pattern of spp. Colonizing European Pond Turtles and Their Natural Environment. First Study from Poland.

Animals (Basel) 2021 Sep 23;11(10). Epub 2021 Sep 23.

Department of Pediatric Orthopedics and Rehabilitation, Faculty of Medicine, Medical University, Gębali 6, 20-093 Lublin, Poland.

The aim of the study was to isolate and identify species belonging to the genus and evaluate the antimicrobial resistance and virulence patterns of isolates colonizing European pond turtles () from natural environment of Eastern Poland. In total, 74 turtles and 15 samples of water from their natural environment were examined. More than 40 strains were isolated and identified: ( = 1), ( = 13), ( = 2), ( = 3), and ( = 23). The highest incidence of resistance was noted for ampicillin (100%) and sulfamethoxazole (62.0%), followed by erythromycin and colistin (both 40.5%). Moreover, eight strains were intermediately resistant to meropenem (19%). Most isolates were found to possess more than one virulence gene among A, , and . We showed that the population of free-living European pond turtles was highly colonized by spp. Such strains may be an infectious agent not only for the population of turtles but also for other species of animals inhabiting their natural environment. Moreover, the undesirable properties of water quality caused by the presence of drug-resistant aeromonads could have a negative impact on human health.
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http://dx.doi.org/10.3390/ani11102772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8532793PMC
September 2021

Last Call for Replacement of Antimicrobials in Animal Production: Modern Challenges, Opportunities, and Potential Solutions.

Antibiotics (Basel) 2020 Dec 9;9(12). Epub 2020 Dec 9.

Department of Pediatric Orthopedics and Rehabilitation, Faculty of Medicine, Medical University, Gębali 6, 20-093 Lublin, Poland.

The constant market demand for easily available and cheap food of animal origin necessitates an increasing use of antibiotics in animal production. The alarming data provided by organizations monitoring drug resistance in indicator and pathogenic bacteria isolated from humans and animals indicate a possible risk of a return to the preantibiotic era. For this reason, it seems that both preventive and therapeutic measures, taken as an alternative to antimicrobials, seem not only advisable but also necessary. Nevertheless, the results of various studies and market analyses, as well as difficulties in the implementation of alternative substances into veterinary medicine, do not guarantee that the selected alternatives will completely replace antimicrobials in veterinary medicine and animal production on a global scale. This publication is a brief overview of the drug resistance phenomenon and its determinants, the steps taken to solve the problem, including the introduction of alternatives to antimicrobials, and the evaluation of some factors influencing the potential implementation of alternatives in animal production. The review also presents two groups of alternatives, which, given their mechanism of action and spectrum, are most comparable to the effectiveness of antibiotics, as emphasized by the authors.
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http://dx.doi.org/10.3390/antibiotics9120883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762978PMC
December 2020

Analysis of the occurrence and molecular characteristics of drug-resistant strains of Enterococcus faecalis isolated from the gastrointestinal tract of insectivorous bat species in Poland: A possible essential impact on the spread of drug resistance?

Environ Pollut 2021 Jan 23;269:116099. Epub 2020 Nov 23.

Wrocław University of Environmental and Life Sciences, Faculty of Veterinary Medicine, Department of Pathology, Division of Microbiology, C. K. Norwida 31, 50-375, Wrocław, Poland.

Bats are poorly understood as a reservoir of multidrug-resistant strains; therefore, the aim of this study was to determine molecular characterization of multidrug-resistant Enterococcus strains isolated from bat species from Poland. A multi-stage analysis based on targeted isolation of drug-resistant strains (selective media with tetracycline, chloramphenicol, gentamicin, streptomycin, and vancomycin), determination of the phenotypic profile of drug-susceptibility using the disc diffusion method, and amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) was used for the isolation and differentiation of strains. The applied strategy finally allowed identification of E. faecalis resistant to at least one antimicrobial in 47.2% of the single-animal group and in 46.9% of the pooled samples of bat's guano. Out of the 36 distinct isolates, 69% met the criteria of multi-drug resistance, with a dominant combination of resistance to tetracycline, erythromycin, and rifampicin. Simultaneously, 41.6% of the strains were high-level aminoglycoside resistant (HLAR). In most strains, phenotypic resistance was reflected in the presence of at least one gene encoding resistance to a given drug. Moreover, our research results show that some genes were detected simultaneously in the same strain statistically significantly more frequently. This may confirm that the spread of some genes (tetM and ermB or aph (3')-IIIa as well as gelE and aac (6')-Ie-aph (2″)-Ia or ant (6)-Ia) is associated with their common occurrence on the same mobile genetic element. To our knowledge, this is the first analysis of multidrug-resistance among E. faecalis isolated from bats. Our research demonstrates that the One Health concept is not associated exclusively with food-producing animals and humans, but other species of wildlife animals should be covered by monitoring programs as well. We confirmed for the first time that bats are an important reservoir of multi-resistant E. faecalis strains and could have a great impact on environmental resistance.
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http://dx.doi.org/10.1016/j.envpol.2020.116099DOI Listing
January 2021

Wildlife omnivores and herbivores as a significant vehicle of multidrug-resistant and pathogenic Escherichia coli strains in environment.

Environ Microbiol Rep 2020 12 28;12(6):712-717. Epub 2020 Sep 28.

Sub-Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Institute of Preclinical Veterinary Sciences, University of Life Sciences, Akademicka 12, Lublin, 20-033, Poland.

The phenomenon of resistance of Escherichia coli strains in free-living animals has been constantly expanding in recent years. However, the data are still fragmented and, due to the growing threat to public health, there is a constant need to search for and analyse new reservoirs and indicate their role and importance in the circulation of resistance genes in the environment. Therefore, the target group in this study were free-living non-predatory animals as reservoirs of drug-resistant and potentially virulent E. coli strains. We obtained 70 different isolates, including 71.4% of multidrug-resistant strains. In strains isolated from all species of animals, we determined high resistance to ampicillin (95.7%), tetracycline (64.3%), streptomycin (51.4%) and chloramphenicol (38.6%). Every third of the E. coli-positive individual was a carrier of more than one resistant clone. Moreover, 11.4% of isolates among the resistant strains had the ExPEC, ETEC, or EHEC pathotype. Our study confirmed that not only free-living predatory animals are reservoirs of resistance but also many synanthropic species of herbivores and omnivores contribute substantially to the spread of resistant and virulent E. coli strains.
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http://dx.doi.org/10.1111/1758-2229.12886DOI Listing
December 2020

Wildlife Carnivorous Mammals As a Specific Mirror of Environmental Contamination with Multidrug-Resistant Strains in Poland.

Microb Drug Resist 2020 Sep 28;26(9):1120-1131. Epub 2020 Apr 28.

Sub-Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Institute of Biological Bases of Animal Diseases, University of Life Sciences, Lublin, Poland.

In recent decades, the number of studies on the occurrence of resistant strains in wildlife animals has increased significantly, but data are still fragmentary. The aim of this study was to evaluate drug resistance of strains isolated from wild carnivorous mammals, common in Poland. Selective media with antimicrobials (tetracycline, kanamycin, chloramphenicol, and cefotaxime) were used for isolation. Of 53 isolates shown to be distinct by the amplification of DNA fragments surrounding rare restriction site-fingerprinting method, 77.8% were multidrug-resistant (multidrug-resistant). All strains were resistant to ampicillin and many of them also exhibited resistance to tetracycline (76.2%), sulfamethoxazole (57.1%), streptomycin and kanamycin (49.2%), chloramphenicol (30.1%), and nalidixic acid (46%). In most cases, the phenotypic resistance profile was confirmed by detection of relevant genes mostly occurring in strains isolated from livestock animals and humans. Extended-spectrum β-lactamase-producing strains were detected in one mink and three martens. The strains were carriers of , and genes. Our research confirmed a high carrier rate of MDR , even more than one MDR strain in a single individual; therefore, wider monitoring in this group of animals should be considered.
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http://dx.doi.org/10.1089/mdr.2019.0480DOI Listing
September 2020

Dermatophytosis with concurrent Trichophyton verrucosum and T. benhamiae in calves after long-term transport.

Vet Dermatol 2020 Oct 26;31(5):414-e111. Epub 2020 Aug 26.

State Veterinary Laboratory, Słowicza 2, Lublin, 20-336, Poland.

Background: Dermatophytosis is a common problem in cattle. The aetiological factors associated with this disease are filamentous fungi with the ability to digest and grow on keratinized substrates. In cattle, and less frequently in other domestic animals and people, the dermatophyte Trichophyton verrucosum is most commonly isolated from skin lesions. The dermatophyte Trichophyton benhamiae is an important zoonotic pathogen, and the main sources of transmission are guinea pigs and other small rodents.

Objectives: In this report, we show multispecies infection in calves (Bos taurus) after long-term transport and vaccination against trichophytosis.

Animals: Sixty animals were imported of which 32 were observed to be affected with superficial infection nine to 12 days after vaccination for dermatophytosis.

Methods And Materials: Diagnosis was made correlating the clinical signs with a micro- and macroscopic examination of cultured fungi. Molecular differentiation was used to confirm the species affiliation.

Results: Eight of the calves were infected with T. verrucosum alone, and 24 calves with both T. verrucosum and T. benhamiae. We suggest that the cause of this large outbreak was immunosuppression of the animals resulting from the stress of transport and administration of vaccine.

Conclusion: Both T. verrucosum and T. benhamiae can be seen concurrently in cattle.
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http://dx.doi.org/10.1111/vde.12880DOI Listing
October 2020

A significant number of multi-drug resistant Enterococcus faecalis in wildlife animals; long-term consequences and new or known reservoirs of resistance?

Sci Total Environ 2020 Feb 2;705:135830. Epub 2019 Dec 2.

University of Life Sciences, Faculty of Veterinary Medicine, Department of Animal Physiology, Akademicka 12, 20-033 Lublin, Poland.

As the last link in the food chain in a complex ecosystem covering at least three different environmental spheres, species of wildlife carnivorous mammals constitute a group accumulating potential pathogens and factors resulting from human activity, including the emergence of drug resistance. Therefore, the aim of this study was to evaluate the level and range of resistance in commensal E. faecalis isolated from wildlife carnivorous mammals and genetic relationships in terms of the source of these strains as well as resistance and virulence genes. Differentiation between strains was performed based on ADSRRS-fingerprinting method. The results showed that almost half of the tested animals (48%) were carriers of at least one multidrug resistant E. faecalis strain. Moreover, 44% of MDR-positive animals showed two or three strains differing in both the genotype and the resistance phenotype. A significant percentage of strains were resistant to high-level aminoglycosides (from 20% to even 57.5%). The resistance and virulence gene profiles showed a rich panel of genes closely related to isolates from nosocomial infection and from livestock animals. The presence of the same genotypes in different hosts reflects not only a possible transfer of genes between E. faecalis strains but also exchange of strains between animals. The obtained results reflect a very high level of contamination of animals that are not subjected to targeted antibiotic therapy, which may suggest the degree of pollution of the environment. Wildlife animals and their environment can be a link closing the circulation cycle of genes and even epidemiologically important strains; therefore, there is a high risk that this pool will never run out and will be maintained at a high level.
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http://dx.doi.org/10.1016/j.scitotenv.2019.135830DOI Listing
February 2020

Bats as a reservoir of resistant Escherichia coli: A methodical view. Can we fully estimate the scale of resistance in the reservoirs of free-living animals?

Res Vet Sci 2020 Feb 30;128:49-58. Epub 2019 Oct 30.

University of Life Sciences, Faculty of Veterinary Medicine, Department of Animal Physiology, Akademicka 12, 20-033 Lublin, Poland.

Bats are a poorly understood reservoir of pathogenic and multi-drug resistant microorganisms; therefore, the aim of the study was to analyze the presence of drug resistance among E. coli isolated from the species of bats occurring naturally in Poland. The strategy of isolation and identification of resistant strains from pooled and single-animal samples was based on selective media with cefotaxime, chloramphenicol, kanamycin and tetracycline, the use of the ADSRRS-fingerprinting method for genomic differentiation of isolates, and the classical methods of evaluation of phenotypic and genotypic resistance. Of the 78 isolated isolates confirmed as E. coli, there were 38 genetically distinct strains resistant at least to one antimicrobial. 71% of these strains met the multi-drug resistance criterion. Moreover, two different multidrug resistant strains were isolated from three single samples. The highest resistance was observed in the case of ampicillin (66%), kanamycin (84%), sulfamethoxazole/trimetoprim (61%/55% respectively), and streptomycin (50%), which in most cases was confirmed by the presence of an adequate gene. Two isolates from single hosts produced extended-spectrum beta-lactamases (bla, bla, bla). With the exception of tetracycline resistance, which was dominant among isolates from single animals, no significant differences in the resistance of the strains from both groups of samples were observed. Bats should not be neglected as another environmental reservoir and as an unpredictable source of potential pathogenic and multidrug resistant bacteria and should be extensively studied to predict the direction of the development and range of spreading resistance.
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http://dx.doi.org/10.1016/j.rvsc.2019.10.017DOI Listing
February 2020

A new locality of the Haemaphysalis concinna tick (Koch, 1844) in Poland and its role as a potential vector of infectious diseases

Ann Parasitol 2019 ;65(3):281-286

Sub-Department of Veterinary Microbiology, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, ul. Akademicka 12, 20-033 Lublin, Poland

The Haemaphysalis concinna tick is a rare species in Poland. To date, it was found only once a few decades ago. During tick collection for epidemiological studies, a stable population of this arachnid was found in a military training area near Nowa Dęba. This report is particularly important, given the role of Haemaphysalis concinna in the spread of dangerous vector-borne diseases.
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October 2019

In search of the source of dermatophytosis: Epidemiological analysis of Trichophyton verrucosum infection in llamas and the breeder (case report).

Zoonoses Public Health 2019 12 19;66(8):982-989. Epub 2019 Sep 19.

State Veterinary Laboratory, Lublin, Poland.

During the last few years, the number of cases of Trichophyton verrucosum isolation from humans suffering from mycoses has been constantly increasing, which is correlated with the presence of an increasing number of outdoor breeding farms. Farmers and their families as well as veterinarians and technicians involved in handling the animals are at a higher risk of infection. One of the most important aims of mycological diagnostics is epidemiological analysis. Typically, the history of the disease is not sufficient to indicate reliably and eliminate the outbreak of infection. PCR fingerprinting methods are a useful tool in this type of analysis, which is presented in this study. The main aim is to present diagnostic and epidemiological analyses of dermatophyte isolates from llamas and their breeder. In two llamas, round alopecia sites or ca. 2-cm excoriations covered with thickened scaling epidermis were noticed at the border of the head and neck with a distinct tendency towards hair loss. Tinea unguium was noticed in a nail of the breeder's right hand. Direct analysis of the material from the clinical lesions revealed the presence of arthrospores. The macro- and micromorphology of the isolates were homogeneous and characteristic for T. verrucosum. The identification analysis based on the ITS sequences confirmed the previous morphological diagnostic examination. The MP-PCR and MSP-PCR analysis indicated high invariability of the genomes of the strains isolated from the human and animals. The epidemiological research has indicated an identical source of dermatophyte infection in the breeder and the lamas. To sum up, the number of pets and farm animals is increasing and dermatologists should always be informed about possible dermatophyte transmission sources. The possibility of transmission of zoophilic dermatophytes from humans to animals is a suggestion for further analysis; therefore, this type of transmission should be considered in dermatological studies.
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http://dx.doi.org/10.1111/zph.12648DOI Listing
December 2019

Host- and pathogen-dependent susceptibility and predisposition to dermatophytosis.

J Med Microbiol 2019 Jun 3;68(6):823-836. Epub 2019 May 3.

2 State Veterinary Laboratory, Droga Męczenników Majdanka 50, 20-325 Lublin, Poland.

Dermatophytes are a highly specialized group of keratinophilic and keratinolytic filamentous fungi causing a ringworm disease called dermatophytosis or superficial mycoses. Although dermatophyte infections do not threaten the host's life, they lower its quality in humans by causing discomfort related to cosmetic problems and through their epidemiological significance, whereas in farm animals they are responsible for economic losses and constitute a source of the spread of spores. Evidence from countless observational studies that have been conducted over the last 90 years indicates that dermatophytes infect humans of every age, race, gender and socioeconomic status with strikingly high rates, as well as both farmed and wild animals in various health conditions and with various epidemiological statuses. However, the prevalence of superficial fungal infections is highly variable, since it depends on several parameters associated with the infected individual and the dermatophyte, their mutual interactions, and epidemiological and geographical factors. The curious disparity in dermatophyte infection patterns has prompted many investigators to search for a link between the host, the host's predispositions and susceptibility to the disease, and the dermatophyte species and virulence. Thus, the question arises as to whether, in addition to the generally recognized factors predisposing hosts to diseases, there are some other predispositions to dermatophyte infections in a species-specific host. In this review, we describe recent findings about the mechanism of dermatophyte infections, focusing on the adaptation of the fungi to the host and conditions predisposing each side to the disease.
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http://dx.doi.org/10.1099/jmm.0.000982DOI Listing
June 2019

Detection of and in Tissues of Wild-living Animals and in Ticks of North-west Poland.

Pol J Microbiol 2018;67(4):529-534

Environmental Protection Regional Directorate, Field Department , Złocieniec , Poland.

This work presents results of the research on the occurrence of Coxiella burnetii and Francisella tularensis in the tissues of wild-living animals and ticks collected from Drawsko County, West Pomeranian Voivodeship. The real-time PCR testing for the pathogens comprised 928 samples of animal internal organs and 1551 ticks. The presence of C. burnetii was detected in 3% of wild-living animals and in 0.45-3.45% (dependent on collection areas) of ticks. The genetic sequences of F. tularensis were present in 0.49 % of ticks (only in one location - Drawa) and were not detected in animal tissues. The results indicate respectively low proportion of animals and ticks infected with C. burnetii and F. tularensis .

This work presents results of the research on the occurrence of and in the tissues of wild-living animals and ticks collected from Drawsko County, West Pomeranian Voivodeship. The real-time PCR testing for the pathogens comprised 928 samples of animal internal organs and 1551 ticks. The presence of was detected in 3% of wild-living animals and in 0.45–3.45% (dependent on collection areas) of ticks. The genetic sequences of were present in 0.49 % of ticks (only in one location – Drawa) and were not detected in animal tissues. The results indicate respectively low proportion of animals and ticks infected with and .
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http://dx.doi.org/10.21307/pjm-2018-059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256700PMC
June 2019

The host range of dermatophytes, it is at all possible? Phenotypic evaluation of the keratinolytic activity of Trichophyton verrucosum clinical isolates.

Mycoses 2019 Mar 1;62(3):274-283. Epub 2019 Jan 1.

State Veterinary Laboratory, Lublin, Poland.

Dermatophytes are fungi that have an ability to invade keratinised structures. Enzymes secreted by dermatophytes can underlie fungal survival on the host and development of infection. It is possible that the range of activity of keratinases from various dermatophytes is limited to specific species of animals and groups of people. The aim of this study was to carry out phenotypic analysis of the degree of keratinolytic activity of Trichophyton verrucosum strains using hairs of humans and various animal species as substrates. Our results indicated that the activity of keratinases is substrate-induced. The host range of T. verrucosum can be defined as wide. The highest activity of keratinases was recorded in media containing keratin from cow (Bos taurus) and sheep (Ovis aries) hairs in comparison with that from other tested species. The production of keratin-degrading enzymes is a function of time, with the peak of their activity occurring on day 15 of incubation. The role of keratin-degrading enzymes in the pathogenesis of dermatophytosis is becoming increasingly clearer. Given the conceptual understanding that keratin breakdown may require more than just one enzyme, the use of phenotypic methods is an optimal approach to in vitro study of the decomposition of species-specific keratin.
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http://dx.doi.org/10.1111/myc.12876DOI Listing
March 2019

Tinea corporis by Microsporum canis in mycological laboratory staff: Unexpected results of epidemiological investigation.

Mycoses 2018 Dec 1;61(12):945-953. Epub 2018 Aug 1.

State Veterinary Laboratory, Lublin, Poland.

Microsporum canis is a zoophilic dermatophyte, which is very contagious, especially to cats and dogs. Asymptomatic animal carriers of M. canis are regarded a critical factor in the epidemiology of the disease. The aim of this study was to investigate the epidemiological origin of M. canis isolates using morphological traits in combination with molecular analysis. Identification of dermatophyte strains was carried out by correlating the clinical manifestation of the infection with a micro- and macroscopic examination. To confirm the species affiliation fully, molecular differentiation methods were used. A positive result of the culture examination was obtained from the samples with arthrospores in the direct analysis, that is, from a symptomatic cat and humans, and from a cat without any signs of infection. The microsatellite-primed PCR fingerprinting (MSP-PCR) electro-profiles were identical for all analysed strains. The melting profile-PCR (MP-PCR) electrophoregram indicated variability of the genomes of the strains. The search for the source of the infection indicated one cat that did not have any signs of dermatophytosis. PCR-fingerprinting techniques are useful tools for epidemiological investigation of the origin of dermatophyte infection. These methods can also be used in many cases for species identification of dermatophytes and clarification of the relationships among varieties of a species.
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http://dx.doi.org/10.1111/myc.12832DOI Listing
December 2018

Infection of Trichophyton verrucosum in cattle breeders, Poland: A 40-year retrospective study on the genomic variability of strains.

Mycoses 2018 Sep 2;61(9):681-690. Epub 2018 Jul 2.

State Veterinary Laboratory, Lublin, Poland.

Trichophyton verrucosum is a zoophilic fungus that is the most frequent aetiological agent of dermatophytosis in cattle. During the last few years, the number of cases of T. verrucosum from humans has been increasing constantly, which is correlated with the presence of cattle-rearing farms. We identified and analysed T. verrucosum strains isolated from humans and cattle. Identification was carried out traditionally by correlating both the clinical manifestations with a micro- and macroscopic examination. To confirm the species affiliation fully, molecular differentiation methods were used. Direct analysis revealed the presence of arthrospores. The macro- and micromorphology of the isolates obtained from material sampled was homogeneous, and characterised for T. verrucosum. The phylogenetic analysis based on the ITS sequences demonstrated that the strains formed a monophyletic group with T. verrucosum ATCC10 695 with a support of 99%. The MP-PCR analysis indicates high invariability of genomes of strains from humans and animals. MSP-fingerprinting analysis gives the same results as the MP-PCR analysis. To sum up, the rDNA ITS sequence analysis in combination with macro- and micro-morphology only facilitated T. verrucosum species identification without the possibility of intraspecific differentiation. Finding and testing methods, especially molecular technique, with sufficient discriminatory power, is the present challenge for mycologists.
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http://dx.doi.org/10.1111/myc.12791DOI Listing
September 2018

Multiple-strain Trichophyton mentagrophytes infection in a silver fox (Vulpes vulpes) from a breeding farm.

Med Mycol 2019 Feb;57(2):171-180

State Veterinary Laboratory, Slowicza 2, 20-336 Lublin, Poland.

Dermatophyte infections are extremely frequent worldwide, and their epidemiological features and distribution make them one of the most frequent infections all over the world. We identified and analysed multiform T. mentagrophytes strains isolated from a silver fox (Vulpes vulpes) kept on a breeding farm. Identification of dermatophyte strains was carried out traditionally by correlating both the clinical manifestations of the infection with a micro- and macroscopic examination. To confirm the species affiliation fully, molecular differentiation methods were used. DNA was isolated from the dermatophytes with the phenol-chloroform method. The reaction of chitin synthase 1 (chs1) amplification was carried out to confirm the dermatophytes. The phylogenetic analysis was based on the ITS sequences. The polymerase chain reaction melting profile (PCR-MP) procedure was used for differentiation of dermatophyte genomes. Direct analysis of the material sampled from the clinical lesions revealed the presence of arthrospores in the samples collected from all animals with skin lesions. The macromorphology of the colonies obtained from material sampled from the same individual was not homogeneous. The PCR-MP electrophoregram indicated high variability of their genomes. Although the dermatophytes were isolated from one infected fox, no two identical genomic profiles were obtained. The PCR-MP result corresponds with the phenotypic diversity of the isolates. The findings about the multiple dermatophyte infection in one individual complicate any future epidemiology work and other clinical investigation. Previously, using only morphological characteristics, it had been assumed that one fungal isolate per patient could be diagnosed. The novel findings encourage application of the newly developed molecular typing methods in the diagnosis of dermatophytosis.
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http://dx.doi.org/10.1093/mmy/myy011DOI Listing
February 2019

Determination of antimicrobial resistance of Enterococcus strains isolated from pigs and their genotypic characterization by method of amplification of DNA fragments surrounding rare restriction sites (ADSRRS fingerprinting).

J Med Microbiol 2017 Mar;66(2):175-183

University of Life Sciences, Faculty of Veterinary Medicine, Institute of Biological Bases of Animal Diseases, Sub-Department of Veterinary Microbiology, Akademicka 12, 20-033 Lublin, Poland.

Purpose: In this study, we analysed phenotypic resistance profiles and their reflection in the genomic profiles of Enterococcus spp. strains isolated from pigs raised on different farms.

Methodology: Samples were collected from five pig farms (n=90 animals) and tested for Enterococcus. MICs of 12 antimicrobials were determined using the broth microdilution method, and epidemiological molecular analysis of strains belonging to selected species (faecalis, faecium and hirae) was performed using the ADSRRS-fingerprinting (amplification of DNA fragments surrounding rare restriction sites) method with a few modifications.

Results: The highest percentage of strains was resistant to tetracycline (73.4 %), erythromycin and tylosin (42.5 %) and rifampin (25.2 %), and a large number of strains exhibited high-level resistance to both kanamycin (25.2 %) and streptomycin (27.6 %). The strains of E. faecalis, E. faecium and E. hirae (n=184) revealed varied phenotypic resistance profiles, among which as many as seven met the criteria for multidrug resistance (30.4 % of strains tested). ADSRRS-fingerprinting analysis produced 17 genotypic profiles of individual strains which were correlated with their phenotypic resistance profiles. Only E. hirae strains susceptible to all of the chemotherapeutics tested had two different ADSRRS profiles. Moreover, eight animals were carriers of more than one genotype belonging to the same Enterococcus spp., mainly E. faecalis.

Conclusion: Given the possibility of transmission to humans of the high-resistance/multidrug resistance enterococci and the significant role of pigs as food animals in this process, it is necessary to introduce a multilevel control strategy by carrying out research on the resistance and molecular characteristics of indicator bacterial strains isolated from animals on individual farms.
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http://dx.doi.org/10.1099/jmm.0.000400DOI Listing
March 2017

Characterization of Multidrug Resistant E. faecalis Strains from Pigs of Local Origin by ADSRRS-Fingerprinting and MALDI -TOF MS; Evaluation of the Compatibility of Methods Employed for Multidrug Resistance Analysis.

PLoS One 2017 30;12(1):e0171160. Epub 2017 Jan 30.

Department of Epizootiology and Clinic of Infectious Diseases, Faculty of Veterinary Medicine, University of Life Sciences, Lublin, Poland.

The aim of this study was to characterize multidrug resistant E. faecalis strains from pigs of local origin and to analyse the relationship between resistance and genotypic and proteomic profiles by amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI -TOF MS). From the total pool of Enterococcus spp. isolated from 90 pigs, we selected 36 multidrug resistant E. faecalis strains, which represented three different phenotypic resistance profiles. Phenotypic resistance to tetracycline, macrolides, phenicols, and lincomycin and high-level resistance to aminoglycosides were confirmed by the occurrence of at least one corresponding resistance gene in each strain. Based on the analysis of the genotypic and phenotypic resistance of the strains tested, five distinct resistance profiles were generated. As a complement of this analysis, profiles of virulence genes were determined and these profiles corresponded to the phenotypic resistance profiles. The demonstration of resistance to a wide panel of antimicrobials by the strains tested in this study indicates the need of typing to determine the spread of resistance also at the local level. It seems that in the case of E. faecalis, type and scope of resistance strongly determines the genotypic pattern obtained with the ADSRRS-fingerprinting method. The ADSRRS-fingerprinting analysis showed consistency of the genetic profiles with the resistance profiles, while analysis of data with the use of the MALDI- TOF MS method did not demonstrate direct reproduction of the clustering pattern obtained with this method. Our observations were confirmed by statistical analysis (Simpson's index of diversity, Rand and Wallace coefficients). Even though the MALDI -TOF MS method showed slightly higher discrimination power than ADSRRS-fingerprinting, only the latter method allowed reproduction of the clustering pattern of isolates based on phenotypic resistance and analysis of resistance and virulence genes (Wallace coefficient 1.0). This feature seems to be the most useful for epidemiological purposes and short-term analysis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171160PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5279778PMC
August 2017

Free-Living Species of Carnivorous Mammals in Poland: Red Fox, Beech Marten, and Raccoon as a Potential Reservoir of Salmonella, Yersinia, Listeria spp. and Coagulase-Positive Staphylococcus.

PLoS One 2016 12;11(5):e0155533. Epub 2016 May 12.

Sub-Department of Veterinary Microbiology, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine University of Life Sciences, Lublin, Poland.

The objective of the study was to examine a population of free-living carnivorous mammals most commonly found in Poland (red fox, beech marten, and raccoon) for the occurrence of bacteria that are potentially pathogenic for humans and other animal species and to determine their virulence potential (the presence of selected virulence genes). From the total pool of isolates obtained (n = 328), we selected 90 belonging to species that pose the greatest potential threat to human health: Salmonella spp. (n = 19; 4.51%), Yersinia enterocolitica (n = 10; 2.37%), Listeria monocytogenes and L. ivanovii (n = 21), and Staphylococcus aureus (n = 40; 9.5%). The Salmonella spp. isolates represented three different subspecies; S. enterica subsp. enterica accounted for a significant proportion (15/19), and most of the serotypes isolated (S. Typhimurium, S. Infantis, S. Newport and S. Enteritidis) were among the 10 non-typhoidal Salmonella serotypes that are most often responsible for infections in Europe, including Poland. Y. enterococlitica was detected in the smallest percentage of animals, but 60% of strains among the isolates tested possessed the ail gene, which is responsible for attachment and invasion. Potentially pathogenic Listeria species were isolated from approx. 5% of the animals. The presence of all tested virulence genes was shown in 35% of L. monocytogenes strains, while in the case of the other strains, the genes occurred in varying numbers and configurations. The presence of the inlA, inlC, hlyA, and iap genes was noted in all strains, whereas the genes encoding PI-PLC, actin, and internalin Imo2821 were present in varying percentages (from 80% to 55%). S. aureus was obtained from 40 individuals. Most isolates possessed the hla, hld (95% for each), and hlb (32.5%) genes encoding hemolysins as well as the gene encoding leukotoxin lukED (70%). In a similar percentage of strains (77.5%), the presence of at least one gene encoding enterotoxin was found, with 12.5% exhibiting the presence of egc-like variants. In two animals, we also noted the gene encoding the TSST-1 toxin. The results of the study showed that free-living animals may be a significant reservoir of bacteria that are potentially pathogenic for humans. The results of the statistical analysis revealed that, among the animals species studied, the red fox constitutes the most important source of infections.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155533PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4865137PMC
July 2017

Coagulase-positive Staphylococcus isolated from wildlife: Identification, molecular characterization and evaluation of resistance profiles with focus on a methicillin-resistant strain.

Comp Immunol Microbiol Infect Dis 2016 Feb 28;44:21-8. Epub 2015 Nov 28.

University of Life Sciences, Faculty of Veterinary Medicine, Institute of Biological Bases of Animal Diseases, Sub-Department of Veterinary Microbiology, Akademicka 12, 20-033 Lublin, Poland.

The aim of the study was molecular analysis of coagulase-positive isolates of Staphylococcus bacteria obtained from wild animals and evaluation of their resistance to antimicrobial agents. A total of 76 rectal swabs were taken from wild animals. The species of the Staphylococcus isolates was determined by MALDI TOF MS, susceptibility to antimicrobials was evaluated by phenotypic and molecular methods, epidemiological analysis (ADSRRS-fingerprinting) was also carried out. MRSA isolate was typed by MLST and spa-typing. The animals tested, were carriers (n=38) of coagulase-positive Staphylococcus (S. aureus, S. pseudintermedius and S. delphini B). Analyzed isolates were resistant to 1 or 2 antimicrobials, which was confirmed by the presence of genes (blaZ, ermA, ermB, msrA, tetK and tetM). A multi-drug resistant and methicillin-resistant isolate of S. aureus was obtained as well (MRSA, ST8, t1635, PVL-positive and ACME-negative). The ADSRRS-fingerprinting method enabled interspecific and intraspecific differentiation of coagulase-positive Staphylococcus isolates, revealing a certain degree of correlation between the species of the isolate, and the degree of similarity between the isolates. The presence of resistance genes in 13% (5/38) of the isolates obtained from wild animals, including one methicillin-resistant isolate, is relatively small in comparison to the degree of colonization by resistant strains in humans, livestock or pets. Nevertheless, due to the possibility of contact between wild animals, domestic animals and humans, transmission of resistant strains is possible, as suggested by our isolation of a MRSA strain typed as ST8 and specific spa type t1635, which had previously been isolated exclusively from humans.
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http://dx.doi.org/10.1016/j.cimid.2015.11.003DOI Listing
February 2016

Molecular identification and classification of Trichophyton mentagrophytes complex strains isolated from humans and selected animal species.

Mycoses 2015 Mar 23;58(3):119-26. Epub 2015 Jan 23.

Sub-Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Institute of Biological Bases of Animal Diseases, University of Life Sciences, Lublin, Poland.

Species differentiation within Trichophyton mentagrophytes complex group currently poses a major diagnostic challenge, with molecular methods increasingly supplementing classical identification based on the morphological and physiological properties of the fungi. Diagnostic and epidemiological research aimed at determining the source and means of transmission of dermatophytoses in both humans and animals requires not only species differentiation of isolates but also differentiation within species. The study was conducted on 24 isolates originating in humans and various animal species with clinical symptoms of dermatophytosis. The analysis included phenotypical identification methods and molecular methods: internal transcribed spacer sequencing and ITS-restriction fragment length polymorphism (RFLP) with multi-enzyme restriction. ITS sequence analysis identified the isolates to species - Trichophyton interdigitale, Arthroderma benhamiae and A. vanbreuseghemii, and ITS-RFLP detected six different genotypes. Genotypes I, II and III characterised strains belonging to A. benhamiae, genotype IV characterised the A. vanbreuseghemii strain, and genotypes V and VI occurred only within the species T. interdigitale. Strains isolated from guinea pigs were dominant within genotype I, while genotype II was found mainly in strains from foxes. Multi-enzyme restriction analysis of this region enables intraspecific differentiation, which may be useful in epidemiological research, particularly in determining the source of infections.
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http://dx.doi.org/10.1111/myc.12284DOI Listing
March 2015

Modified 16S-23S rRNA intergenic region restriction endonuclease analysis for species identification of Enterococcus strains isolated from pigs, compared with identification using classical methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

J Med Microbiol 2015 Mar 13;64(Pt 3):217-223. Epub 2015 Jan 13.

University of Life Sciences, Faculty of Veterinary Medicine, Department of Preclinical Veterinary Sciences, Sub-Department of Pharmacology, Akademicka 12, 20-033 Lublin, Poland.

Fast and reliable identification of bacteria to at least the species level is currently the basis for correct diagnosis and appropriate treatment of infections. This is particularly important in the case of bacteria of the genus Enterococcus, whose resistance profile is often correlated with their species (e.g. resistance to vancomycin). In this study, we evaluated restriction endonuclease analysis of the 16S-23S rRNA gene intergenic transcribed spacer (ITS) region for species identification of Enterococcus. The utility of the method was compared with that of phenotypic methods [biochemical profile evaluation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)]. Identification was based on 21 Enterococcus reference strains, of the species E. faecalis, E. faecium, E. hirae, E. durans, E. casseliflavus, E. gallinarum, E. avium, E. cecorum and E. columbae, and 47 Enterococcus field strains isolated from pigs. Restriction endonuclease analysis of the ITS-PCR product using HinfI, RsaI and MboI, in the order specified, enabled species differentiation of the Enterococcus reference and field strains, and in the case of the latter, the results of species identification were identical (47/47) to those obtained by MALDI-TOF MS. Moreover, as a result of digestion with MboI, a unique restriction profile was also obtained for the strains (3/3) identified by MALDI-TOF MS as E. thailandicus. In our opinion, restriction endonuclease analysis of the 16S-23S rRNA gene ITS region of Enterococcus may be a simple and relatively fast (less than 4 h) alternative method for identifying the species occurring most frequently in humans and animals.
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http://dx.doi.org/10.1099/jmm.0.000008DOI Listing
March 2015

Aerobic bacterial microbiota isolated from the cloaca of the European pond turtle (Emys orbicularis) in Poland.

J Wildl Dis 2015 Jan;51(1):255-9

1 University of Life Sciences, Faculty of Veterinary Medicine, Institute of Biological Bases of Animal Diseases, Sub-Department of Veterinary Microbiology, Akademicka 12, 20-033 Lublin, Poland.

We conducted a comparative analysis of the aerobic cloacal bacteria of European pond turtles (Emys orbicularis) living in their natural environment and juvenile turtles reared under controlled conditions in a breeding center. We included 130 turtles in the study. The aerobic bacteria isolated from the cloaca of the juvenile turtles were less diverse and more prevalent than the bacteria isolated from free-living adults. We isolated 17 bacterial species from juvenile captive turtles, among which the dominant species were Cellulomonas flavigena (77/96), Enterococcus faecalis (96/96), Escherichia coli (58/96), and Proteus mirabilis (41/96). From the adult, free-living turtles, we isolated 36 bacterial species, some of which are a potential threat to public health (e.g., Salmonella enterica serovars Newport, Daytona, and Braenderup; Listeria monocytogenes; Yersinia enterocolitica; Yersinia ruckeri; Klebsiella pneumoniae; Vibrio fluvialis; and Serratia marcescens), and pathogens that are etiologic agents of diseases of ectothermic animals (e.g., Aeromonas sobria, Aeromonas caviae, Hafnia alvei, Edwardsiella tarda, and Citrobacter braakii; the last two species were isolated from both groups of animals). The cloacal bacterial biota of the European pond turtle was characterized by numerous species of bacteria, and its composition varied with turtle age and environmental conditions. The small number of isolated bacteria that are potential human pathogens may indicate that the European pond turtle is of relatively minor importance as a threat to public health.
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http://dx.doi.org/10.7589/2013-07-157DOI Listing
January 2015

Undomesticated animals as a reservoir of multidrug-resistant Enterococcus in eastern Poland.

J Wildl Dis 2014 Jul 7;50(3):645-50. Epub 2014 May 7.

1  University of Life Sciences, Faculty of Veterinary Medicine, Institute of Biological Bases of Animal Diseases, Sub-Department of Veterinary Microbiology, Akademicka 12, 20-033 Lublin, Poland.

To assess implications for public health we compared the resistance of Enterococcus spp. strains to antibacterial drugs in wild and exotic animals with strains originating in domesticated animals and characterized correlations between Enterococcus species, the source of the isolate, and the degree of resistance to selected antibiotics. All strains, regardless of source, were susceptible to β-lactams, gentamicin, linezolid, and teicoplanin; the highest resistance was to kanamycin, quinupristin, and rifampicin. Thirteen strains from undomesticated animals were resistant to vancomycin, and one strain, from a fox, was resistant to streptomycin (high-dose). Multidrug-resistant strains accounted for 46% of the strains from wild animals and 59% of the strains from an exotic animal (the Russian tortoise; Testudo horsfieldii). Despite the relatively low level of resistance in the strains isolated from wild and exotic animals, the large number of intermediately susceptible strains in these groups is an indication of the evolutionary character of the development of resistance, suggesting that these animals may be potential reservoirs of Enterococcus strains resistant to a wide panel of currently used antibiotics.
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http://dx.doi.org/10.7589/2013-09-240DOI Listing
July 2014

Russian tortoises (Agrionemys horsfieldi) as a potential reservoir for Salmonella spp.

Res Vet Sci 2012 Apr 12;92(2):187-90. Epub 2011 Apr 12.

University of Life Sciences, Faculty of Veterinary Medicine, Institute of Biological Bases of Animal Diseases, Sub-Department of Veterinary Microbiology, Akademicka 12, 20-033 Lublin, Poland.

A total of 80 Russian tortoises brought in Poland were examined for presence of Salmonella. Salmonella was detected in 15 out of all the animals tested (18.75%). Of the total of 56 strains, 30 (53.57%) belonged to Salmonella enterica subsp. enterica (I) and 26 to Salmonella enterica subsp. salamae (II). The predominant serotype within subspecies I was S. Newport, which is one of the most serotypes causing salmonellosis in humans and warm-blooded animals. In vitro determination of the susceptibility of Salmonella to the 10 medicinal preparations showed that all tested strains were sensitive to norfloxacin, sulfamethoxazole with trimethoprim, florfenicol, gentamicin, tetracycline and ampicillin, resistance was noted only to amoxicillin with clavulanic acid (12 strains), and intermediate sensitivity to colistin (7 strains), enrofloxacin (2 strains) and cephalexin (5 strains). These studies confirmed that Russian tortoises are a significant reservoir for Salmonella and may represent a potential source of infection for humans.
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http://dx.doi.org/10.1016/j.rvsc.2011.03.019DOI Listing
April 2012
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