Medycyna doswiadczalna i mikrobiologia最新文献

Introduction: Adhesion of pathogenic bacteria to host cells is a crucial step during infection. The presence of specific adhesion factors on the bacterial cell surface determines the tropism of the pathogen to the tissues expressing certain surface receptors. The adhesion is mediated primarily by filamentous structures called pili. Three distinct pilus structures can be produced by Corynebacterium diphtheriae: SpaA-, SpaD- and SpaH-type pili. Pilus genes encode a total of nine pilus proteins, named SpaA through SpaI, and six sortases, named SrtA through SrtF. All the pilus genes are located on pathogenicity islands and can be acquired and lost by different strains. The aim of presented studies was assessment of occurence of pili genes among C. diphtheriae strains isolated from different infections, including invasive infections, in Poland.

Methods: Thirty-one toxigenic and nontoxigenic C. diphtheriae strains isolated from wounds, blood, nose and pharynx were investigated for presence of 15 pili genes. The studies were conducted using PCR. Gene specific primers were designed on the basis of the complete genome sequence of C. diphtheriae NCTC 13129.

Result: All the nontoxigenic C. diphtheriae strains isolated from invasive infections possess every tested genes in contrast to toxigenic strains that revealed highly mosaic structure of pili gene clusters. Differences in gene content were detected in SpaA- and SpaH-type pili gene clusters. Complete set of genes in SpaD-type pili gene cluster was detected in all but two strains. The two strains did not possess any of SpaD-type pili genes.

Conclusions: Invasiveness of C. diphtheriae strains could be related to adhesive factors. Results of our studies suggest that ability to express all types of pili is indispensable for causing invasive infections by nontoxigenic C. diphtheriae. Whereas full set ofpili genes is not necessary for causing classical diphtheria.

Introduction: Gram-negative bacteria belonging to the family Enterobacteriaceae cause severe and difficult to treat nosocomial infections. Strains of different species that produce extended-spectrum beta-lactamases (ESBL) were classified as alert pathogens. The purpose of this study was to analyze the occurrence and determination of antimicrobial susceptibility patterns of 134 ESBL-producing Enterobacteriaceae strains.

Methods: 96 (72%) isolates of Klebsiella pneumoniae and 38 (28%) isolates of Escherichia coli, were cultured from patients of Specialistic Hospital in Krakow, in the period from 2008 to 2010. Bacterial identification and antimicrobial susceptibility testing were performed by automated system Vitek 2 Compact (bioMerieux, Poland). Condition for inclusion in the study was the production by the strains of beta-lactamases with extended substrate spectrum (ESBL), which was confirmed using the automated method (Vitek 2 Compact system) and the disc-diffusion assay (DDST). Taken into consideration the first isolate from the patient.

Results: Bacilli of the species K. pneumoniae ESBL(+) were mainly isolated from respiratory tract samples (46%), urine (27%) and blood (12%). The dominant divisions in terms of frequency of isolation of these pathogens were anesthesiology and intensive care (42%), neurology and brain strokes (16%) and internal medicine (11%). Drugs with the highest efficiency against K. pneumoniae ESBL(+), in our in vitro studies, were: imipenem (100%), meropenem (100%), amikacin (90%) and tetracycline (75%). E. coli ESBL(+) isolates derived from patients of Anesthesiology and Intensive Care Unit (32%), Internal Medicine Unit (16%) and Division of Hematology (13%). Among all tested strains majority were obtained from respiratory tract samples, urine, swabs from wounds and blood (respectively 24%, 24%, 21% and 18%). Isolates of E. coli ESBL(+) demonstrated the greatest susceptibility in case of amikacin (92%) and piperacillin with tazobactam (76%), which suggests the highest activity of that antimicrobials against infections caused by examined strains. None of the analyzed bacilli were resistant to carbapenems.

Conclusions: Our study highlights the importance of characteristics of distribution of ESBL-producing K. pneumoniae and E. coli strains among hospitalized patient. Good antibiotic policies based on antibiotic resistant patterns can decrease the risk of ESBL infection.

Verocytotoxigenic E. coli (VTEC) are one of the most common foodborne pathogen in human worldwide. High pathogenic potential of these organisms makes it often the cause of international outbreaks with numerous fatalities. This study presents the current knowledge on verocytotoxigenic E. coli: pathogenicity, drug resistance as well as the epidemiology of infections.

Introduction: Multidrug-resistant gram-negative non-fermenting bacilli are an important cause of nosocomial infection. Aim of this study was to analyze the prevalence and antimicrobial susceptibility of rods of the species Acinetobacter baumannii and Pseudomonas aeruginosa, belonging to multidrug-resistant alert pathogens.

Methods: 105 (70%) strains of A. baumannii and 46 (30%) strains of P. aeruginosa were isolated from 125 patients hospitalized in the Specialistic Hospital in Krakow, in the years 2008-2010. Taken into account first isolate from the patient. The condition for inclusion in the study was the resistance or reduced susceptibility to selected groups of antibiotics, such as beta-lactams, aminoglycosides and fluoroquinolones. Bacterial identification and antimicrobial susceptibility testing were performed by automated system Vitek 2 Compact (bioMerieux, Poland). All strains were tested with phenotypic method Etest MBL (AB Biodisk, Sweden) for the presence of resistance mechanism associated with the production of metallo-beta-lactamases.

Results: Bacilli of the species A. baumannii were isolated most frequently from patients from the Department of Anesthesiology and Intensive Care (52%) and Burn Therapy Unit (25%), with clinical materials collected from the respiratory tract (51%), the wound swabs (18%), urine (11%) and blood (11%). Production of metallo-beta-lactamases was found in 24 (22.9%) strains of A. baumannii. Drugs effective against multidrug-resistant isolates of A. baumannii were colistin and amikacin. Department of anesthesiology and intensive care (59%) and unit of internal medicine (11%) were the main source of multidrug-resistant strains of P. aeruginosa. Pathogens were mainly isolated from clinical specimens collected from the respiratory tract (61%), urine (15%) and wound swabs (13%). Seven (15.2%) strains of P. aeruginosa produced the metallo-beta-lactamases. With regard to colistin and piperacillin with tazobactam was noted the highest percentage of susceptible isolates.

Conclusions: MDR bacteria belonging to alert pathogens are an important cause of many severe and difficult to treat infections which greatly increases the morbidity and mortality among hospitalized patients worldwide. Epidemiological studies and detection of local resistance patterns can provide useful information which can be used in the development of strategies to combat the rising tide of microbial antibiotic resistance.

Introduction: Bacillus genus comprises about 215 species. The most closely related are B. cereus, B. thuringiensis, B. anthracis, B. mycoides, B. pseudomycoides and B. weihenstephanensis. These bacterial species belong to the Bacillus cereus group. Identification and differentiation of Bacillus cereus group bacteria is difficult because of genetic and phenotypic similarity. Many molecular methods have already been suggested to differentiate B. cereus group members. However easier and more convenient methods are still sought. The aim of this study was to evaluate of multiplex PCR method to identify and distinguish strains of Bacillus cereus group, as proposed by Park et al. (J Microbiol Biotechnol 2007; 17: 1177-82).

Materials and method: Twenty four strains of Bacillus cereus group included B. cereus, B. anthracis, B. thuringiensis and B. weihestephanensis was examined. A multiplex-PCR assay for the differentiation of the species has been applied by using three pairs specific oligonucleotide primers based on sequences of gyrB genes which identify species from Bacillus cereus group and one pair specific primers based on sequences of groEL gene, which are used to identify Bacillus cereus group.

Results: Using a specific primers complementary to fragment of groEL gene, we received all PCR products and thus we identified Bacillus cereus group. We have not recived a specific products characteristic for each of the species. Oligonucleotide primers recognized by Park et al. as specific for each species were complementary (often 100%) for the gyrB gene sequence in almost all species of the B. cereus group.

Conclusions: The multiplex PCR method proposed by Park et al. multiplex PCR method for identification ofB. cereus group and individual bacterial species has been proved to be useful only for identification the entire group of B. cereus. This method does not provide specific identification of the individual species. Lack of specificity of the primers used in this study creates a risk of obtaining PCR product in more than one species of the entire examined group of bacteria and does not allow the precise identification to the species level.

Introduction: Among different laboratory techniques used in influenza surveillance, methods based on molecular biology, such as real-time PCR, play increasingly important role. They allow detection and identification of viruses currently circulating in human population and responsible for infections and diseases. The aim of the study was to develop duplex real-time PCR assay for detection and differentiation of influenza virus subtype A(H1N1) pdm09 and A(H3N2).

Methods: Specific primers targeting a highly conservative region ofhaemagglutinin gene and a dual-color TaqMan probes, labeled with fluorophore JOE (560 nm) and quencher BHQ-1 or CalFluor Red 610 (610 nm) and BHQ-2 were designed. The specificity of duplex reaction was evaluated using RNA isolated from reference strains of influenza virus A(H1N1)pdm09, A(H3N2), A(H1N1) and A(H5N1), B and other respiratory pathogens. Sensitivity of the assay was tested using serial dilutions ofplasmid constructs carrying fragment of specific viral HA gene, in range between 10 and 10(5) copies/sample. A number of 116 clinical samples were tested using developed duplex qPCR assay in comparison with the CDC monoplex assay,

Results: Positive duplex qPCR results were obtained only in samples containing RNA of influenza viruses of a specific subtype. The limit of detection (LOD) of developed method amounted up to 27 copies/sample for A(H1N1)pdm09 and up to 37 copies/sample for A(H3N2).

Conclusions: The results show that developed TaqMan-based duplex qPCR assay is a valuable diagnostic tool in influenza virus surveillance for using in direct study of clinical specimens as well as identification of isolated strains of influenza virus.

Introduction: Majority of nosocomial Acinetobacter baumannii strains are highly resistant to many available groups of antibiotics, causing therapy of infections the clinical challenge. The aim of study was to estimate of resistance development to sulbactam, cefoperazone and cefoperazone/sulbactam in Acinetobacter baumannii clinical strains.

Methods: Five Acinetobacter baumannii strains (Acb1, Acb2, Acb4, Acb13 and Acb25) were identified by the VITEK 2 GN card and the automatic system VITEK 2 according to the procedure and following the producer's instructions. Additionaly, the belonging of the strains to the species was confirmed by the presence of the bla(OXA-51-like) gene. Initial and after antibiotic exposure MIC values of sulbactam, cefoperazone and cefoperazone/sulbactam were determined by using a broth microdilution method. Antibiotic pressure of examined strains was performed in Mueller-Hinton broth containing 0,5x, 0,9x and 2x initial MIC of individual compounds during six-day passages and next six-day passages without antibiotic presence. The Mann-Whitney U test and Kruskal-Wallis non-prarametric Anova test were used to statistical analysis.

Results: Serial passaging of Acinetobacter baumannii strains in the presence of antibiotics caused permanent increasing MIC value independently of used concentrations in the majority of examined strains. The highest MIC value increase of sulbactam was found in Acb4 strain. Even after two passages this isolate changed MIC from 0.5 microg/ml to 4 microg/ml (increase about four levels of concentration). Moreover, after incubation in 0.9x MIC concentration similar observation was noted. No normalization of MIC value of sulbactam after incubation during next six passages without sulbactam was observed. In case of cefoperazone the highest levels of induction were noted in Acb1, Acb13 and Acb25 strains. In these strains, after two passages in presence of cefoperazone (2xMIC) the exceedance of minimal of growth concentration over the highest examined concentration was observed. Similar effects were observed in Acbl strain after stimulation with 0.9x and 0.5x MIC cefoperazone. Return of initial MIC values was received only after induction with 0.5 x MIC cefoperazone. In some cases, no opportunities for evaluation of resistance development was noted, because during stimulation with 2x MIC of used antibiotics concentarations, bactericidal effect was found.

Conclusions: Sulbactam, cefoperazone and cefoperazone/sulbactam rapidly induce increasing of resistance in Acinetobacter baumannii clinical isolates. Statistically essential MIC increase after using higher concentration than lower was showed. This effect was particularly visible in the case of stimulation of cefoperazone/sulbactam combination.

Introduction: Yersinia enterocolitica includes both human pathogenic and non-pathogenic strains. The pathogenic strains belong to two evolutionary lineages: European and American, of mild- and high- pthogenicity, respectively. Y. enterocolitica European bioserotypes 4/O3 and 2/O9 are one of the major etiological agents of human yersiniosis worldwide. American lineage Y. enterocolitica bioserotype 1B/O8 has recently emerged in Europe. Since 2004 this high-pathogenicity bioserotype is increasingly isolated from humans in Poland. The rapid and accurate identification of pathogenic Y. enterocolitica strains is essential for diagnostic purposes. The aim of this study was to assess the usefulness of commercially available MALDI-TOF mass spectroscopy for Y. enterocolitica identification and subtyping.

Methods: A total of 33 strains of Y. enterocolitica belonging to bioserotype: 1B/O8 (n=2), 4/O3 (n=25) and 2/O9 (n=6) isolated from clinical specimens in Poland and 10 reference Y. enterocolitica 1B/O8 strains (Institute Pasteur, France) were used in this study. The identification of the Y. enterocolitica strains by MALDI-TOF MS was performed on MALDI Biotyper system (Bruker Daltonics, USA) with flexControl 3.0 software (Bruker Daltonics) according with the manufacturer's instruction.

Results: All of the tested Y. enterocolitica strains were correctly identified at the species level. However, American and European strains were not differentiated.

Conclusions: Our data indicate that MALDI-TOF MS can be used as a first-line method for rapid identification of Y. enterocolitica strains at the species level in clinical microbiology laboratories.

Introduction: Intensive use of broad-spectrum antibiotics is responsible for conversion of enterococci from commensal bacteria to opportunistic nosocomial pathogenes. The serious infections caused by them are often treated with synergistic bacteriocidal combination of beta-lactam and aminoglycoside. The presence of high-level aminoglikosyde (HLAR) and/or beta-lactam (HLPR) resistance makes this treatment ineffective.

Methods: This study investigated the differences in occurance of HLAR and/or HLPR among groups of enterococi isolated from: rectal swabs of healthy volunteers, samples of food and clinical specimens. Enterococci were identified by 6.5% NaCl tolerance and growth on bile-esculin agar with esculin hydrolysis. Species-level identification was made on the basis of motility, pigmentation, arginine hydrolysis, growth on tellurite and formation of acid in mannitol, sorbitol, sucrose, arabinose, raffinose and sorbose broth (according to the Facklam's and Sahm's scheme). High-level resistance were determined to penicillins (> 100 microg/ml), gentamycin (> 500 microg/ml) and streptomycin (> 2000 microg/ml).

Results: Although differences in species prevalence varied by source, E. faecalis was the common enterococcal species in all samples (> 70%), followed by E. faecium (10%-20.4%) and E. durans (4.2%-8.45%). E. faecalis varians, E. pseudoavium and E. raffinossus were isolated rarely. Analysis of 452 enterococcal strains revealed the presence HLAR in all groups, but HLPR was not present in samples of food. In community the HLR was greatest for streptomycin, but among hospital isolations-HLR both to streptomycin and gentamycin were most common. Such resistance was more frequent in E. faecium than in E. faecalis. HLR to kanamycin was not found.

Conclusions: This study shows that high-level resistance to aminoglycoside and/or beta-lactam of enterococci may play an important role in public health. Little is known about the prevalence, risk factors and reservoirs for resistance enterococci outside of the hospital. A comparison of the resistance determinants in isolates from healthy volunteers with the genes responsible for resistance in isolates from patients.

Introduction: In 2011, the Polish Ministry of Health introduced Candida sp. resistant to fluconazole and Aspergillus sp. to the list of Alarm Factors as alert pathogens. The purpose of this paper is to confirm the validity of continuous monitoring of fungal infections caused by the pathogens mentioned above. The role offluconazole therapy in the Candida sp. infections is also discussed. The analysis of the fungal infections is performed based on the results obtained in the University Clinic Hospital (UCH) No. 1 in Lodz in 2009-2011.

Methods: The swabs were plated on Sabouraud's agar. Body fluids and blood were incubated in an automated system Bactec 9050. Yeast ID Phoenix BD panels were used to determine the species of fungi. In turn, antimicrobial susceptibility testing was carried out by E-tests (bioMerieux).

Results: In the analysis of fungal infections occurring among patients in the UCH No. 1 in Lodz in 2009-2011, C. albicans, C. non-albicans and Aspergillus sp. infections are taken into account. This analysis is performed based on relations of the number of infections (per 100 patients) versus six-month periods. As one can see in Fig. 1, a clear, linear and statistically significant increase in the number of C. albicans and C. non-albicans infections is observed throughout the entire time period under discussion. On the other hand, the number of Aspergillus sp. infections remains at an almost constant low level. The more detailed analysis of fungal infections in the different hospital units, which are particularly exposed to this type of infections (Figs. 2-6), shows that there is a clear correlation between the number of C. non-albicans infections and the frequency of therapy with fluconazole.

Conclusions: The results presented in this paper show in the example of the UCH No. 1 in Lodz that the number of infections caused by C. albicans and C. non-albicans resistant to fluconazole is clearly increasing in a hospital environment in recent years, which is a great clinical problem. Although the number of Aspergillus sp. infections is relatively much lower in comparison to that of Candidia sp., these infections also constitute a problem of clinical importance. In light of the presented analysis, it should be assessed positively the fact that Candida sp. resistant to fluconazole and Aspergillus sp. are considered to be alert pathogens that require the continuous monitoring.