Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii Blood Culture Isolates from Three Hospitals in Turkey.

We aimed to investigate the clonal relationships, common sequence types, and carbapenemase genes in 177 non-repetitive blood culture isolates of Acinetobacter baumannii collected from patients at three university hospitals in Turkey in 2016. Molecular epidemiological characteristics of the isolates were examined using pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) (Pasteur scheme-cpn60, fusA, gltA, pyrG, recA, rplB, and rpoB). Multiplex PCR was used to investigate the carbapenemase genes, including blaOXA-23-like, blaOXA-24-like, blaOXA-48-like, blaOXA-58-like, blaIMP, blaVIM, and blaNDM. PFGE genotyping yielded 92 pulsotypes with a clustering ratio of 69.7%. As per a ≥85% similarity coefficient, 159 (90.9%) isolates were found to be clonally related. The blaOXA-23-like and blaOXA-58-like genes were identified in 100% and 28.2% of the isolates, respectively. The blaNDM gene was identified in two isolates. The MLST analysis included 54 isolates with different pulsotypes, and 29 sequence types (STs). Most of the isolates (n = 36) belonged to the clonal complex (CC)2, one isolate belonged to CC1, and one isolate belonged to CC164. Sixteen new STs (ST1235-ST1250) were identified. Identifying both global ST2 and a large number of new STs, revealed high genetic diversity in A. baumannii isolates in the study population.

[The Identification of Acinetobacter baumannii Blood Isolates by MALDI-TOF MS, ARDRA and blaOXA-51-like Gene-Specific Real-Time PCR]. / Acinetobacter baumannii Kan Izolatlarinin MALDI-TOF MS, ARDRA ve blaOXA-51-benzeri Gene Özgül Gerçek Zamanli Polimeraz Zincir Reaksiyonu ile Tanimlanmasi.

The Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex consists of phenotypically very similar nosocomial species; A.baumannii, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii and Acinetobacter djikshoorniae and one environmental species A.calcoaceticus. The rapid and accurate identification of the members of Acb complex is critical as these nosocomial pathogens can show differences in antimicrobial susceptibility and clinical outcomes. The conventional phenotypic methods are slow, unreliable and less efficient for the differentiation of Acb complex species, including the A.baumannii species within the Acb complex. Although various molecular methods are available, such as amplified ribosomal DNA restriction analysis (ARDRA) and blaOXA-51-like gene specific PCR, they are usually inconvenient for the routine diagnostic laboratories. Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) offers an opportunity for rapid, cost-effective and convenient bacterial identification in routine diagnostic procedures conducted in clinical laboratory. In this study, we aimed to evaluate the diagnosis performance of MALDI-TOF MS system to identify blood isolates of A.baumannii. A total of 180 nonduplicate carbapenem resistant Acb complex (strain numbers; TR1-TR60) and A.baumannii (TR61-TR180) blood isolates were collected from the intensive care units of the three university hospitals in Turkey from January 2016 to December 2016. All isolates were evaluated by using blaOXA-51-like gene specific real time (Rt-PCR) analysis, ARDRA (restriction enzymes-AluI, CfoI, MboI, MspI, RsaI) method and MALDI-TOF MS (VITEK® MS, bioMérieux, France) system. All the strains except TR10, TR31, TR35 and TR52 were identified as A.baumannii by ARDRA method. Out of 177 of all the isolates, presence of blaOXA-51-like gene was found except for TR10, TR31 and TR52 isolates. However, TR31 without the presence of blaOXA-51-like gene was identified as A.pittii using the ARDRA. Totally 176 isolates which were identified as A.baumannii by both of the methods, ARDRA and Rt-PCR- blaOXA-51-like, were accepted as a reference for the evaluation of the diagnosis performance capacity of the MALDI-TOF MS. Overall, for all 176 isolates tested, the sensitivity obtained with the MALDI-TOF MS were 99.4% with 75% specificity. The accuracy value of the method was determined as 98.9% for the identification of A.baumannii to the species level. MALDI-TOF MS is increasingly used in diagnostic microbiology for the routine identification of bacteria to the genus, species or subspecies level with high rates of sensitivity and specificity. In future, by expanding the database, MALDI-TOF MS system would possibly become the ideal method for routine diagnostic laboratories that could potentially identify more species and even determine some characteristics of antimicrobial resistance and virulence determinants.

Detection of human papillomavirus subtypes harbored in the foreskin of asymptomatic boys: Controlled study.

INTRODUCTION: Human papillomavirus infection (HPV) is one of the most common sexually transmitted infections and can cause penile and anal cancer in men, and invasive cervical cancer in women. OBJECTIVE: To evaulate the colonization of 32 HPV subtypes in the foreskin of boys. STUDY DESIGN: A prospective analysis was made of the data of 62 healthy boys who had undergone standard circumcision. Deoxyribonucleic acid (DNA) was isolated from the foreskin tissues, and the integrity of DNA was tested. The DNA of each patient was further assessed with real-time polymerase chain reaction (PCR) and the presence of 32 subtypes of HPV was explored. To confirm the results, melting curve analysis and agarose gel electrophoresis (AGE) were performed for all samples. Further analysis was made using LCD-array on six randomly selected samples to confirm the results together with negative and positive controls. RESULTS: The mean age of the boys was 6.8 ± 2 years at the time of surgery. All positive controls and samples were positive, all negative controls were negative in the first HPV amplification assay. All positive controls had typical melting curve peaks, whereas all sample amplifications had non-specific, atypical melting curves not fitting with those of the positive controls. Two bands of expected sizes (124 and 405 bp) were only observed in positive controls, but not in negative controls or samples on AGE. The same results were observed on the 6 randomly selected samples using LCD-array. Consequently, all the foreskin samples were evaluated as negative for the 32 HPV types investigated in the study. DISCUSSION: Literature shows a high prevalence of genital HPV in newborns, in early infancy, late adolescence and adulthood. However there is a lack of data in literature on the prevalence in early and late childhood. The negative results of HPV colonization on the foreskin in the current study may be attributed to the conservative and mostly monogamous nature of most family structures in Turkey. CONCLUSION: The results of the present study have shown that foreskin tissue is not a natural reservoir for HPV and subclinical HPV infection is not likely in the absence of suspected sexual contact.

Optimization and validation of a real-time polymerase chain reaction protocol for the diagnosis of human brucellosis.

Due to limitations in commercial diagnostic methods, this study aimed to develop a reliable real-time polymerase chain reaction (Rt-PCR) assay for early diagnosis of brucellosis. Optimization of the Rt-PCR method was performed on serum samples spiked by Brucella melitensis with different densities ranging from 101 to 108 colony-forming units (cfu)/mL; each density was prepared in ten samples. The limit of detection was investigated by using Thermo DNA extraction kit with Maxima SYBR Green Rt-PCR and two TaqMan probe-based Rt-PCR protocols performed by QuantiTect and TEMPase multiplex PCR master mixes in two thermal cyclers, which were Rotor-Gene and Bio-Rad. The validation of the optimized protocol was carried on 20 brucellosis-negative samples and 20 samples spiked with B. melitensis by using a combination of Thermo DNA extraction kit with TEMPase PCR master mix. SYBR Green Rt-PCR yielded positive results on all samples having ≥ 104 cfu/mL of B. melitensis in both thermal cyclers. Its limit of detection was 112 DNA copies per reaction. The positivity of both probe-based Rt-PCR protocols was 100% and 80% on the samples having 103 cfu/mL and 102 cfu/mL of B. melitensis, respectively. The limit of detection of probe-based protocols was defined as 4 DNA copies per reaction. The optimized Rt-PCR protocol showed high-level accuracy, precision, specificity, and sensitivity, each having a rate of 100%. The current study indicated that the TaqMan probe-based Rt-PCR protocol optimized and validated with serum samples can be reliably used for early diagnosis of brucellosis.

Risk factors for rectal colonization of carbapenem-resistant Enterobacteriaceae in a tertiary care hospital: a case-control study from Turkey

Background/aim: This study aimed to evaluate the risk factors of patients colonized with carbapenem-resistant Enterobacteriaceae (CRE). Materials and methods: The study was conducted between January 2010 and March 2016. The colonized group consisted of patients who had a CRE strain in their rectal swab cultures, whereas patients with negative rectal surveillance cultures for CRE who were concurrently hospitalized in the same units with the colonized group patients were included in the control group. Results: The number of patients in the colonized and the control group was 71 and 120, respectively. Both groups were evaluated for demographic and healthcare-associated characteristics. Isolated microorganisms in rectal surveillance cultures for CRE were Klebsiella pneumoniae (75.5%), Escherichia coli (15.5%), Enterobacter cloacae (4.2%), Klebsiella oxytoca (1.4%), and Klebsiella terrigena (1.4%). The isolates were resistant to imipenem, meropenem, and ertapenem (52.1%, 73.2%, and 100%, respectively). In multivariate analysis, presence of decubitus, colistin usage, glycopeptide usage, and fluoroquinolone usage were found to be independent risk factors for CRE colonization. There was no significant difference between the two groups with regards to mortality (P = 0.070). Conclusion: These results are in agreement with the current literature. The findings of this study could be useful for improvement of infection control strategies related to CRE

Antimicrobial Activity of Garlic Derivatives on Common Causative Microorganisms of the External Ear Canal and Chronic Middle Ear Infections.

OBJECTIVE: Today, antibiotic resistance is increasing and evolving into an important health problem. Therefore, it is important to research on alternative therapies to antibiotics. This study aimed to investigate the inhibitory effect of four garlic derivatives on microorganisms commonly isolated in ear infections. METHODS: The antimicrobial activities of allicin, s-allyl cysteine (SAC), diallyl disulfide (DADS), and s-allyl mercaptocysteine (SAMC) were investigated on standard strains of commonly isolated microorganisms using the broth microdilution method. The test strains were selected among the microorganisms responsible for chronic suppurative otitis media and otitis externa. These microorganisms were Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, Candida albicans, and Candida tropicalis. RESULTS: Minimum inhibitory concentration (MIC) values of allicin and SAC ranged from 0.125 to 20 µg/mL for fermentative bacteria (E. coli and K. pneumoniae), 20 to 80 µg/mL for non-fermentative bacteria (P. aeruginosa and A. baumannii), 5 to 10 µg/mL for gram-positive cocci (S. aureus and E. faecium), and 40 to 80 µg/mL for yeasts (C. albicans and C. tropicalis). MIC values of DADS ranged from 40 to 80 µg/mL for fermentative bacteria, 40 to 160 µg/mL for non-fermentative bacteria, 40 to 80 µg/mL for gram-positive cocci, and 20 to 40 µg/mL for yeasts. The MICs of SAMC were >640 µg/mL for the tested bacteria and yeasts. CONCLUSION: Both allicin and SAC showed antimicrobial activity against the tested microorganisms, even at low concentrations. These two derivatives may be used to treat infections in the future.

Comparison of multiplex real-time polymerase chain reaction with serological tests and culture for diagnosing human brucellosis.

OBJECTIVE: Brucellosis is a zoonotic disease with various clinical presentations and early diagnosis is crucial to avoid severe complications. Due to limitations of conventional diagnostic methods, polymerase chain reaction (PCR) based approaches have gained importance in diagnosis.We aimed to evaluate diagnostic value of multiplex real time-PCR (mRT-PCR) in serum samples collected from brucellosis suspected patients by comparision sensitivity of mRT-PCR with those of conventional diagnostic methods. MATERIAL AND METHODS: A total of 249 serum samples collected from the suspected brucellosis patients admitted to the hospitals in three different provinces were analyzed by serological tests, culture and mRT-PCR. In laboratories of the participating hospital, serum samples were tested for the Brucella specific antibody by commercial serological kits including standart tube agglutination test (STAT), Coombs' test, and immunocapture test (ICT). Blood culture was performed for 153 of the patients in the participating hospital. All serum samples were analyzed for the presence of Brucella DNA by mRT-PCR. RESULTS: According to laboratory test results, 215 of the 249 suspected cases having comparible clinical data were identified as brucellosis cases. Of the 215 brucellosis cases, 36 were diagnosed as definitive cases, the remaning 179 patients were presumptive cases. Sensitivity of mRT-PCR in the samples that were positive by ICT, STAT, Coombs' test, and blood culture was 70.2%, 77.3%, 83%, and 97.2%, respectively. By using mRT-PCR, additional 17 suspected patients were diagnosed as presumptive cases. Among the mRT-PCR positive serum samples, Brucella abortus was detected in 3 samples (1.9%), the remaining 156 samples (98.1%) had B. melitensis DNA. CONCLUSION: Our results indicate that mRT-PCR can be considered a useful diagnostic tool in patients who have negative serologic test results, and in detection of Brucella species.

[Comparison of two commercial DNA extraction kits and PCR master mixes for the detection of Brucella from blood samples and blood culture bottles]. / Kandan ve kan kültür siselerinden Brucella saptanmasinda iki farkli ticari DNA ekstraksiyon kiti ve PCR master kiksinin karsilastirilmasi.

The diagnosis of human brucellosis requires culture or serological tests for the conformation of the clinical findings. Isolation of the bacteria is used as a gold standard, however it is time consuming and processing of positive cultures has a potential risk for laboratory acquired human brucellosis. Polymerase chain reaction (PCR) based methods have offered new approaches for early diagnosis of brucellosis and reduce the risk of laboratory acquired human brucellosis. A major limitation of the PCR method is the difficulty to remove the inhibitors in specimens. The aim of this study was to determine the performance of two DNA extraction kits by using two separate PCR master mixes and to determine appropriate "extraction kit - PCR master mix" combination for the diagnosis of Brucella from whole blood samples and blood culture bottles. Two commercial DNA extraction kits, NORGEN Blood DNA isolation kit (Norgen) and Thermo Scientific GeneJet Whole blood genomic DNA purification kit (Thermo Fisher Scientific, USA) and two PCR master mixes, QuantiTect multiplex PCR (QuentiTect, Qiager, Almanya) and Ampliqon Multiplex TEMPase (Amliqon, Denmark) were assessed on 30 simulated blood samples with known concentrations (102-104 cfu/ml) of Brucella melitensis ATCC 23456 strain and 10 blood culture bottles that gave positive signal. By using different combinations of extraction kits and PCR master mixes, a total of 160 different multiplex real-time PCR (Rt-PCR) trials were performed with probes and primers specific to Brucella spp., B.melitensis, and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All the 120 Rt-PCR trials performed on the DNA samples extracted from blood samples gave positive results with GAPDH probe/primers. The rate of positive PCR results for Brucella spp. was 96.7% for the combination of Norgen-QuantiTect, 93.3% for Thermo-Ampliqon, 93.3% for Thermo-QuantiTect, and 86.7% for Norgen-Ampliqon. The frequency of positive B.melitensis results for these combinations were 96.7%, 93.3%, 56.7% and 90%, respectively. In the samples with the bacterial density of 102 cfu/ml, Brucella spp. detection rates were 80% for Thermo-Ampliqon and Norgen-Ampliqon, and 90% for Thermo-QuantiTect and Norgen-QuantiTect; and for B.melitensis positivite rates were 90%, 70%, 20%, and 90%, respectively. Rt-PCR assays with the DNA samples extracted from blood culture bottles using Norgen isolation kit yielded 80% positivite result. However, the frequency of PCR positivite results was only 20% in the DNA samples extracted by Thermo DNA extraction kit. PCR result for GAPDH gene was also negative in 80% of the samples extracted by Thermo kit. Our results revealed that for the removal of inhibitors and detection of even low number of Brucella spp./B.melitensis in blood samples and blood culture bottles, NORGEN Blood DNA isolation kit can be used with a combination of QuantiTect multiplex PCR or Ampliqon Multiplex TEMPase.

Role of efflux pump and OprD porin expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates.

INTRODUCTION: In recent years, the prevalence of multidrug-resistant P. aeruginosa has remarkably increased. Thus, we wanted to investigate the carbapenem resistance mechanisms and clonal relationship among 80 carbapenem-resistant P. aeruginosa strains. METHODOLOGY: Carbapenemase production was detected using the Modified Hodge Test (MHT), EDTA combined disc method (ECD), and PCR. Expression levels of efflux and porin genes were mesured by real-time reverse transcription PCR. Clonal relationship of the isolates was investigated by pulsed-field gel electrophoresis (PFGE). RESULTS: Carbapenemase production was detected in 7.5% of the isolates with MHT/ECD tests and in 11.3% of the isolates with PCR. Although the specificity of MHT/ECD was high, the sensitivitivity was low. oprD downregulation and mexB, mexY, and mexD overexpression were demonstrated in 55%, 16.3%, 2.5%, and 2.5% of the isolates, respectively. Multiple carbapenem resistance mechanisms were found in nearly a quarter of the isolates. PFGE typing of the 80 P. aeruginosa isolates yielded 61 different patterns. A total of 29 isolates (36.3%) were classified in 10 clusters, containing 2 to 7 strains. We could not find a strict relationship between PFGE profile and carbapenem resistance mechanisms. CONCLUSIONS: Although oprD downregulation and MexAB-OprM overexpression were the most common mechanisms, carbapenem resistance was associated with multiple mechanisms in the study. MHT/ECD tests should not be used alone for investigation of carbapenemase production in P. aeruginosa. Rapid tests with high sensitivity and specificity should be developed for the detection of carbapenemase production in P. aeruginosa.

Investigation of an algorithm for anti HCV EIA reactivity in blood donor screening in Turkey in the absence of nucleic acid amplification screening.

PURPOSE: In this study we aimed to propose an algorithm for initial anti HCV EIA reactive blood donations in Turkey where nucleic acid amplification tests are not yet obligatory for donor screening. METHODS: A total of 416 anti HCV screening test reactive donor samples collected from 13 blood centers from three cities in Turkey were tested in duplicate by Ortho HCV Ab Version 3.0 and Radim HCV Ab. All the repeat reactive samples were tested by INNO-LIA HCV Ab 3.0 or Chiron RIBA HCV 3.0 and Abbott Real Time HCV. Intra-assay correlations were calculated with Pearson r test. ROC analysis was used to study the relationship between EIA tests and the confirmatory tests. RESULTS: The number of repeat reactive results with Ortho EIA were 221 (53.1%) whereas that of microEIA, 62 (14.9%). Confirmed positivity rate was 14.6% (33/226) by RIBA and 10.6% (24/226) by NAT. Reactive PCR results were predicted with 100% sensitivity and 95% specificity with S/CO levels of 8.1 with Ortho EIA and 3.4 with microEIA. CONCLUSIONS: Repeat reactivity rates declined with a second HCV antibody assay. Samples repeat reactive with one HCV antibody test and negative with the other were all NAT negative. All the NAT reactive samples were RIBA positive. None of the RIBA indeterminate or negative samples were NAT reactive. Considering the threshold values for EIA kits determined by ROC analysis NAT was decided to be performed for the samples above the threshold value and a validated supplemental HCV antibody test for the samples below.

Risk factors for infection with colistin-resistant gram-negative microorganisms: a multicenter study.

BACKGROUND: Knowing risk factors for colistin resistance is important since colistin is the only remaining choice for the treatment of infections caused by multi-drug resistant microorganisms. OBJECTIVE: Evaluate risk factors associated with infection by colistin-resistant microorganisms. DESIGN: Retrospective study. SETTING: Tertiary healthcare centers. PATIENTS AND METHODS: An e-mail including the title and purpose of the study was sent to 1500 infec.tious disease specialists via a scientific and social web portal named "infeksiyon dunyasi (infection world)". Demographic and clinical data was requested from respondents. MAIN OUTCOME MEASURE(S): Colistin-resistance. RESULTS: Eighteen infectious disease specialists from twelve tertiary care centers responded to the invitation data was collected on 165 patients, 56 cases (39.9%) and 109 (66.0%) age- and sex-matched controls. The colistin-resistant microorganisms isolated from cases were 29 Acinetobacter baumannii (51.8%), 18 Pseudomonas aeruginosa (32.1%) and 9 Klebsiella spp. Colistin, carbapenem, and quinolone use in the last three months were risk factors for colistin resistance in the univariate analysis. Previous quinolone use in the last three months (P=.003; RR:3.2; 95% Ci:1.5-6,7) and previous colistin use in the last three months (P=.001; RR: 3.6; 95% CI: 1.63-7.99) were significant risk factors in the multivariate analysis. CONCLUSION: Clinicians should limit the use of quinolones and remain aware of the possibility of resistance developing during colistin use. LIMITATIONS: The lack of a heteroresistance analysis on the isolates. no data on use of a loading dose or the use of colistin in combination.

[Bacteremia caused by ciprofloxacin-resistant Salmonella serotype Kentucky: a case report and the review of literature]. / Siprofloksasine dirençli Salmonella serotip Kentucky bakteriyemisi: Olgu sunumu ve literatürün gözden geçirilmesi.

Salmonella infections can be seen in four clinical types, namely gastroenteritis, bacteremia/sepsis, enteric fever and carriage. These infections can result in uncomplicated diarrhea in most cases, but can lead to invasive disease requiring antimicrobial therapy and can be life-threatening in elderly or immunocomprimised patients. Broad-spectrum cephalosporins and fluoroquinolones are crucial options in the treatment of the invasive infections. Ciprofloxacin resistance is rarely seen in non-typhoid Salmonella enterica isolates, and only in S. Typhimurium, S. Choleraesuis and S. Schwarzengrund. In this report, we aimed to discuss a patient infected with ciprofloxacin-resistant Salmonella Kentucky under the light of data from our country and the world. A 52-year-old male patient wih acute myocardial infarction was hospitalized in intensive care unit of cardiovasculer surgery for left ventricular assist device (LVAD) implantation for the treatment of left ventricular disfunction. On the seventh day of LVAD and coronary artery bypass grafting (CABG), the patient presented high fever and productive cough. His physical examination revealed hyperemia around the insertion point of right jugular central venous catheter (CVC) and a serous discharge from the insertion point of LVAD located just below the inferior edge of sternum. Empiric IV cefoperazone/sulbactam (SCF) therapy was started with the prediagnosis of pneumonia and bloodstream infection. The blood samples taken from peripheral veins and CVC, and swabs taken from LVAD insertion point for culture when the patient was febrile, revealed the growth of bacteria with S type and lactose-negative colonies on EMB and SS media. Biochemical characteristics of the isolate were as follows: lactose fermentation negative, H2S positive, IMVIC (-,+,-,+), urease negative, lysine/ornithine decarboxylase positive and motile. The bacteria was then identified as Salmonella enterica serotype Kentucky (8,20;i;z6) by agglutination tests. Antibiotic susceptibility tests were conducted according to CLSI guidelines and it was found as ampicillin- and ciprofloxacin-resistant. Ciprofloxacin resistance of the isolate was confirmed with E-test. Stool culture was performed to investigate the source of infection, and S. Kentucky was isolated. On the 15th day of SCF treatment, LVAD was taken out, and tissue cultures taken from the fibrillar tissues between pericardial layers during surgery, also yielded S. Kentucky growth. On the second day of SCF therapy the patient's fever returned normal and on the seventh day, CBC and CRP values were normalized. Nevertheless, the clinical situation of the patient worsened gradually and on the 40th day he was intubated due to low oxygen saturation and pleural effusion. His antibiotherapy was stopped on 42nd day as the blood cultures were negative and his clinical situation was attributed to cardiac failure. The patient died four days after the antibiotherapy has stopped due to cardiac reasons. To our knowledge, this is the first reported case infected with ciprofloxacin-resistant Salmonella Kentucky in our country.

[Extended-spectrum beta-lactamase production by Enterobacteriaceae isolates from urine cultures of outpatients: results of a 7-year follow-up]. / Ayaktan basvuran hastalarin idrar kültürlerinde üretilen Enterobacteriaceae izolatlarinda genislemis spektrumlu beta-laktamaz üretimi: 7 yillik izlem sonuçlari.

The aim of the study was to evaluate the change of the frequency of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae isolates from urine samples of outpatients in years and to analyse the antibiotic resistance profiles for a rational drug use. The urine samples cultured in our laboratory from the patients who were admitted to outpatient clinics of our hospital between years 2007-2013 were included in this study. Enterobacteriaceae strains were isolated and identified by conventional methods and API 20E system (BioMérieux, France). The standard antimicrobial susceptibility tests were performed by Kirby Bauer disk diffusion method. ESBL production were screened by double-disk synergy method according to CLSI guidelines. E-test method (BioMérieux, France) were used for the verification of suspicious ESBL production. The identification and antimicrobial susceptibility testing were performed for a total of 12.535 isolates. Of the isolates 8716 were identified as Escherichia coli (69.3%), 1514 were Klebsiella pneumoniae/oxytoca (12.1%), 257 were Proteus mirabilis (2.1%), 345 were other Enterobacteriae members (8%), 411 were various non-fermentative gram-negative bacteria (3.3%) and 1292 were various gram-positive bacteria (10.3%). The total positivity rate of ESBL was found as 21.8% (2.283/10.487), and the ESBL positive rates for E.coli, K.pneumoniae/oxytoca and P.mirabilis were 21.2%, 28.2% and 4.7%, respectively. Other Enterobacteriaceae isolates were not evaluated because of the absence of standardized methods and breakpoint values. There was no statistically significant difference among ESBL producing isolates within seven years (p= 0.364). The antibiotic resistance rates of the ESBL-positive isolates were statistically higher than ESBL-negative isolates [amoxicillin-clavulanate (73.1%/11.3%), trimethoprim-sulfamethoxazole (63.1%/31.0%), nitrofurantoin (17.3%/8.6%), gentamicin (42.2%/10.1%), amikacin (3.5%/0.9%), tobramisin (56.8%/10.5%), imipenem (0.3%/0.1%), ofloxacin (66.8%/19.8%), ticarcillin-clavulanate (73.5%/19.8%), piperacillin-tazobactam (28.8%/5.0%)] (p< 0.05). Statistically significant variations were detected within the years for the resistance rates of amoxicillin-clavulanate (p= 0.001), tobramycin (p=0.003), ofloxacin (p= 0.001), ticarcillin-clavulanate (p= 0.001) and piperacillin-tazobactam (p= 0.001) were detected within the years. Although a quite high percentage of ESBL positivity in Enterobacteriaceae isolates was determined, there was a slight but not statistically significant increase of this value during the seven-year period. The stability of the percentage of ESBL positivity may indicate a positive change in the habit of the usage of beta-lactam antibiotics. According to the results of our study, the most effective drugs for ESBL-producing isolates were piperacillin-tazobactam among inhibitor combinations, amikacin among aminoglycosides and nitrofurantoin among orally-used drugs.

Colistin alone or combined with sulbactam or carbapenem against A. baumannii in ventilator-associated pneumonia.

INTRODUCTION: Colistin use has increased over the last ten years because of multidrug-resistant microorganisms. The aim of this study was to compare the clinical and microbiological efficacy of colistin alone or in combination with sulbactam or carbapenem in the treatment of ventilator-associated pneumonia (VAP) due to multidrug-resistant (MDR) and extremely drug-resistant (XDR) A. baumannii. METHODOLOGY: Cases treated for VAP because of MDR and XDR A. baumannii between January 2011 and January 2013 were included in the study. The primary and secondary outcome for colistin alone, colistin with sulbactam, and colistin with carbapenems were evaluated. The primary outcomes were clinical efficacy and microbiological efficacy; the secondary outcomes were nephrotoxicity, length of hospitalization, and mortality. RESULTS: A total of 70 VAP patients were evaluated. A total of 17 patients (24.3%) were administered colistin alone, 20 patients (28.6%) were administered colistin and sulbactam, and 33 patients (47.1%) were administered colistin and carbapenem. Clinical and microbiological response rates were higher in the carbapenem combination group (63.6% and 63.6% in both) than in the sulbactam combination group, which registered 55.0% and 60.0%, respectively. However, this did not represent a significant difference statistically (p > 0.05). There was also no significant difference between colistin alone and the combination groups regarding clinical and microbiological efficacy and mortality. CONCLUSIONS: Neither the administration of colistin alone nor colistin combined with either sulbactam or carbapenem had any noticeable advantage in the treatment of VAP in terms of clinical response, microbiological response, nephrotoxicity, length of hospitalization, and mortality.

Molecular characterization of vancomycin-resistant Enterococcus faecium strains isolated from carriage and clinical samples in a tertiary hospital, Turkey.

This study aimed to determine the presence of vancomycin resistance (vanA and vanB) and virulence genes (esp, asa1, gelE, ace, hyl, cylA, cpd and ebpA) in vancomycin-resistant Enterococcus faecium (VREfm) strains and to analyse the clonal relationships among the strains. E. faecium strains were identified from rectal and clinical specimens by biochemical tests and the API-20 Strep kit. Susceptibility testing was performed using disc-diffusion and broth-dilution methods. PFGE was used for molecular typing of the VREfm strains. The vancomycin resistance and virulence genes were amplified by two-step multiplex PCR. All 55 VREfm isolates were resistant to penicillin G, ampicillin and high-level gentamicin but were susceptible to quinupristin/dalfopristin and linezolid. Multiplex PCR analysis indicated that all isolates harboured vanA and that 41 (75 %) were positive for virulence genes. The esp gene was the most common virulence factor and was detected in nine (41 %) invasive and 32 (96.7 %) non-invasive isolates. Multiple virulence genes were observed only in two non-invasive isolates; one harboured esp and ebpA and the other harboured esp, ebpA, asa1, gelE and cpd. PFGE typing yielded 16 different types, seven of which were clusters with two to 14 strains each. The clustering rates of the rectal swab, blood and urine isolates were 72.7 %, 61.5 % and 87.5 %, respectively. The genetic similarity observed among the VREfm isolates indicated cross-transmission in the hospital. Further studies on the virulence factors present in the strains might provide insight into the acquisition of these traits and their contribution to increased prevalence of VREfm.

Brucellosis as an aetiology of septic arthritis.

Brucellosis is a zoonotic disease caused by a Gram-negative coccobacillus from the Brucella genus. The disease has a broad spectrum of clinical manifestations. The musculoskeletal system involvement is frequent and, rarely, arthritis can be the only clinical feature of the disease. We report a case of monoarthritis caused by Brucella melitensis.

[Fungal keratitis caused by Scedosporium apiospermum: first report from Turkey]. / Scedosporium apiospermum'a Bagli Fungal Keratit: Ülkemizden Ilk Olgu.

Fungal keratitis, an eye infection with poor prognosis, is difficult to treat and can lead to loss of vision. Among filamentous fungi Scedosporium spp. rarely lead to fungal keratitis. Here we present a case of keratitis caused by Scedosporium apiospermum. A 61-year-old female patient was admitted to our hospital with the complaints of right eye pain and decreased vision after a foreign body trauma to the right eye. The patient was diagnosed as keratitis by biomicroscopic examination. Conjunctival swabs collected from both eyes were inoculated onto sheep blood agar, chocolate agar, eosin methylene blue agar and Sabouraud dextrose agar. Corneal scrapings from the right eye were inoculated onto the same solid media by "C-streak" method, and in brain-heart-infusion broth by immersion. While gram-stained smears of conjunctival swabs showed no significant finding, smears of corneal scrapings revealed abundant neutrophils and profuse septate hyphae. Fungal keratitis was diagnosed and topical enhanced amphotericin B (0.5 mg/ml) therapy was initiated with netilmicin sulfate and oxytetracycline HCl plus polymyxin B sulfate. At the 10th day of therapy a mold growth was detected in corneal scraping cultures and was identified microscopically as S.apiospermum. Based on the relevant literature, therapy was changed to enhanced topical voriconazole (2 mg/ml) applied hourly, plus systemic voriconazole administration. At the third day of treatment, reduction of epithelial defect and decline in the focus of keratitis were observed. In the following days, however, a progression occurred in the focus of keratitis and 5% natamycin ophthalmic suspension was added to the therapy. Since the patient did not respond to any of the medical treatments, therapeutic penetrating keratoplasty was planned; yet, the patient refused the operation and was discharged with her own request. As far as the local literature was concerned, this is the first report of keratitis caused by S.apiospermum in Turkey. Though a very rare causative agent of keratitis, S.apiospermum is generally resistant to antifungal therapy and often require surgical treatment. Especially in patients with predisposing factors, this organism should be kept in mind as a potential causative agent and relevant microbiological examinations should be performed.

Does treatment affect the levels of serum interleukin-6, interleukin-8 and procalcitonin in diabetic foot infection? A pilot study.

AIMS: To investigate about serum PCT, IL-6 and IL-8 levels and how they are affected by the treatment in diabetic foot patients. METHODS: Fifty patients' blood samples were taken to study ESR and CRP, IL-6, IL-8 and PCT before and at the 14th day of the treatment. RESULTS: The pretreatment results of the 50 patients showed positive correlations between PCT and either ESH (r=0.49, p<0.001), or CRP (r=0.56, p<0.001). Similarly, there was a positive correlation between IL-6 and ESH (r=0.46, p=0.001), just like as it was between IL-6 and CRP (r=0.54, p<0.001). At the 14th day, the levels of ESR (70 ± 30.2 and 58.4 ± 26.2, p=0.02), CRP (63.8 ± 73.1 and 18.1 ± 19.7, p<0.001) and PCT (0.6 ± 2.1 and 0.05 ± 0.02, p=0.007) were significantly decreased while IL-6 was decreased at a close range to statistical significancy at healing patients (97.5 ± 147.2 and 47.1 ± 77.6; p=0.05), but they did not at nonhealing patients. IL-8 levels were not changed anyhow. CONCLUSIONS: PCT was significantly decreased such as ESR and CRP were in the early phase of healing; IL-6 and IL-8 levels were also decreased by the treatment, but not statistically significantly. IL-6 and PCT were affected in correlation with the other inflammatory parameters in the beginning, but IL-8 was not. PCT and IL-6 may be useful like CRP and ESR in the diagnosis and follow up of diabetic foot infection, but IL-8 is not. Further investigation is needed.

[In vitro susceptibility of methicillin-resistant Staphylococcus aureus isolates to vancomycin, teicoplanin, linezolide and daptomycin]. / Metisiline dirençli staphylococcus aureus izolatlarinin vankomisin, teikoplanin, linezolid ve daptomisine in vitro duyarliliklari.

In this study, vancomycin, teicoplanin, linezolide and daptomycin susceptibility rates of 67 methicillin-resistant Staphylococcus aureus (MRSA) isolates obtained from various clinical samples between November 2006 and August 2010 in our laboratories, were investigated by E-test method and MIC values of the drugs were determined. Seventeen (25%) of the samples were from outpatient wards, 50 (75%) from inpatients of which 24 (48%) were from intensive care units. Distribution of MRSA isolated clinical samples were as follows: 16 (23.4%) blood, 28 (42.2%) wound swab, 15 (21.8%) tracheal aspirate, 2 (3.1%) urine, 2 (3.1%) urethral discharge, and one for each (1.6%) cerebrospinal fluid, joint fluid, catheter tip and nasal swab. Except one (1.5%) which was probably intermediate-resistant to vancomycin (since not confirmed by microdilution test or population analysis, this isolate was considered as "probable" intermediate-resistant), all of the isolates were found susceptible to all tested antibiotics. MIC(50) and MIC(90) values were determined as 0.75 and 1.5 µg/ml for vancomycin, 2 and 3 µg/ml for teicoplanin, 0.38 and 0.5 µg/ml for linezolide and 0.094 and 0.19 µg/ml for daptomycin, respectively. The MIC ranges were 0.25-3 µg/ml for vancomycin, 0.125-4 µg/ml for teicoplanin, 0.094-3 µg/ml for linezolide and 0.047-0.25 µg/ml for daptomycin. There was no statistically significant difference between MICs of outpatient, inpatient and intensive care unit isolates for any of the tested drugs (p> 0.05). Based on MIC90 values, daptomycin seems 4-16 times more effective than the other three drugs. It was concluded that considering their in-vitro antibacterial activity, these antibiotics can be used as alternatives to each other for the treatment of MRSA infections.

[Antibiotic resistance profiles of Acinetobacter species isolated from several clinical samples between 2007-2010]. / Çesitli klinik örneklerden 2007-2010 yillarinda izole edilen acinetobacter türlerinin antibiyotik direnç profilleri.

This study was aimed to investigate the changes in antibiotic resistance profiles of Acinetobacter spp. in our hospital during a four-year period. The study included a total of 465 non-duplicate Acinetobacter spp. isolated from various samples sent from intensive care (n= 274, 58.9%), inpatient (n= 141, 30.3%) and outpatient (n= 49, 10.5%) units of our hospital between 2007 and 2010. Sample distribution was as follows: 184 tracheal aspirates (39.5%), 70 blood (15.3%), 92 (19.8%) wound, 40 urine (8.6%), 24 sputum (5.1%), 22 (4.7%) bronchial lavage and 22 (4.7%) other (catheter tip, cerebrospinal fluid, thorasynthesis material) samples. The isolates were identified as A.baumannii (n= 340, 73.1%), A.lwoffii (n= 64, 13.7%) and Acinetobacter spp. (n= 61, 13.1%). The susceptibility profiles were investigated by Kirby-Bauer disc diffusion method. Overall, the results indicated an increase in resistance against all tested drugs since 2007. A steady increase of resistance from 2007 to 2009, followed by a tendency to decrease in 2010 was also noted for all drugs, except for ceftazidime (CAZ), trimethoprim-sulfomethoxazole (SXT), netilmicin (NET), imipenem (IPM), meropenem (MER) and gentamicin (CN). NET, IPM, cefepime and MER resistance rates increased regularly from 2007 to 2010. CAZ resistance followed a fluctuating course, while CN resistance displayed a decreasing trend since 2009. According to the statistical analyses (X2 and Fisher’s exact test), there was a regular resistance increase between 2007-2009 except for amikacin (AK), SXT and PIP. Resistance rates were also increased for AK and PIP, but only between 2007 and 2009; as well as for piperacillin-tazobactam, ticarcilin-clavulanate, NET, MER and IPM between 2008 and 2009. A significant increase from 2008 to 2010 was observed for NET; and a significant resistance decrease in 2010 was noted for only sultamicillin, cefotaxime, CN and tobramycin (TOB) (p< 0.05). As of 2010, the results indicated high resistance rates against ciprofloxacin [resistance rate (RR): 79%], NET (RR: 60%) and all beta-lactam drugs, including carbapenems (mean RR: 80%). Moreover, there was a progressive increase in resistance to carbapenems and NET, two very important treatment alternatives. Tigecycline (RR: 5.5%), TOB (RR: 19%), CN (RR: 34%) and cefoperazone-sulbactam (RR: 38%) appeared to remain as relatively effective treatment choices. The resistance rates of inpatient and outpatient isolates which were usually lower than those of the intensive care unit isolates, also displayed a noteworthy increase over the past four years. Evidently, pan-resistant Acinetobacter spp. will become a serious health problem in the near future, unless efficient and appropriate precautions are taken.