Medycyna doswiadczalna i mikrobiologia最新文献

Mycoplasma pneumoniae is one of the most common causes of community-acquired pneumonia in children and adults. Correct and rapid laboratory diagnosis of M pneumoniae infections is important to introduce appropriate antibiotic treatment. Diagnosis for M. pneumoniae usually relies on serological tests and/or molecular investigations. Both methods have some advantages but also limitations. This paper presents advantages and disadvantages of microbiological methods used in M. pneumoniae infection an example of case of patient with mycoplasmosis.

Introduction: Clostridium difficile is main reason of antibiotic-associated diarrhea in hospitalized patients. Diagnostic method for detection of Clostridium difficile infection (CDI) are limited to an enzyme immunoassays (EIAs), while the culture of toxigenic strains is still seen as the gold standard for the laboratory diagnosis. The aim of this study was to compare growth of C. difficile strains belonging to different polymerase chain reaction (PCR) ribotypes on new ChromID C. difficile Agar (CDIFF, bioMérieux, Marcy l'Etoile, France).

Materials and methods: One hundred thirty one of clinical C. difficile strains stored. in Anaerobic Laboratory were cultured on ChromID C. difficile Agar. Ten faecal samples were cultured on the same chromogenic medium and incubated at 37°C for 24 h under anaerobic conditions. Isolates were confirmed as C. difficile on the basis of well-known criteria. PCR-ribotyping was performed by visually comparison of patterns of PCR products of the 16S-23S rRNA intergenic spacer region. We examined the occurrence of beta-glucosidase gene, responsible for the dark color of the colony C. difficile on ChromID C.difficile Agar using a pair of primers: gluF (5'-AAGGT GTAAATTTAGGAGGTTGGTT-3') i gluR (5'-AGGTCCCAACTATCCC ATCC-3').

Results: Among ten C. dfficile isolates obtained from stool specimens one formed colorless colonies. We received 8 colorless isolates from 131 additional examined strains. All C. difficile isolates forming colorless colonies belonged to PCR ribotype 023. The prevalence of PCR-ribotype 023 was about 6%. We detected lack of beta-glucosidase gene in PCR-ribotype 023 isolates.

Conclusions: There are some C. difficile strains forming colorless colonies on ChromID C.difficile Agar. This appearance is important in routine diagnostic use this chromogenic culture medium.

Introduction: Gonorrhoea is one of the most common sexually transmitted diseases in the world. Fast and effective laboratory diagnostics of the infection is very important. The aim of the study was to compare Real-Time PCR and bacterial culture in diagnostics of Neisseria gonorrhoeae infections in patients of Department of Dermatology and Venereology Medical University of Warsaw.

Methods: The urethral, cervical, anal and pharyngeal specimens from 93 patients of Department of Dermatology and Venereology Medical University of Warsaw were examined by two methods. The bacterial culture was performed on chocolate agar plates with antibiotics in the 5% CO2 atmosphere at 37°C. N. gonorrhoeae was identified by colony-morphology, Gram stain and oxidase reaction, followed by carbohydrate utilization test. The DNA Bact Extra Pure Kit (Euroclone®) to isolate DNA was used. Real-Time PCR DUPLICα® Real-Time Neisseria gonorrhoeae 2nd Generation Detection Kit (Euroclone®) was performed using Smart Cycler® Dx.

Results: In 85.9% of results of Real-Time PCR and culture were identical. The remaining 14.1% of samples were culture negative and PCR positive. The results ofthe Real-Time PCR and culture were more often concordant in the case of samples obtained from men (96.8%) than from woman (85%). In patients after treatment with antibiotics the concordance of the results obtained with these two methods was 81%.

Conclusions: In laboratory diagnostics of N. gonorrhoeae infections, Real-Time PCR was more sensitive than the culture, but in some cases it was less specific.

Introduction: A rapid growth of the antibiotic resistance among Neisseria gonorrhoeae strains was recently observed in many countries. The common resistance or decreased susceptibility to penicillin, tetracyclines, ciprofloxacin, azithromycin as well as emergence of the first strains resistant to ceftriakson and cefixim is a cause of an anxiety worldwide. Spectinomycin may constitute an alternative therapy of gonorrhoea except of pharyngeal infection.

Methods: The susceptibility to spectinomycin of 65 N. gonorrhoeae strains isolated in the second half of 2012 and the first half of 2013 in Dermatology and Venereology Clinic in Warsaw was investigated. The E-Tests (bioMerieux) were performed and the results (sensitive or resistant) were interpreted according to EUCAST and CLSI recommendations.

Results: The MIC (Minimal inhibitory concentration) values of spectinomycin for the investigated strains ranged from 4,0 mg/L to 32 mg/L, MIC50=16,0 mg/L and MIC=90=16,0 mg/L. It was shown that 100% of the strains was sensitive to spectinomycin according to EUCAST as well CLSI tables.

Conclusions: High susceptibility of the investigated strains to spectinomycin suggests that the drug can be used in the therapy of the resistant gonorrhoea in monotherapy or combined with other drugs.

Introduction: Didecyldimethylammonium chloride is an active substance which is part of variety of formulations used for the disinfection and antisepsis, both in the medical area as well as in the food, industrial and institutional area. Because of the widespread use of this substance and the development of bacterial resistance to quaternary ammonium compounds (QACs), the aim of this study was determination of the susceptibility of the standard strains used for the evaluation of the effectiveness of disinfectants and standard antibiotic-resistant strains to didecyldimethylammonium chloride in 2-propanol and its bactericidal activity.

Methods: Susceptibility of standard strains used for the evaluation of the effectiveness of disinfectants (Staphylococcus aureus ATCC 6538-SA; Pseudomonas aeruginosa ATCC 15442-PA) and standard antibiotic-resistant strains (Staphylococcus aureus ATCC 43300-MRSA; Pseudomonas aeruginosa ATCC 47085-PAO-LAC) to CMAP was determined by minimum inhibitory concentrations (MICs) and minimum bactericidal concentration (MBCs). The bactericidal efficiency of CMAP against these strains was evaluated by using phenol coefficient (PC).

Results: Susceptibility of Gram-positive tested strains SA and MRSA to CMAP was similar (P>0,05). Significant difference in susceptibility of tested Gram-negative strains to CMAP was evaluated between PA and PAO-LAC strains (P<0,05). However,.higher resistance of PAO-LAC to CMAP was not significant when parameters such as concentration and contact time were applied in PC method.

Conclusions: The correct determination and application of "in use" parameters (i.e. concentration, contact time, temperature and interfering substances) in disinfection process prevents the spread of resistant strains in.the environment.

Introduction: Number of infection caused by Clostridium difficile in hospitalized children is increasing. Even though children unlike adults seldom develop complications after being ill, cases of persistent diarrhoea triggered by this pathogen and mortality from this origin have been reported. At present the important problem constitute differentiation between the colonization and infection limiting the proper diagnosis of C. difficile infections (CDI) in this age group. The aim of this study was to evaluate the presence of lactoferrin in faeces as the inflammatory marker confirming C. difficile infections in children.

Methods: Seventy seven samples of faeces where examined. Among them in 55 toxin A/B or C. difficile toxinogenic strain and in 15 nontoxinogenic C. difficile had been detected, 7 were collected from healthy children. Stool samples were tested with the use of method routinely applied in laboratory: automatic VIDAS C. difficile Toxin A&B test (bioMerieux, France), culture and GDH test. Lactoferrin in stool has been identified with ELISA IBD-SCAN test (Techlab, Blacksburg, VA). The CRP protein was detected with VITROS 5600.

Results: Among 55 children with CDI lactoferrin was detected in 45,5% (25/55) of them. In 30 (54,5%) CDI patients the inflammatory biomarker was not identified. In 15 persons with nontoxinogenic strain cultured, one child had lactoferrin present. CDI was detected most frequently (51%) in 6-11 years old children. The increase of CDI cases was observed in period 2013-2014. Neither differences in frequency of raised CRP level in examined groups of children nor correlation between presence of lactoferrin and CRP was observed.

Conclusions: Lactoferrin an intestinal inflammatory biomarker may be a useful tool in distinguishing between C. difficile infection and colonization. More studies including clinical observations are needed.

The phenomenon of multidrug. resistance of bacteria is a serious problem of modern medicine. This resistance largely is a consequence of abuse and improper use of antibacterial substances, especially antibiotics and chemotherapeutics in hospital settings. Multidrug resistance is caused by a number of interacting mechanisms of resistance. Recent studies have indicated that efflux pumps and systems of efflux pumps are an important determinant of this phenomenon. Contribute to this particular RND efflux systems of Gram-negative bacteria, which possess a wide range of substrates such as antibiotics, dyes, detergents, toxins and active substances of disinfectants and antiseptics. These transporters are usually encoded on bacterial chromosomes. Genes encoding efflux pumps' proteins may also be carried on plasmids and other mobile genetic elements. Such pumps are usually specific to a small group of substrates, but as an additional mechanism of resistance may contribute to the multidrug resistance.

Introduction: Traditionally Salmonella enterica subsp. enterica serotypes are identified by slide agglutination with specific antisera for somatic, flagellar and sometimes capsular antigens. An alternative way is genoserotyping using for example a microarray, eg. commercially available test Check&Trace Salmonella. The goal of this study was to evaluate the Check&Trace Salmonella microarray for Salmonella enterica subsp. enterica serotype identification, using Salmonella strains provided by reference laboratories during External Quality Assurance Systems organized for national reference laboratories by ECDC and WHO GFN.

Material and methods: 80 Salmonella enterica subsp. enterica have been tested using Check & Trace Salmonella (Check-Points BC, Netherlands). Also classical slide agglutination was performed according to EN ISO 6579:2003/Al:2007 norm, used as reference method.

Results: In the group of 80 tested strains, 66% were identified correctly, 4% gave uncertain results and 29% showed "Salmonella, genovar" without a serotype, of which 69% were not included in the CTS list of serotypes. Finally one strain has been recognized incorrectly.

Discussion: Because of IVD certification lack, the CTS test could not be recommended to clinical laboratories. AOAC-RI and OIE certification for test cause, that CTS could be used in most food, environmental and veterinary laboratories with the condition, that all unrecognized strains should be sent to a reference laboratory, to type according to EN ISO 6579:2003/Al:2007 norm, by KWM serotyping or other equal alternative methods.

Introduction: Since the 1990s pertussis re-emergence has been observed in many highly immunized countries. Genetic divergence between circulating B. pertussis isolates and vaccine strains has been suggested as one of the reasons responsible for the resurgence of pertussis. This divergence was observed in some studies to affect the effectiveness of pertussis vaccine when tested in murine model. In the study, using the murine intranasal challenge model we evaluated the effectiveness of four experimental wP vaccines, prepared with B. pertussis isolates belonging to different PFGE groups, in the elimination of the bacterial infection induced with mixture of the four B. pertussis isolates.

Methods: The experimental wP vaccines were prepared with clinical isolates belonging to PFGE groups V, IVγ and C, used individually or together. The mixture of four isolates classified to PFGE groups V, IVγ, III and C was used as intranasal mice challenge. The chosen strains represent PFGE groups characteristic for isolates currently circulating in Europe (PFGE groups IV and V), specific for Poland (PFGE group C) and vaccine strains of Polish wP vaccine (PFGE group III). Additionally, to study bacterial fitness, changes in the proportions of four isolates used as the challenge within the course of infection in mice lungs were monitored.

Results and conclusions: All experimental wP vaccines were found to be equally effective in eliminating B. pertussis from mice lungs. Their effectiveness was independent on PFGE group of vaccine strain. The results on bacterial fitness during mixed infections induced in the non-immunized mice found the isolate of PFGE group IVγ dominating among the other isolates used in the mixture belonging to PFGE group III, V, and C. This data might suggest that the isolates belonging to PFGE group IV, so commonly seen in Europe, might be more fitted to explore in conditions of waning immunity.

Introduction: Highly pathogenic Y. enterocolitica bioserotype 1B/O8 is considered to be an important etiological agent of yersiniosis in Poland. Infections caused by Y. enterocolitica 1B/O8 became an important public health problem in Poland, especially because of their high potential of virulence and the unknown source of the bacteria. Y. enterocolitica 1B/O8 isolates recovered in Poland are genetically highly related and constitute single epidemic sensu stricto strain. The aim of the present study was to develop a time- and money-effective molecular assay for rapid identification of pathogenic Y. enterocolitica 1B/O8 isolates belonging to the epidemic strain.

Methods: In the first stage we performed a multiplex-PCR for four genetic markers: ail, ystA, irp1 and 16S rDNA sequence. In the next stage we designed a duplex-PCR-RFLP assay with BtsI endonuclease to detect/identify specific variant of an ysrR gene that is characteristic for epidemic strain of Y. enterocolitica 1B/O8 strain. The assay was tested against a panel of a consisted of a variety Yersinia enterocolitica and Y. pseudotuberculosis strains.

Results: All the tested Y. enterocolitica 1B/O8 strains were positive for all the genetic markers in multiplex-PCR assay what distinguished them from other tested Yersinia strains. In duplex-PCR-RFLP test all tested isolates of the epidemic strain were negative for ysrR digestion with BtsI endonuclease, while all tested reference strains of Y. enterocolitica 1B/O8 were positive.

Conclusions: The assay developed in this study was two-stage/two-step molecular test efficiently distinguishing wild-type and the epidemic Y. enterocolitica 1B/O8 strain. Such test can be a useful screening tool for clinical, veterinary and food diagnostics, as well as for the purposes of epidemiological investigation.