Are dogs and cats a reservoir of resistant and virulent Enterococcus faecalis strains and a potential threat to public health?

AIMS: The aim of the study was to assess resistance and virulence of Enterococcus faecalis isolated from the gastrointestinal tract of dogs and cats, analyse their genotypic variability and estimate the correlation between the occurrence of antimicrobial resistance, virulence determinants and genotypic profiles. METHODS AND RESULTS: The susceptibility of E. faecalis to penicillin, ampicillin, vancomycin, erythromycin, tetracycline, ciprofloxacin, gentamicin, streptomycin and kanamycin was determined by the broth microdilution method. The isolates were tested for the presence of selected genes encoding resistance to macrolides, tetracyclines, aminoglycosides and glycopeptides as well as genes encoding virulence factors. Genotyping was performed using the ADSRRS-fingerprinting method. The highest percentage of resistant strains was observed in relation to erythromycin (96%), ciprofloxacin (93%) and tetracycline (82%). High percentage of strains resistant to high-level aminoglycosides was noted (kanamycin-33%, gentamicin-29%, streptomycin-24%), as well as multidrug-resistant (78%). The genotypic analysis of E. faecalis showed high heterogeneity of genotypic profiles (37) correlating with some resistance profiles. The most common virulence genes amongst E. faecalis were efaAfs (93%), cpd, ccf and cob (86%). SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study confirm that companion animals should be considered as a reservoir of E. faecalis carrying resistance and virulence determinants.

Assessment of the subtilisin gene profile in Trichophyton verrucosum isolated from human and animal dermatophytoses in two-stage multiplex PCR.

AIMS: Keratin is a fibrous and recalcitrant structural protein and the third most abundant polymer in nature after cellulose and chitin. Subtilisin-like proteases (SUB) are a group of serine endoproteases, coded by seven genes (SUB1-7), which decompose keratin structures and have been isolated from dermatophytes. Herein, we identified the SUB genes in 30 clinical isolates of Trichophyton verrucosum obtained from human and animal dermatophytosis as well as asymptomatic animal carriers. METHODS AND RESULTS: We designed and proposed a two-stage multiplex PCR technique to detect all seven genes encoding serine proteases in dermatophytes. The analysis revealed the presence SUB1 and SUB2 amplicons in all strains regardless of the host. In the group of isolates obtained from humans, all seven subtilisin genes were shown in 40% of the strains. In T. verrucosum from asymptomatic animals, none of the isolates showed the presence of all seven subtilisin genes, and only 30% had six genes. In turn, 10% of the isolates from symptomatic animals demonstrated all seven subtilisins amplicons. CONCLUSIONS: In conclusion, the severity of infection and ability of T. verrucosum to cause dermatophytosis in humans may not be related to specific genes but their accumulation and synergistic effects of their products. SIGNIFICANCE AND IMPACT OF THE STUDY: Dermatophytes are pathogenic filamentous fungi with capacity to attack keratinized structures such as skin, hair and nails, causing cutaneous superficial infections. Indeed, a biological characteristic of dermatophytes is their ability to invade keratin-rich tissues by producing enzymes. Various degrees of inflammatory responses can be induced exactly by the enzymes. Subtilisin-like proteases are endoproteases, which decompose keratin structures. Our study identifies SUB genes in clinical isolates of T. verrucosum obtained from human and animal dermatophytosis as well as asymptomatic animal carriers.

Detection and identification of dermatophytes based on currently available methods - a comparative study.

AIMS: Accurate identification of dermatophytes is essential for implementing appropriate antifungal treatment and epidemiological analysis. However, the limitations of conventional diagnostics are a frequently discussed topic, and new diagnostic techniques are constantly expanding. In this study, we assess the suitability of conventional diagnostic techniques in comparison to the real-time PCR assay and MALDI-TOF MS in detection and identification of dermatophytes. METHODS AND RESULTS: Strains included in this study were obtained from human and animals with symptomatic, and asymptomatic infection. A direct examination revealed that 31·7 and 60·9% of samples from symptomatic patients, and 25·7 and 60% from asymptomatic animals were positive, as shown by light and fluorescence microscopy respectively. In turn, dermatophytes were isolated from 90·2 and 71·4% of these samples. The pan-dermatophyte primers in real-time PCR assay facilitated detection in 85·3 and 82·9% of the symptomatic and asymptomatic dermatophytoses respectively. Additionally, species-specific PCR assays were positive in 70·7 and 37·1% of these samples. The MALDI-TOF MS analysis yielded positive results consistent with conventional techniques in 97·2 and 72% of symptomatic and asymptomatic infections respectively. CONCLUSIONS: Our study revealed that there is no universal diagnostic method that would be ideal in each of the cases considered. Nonetheless, conventional techniques are still the most effective and reliable tools for mycological diagnostics. SIGNIFICANCE AND IMPACT OF THE STUDY: Dermatologists and veterinarians have difficulties in making a diagnosis of dermatophytoses based only on observed symptoms of fungal infections, as they mimic symptoms of other dermatoses. In this context, a comparative analysis of the results of diagnostics performed using conventional methods and new technologies are crucial for implementing these pioneer methods into routine laboratory practice.

Site-directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote Paramecium octaurelia.

Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue - distinct from that of Rab7a directly involved in phagocytosis - was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14-]UDP-glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non- mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC-MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14-]UDP- glucose, the suggested composition of the adduct attached to Thr200 might be (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.

Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues.

Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein) and LAMP-2 (lysosomal membrane protein 2) as well as alpha7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2) in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100,000 x g) of equal load were quantified by immunoblotting. LAMP-2 cross-reacting polypeptide of approximately106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The alpha7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILP-related polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.

Antibacterial activity of gentamicin-bonded gelatin-sealed polyethylene terephthalate vascular prostheses.

OBJECTIVES: To create an antibiotic-modified vascular prosthesis with a prolonged bactericidal activity, susceptible to endothelialisation. METHODS: We used a covalent method of gentamicin sulphate immobilisation to polyethylene terephthalate prosthesis sealed with gelatin. Antibacterial activity was assayed in Luria-Bertani medium against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa strains. Prosthesis endothelialisation was performed using bovine aorta endothelial cells (BAEC). RESULTS: Gentamicin was bound to vascular prostheses in the amount of 12g per kg of prosthesis. Ninety-seven percent of antibiotic bound in covalent way and remained on the biomaterial for at least 30 days during shaking in PBS solution. Gentamicin-modified prostheses exerted bactericidal or bacteriostatic effect on growth of clinical and reference bacterial strains, prevented biofilm formation and were highly susceptible to endothelialisation. BAEC viability exceeded 90%, which indicated that gentamicin-vascular prostheses were not toxic for these cells. CONCLUSIONS: Covalent gentamicin immobilisation resulted in effective antibacterial protection of vascular prostheses against clinical and reference strains of S. aureus, E. coli and P. aeruginosa and allowed for a strong adherence of endothelial cells to antibiotic-modified prostheses.

Children with acute lymphoblastic leukemia: is there any subgroup of children without elevated thrombin generation? A preliminary study utilizing measurements of thrombin-antithrombin III complexes.

UNLABELLED: The mechanisms contributing to thromboembolic complications in children with acute lymphoblastic leukemia (ALL) are complex, but it is believed that two factors are of critical importance, i.e. increased thrombin generation and decreased antithrombotic potential of the blood plasma. We evaluated generation of thrombin in three periods of observation of the children: a) prior to chemotherapy, b) after remission-inducing chemotherapy, and c) after infusion of L-asparaginase in the consolidation phase. The study group consisted of 23 children (x = 6.8 years of age), and a control group of 11 children (x = 7.3 years of age). Thrombin-antithrombin III complex (TAT) was selected as a marker of thrombin generation and it was measured by ELISA method. TAT levels prior to chemotherapy were found to be normal in a small subgroup of children (7/23--ca 30%), i.e. they were within the control range (1.5-4.5 micrograms/l), but all the levels increased following remission-inducing chemotherapy. In contrast, in the major subgroup of children whose TAT levels were elevated at presentation (16/23--ca 70%) no significant changes were observed following chemotherapy. CONCLUSION: There is a subgroup of children with ALL whose thrombin generation is normal as measured by its marker--thrombin-antithrombin III (TAT). Only in those children thrombin generation increases following chemotherapy.