Medycyna doswiadczalna i mikrobiologia最新文献

Introduction: Clostridium difficile infection (CDI) is a serious problem in hospitalized patients. Rapid and accurate laboratory diagnosis is the key to reducing of CDI. The suboptimal sensitivity and specificity of many commercial enzyme immunoassays have limited their utility. The aim of this study was analysis of faecal samples obtained from patients with clinical evidence of CDI, with non-detectable or questionable result of toxins A/B C. difficile recognized by toxins A/B EIA test.

Methods: A two-step algorithm for diagnostics of C. difficile infection (CDI) in patients with non-detectable or questionable result of toxins A/B C. difficile confirmed by C. difficile enzyme immunoassay (EIA) (Wampole, TOX A/B II, TechLab, USA) was used. Sixty nine faecal samples obtained from patients with nosocomial diarrhea were retested. All faecal samples were cultured on selective medium CLO C. difficile (BioMérieux, Francja). The positive samples on selective medium were tested by using Real Time-PCR (Xpert CD assay, Cepheid, Sunnyvale, CA, USA). Xpert CD assay is a real time multiplex PCR that can be used to detect toxigenic C. difficile strains and differentiate the C. difficile presumptive NAP1/BI/027 strain. All results when faecal samples were negative in culture growth on selective medium and result of EIA test were questionable was confirmed by use a RT-PCR test.

Results: Among 69 faecal samples 56 were negative for toxins A/B using EIA test and 13 gave questionable results. By anaerobic culture 60 of 69 specimens yielded C. difficile isolates. Among 69 faecal samples 55 were positive using RT-PCR. Thirty four (62%) of patients was infected by presumptive C. difficile NAP1/BI/027.

Conclusions: C. difficile testing by use of culture and Real Time PCR (RT-PCR) increases diagnostic yield in a hospital patients with non-detectable or low level of toxins A/B in stool samples of patients infected by toxigenic C. difficile strains including presumptive C. difficile NAP1/BI/027.

Introduction: The present study was aimed at determining the IgG subclass distribution against pertussis toxin (PT) and filamentous hemagglutinin (FHA) of Bordetella pertussis in patients with whooping cough.

Methods: The total number of 222 serum samples obtained from patients suspected in clinical investigation for pertussis were tested separately by in-house ELISA for the presence of IgG antibodies to pertussis toxin and filamentous hemagglutinin. The percentage distribution of specific anti-PT and anti-FHA IgG subclass response was calculated only on the basis of group of sera confirmed in the present study as positive for total IgG antibodies (183 sera to PT antigen and 129 to FHA antigen). Paired serum specimens were obtained from 36 patients. Based on the results of determining the level of antibodies in the sera of 40 blood donors, the cut-off limit of serum antibodies for each subclass was set at arithmetic mean plus two standard deviations.

Results: Antibodies of IgG1 to pertussis toxin and filamentous hemagglutinin were diagnosed in 151 (82.5%) and 99 (76.7%), IgG2 in 72 (39.0%) and 50 (38.8%), IgG3 in 17 (9.3%) and 43 (33.3%), IgG4 in 55 (30.1%) and 53 (41.1%) serum samples, respectively. There were no significant differences in percentage of sera with IgG1, IgG2 and IgG3 in relation to age of the patients. However, the frequency of occurrence of IgG4 antibodies was highest in the group of the youngest children to the age of 6 years old (61.8% for PT and 68.0% for FHA), and decrease with age, reaching the minimum in the group of patients above 40 years old (13.2% and 4.2% for PT and FHA, respectively). We also found significantly higher frequency of IgG4 to PT and FHA antigens in men than in women. Statistically significant, essential changes in the pattern of IgG subclass during the course of infection were not found.

Conclusions: In conclusion, this study showed that all four subclasses of IgG antibodies to pertussis toxin and filamentous hemagglutinin are produced during whooping cough.

Introduction: Serotyping is a commonly used method to characterize the presence of Fimbriae 2 and 3 in Bordetella pertussis strains for epidemiological purposes and optimal choice of strain composition of the pertussis whole-cell vaccine. Monoclonal antisera against Fim2 and Fim3 are recommended to be used for microplate serotyping instead ofpolyclonal. Reliable evaluation offimbriae expressed by B. pertussis strains influence interpretation of vaccine-driven strain evolution.

Methods: To evaluate the impact of tests conditions on the reproducibility of serotyping, results of serotyping based on a standardized protocol for microplate agglutination with monoclonal antisera performed in three different accredited laboratories were compared. For the study isolates of three vaccine strains of B. pertussis deposited within seed lot system originating from different liofilization lots were compared.

Results: Lack of the complete agreement on serotyping results among three labs might relates to the differences of media used, subjective reading, test conditions, and specificity of the reagents.

Conclusions: Serotyping results should be interpreted with caution and the type of media and culture conditions used should be precisely recommended after validation studies. Inconsistent results should be confirmed using an alternative technique, eg. ELISA or by reference laboratory.

Introduction: According to common belief, supported by the authority of the World Health Organization - WHO, the common (social) hand washing is the simplest, cheapest and the most effective way of reduction the hospital-acquired infections. For this purpose products of"liquid soaps", present in a large number on the market, are most often applied. Microbiological status (microbiological purity and antimicrobial activity) of"liquid soaps" available on the Polish market is not known, because relevant routinely studies have not been performed. Only the antibacterial and / or antifungal activity of certain formulations is sometimes assessed, especially when the manufacturer suggests the standardized application of the products for surgical or hygienic procedures. The aim of this study was to determine the microbiological quality, especially microbiological purity and antimicrobial activity of the selected hands washing products, presents on the Polish market.

Methods: The 12 selected commercial products, available on the market in Poland, dedicated for hands washing were included into study. Microbiological purity test was carried out in accordance with the Polish Pharmacopoeia (FP) monograph (FP monograph numbers correspond to numbers of the European Pharmacopoeia monograph- Ph. Eur.) No 2.6.12 "Microbiological examination of non-sterile products: microbial enumaration tests", and the monograph of FP No. 2.6.13 "Microbiological examination of non-sterile products: test for specified microorganisms". The following physico-chemical properties of soaps were examined: the pH of the formulations was measured according to the monograph FP No. 2.2.3. "Potentiometric determination of pH", the density of products was assayed according to the monograph FPNo. 2.2.5. "Relative density" and determination the water activity was performed by monograph FP No 2.9.39 "Water-solid interactions: determination of sorption-desorption isotherms and of water activity". Next, antibacterial and antifungal protection was determined in accordance with the monograph FP No 5.1.3. "Efficacy of antimicrobial preservation". The study of antimicrobial activity was carried out in accordance with PN-EN 1040 "Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of basic bactericidal activity of chemical disinfectants and antiseptics - Test method and requirements (phase 1)". Finally, using the "time-kill" method the survival of microorganisms after different contact times of the products with bacteria and fungi were determined.

Results: All the examined products showed a very high microbiological purity. None of the formulations was characterized by a high acidity or alkalinity. All the analyzed products were slightly thicker than water, but such density of the preparation does not seem to be important parameter in the growth of microorganisms. The results of water activity estimation

Introduction: The aim of the study was evaluation results of molecular typing L. pneumophila strains that was carried by using SBT (Sequence-Based Typing) method, obtained by laboratory of Department of Bacteriology NIZP-PZH within the framework of the ninth international external quality assessment (ELDSNet Legionella pneumophila DNA SBT) and their comparision with the results obtained by other reference laboratories in EU.

Material and methods: The panel of five coded isolates of L. pneumophila was investigated. Genomic DNA of Legionella were extracted and defined regions of seven genes were amplified by PCR and sequenced. Then, consensus sequence of the correct length were generated. In order to determine the complete allelic profile and Seqence Type (ST) forward and reverse sequence for each allele were submitted online by using the L. pneumophila database.

Results: All of L. pneumoniae isolates sent to genotyping by SBT method were correctly identified in our laboratory.

Conclusions: Results of the ninth international external quality assessment have confirmed competences of laboratory of Department of Bacteriology NIZP-PZH in typing of L. pneumophila isolates accordance with the requirements of the international classification.

Introduction: The high pathogenicity Yersinia enterocolitica 1B/O8 produce variety of virulence factors including chromosomal T3SS known as Ysa-Ysp system that is considered to act at the early stage of infection. The aim of the study was to examine the ability to produce Ysa-Ysp proteins in vitro by human clinical isolates of the epidemic Y. enterocolitica 1B/O8 strains in native conditions and immunological characterization of expressed proteins.

Methods: Seven Y. enterocolitica 1B/O8 isolates with known epidemiological link and the reference high pathogenicity strain WA-314 and six strains from the Institute Pasteur (France) were examined for production of Ysa-Ysp proteins according with procedure described by Matsumoto and Young (Mol. Microbiol., 2006, 59:689-76). All the isolates and strains were characterized by SDS-PAGE to determined Ysa-Ysp proteins profile. The immunological characterization was performed by using western-immunobloting method using sera from two immunized rabbits and from two patients with bacteriology confirmed Y. enterocolitica 1B/O8 infection.

Results: The reference strain WA-314 yielded typical Ysa-Ysp proteins profile. In contrast all the tested Y. enterocolitica 1B/O8 human isolates yielded the same SDS--PAGE profile that was apparently distinct from profile of Ysa-Ysp proteins of reference strain WA-314.

Conclusions: The Y. enterocolitica 1B/O8 isolates of the epidemic strain circulating in Poland were found to be unable to produce Ysa-Ysp proteins in vitro under conditions sufficient to stimulate expression of the Ysa-Ysp proteins in the reference strain WA-314 and strains from the Institute Pasteur (France). Our results may suggest that the ability to produce Ysa--Ysp proteins in concentrations sufficient to induce production of specific antibodies is not indispensible for Y. enterocolitica 1B/O8 infection in humans. The western-immunoblotting analysis of human serum samples showed that the antibodies were not induced by Ysa and Ysp proteins during infection caused by the epidemic strain of Y. enterocolitica 1B/O8 circulating in Poland. Similar, negative result was found with serum of a rabbit immunized intravenously by the reference strain WA-314. The project was funded by the National Science Centre in Cracov, Poland, grant N N401 076039.

Introduction: The aim of this study was to use molecular methods to identify selected strains of C. difficile isolated from patients hospitalized at Independent Public Central Teaching Hospital [SP CSK] between 2008 and 2011 in order to demonstrate their toxicgenic character and to determine their epidemic potential, including the incidence of a suspected C. difficile strain 027/NAP1/B1.

Material and methods: Originally evaluated material consisted of freshly collected stool samples from patients who had developed diarrhea. Stool samples were assessed for toxins A and B via an immunoenzymatic method and for the presence of C. difficile via the first culture method. The isolated strains were stored on MICROBANK mediums, at -70 degrees C. From this sample collection, 48 strains isolated in 2008 and 28 strains isolated in 2011 were selected for molecular analysis.

Results: Among the C. difficile isolates that underwent molecular analysis there were 6 strains 027/NAP1/BI out of the 48 evaluated strains isolated in 2008, which constituted 12.5% and 24 strains 027/NAP1/BI out of the 28 strains isolated in 2011, which constituted 85.7%.

Conclusions: Identification of a possible hyperepidemic strain of C. difficile is crucial for undertaking any anti-epidemic activities in health care facilities, where such activities are more and more common and are responsible for nosocomial foci of infection.

Introduction: Many pathogenic bacterial species have the ability to biofilm formation. In our study we determined the influence of Lactobacillus casei on biofilm formation by enteroaggregative Escherichia coli (EAEC) strains obtained from irritable bowel syndrome patients.

Methods: The ability of EAEC isolates to biofilm formation was assessed in the presence of various concentrations of the probiotic L. casei strain in an a semi- quantitative microtitre plate assays under culture conditions, similar to those prevailing in the human intestine.

Results: Depending on the concentrations L. casei inhibited biofilm formation of the majority (> 80%) of the EAEC strains. Concentration of 4.5 x 10(7) cfu/ml of L. casei was the most effective inhibitory dose, although a few strains (approximately 18%) formed the biofilm regardless of the presence and concentration of the probiotic L. casei strain.

Conclusion: The inhibitory effect of L. casei on biofilm formation at most of studied EAEC strains suggest that L. casei may reduce the risk of developing persistent intestinal infections in humans.

Introduction: Uncontrolled bacteria of dental plaque generate formation of oral biofilm located on teeth and subgingival surfaces. It may induce local inflammation (gingivitis) with further development of periodontal diseases. A variety of oral bacteria such as Streptococcus mutans and Porhyromonas gingivalis are involved in pathogenesis of dental carries and periodontitis. Very often bacterial infections are associated with candidiasis (Candida albicans). Chlorhexidine (CHX) is the most commonly used antiseptic in dentistry due to its strong antibacterial activity and capacity to reduce the accumulation of oral biofilms. However, other antiseptics, especially endodontic irrigants, are still tested to improve their preventive and therapeutic effects in oral cavity infections. In this in vitro study we have compared antimicrobial activity of CHX with that of taurine chloramine (TauC1) and taurine bromamine (TauBr), natural taurine derivatives with known antibacterial and anti-inflammatory properties.

Methods: Antimicrobial activity of CHX, TauC1 and TauBr was tested by incubation of the compounds with S. mutans, P gingivalis and C. albicans. The agents were incubated in low (105/ml) and high (108/ml) density microbe suspensions, related to early and late biofilm infections, respectively. In some experiments bacteria were incubated with a combination of CHX + NaOCl and CHX + TauBr. MIC was determined by the pour-plate method.

Results: CHX showed the strongest antimicrobial activity against all tested pathogens. On the contrary, TauC1 was the weakest antiseptics used without effect on the growth of C. albicans. TauBr at non-cytotoxic concentrations inhibited the growth of S. mutans and P gingivalis with slight effect on the low density C. albicans. All tested agents showed weaker antiseptic properties in the presence of serum. Moreover, we have shown that interactions between CHX and sodium hypochlorite (NaOC1), the main endodontic irrigant, but not between CHX and TauBr,resulted in precipitation. Therefore, it may restrict their simultaneous application in root canal treatment. However, in spite of this unwanted reaction, the mixture of CHX with NaOCl kills pathogens more effectively then CHX alone.

Conclusions: The results confirmed CHX exceptional potential as primary antiseptic in dentistry, especially in prevention and treatment of dental carries, periodontal diseases and mouth candidiasis. Moreover, our study shows that TauBr may be used alternatively or in combination with CHX in killing of oral pathogens, due to its strong antibacterial and anti-inflammatory properties.

Introduction: Dental caries is a bacterial disease. The most important element used in caries prevention is fluoride, which is derived from the air, diet or fluoride-containing preparations and materials, e.g. glass-ionomer restorations. Fluoride can inhibit metabolism and bacterial growth in the dental plaque. The aim of the study was to evaluate the effect of topical fluoridation of the enamel on the growth of Lactobacillus spp. in the dental plaque.

Methods: The study was carried out in 15 patients with good oral hygiene, in whom three-day dental plaque from the enamel was examined. Next, fluoride was rubbed on the same surface and the examination of three-day dental plaque was repeated.

Results: No statistically significant differences (p = 0.475) in the amounts of Lactobacillus spp. in the plaque collected prior to and after the topical fluoridation were revealed.

Conclusions: Fluoride rubbed in the enamel, did not affect the amount of Lactobacillus spp. in the dental plaque growing on this material.