Katarzyna Wolska

Virulence gene profiles in Staphylococcus aureus isolated from cows with subclinical mastitis in eastern Poland.

Staphylococcus aureus is arguably the most important pathogen involved in bovine mastitis. The aim of this study was to determine the virulence gene profiles of 124 Staph. aureus isolates from subclinical mastitis in cows in eastern Poland. The presence of 30 virulence genes encoding adhesins, proteases and superantigenic toxins was investigated by PCR. The 17 different combinations of adhesin genes were identified. Occurrence of eno (91·1%) and fib (82·3%) genes was found to be common. The frequency of other adhesion genes fnbA, fnbB, ebps were 14·5, 50, 25%, respectively, and for cna and bbp were 1·6%. The etA and etD genes, encoding exfoliative toxins, were present in genomes of 5·6 and 8·9% isolates, respectively. The splA and sspA, encoding serine protease, were detected in above 90% isolates. The most frequent enterotoxin genes were sei (21%), sem (19·4%), sen (19·4%), seg (18·5%) and seo (13·7%). The tst gene was harboured by 2·4% isolates. The 19 combinations of the superantigenic toxin genes were obtained and found in 35·5% of isolates. Three of them (seg, sei, sem, sen, seo; sec, seg, sei, sem, sen, seo and seg, sei, sem, sen) were the most frequent and found in 16·1% of the isolates. The most common virulotype, present in 17·7% of the isolates, was fib, eno, fnbB, splA, splE, sspA. The results indicate the variation in the presence of virulence genes in Staph. aureus isolates and considerable diversity of isolates that are able to cause mastitis in cows.

Genotyping Techniques for Determining the Diversity of Microorganisms

Typing of microbial pathogens, or identifying bacteria at the strain level, is particularly important for diagnosis, treatment, and epidemiological surveillance of bacterial infections. This is especially the case for bacteria exhibiting high levels of antibiotic resistance or virulence, and those involved in nosocomial or pandemic infections. Strain typing also has applications in studying bacterial population dynamics. The part that molecular methods have to play in elucidating bacterial diversity is increasingly important. The shortcomings of phenotypically based typing methods (generally these methods are viewed as being too time consuming and lacking in sufficient resolution amongst related strains) have led to the development of many DNA – based techniques. A suitable typing method must have high discrimination power combined with good to moderate interand intra-laboratory reproducibility. In addition, it should be easy to set up, to use and to interpret, and inexpensive (Olive & Bean, 1999). In this chapter, we review the current bacterial genotyping methods and classify them into six main categories: (1 and 2) DNA banding pattern-based methods, which classify bacteria according to the size of fragments generated respectively by enzymatic digestion of genomic/plasmid DNA, and PCR amplification, (3) DNA hybridization–based methods using nucleotidic probes, (4) DNA sequencing-based methods, which study the polymorphism of DNA sequences, (5) differentiation of isolates on the basis of presence or absence of particular genes and (6) high resolution melting analysis–real–time monitoring of melting process of PCR amplified polymorphic DNA fragment. We described and compared the applications of genotyping methods to the study of bacterial strain diversity. We also discussed the selection of appropriate genotyping methods and the challenges of bacterial strain typing and described the current trends of genotyping methods.

BOX-PCR is an adequate tool for typing of clinical Pseudomonas aeruginosa isolates

In this study, the BOX-PCR fingerprinting technique was evaluated for the discrimination of clinical Pseudomonas aeruginosa isolates. All isolates were typeable and nearly half showed unique banding patterns. According to our results, BOX-PCR fingerprinting is applicable for typing of Pseudomonas aeruginosa isolates and can be considered a useful complementary tool for epidemiological studies of members of this genus.

Virulence factors, biofilm-forming ability, and antimicrobial resistance of urinary Escherichia coli strains isolated from hospitalized patients

BACKGROUND/AIM Escherichia coli is the most frequent cause of urinary tract infections. We investigated the possible associations between the origin of strains, antimicrobial resistance, the presence of urovirulence factors, and biofilm-forming ability. MATERIALS AND METHODS Antibiotic susceptibility of E. coli strains was tested by disk diffusion method. Hemagglutination assays were performed for phenotypic characterization of the cell surface. Multiplex PCR was used for detection of virulence genes and for determination of phylogenetic relationships. RESULTS The resistance to ampicillin (55.5%) and tetracycline (39.3%) was significantly more frequent than to other antimicrobial agents. The fim gene was present in 92.5% of strains. The sfa and pap genes were found in 53.8% and 38.7% of strains, respectively. The pap gene was significantly less frequently detected in strains from dialysis patients. The hly gene was present in 18.5% of strains. The aer gene was detected in 52.6% and cnf in 12.1%, while afa was detected in 4.6% of strains. Most strains belonged to the B2 and D phylogenetic groups. The aer gene was significantly associated with strains producing strong biofilms. CONCLUSION The E. coli strains causing cystitis in hospitalized patients differed in terms of resistance to antibiotics, virulence genes, and potential for biofilm formation.

Occurrence of the nan1 gene and adhesion of Pseudomonas aeruginosa isolates to human buccal epithelial cells

Abstract This study shows an association between the frequency of the nan1 gene (encoding neuraminidase) among 62 clinical Pseudomonas aeruginosa isolates and adhesion of these bacteria to human buccal epithelial cells. The 52 strains in which the gene was present (83.9%) were characterized by a higher adhesiveness (the mean number of adhering bacteria was 23.51 per cell) than strains in which the gene was not detected (16.23 per cell) and the difference was significant (P = 0.009, Mann-Whitney U test). Thus we found that the nan1 gene may play a role in the binding of clinical P. aeruginosa strains to buccal cells.