Search

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (© 2015 The Authors.)

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., (© 2015 Blackwell Verlag GmbH.)

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.

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.

A new linear amidrazone derivative, 6-acetyl-cyclohex-3-enecarboxylic acid [1-pyridin-2-yl-1-(pyridyn-2-yloamin)meth-(Z)-ylidene] hydrazide, H(2)L (2) and its Cu(II) complex, [Cu(2)L(2)]·4H(2)O (3) were synthesized and characterized by elemental analysis, IR and (1)H NMR spectroscopy and cyclic voltammetry. Compound 2 was synthesized in the equimolar reaction of N(3)-substituted amidrazone with cis-1,2,3,6-tetrahydrophthalic anhydride. The Cu complex of 2 was obtained in the reaction with copper(II) acetate. The molecular structures of 2 and 3 were determined by X-ray crystallography. The parent ligand exists in its amide-hydrazone form in the solid state. The central amidrazone moiety has a Z configuration with respect to the double C=N bond. Coordination to the metal center promotes Z/E isomerization of the hydrazone group of the ligand. Compound 3 is a dinuclear four-coordinated Cu(II) complex with the amidrazone ligand behaving as a tetradentate double deprotonated chelating one. Several biological activities of 2 and 3 were examined in vitro; they were: antimicrobial properties against selected bacterial and fungal strains, suppression of phytohemagglutinin A (PHA)-induced proliferation of human peripheral blood mononuclear cells (PBMC) and their effects on tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) production. The cytotoxic activity of Cu(II) complex was determined with respect to the four carcinoma cell lines (SW 984, CX-1, L-1210, A-431). The studied complex exhibited significant cytotoxic effects (particularly against CX-1 colon carcinoma), comparable to those reported for cisplatin. Both compounds have shown a relatively low antibacterial activity and were devoid of antifungal properties., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The purpose of the work was to assess the immune response of guinea pigs after the experimental infection with Microsporum canis, and after immunization with a specific live vaccine. The guinea pigs after the recovery from infection showed a delayed type of hypersensitivity and in addition, 30 per cent of the animals were characterized by the presence of immediate hypersensitivity reactions. All the animals were resistant to reinfection with M. canis and to some extent to Trichophyton verrucosum, however, no cross-immunity against T. mentagrophytes was found. The vaccinated animals were characterized mainly by delayed type of hypersensitivity and the resistance to approximately 1000 infectious doses of M. canis.

Using 6 samples of pathologically altered epidermis of guinea pigs and cattle, chosen strains Trichophyton verrucosum (6 strains) and the two layer method of inoculation on a classic Sabouraud's medium, arthropogenesis of the fungus was investigated. The authors described successive stages of the developmental cycle of T. verrucosum and they found that in all cases the process of arthropogenesis was very similar irrespective of the source of the material used for inoculation. Using various modifications of Sabouraud's medium and a rice medium microscopic examinations revealed in the examined strains of T. verrucosum the presence of chlamydospores (Sabouraud's medium without sugar) macroconidia (rice medium) and spiral forms (Sabouraud's medium with gluconic acid or natrium sulphite) analogical to the forms noted in Trichophyton mentagrophytes.

The investigations demonstrated that the strains under study, either virulent or in the form of inactivated vaccines, induced a delayed type allergy. The response depended upon the affiliation to a determined species and individual properties of a strain. The strain T. mentagrophytes 1 displayed the highest biological activity, since it was a good inducer of allergy and a manufacturer of active trichophytin in tests performed both in vivo and in vitro.

Neutral lipid composition and that of phospholipids of mycelial and spore forms of Trichophyton verrucosum were examined. It was found that arthrospores had more than twice as high content of lipids (99.3 mg/g) than the corresponding mycelial form (44.7 mg/g). Differences were also found in the qualitative composition: almost two times more neutral lipids (58.5 mg/g) and three times more phospholipids (40.8 mg/g) occurred in the spores than in the mycelial form 30.6 and 14.1 mg/g, respectively). Analysis of the neutral lipid fraction composition of both examined forms of T. verrucosum showed that the basic component were triglycerides, constituting about 70% in the spores and 44% in the mycelium of all the lipids. In the case of phospholipids no significant differences were observed between the spore and mycelial forms, phosphatidylcholine and phosphatidylethanolamine were predominating in both forms. A much higher content of lipids in the infectious form of the fungus, the arthrospores, suggest a possibility of participation of this fraction in the pathogenicity of the fungus.