[Infectivity-resistotype-genotype clustering of methicillin-resistant Staphylococcus aureus strains in the Central Blacksea Region of Turkey]. / Türkiye'nin Orta Karadeniz Bölgesinde metisiline dirençli Staphylococcus aureus kökenlerinin enfeksiyözite-rezistotip-genotip kümelenmesi.

The increase in the prevalence of epidemic strains of methicillin resistant Staphylococcus aureus (MRSA) in hospitals and community requires special attention of infection control. The aim of this study was to determine the pathogenic phenotype (i.e. infectivity and resistotype) and genotypic characteristics (i.e. PFGE-pulsotyping, SLST-spa typing, MLST-sequence typing, eBURST-clonal complex detection algorithm) of clinical MRSA isolates in the Central Blacksea region of Turkey, in order to understand their short- and long-term epidemiological and evolutionary dynamics, and to investigate any probable presence of a significant clustering. This prospective study included consecutive but non-repetitive 48 MRSA isolates (of them 18 were colonized strains and 30 were causes of nosocomial infection) and seven methicillin-susceptible S.aureus (MSSA, all were isolated from nosocomial infection), collected between December 2006-February 2007 period from hospitalized patients. Identification of the isolates were performed by Vitek-2 automated system (BioMérieux, USA), and in vitro antimicrobial susceptibility testing by broth microdilution method and Vitek-2 automated system. The MRSA isolates found susceptible to erythromycin (n= 10) were further investigated for the presence of ermA gene by the PCR method. All the strains were typed by spa-typing and PFGE-pulsotyping methods. Among the isolates with different spa-types, representatives were selected (3 MRSA, 7 MSSA) and typed with MLST typing method. Among the isolates with different spa-types, representatives with different antimicrobial susceptibility patterns were selected (n= 8), and SCCmec types were determined by the multiplex PCR method. Antimicrobial resistance patterns of the isolates were digitized to get standardized antimicrobial resistance phenotypes. Clustering of MRSA isolates in pattern groups on the basis of discriminatory characteristics, namely infectivity, phenotype and genotype were statistically analyzed with specific inclusion and exclusion criteria. As a result, three different antimicrobial resistance phenotypes were found in MSSA isolates, whereas 13 were identified in MRSA isolates. In MSSA isolates, seven different PFGE-pulsotypes were detected, as compared to 14 pulsotypes in MRSA isolates. Among MRSA isolates, 10 sporadic strains with single PFGE-pulsotypes were detected. All MRSA isolates, with two exceptions (t459, t632), were of t030 spa-type; in the MLST analysis of the representatives of different spa-types (n= 3), a single type of MLST-clonal complex (CC8) and single MLST-sequence type (ST239) were identified. Each of the seven MSSA isolates yielded different spa-types, MLST-clonal complex types and MLST-sequence types (t777-ST5-CC5; t660-ST25-CC5; t153-ST34-CC30; t015-ST45-CC45; t267-ST97-CC97; t377-ST360-CC8; t084-ST15-C15). In the statistical analysis of 38 non-sporadic MRSA isolates, the isolates in Group-13 (n= 16; infectious, resistotype 14, pulsotype 4; antimicrobial resistance score= 24) displayed significant infectivity-phenotype-genotype clustering (p< 0.001). In 27 of the MRSA isolates, decreased susceptibility to teicoplanin (MIC= 4 µg/mL) was detected. Although, global MRSA isolates belonging to MLST-CC8, MLST-ST239, t030 spa-type were usually expected to be resistant to erythromycin, 10 such strains were erythromycin susceptible. However, ermA gene was found in six of these 10 strains, leading to a conclusion that the ermA gene of these isolates might be dysfunctional due to a point mutation or deletion. Selected representatives of MRSA isolates with different antimicrobial susceptibility patterns (n= 8) were detected to be SCCmec type III. In conclusion, S.aureus isolates in the patient population of our hospital representing the Central Blacksea region showed statistically significant clustering in infectivity, antimicrobial resistance phenotype and clonal genotype (p< 0.001). The dominant MRSA clone was ST239 which was one of the five major pandemic MRSA clones. Nosocomial MSSA isolates displayed long-term clonal diversity. This study produced regional evolutionary-epidemiological data that may support further regional, national and international long-term surveillance studies of S.aureus strains.

[Performance evaluation of VITEK 2 system in meropenem susceptibility testing of clinical Pseudomonas aeruginosa isolates]. / Pseudomonas aeruginosa klinik izolatlarinda VITEK 2 sistemiyle yapilan meropenem duyarlilik testinin performans degerlendirmesi.

Pseudomonas aeruginosa is an important opportunistic pathogen associated with various community-acquired or nosocomial infections. Multi-drug resistant P.aeruginosa strains increasingly cause epidemics and spread in various hospital wards and geographic regions. Carbapenems are among the most effective antimicrobials in the treatment of multi-drug resistant P.aeruginosa infections, and meropenem is the most successful among alternatives in initial therapy. Particularly in severe infections, inappropriate or inadequate initial antimicrobial therapy is independently associated with adverse clinical and economic outcomes. Availability of accurate and rapid susceptibility testing is a priority. Most of the automated microbiology systems can provide rapid results within 8 to 12 hours. In comparison to standard methods, problems in the antimicrobial susceptibility testing of particular microorganisms and antimicrobial agents have been reported for automated microbiology systems. Failures have been reported previously especially in the susceptibility testing of P.aeruginosa versus carbapenem. Most of these studies are designed according to the Food and Drug Administration (FDA, USA) performance analysis scheme (Class II Special Controls Guidance Document: Antimicrobial Susceptibility Test Systems) in a simplified form. However, there are many lacking issues in the design of most of these studies. Among these, insufficient sample size, use of inappropriate reference method, lack of reproducibility testing, and inadequate distribution of study isolates in interpretative categories are of notice. There are only few studies in the literature that evaluate the performance of automated systems in antimicrobial susceptibility testing of carbapenems in clinical P.aeruginosa isolates with a sufficient sample size (n ? 100). However, most of these studies still have one or more major deficiencies in the study design. Furthermore, none of these studies evaluate the performance of VITEK 2 system without a major deficiency in study design. Therefore, we aimed to evaluate the performance of VITEK 2 system (bioMérieux, France) in antimicrobial susceptibility testing of carbapenems in clinical P.aeruginosa isolates in a well-designed study. The study was conducted on nonrepetitive P.aeruginosa isolates (n= 142) of clinical origin. Isolates were selected from the isolate collections of Culture Collection Unit of the Medical Laboratories at Ondokuz Mayis University Hospital. The study collection was characterized with conventional tests and the VITEK 2 automated microbiology system. Broth microdilution method standardized by Clinical and Laboratory Standards Institute was used as the reference method. P.aeruginosa ATCC 27853 was used as the quality control strain in all experimental steps. Twofold dilutions of meropenem (AstraZeneca, USA) concentrations between 64 mg/L and 0.125 mg/L were tested. In compliance with FDA recommendations, minimum inhibitory concentrations of study isolates were shown to be on-scale and distributed within the range of five sequential dilutions in both methods. In reproducibility testing, 15 organisms were tested with VITEK 2 system in triplicate. Results of the reproducibility tests were evaluated in comparison to the test mode (the most frequent test result for the isolate) as a reference. Overall reproducibility was 100%. Essential and categorical agreements of the VITEK 2 system in comparison to the reference method were 83.8% and 96.5%, respectively. Very major and minor discrepancy rates were 1.4% and 2.8%, respectively. There was no major discrepancy. While the results of the essential agreement was not acceptable, categorical agreement was acceptable according to the FDA performance criteria. There was very good agreement between methods as shown by the kappa value (?= 0.938). In conclusion, VITEK 2 system exhibited acceptable performance in the meropenem susceptibility testing of clinical P.aeruginosa isolates. As pre-market approval may not guarantee proper validation, performance of the automated microbiology systems in antimicrobial susceptibility testing should at least be verified and the literature that reports performance evaluation results should be read critically before implementation for routine use in laboratory.

Colorimetric-plate method for rapid disk diffusion susceptibility testing of Escherichia coli.

Here, we report a laboratory-developed colorimetric-plate method for rapid disk diffusion susceptibility testing of Escherichia coli. One hundred isolates were evaluated. Categorical agreement between the colorimetric plate and the standard disk diffusion method was 99%. Mean time to results was 7.07 h (95% confidence interval, 5.96 to 8.19).

[Evaluation of phenol ammonium sulfate sedimentation method for diagnosis of pulmonary tuberculosis]. / Fenol amonyum sülfat sedimentasyon yönteminin pulmoner tüberküloz tanisi için degerlendirilmesi.

In the study, the results of culture have been accepted as a gold standard and the specificity and sensitivity of phenol ammonium sulfate sedimentation method has been evaluated. When it is evaluated according to the results of culture, it has been found that the specificity and sensitivity of phenol ammonium sulfate sedimentation method is 90%, the specificity of the process which is made through the N-acetyl-L-cysteine NaOH method is 90% and the sensitivity of it is 85%. In conclusion, the phenol ammonium sulfate method seems to be as a secure method that can only be used in the laboratories in which microscopic studies are made.

[Meningitis due to Pseudomonas stutzeri: a case report]. / Pseudomonas stutzeri'nin neden oldugu bir menenjit olgusu.

Pseudomonas stutzeri is a saprophytic microorganism that rarely causes severe infections. In this report, a 28 days old male patient with meningomyelocele at birth was presented. The patient was admitted to the hospital with fever, and diagnosed as meningitis on the basis of physical examination and leukocytosis (blood: 16.380/mm3, cerebrospinal fluid (CSF): 130/mm3; 90% PMNL). Following diagnosis ceftriaxone therapy was started led to improvement in clinical and laboratory findings. However on the 20th day, the clinical signs and symptoms became worse, and the patient was diagnosed to develop a second meningitis attack by laboratory examination of CSF. P. stutzeri was isolated from the CSF culture, and the isolate was found to be resistant to ceftriaxone. Upon this result the therapy has changed to meropenem. On the 5th day of the therapy, the patient has slightly improved and he was discharged due to the wishes of his parents, however he died two days after discharge. This first case of P. stutzeri meningitis in neonates was presented to withdraw attention to this clinical entity.