Multiple-locus variable-number tandem repeat fingerprinting as a method for rapid and cost-effective typing of animal-associated Staphylococcus aureus strains from lineages other than sequence type 398.

In veterinary medicine, Staphylococcus aureus is associated with a range of mild to severe infections. The high density of livestock in intensive farming systems increases the risk of disease spread and hampers its control and measures of prevention, making S. aureus one of the most important animal pathogens. Multiple-locus variable-number tandem repeat fingerprinting (MLVF) has been successfully applied to the characterization of livestock-associated meticillin-resistant Staphylococcus aureus (MRSA) ST398 but not to the characterization of a wide range of other animal isolates. The objective of the current study was to examine the effectiveness of MLVF for studying S. aureus strains isolated from households, farms and exotic animals in three regions of Poland. MLVF, random amplification of polymorphic DNA (RAPD), spa typing and diagnostic microarrays were compared to determine the most suitable combination of methods for veterinary purposes. MLVF generated results consistent with host and geographic origins, reflecting population structures with a high concordance to spa typing results. MLVF has been proven to be a rapid, highly discriminatory and cost-effective method suitable for molecular typing in veterinary settings.

Biofilm production and presence of ica and bap genes in Staphylococcus aureus strains isolated from cows with mastitis in the eastern Poland.

The aim of the study was phenotypic and genotypic analysis of 132 S. aureus strains isolated from mastitis in eastern Poland in respect to their biofilm formation ability. The analysis of the size polymorphism of fragment X in the gene encoding protein A (spa) revealed high genetic differentiation of the analyzed group of isolates. The ability of biofilm formation by the isolates was tested using two phenotypic methods. The Congo Red plate assay was found to be irreproducible and very subjective. More objective results were obtained using the spectrophotometric, microtiter plate assay. Most of the isolates, namely 76/132 (57.6 %) were classified as biofilm producers depending on the value of absorbance in the microtiter plate test. All of the isolates tested were found to possess both icaA and icaD genes, while the bap gene was absent in all strains.

Phenotypic and genotypic diversity of Pseudomonas aeruginosa strains isolated from hospitals in siedlce (Poland)

A total of 62 Pseudomonas aeruginosa strains isolated from two hospitals in Siedlce (Poland) were studied by repetitive element based PCR (rep-PCR) using BOX primer. BOX-PCR results revealed the presence of 7 numerous genotypes and 31 unique patterns among isolates. Generally, the strains of P. aeruginosa were characterized by resistance to many antibiotics tested and by differences in serogroups and types of growth on cetrimide agar medium. However, the P. aeruginosa strains isolated from faeces showed much lower phenotypic and genotypic variations in comparison with strains obtained from other clinical specimens. It was observed that genetic techniques supported by phenotypic tests have enabled to conduct a detailed characterization of P. aeruginosa strains isolated from a particular environment at a particular time.

Phenotypic and genotypic diversity of Pseudomonas aeruginosa strains isolated from hospitals in siedlce (Poland).

A total of 62 Pseudomonas aeruginosa strains isolated from two hospitals in Siedlce (Poland) were studied by repetitive element based PCR (rep-PCR) using BOX primer. BOX-PCR results revealed the presence of 7 numerous genotypes and 31 unique patterns among isolates. Generally, the strains of P. aeruginosa were characterized by resistance to many antibiotics tested and by differences in serogroups and types of growth on cetrimide agar medium. However, the P. aeruginosa strains isolated from faeces showed much lower phenotypic and genotypic variations in comparison with strains obtained from other clinical specimens. It was observed that genetic techniques supported by phenotypic tests have enabled to conduct a detailed characterization of P. aeruginosa strains isolated from a particular environment at a particular time.

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.

Extended multiple-locus variable-number tandem-repeat analysis of Bacillus anthracis strains isolated in Poland.

Twenty-one variable-number tandem-repeat (VNTR) marker loci were used for extended multiple locus VNTR analysis (MLVA) of 14 laboratory strains of Bacillus anthracis isolated in Poland and vaccine strain Sterne 34F2A. The extended MLVA (MLVA-21) distinguished six genotypes clustered in three main branches. Monomorphic branch 1 consisted of the vaccine strain and six isolates from distinct samples of a cow died from anthrax. This group also encompassed three haemolytic isolates of B. anthracis. Branches 2 and 3 were heterogeneous and consisted of five and three isolates of the phylogenetic lineages B2 and A1, respectively. MLVA-21 supported thesis on the anthrax agent heterogeneity in Poland. This study brought an additional evidence that haemolytic B. anthracis strains isolated in Poland are closely related to the vaccine strain Stere 34F2 and may together constitute the same sensu stricto strain. No epidemiological link could be however traced between both the vaccine and the haemolytic strains.

[Antibiotic susceptibility and occurrence of ESBL, IBL and MBL in Pseudomonas aeruginosa strains]. / Wrazliwosc na antybiotyki oraz wystepowanie beta-laktamaz typu IBL, ESBL i MBL u szczepów Pseudomonas aeruginosa.

The aim of this study was to evaluate the drug susceptibility of P. aeruginosa strains and to detect strains producing inducible beta-lactamases (IBL), extended-spectrum beta-lactamases (ESBL), and metallo-beta-lactamases (MBL). During 6 month (October 2005 - March 2006), 66 strains of P. aeruginosa strains were cultured from clinical specimens obtained from patients of two of hospitals in Siedlce and from patients of outpatient clinics. All the strains were identified in the automatic ATB (bio Mérieux). The susceptibility of bacteria to antibiotics was tested by standard disc diffusion method. The majority of strains were susceptible to meropenem (89.4%), piperacillin combined with tazobactam (84.8%), ciprofloxacin (84.8%) and piperacillin (83.3%). Many of our strains were resistant to carbenicillin (69.7%), mezlocillin (45.5%), gentamicin (42.4%) and netylmicin (30.3%). 6 strains (9.1%) were multidrug-resistant (MDR). Inducible beta-lactamases were detected with the use double disc method according to Sanders and Sanders. ESBL-producing strains were detected with double disc test (DDST) according to Jarlier et al. These strains were identified as ESBL-positive on the basis of the DDST were also determined using a double disc (DD) test according to Appleton. Production of metallo-beta-lactamases (MBL) was examined with the use of Etest MBL (AB Biodisk, Sweden) and the double disc test according to Arakava et al. Sixty-five IBL-producing strains (98.5% of all strains) and three strains (4.5%) with MBL activity were detected. Strains producing extended beta-lactamases (ESBL) were not found.

Application of multiplex PCR for monitoring colonization of pig tonsils by Yersinia enterocolitica, including biotype 1A, and Yersinia pseudotuberculosis.

A multiplex PCR assay was developed for the detection and differentiation of the Yersinia enterocolitica and Yersinia pseudotuberculosis isolates in both pure bacterial cultures and pig tonsils. The assay was based on the amplification of the ail, inv, yadA, and ystB genes. The PCR products, corresponding to the ail gene and the plasmid-borne yadA gene or only one product corresponding to the ail gene, were detected in Y. enterocolitica 4 biotype isolates. All of the Y. pseudotuberculosis isolates (n=6) tested gave a positive PCR reaction for the inv gene. For all tested Y. enterocolitica 1A biotype isolates (n=31), one product corresponding to the ystB gene was observed. The multiplex PCR assay was used to detect Y. enterocolitica and Y. pseudotuberculosis strains in pig tonsil samples obtained from 80 slaughtered pigs from three different herds. The presence of at least one of the specific PCR amplification products of ail-, ystB-, yadA-, and inv-specific sequences was observed in 11 samples (13.75%). These results of the multiplex PCR assay were compared with the results of conventional, microbiological testing. Y. enterocolitica isolates were cultured from only 3 (3.75%) of the 80 pig tonsils examined. The multiplex PCR assay was shown to be an efficient tool for differentiation between the pYV plasmid-bearing Y. enterocolitica isolates, the plasmidless Y. enterocolitica isolates, the Y. enterocolitica biotype 1A isolates, and the Y. pseudotuberculosis isolates with and without the pYV plasmid in naturally contaminated pig tonsils. This indicates that this assay is useful to control food processing and track the source of contamination.

Effect of neuraminidase on adherence of Pseudomonas aeruginosa to human buccal epithelial cells. Inhibition of adhesion by monosaccharides.

The aim of this study was to evaluate the actiion of Clostridium perfringens neuraminidase on the adherence of 28 strains of Pseudomonas aeruginosa which were isolated from humans, different animals and environment to human buccal epithelial cells (BECs). Two reference strainns--NCTC 6749 and ATCC 27853 were also examined. Incubation of cells with the enzyme significantly increased bacterial adherence (a mean number of bacteria adhering to cells amounted 19.62 +/- 9.20, for controls - 7.54 +/- 5.86). The reference strains of Pseudomonas aeruginosa showed the following adherence: NCTC 6749-43.04 (control 20.83) and ATCC 27853-22.21 (control 5.51). This study demonstrates that asialogangliosides function as receptors on buccal epithelial cells for P. aeruginosa strains. Monosaccharides inhibition studies showed an inhibition of adhesion of P. aeruginosa (two reference strains - NCTC 6749 and ATCC 27853, two hospital strains - 80/85 and 351) to normal BECs in the presence of N-acetylneuraminic acid and N-acetylgalactosamine. D-galactose is the best inhibitor of bacterial adhesion to neuraminized BECs. All monosaccharides used had a significant effect on P. aeruginosa adherence to trypsinized BECs. These data suggest a difference in the receptors on the three types of BECs.

Reduction in the adherence of Pseudomonas aeruginosa to human buccal epithelial cells with neuraminidase inhibition.

The aim of this study was to evaluate the reduction in the adherence of 33 strains of Pseudomonas aeruginosa isolated from humans and different animals to human buccal epithelial cells with neuraminidase inhibition. Buccal epithelial cells were incubated with strains of Pseudomonas aeruginosa in the presence or absence of the neuraminidase inhibitors, 2,3-dehydro-2-deoxy-N-acetyl-neuraminic acid (DANA) or N-acetyl-neuraminic acid (NANA). Incubation of cells with bacteria in the presence of either DANA or NANA reduced bacterial adherence significantly by 35.24 +/- 23.90%, and 68.00 +/- 22.51 %, respectively. We suggest that the in vivo effects of such interventions should be explored as potential mechanisms reducing Pseudomonas aeruginosa in the binding to buccal cells.

[Inhibition of adhesion to buccal epithelial cells of Pseudomonas aeruginosa by dextran]. / Hamujace dzialanie dekstranu na adhezje Pseudomonas aeruginosa do komórek nablonka policzka.

Adhesion of Pseudomonas aeruginosa strains to buccal epithelial cells appears to be a necessary precondition for colonization and infection of respiratory tract. There are many strategies to prevent host organisms for Pseudomonas aeruginosa. The purpose of these studies was to evaluate the potential for preventing adhesion of Pseudomonas aeruginosa to epithelial cells with dextran. Dextran (5,000 MW) inhibited adhesion of Pseudomonas aeruginosa to buccal cells, at 20 mM was most inhibitory. The inhibitory effect appeared to be nonspecific because other neutral polysaccharides (glycogen and mannan) were also inhibitory. Dextran is an inexpansive and nontoxic agent and may be useful to prevent colonization and infection of respiratory tract with Pseudomonas aeruginosa.

Intriguing diversity of Bacillus anthracis in eastern Poland--the molecular echoes of the past outbreaks.

The multiple locus VNTRs analysis (MLVA) revealed the presence of five genotypes in a group of 10 Bacillus anthracis isolates from epidemiologically unrelated cases of bovine-anthrax in eastern Poland. Eight tested isolates possessed the pagA and capB genes indicating the presence of both virulence plasmids, while two isolates revealed only pagA and lacked pXO2. The MLVA and DNA sequence analysis indicated that seven tested isolates represent four novel genotypes. Five tested strains revealed a unique 144 bp vrrB2 variant as well as 220 bp variant of vrrB1, implying the relatedness to the lineage B2. Consequently, we propose establishing of novel B2 strains sub-lineage. Multiple anthrax outbreaks, which took place in Poland several decades ago were proposed as a cause of intriguing diversity of B. anthracis observed in this study.

Adherence of Pseudomonas aeruginosa to human buccal epithelial cells.

The aim of this study was to evaluate adherence of 83 strains of Pseudomonas aeruginosa isolated from humans and different animals to trypsin-treated buccal cells. We have demonstrated that Pseudomonas aeruginosa attached to trypsin-treated buccal cells in far greater numbers than to cells from controls (normal buccal epithelial cells). The mean number of bacteria adhering to trypsin-treated cells amounted 107.05 +/- 102.16 and to normal cells - 6.97 +/- 3.53. We conclude that exposure of cells to proteolytic enzymes increases Pseudomonas aeruginosa binding to buccal cells.

[Detection of Pseudomonas aeruginosa biofilm on medical biomaterials]. / Wykrywanie biofilmu Pseudomonas aeruginosa na biomaterialach medycznych.

The adhesion and biofilm formation of Pseudomonas aeruginosa strains on the surface of catheters made of various polymers (PU, SL, PCW) were determined in vitro. It was used the method by Richards et al. with modification of Rózalska et al. (1998), in which soluble colourless TTC is reduced to insoluble red formazan. The results of this study indicate that 80.3% of this strains adhered and 94.6% formed biofilm on the Nelaton catheter, 86% strains adhered and 76.1% formed biofilm on the polyurethane catheter, and 73.2% strains adhered, and 78.9% formed biofilm on the Foley catheter.

[The effect of culture conditions on hydrophobic properties of Pseudomonas aeruginosa]. / Wplyw warunków hodowli na hydrofobowe wlasciwosci paleczek Pseudomonas aeruginosa.

The cell surface hydrophobicity (CSH) plays an important role in a adhesion of bacteria on solid surfaces. CSH of 62 Pseudomonas aeruginosa strains isolated from humans and different animals was assessed using the ammonium sulfate salt aggregation test. Bacteria were grown for 24 h and 48 h at a room temperature (22 degrees C) and 37 degrees C on enrichment broth and agar (Biomed) and tryptic soy agar (Difco). The hydrophobic properties of the Pseudomonas aeruginosa strains were depended on the temperature, time of the culture of bacteria and the kind of media. CSH properties were most frequently expressed when the analyzed strains were cultured in enrichment broth. In a such conditions Pseudomonas aeruginosa strains were more hydrophobic when grown at 22 degrees C (94% after 24 h and 87% after 48%) than those at 37 degrees C (72% after 24 h and 71% after 48 h). Among strains cultured in tryptic soy agar at 37 degrees C, 48% after 24 h and 75% after 48 h were autoaggregating, representing very strong hydrophobic properties.