Grażyna Ziółkowska

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

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.

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

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.

Immunological Response in Guinea Pigs and Calves Infected Experimentally with Trichophyton verrucosum:† Immunologische Reaktion bei experimentell mit Trichophyton verrucosum infizierten Meerschweinchen und Kälbern

Summary: In the guinea pigs experimentally infected with Trichophyton verrucosum acquired immunity determined by the “challenge” method, delayed hypersensitivity measured by the skin test and specific inhibtion of spleen cells migration are formed between 10–20 days after infection. Immunity and hypersensitivity last at least 5 months after infection, whereas specific inhibition of cell migration lasts until the second month after infection. Statistical analysis showed an essential interdependance of the cell inhibition extent on the time elapsing from the moment of infection.

Evaluation of growth conditions and DNA extraction techniques used in the molecular analysis of dermatophytes

Recent molecular methods for diagnosis of superficial mycoses have determined the need for a rapid and easy method of extracting DNA. The aim of study was to determine growth conditions and techniques of DNA extraction for Microsporum canis, Trichophyton mentagrophytes and T. verrucosum.

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

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.

Fatty acid profile and influence of oxythiamine on fatty acid content in Malassezia pachydermatis, Candida albicans and Saccharomyces cerevisiae.

Malassezia pachydermatis and Candida albicans are fungi involved in the skin diseases and systemic infections. The therapy of such infections is difficult due to relapses and problems with pathogen identification. In our study, we compare the fatty acids profile of M. pachydermatis, C. albicans and S. cerevisiae to identify diagnostic markers and to investigate the effect of oxythiamine (OT) on the lipid composition of these species. Total fatty acid content is threefold higher in C. albicans and M. pachydermatis compared with S. cerevisiae. These two species have also increased level of polyunsaturated fatty acids (PUFA) and decreased content of monounsaturated fatty acids (MUFA). We noted differences in the content of longer chain (>18) fatty acids between studied species (for example a lack of 20 : 1 in S. cerevisiae and 22 : 0 in M. pachydermatis and C. albicans). OT reduces total fatty acids content in M. pachydermatis by 50%. In S. cerevisiae, OT increased PUFA whereas it decreased MUFA content. In C. albicans, OT decreased PUFA and increased MUFA and SFA content. The results show that the MUFA to PUFA ratio and the fatty acid profile could be useful diagnostic tests to distinguish C. albicans, M. pachydermatis and S. cerevisiae, and OT affected the lipid metabolism of the investigated species, especially M. pachydermatis.

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.

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.

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)

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.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. Samples were collected from five pig farms (n=90 animals) and tested for Enterococcus. Minimum inhibitory concentrations of 12 antimicrobials were determined using the broth microdilution method, and epidemiological molecular analysis of strains belonging to selected species (E. faecalis, E. faecium and E. hirae) was performed using the ADSRRS-fingerprinting method with a few modifications. The highest percentage of strains were resistant to tetracycline (73.4%), erythromycin and tylosin (42.5%), and rifampicin (25.2%) as well as a large number 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 multi-drug 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. Given the possibility of transmission to humans of the high resistance/multi-drug resistance enterococci and the significant role of pigs as food animals in this process, it is necessary to introduce a multi-level control strategy by carrying out research on the resistance and molecular characteristics of indicator bacterial strains isolated from animals on individual farms.

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

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.