Characterization of resistance genes and replicon typing in Carbapenem-resistant Klebsiella pneumoniae strains.

OBJECTIVE: In our study, K. pneumoniae strains (non-susceptible to carbapenem) (n = 60) were obtained from various clinical samples from Rize State Hospital between 2015 and 2017 and it is aimed to identify antibiotic resistance genes and replicon typing. METHODS: Antibiotic susceptibility tests of the strains were performed with Kirby-Bauer disk diffusion test and the Vitek-2 automated system (BioMerieux, France). Antibiotic resistance genes and replicon typing was characterized by PCR method. RESULTS: It was determined that K. pneumaniae isolates were mostly isolated from the samples of the intensive care unit. All of the K. pneumoniae strains examined in this study were found to be ampicillin/sulbactam and ertapenem resistant but colistin susceptible. Amoxacillin/clavulonic acid resistance was detected at 98.14% of strains. The blaOXA-48 gene was mostly detected in isolates. The most common type of plasmid was I1 and 3 different plasmid types were found in five different strains together. CONCLUSION: This study also shows that the distribution of NDM-1 and OXA-48 carbapenemases has increased since the first co-display in Türkiye and that IncHI1 is the first record in our country. This study provides an overview of the major plasmid families occurring in multiple antibiotic-resistant strains of K. pneumoniae. To our knowledge, this study represents the first report of IncHI1 record in Türkiye.

Stenotrophomonas maltophilia outbreak originating from a pull-out faucet in a pediatric intensive care unit in Turkey: Insights from clinical records and molecular typing.

BACKGROUND: Nosocomial Stenotrophomonas maltophilia-related cases are rising and pose a threat to immunocompromised patients. Twelve patients from our pediatric intensive care unit (PICU) presented with S maltophilia-associated bloodstream infection. METHODS: This outbreak investigation includes 12 patients from PICU between the ages of 2 months and 4 years (mean 16 months, 7 male). To identify the origin, samples from all possible sources throughout the hospital were collected and ran through DNA isolation and Pulse Field Gel Electrophoresis. RESULTS: 120 samples were collected during the outbreak. 31 samples (26%) were positive for S maltophilia. 30 S maltophilia isolates were analyzed, 10 different genotypes were identified. Clustering isolates were grouped into 3 different clusters (tolerance and optimization 1.0, cutoff 90%). The largest cluster was genotype 1, which included 19 isolates, those belong to patients' samples and a sample from a pull-out faucet inside the PICU. The Pull-out faucet was the origin of the bloodstream infection. DISCUSSION: Pull-out faucets allow biofilm production, due its structure. Pulse Field Gel Electrophoresis identifies the transmission dynamics of the outbreak, with its high discriminatory power. CONCLUSIONS: Water sources should be monitored on a regular basis. Pull-out faucets enable bacterial overgrowth; therefore, we recommend water surveillance during outbreak investigations.

Evaluating molecular epidemiology of carbapenem non-susceptible Klebsiella pneumoniae isolates with MLST, MALDI-TOF MS, PFGE.

BACKGROUND: This study aimed to evaluate antibiotic resistance genes and virulence genes and the clonal relationship of the carbapenem-nonsusceptible Klebsiella pneumoniae strains by molecular methods which are isolated from various clinical specimens from patients treated in tertiary care hospital in Turkey. METHODS: Identification of 32 carbapenem non-susceptible K. pneumoniae were determined by VITEK-2 (BioMérieux, France) automated system. Thirteen colistin-resistant strains were tested with the broth microdilution method. Various antibiotic resistance genes and virulence genes frequently seen in carbapenem-resistant strains were screened by PCR. Immunochromatographic tests used in the rapid diagnosis of carbapenemases were compared with PCR results. In addition, PFGE, MLST and MALDI-TOF MS methods were used to determine the clonal relationship among these strains. RESULTS: PCR demonstrated that 31 of the strains carried at least one of the carbapenemase genes. In one strain, the coexistence of blaOXA-48+NDM was shown. The most common resistance genes were determined as blaSHV (84.3%), blaCTX-M-1 (46.8%), blaOXA-48 (40.6%), blaKPC (40.6%), blaTEM (31.2%), blaNDM (18.8%) respectively. Among the virulence genes; magA (68.7%) was the most common, followed by kpn (59.3%) and K2 (9.3%). Immunochromatographic tests were found to be 100% compatible with PCR results. All colistin-resistant isolates were also found to be resistant by colistin broth microdilution. In PFGE analysis, 25 different genotypes were determined and clustering isolates were collected in 5 different clusters and the clustering rate was 35.4%. In MLST analysis, ST101 type was determined as the most common ST type with a rate of 29%. ST101 is followed by ST16, ST307, ST14, ST147, ST309, ST377, ST395 and ST2096, respectively. The compatibility rate between MALDI-TOF MS and VITEK-2 was found 94.3%, in bacterial identification. In MALDI-TOF MS typing, the maximum similarity between the strains was less than 70% and clustering not shown. CONCLUSION: In addition to OXA-48, which is endemic in our country, it has been determined that KPC, which is more common in the world, is becoming increasingly common in our region. ST101 type was determined as the most common type between the strains. To the best of our knowledge, this is the first study that compares these three methods in our country. There may be differences between bacterial identifications made with VITEK-2 and MALDI-TOF MS. In this study, it was observed that MALDI-TOF MS analyses were not compatible with the typing of strains according to PFGE and MLST analysis results.

Comparison of a novel antigen detection test with reverse transcription polymerase chain reaction assay for laboratory diagnosis of SARS-CoV-2 infection.

Molecular diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcription polymerase chain reaction (RT-PCR) in respiratory specimens is considered the gold standard method. This method is highly sensitive and specific but it has some limitations such as being expensive and requiring special laboratory equipment and skilled personnel. RapidFor™ Antigen Rapid Test Kit is a commercially available Ag-RDT which is produced in Turkey and designed to detect the nucleocapsid antigen of SARS-CoV-2 in nasopharyngeal swab samples. The aim of this study was to evaluate the performance of this novel SARS-CoV-2 antigen detection considering the RT-PCR method as the gold standard. Four hundred forty-four nasopharyngeal swab samples which were collected from the patients who met clinical criteria of COVID-19 from ten centers in Turkey between September 2020 and February 2021 were included in the study. All the nasopharyngeal swab samples were tested for SARS-CoV-2 RNA using commercial RT-PCR kits (Bioeksen and A1 Lifesciences, Istanbul, Turkey) according to the manufacturer's instructions. Viral loads were assessed according to the cycle threshold (Ct) values. RapidFor™ SARS-CoV-2 antigen test (Vitrosens Biotechnology, Istanbul, Turkey) was used to investigate the presence of SARS-CoV-2 antigen in all samples following the manufacturer's instructions. Out of 444 nasopharyngeal swab samples tested, 346 (77.9%) were positive and 98 (22.1%) were negative for SARS-CoV-2 RNA by RTPCR. Overall sensitivity of the RapidFor™. Antigen Rapid Test Kit was 80.3% whereas specificity was found to be 87.8%. Positivity rate of rapid antigen test in samples with Ct values over 25 and below 30 was 82.7%, while it increased to 95.7% in samples 20 ≤ Ct < 25 and reached 100% in samples with Ct values below 20. RapidFor™ SARS-CoV-2 Ag test might be a good choice in the screening of symptomatic and asymptomatic patients and their contacts for taking isolation measures early, with advantages over RT-PCR as being rapid, easy and being applicable in every laboratory and even at point of care.

Molecular epidemiological analysis of integron gene cassettes and tetA/tetB/tetD gene associations in Escherichia coli strains producing extended-spectrum ß-lactamase (ESBL) in urine cultures.

BACKGROUND: Epidemiological studies of tetracycline (TE) resistance genes and integron gene cassettes, particularly in urine samples, are limited in Turkey. OBJECTIVES: To investigate antibiotic susceptibility profiles, extended-spectrum beta-lactamase (ESBL) positivity, tet gene types, class-I/-II integron gene cassettes, and clonal relationships among tet-resistant isolates of Escherichia coli from urine cultures of outpatients. MATERIAL AND METHODS: Isolates were identified using conventional methods and the automated Vitek® 2 Compact system. Antimicrobial susceptibility was performed for 19 antibiotics. The ESBL production was performed using the Kirby-Bauer disk diffusion test. The double disk synergy test was used for confirmatory testing. Polymerase chain reaction (PCR) was used to determine the presence of class-I/-II integron gene cassettes and tetA, tetB and tetD resistance genes. The pulsed-field gel electrophoresis typing was performed to identify clonal relations. RESULTS: A total of 121 isolates were obtained and found to be resistant or sensitive to ampicillin and amikacin/imipenem. Resistance to ceftazidime, cefotaxime and ceftriaxone was determined to be 31.3%, 77.6% and 83.1%, respectively. Tetracycline resistance was detected in 82 isolates, mostly caused by the tetB gene. No tet gene was detected in the remaining 39 isolates. Although 64 out of 82 isolates carried a class-I integron, only 4 had a class-II integron (with sizes of 800-2900 base pairs). Furthermore, tet genes were identified with different size class-I integron gene cassettes. However, tet genes were not detected in any isolate identified with integron gene cassette II. Clonally, the isolates were found to be related in subgroups because they were community-acquired. CONCLUSIONS: This study showed that the tetB gene is most commonly found in E. coli isolates grown in urine samples from the Turkish population.

Screening of Antimicrobial Resistance Genes and Epidemiological Features in Hospital and Community-Associated Carbapenem-Resistant Pseudomonas aeruginosa Infections.

INTRODUCTION: Researching carbapenem-resistant isolates enables the identification of carbapenemase-producing bacteria and prevents their spread. METHODS: P. aeruginosa isolates were recovered from Medicine Faculty of Recep Tayyip Erdogan University and identified by conventional methods and the automated Vitek 2 Compact system. Antimicrobial susceptibility experiments were performed in accordance with CLSI criteria and the automated Vitek 2 Compact system. The PCR method was investigated for the presence of ß-lactamase resistance genes. PFGE typing was performed to show clonal relation among samples. RESULTS: Seventy P. aeruginosa isolates were isolated from seventy patients. Of the patients, 67.1% had contact with the health service in the last 90 days and 75.7% of the patients had received antimicrobial therapy in the previous 90 days. Twenty-four isolates were carbapenem resistant, 2 isolates were multidrug-resistant except colistin, and none of the samples had colistin resistance. The gene encoding ß-lactamase or metallo-ß-lactamase was found in a total of 36 isolates. The bla VEB and bla PER genes were identified in 1 and 5 isolates alone or 17 and 13 isolates in combination with other resistance genes, respectively. The bla NDM was the most detected metallo-ß-lactamase encoding gene (n=18), followed by bla KPC (n=12). bla IMP and bla VIM were detected in 5 and 1 isolates, respectively. Also, the association of bla VEB-bla PER and bla VEB-bla KPC-bla NDM was found to be very high. Much more resistance genes and co-occurrence were detected in hospital-acquired samples than community-acquired samples. No difference was found between the community and hospital-associated isolates according to PFGE results. Simultaneously from 6 patients, other microorganisms were also isolated and 5 of them died. CONCLUSION: The average length of stay (days) was found to be significantly higher in HAI group than CAI group. The death of 5 patients with fewer or no resistance genes showed that the co-existence of other microorganisms in addition to resistance genes was important on death.

Determination of antibiotic resistance genes and virulence factors in Escherichia coli isolated from Turkish patients with urinary tract infection.

INTRODUCTION: : Escherichia coli ranks among the most common sources of urinary tract infections (UTI). METHODS: Between November 2015 and August 2016, 90 isolates of E. coli were isolated from patients at Rize Education and Research Hospital in Turkey. Antibiotic susceptibility was determined for all isolates using the Kirby-Bauer disk diffusion method. These E. coli isolates were also screened for virulence genes, ß-lactamase coding genes, quinolone resistance genes, and class 1 integrons by PCR. RESULTS: With respect to the antibiotic resistance profile, imipenem and meropenem were effective against 98% and 90% of isolates, respectively. A high percentage of the isolates showed resistance against ß lactam/ß lactamase inhibitor combinations, quinolones, and cephalosporins. PCR results revealed that 63% (57/90) of the strains carried class 1 integrons. In addition, a high predominance of extended-spectrum ß-lactamases (ESBLs) was observed. The qnrA, qnrB, and qnrS genes were found in 24 (26.6%), 6 (6.6%), and 3 (3.3%), isolates, respectively. The most common virulence gene was fim (82.2%).The afa, hly, and cnf1 genes were detected in 16.6%, 16.6%, and 3.3% of isolates, respectively. Moreover, we observed eleven different virulence patterns in the 90 E. coli isolates. The most prevalent pattern was fim, while hly-fim, afa-aer-cnf-fim, aer-cnf, afa-aer, and afa-cnf-fim patterns were less common. CONCLUSIONS: Most of the E. coli virulence genes investigated in this study were observed in E. coli isolates from UTI patients. Virulence genes are very important for the establishment and maintenance of infection.

Absence of the mecC gene in methicillin-resistant Staphylococcus aureus isolated from various clinical samples: The first multi-centered study in Turkey.

BACKGROUND: mecA is a predefined gene causing methicillin resistance in Staphylococcus aureus (S. aureus) isolates; however, it has been shown that some methicillin-resistant S. aureus (MRSA) strains do not carry this gene. Recently, in isolates found to be MRSA-positive but mecA-negative, a new resistance gene called mecC, which is a homolog of mecA, has been reported. This study aimed to investigate the mecC and mecA genes in MRSA strains isolated from different geographic regions in Turkey. METHODS: The sample of the study consisted of 494 MRSA strains isolated from seven geographical regions in Turkey between 2013 and 2016. The strains were obtained from 17 centers, comprising 13 university hospitals, three education and research hospitals, and one state hospital. Methicillin resistance in S. aureus strains was determined using the agar disk diffusion method with a cefoxitin disk and the agar dilution method with oxacillin. The mecC and mecA genes in MRSA strains was investigated by Polymerase Chain Reaction (PCR). RESULTS: Of the MRSA strains investigated, 47.9% were isolated from intensive care units. Concerning sample type, 36.7% were detected in the respiratory tract (tracheal aspirate, sputum, etc.), 24.8% in blood, 18.7% in skin and soft tissues, 9.3% in nasal swabs, 5.4% in urine, 4.1% in ears, and 1% in sterile body fluid. Using PCR, mecC was not identified in any of the S. aureus strains isolated from different clinical microbiology laboratories. mecA gene positivity was found in 315 of the MRSA strains (63.8%). Staphylococcal Cassette Chromosome mec (SCCmec) type was identified in 232 strains (46.9%), of which 136 (58.7%) were type II, 75 (32.4%) were type IV, 12 (5.1%) were type IIIb, six (2.5%) were type I, and three (1.3%) were type III. CONCLUSION: This is the first multi-centered study to investigate MRSA strains isolated from different regions in Turkey. The mecC gene was not detected in any of the MRSA strains. We believe that this study will constitute an important basis for monitoring possible future changes.

The Antibiotic sensitivity of Stenotrophomonas maltophilia in a 5-year period and investigation of clonal outbreak with PFGE.

INTRODUCTION: Stenotrophomonas maltophilia, which is able to form a biofilm, has mostly been related to catheters when it is the agent in hospital infections; these infections generally present as bacteremia and pneumonia, which may progress with complications and result in death. METHODOLOGY: The study included 153 S. maltophilia strains isolated from clinical samples sent to our hospital laboratory between 1 January 2014 and 30 June 2018. The bacteria were identified and their antibiotic sensitivity was determined using the VITEK-2 automated system. PFGE (Pulsed Field Gel Electrophoresis): The strains isolated from 34 patient clinical samples and from 1 patient bedcover were taken for PFGE examination. RESULTS: The TMP/SXT and levofloxacin sensitivity of 153 S. maltophilia strains was examined. TMP/SXT resistance was determined to be 39% and levofloxacin resistance at 5%. Among 35 S. maltophilia strains, seven genotypes were identified using the PFGE method. While three strains showed a specific genotype profile, the other 32 were determined to consist of four clusters. The cluster rate was therefore 91.4% (32/35). CONCLUSIONS: There was a clonal relationship between the vast majority of the 35 S. maltophilia isolates, which suggests that there was a cross-contamination problem in the hospital. One strain (#4) was identified by dendrogram analysis showed a high rate of similarity to the other strains and was determined to be the common source of the cross-contamination.

Determination of antibiotic resistance genes and virulence factors in Escherichia coli isolated from Turkish patients with urinary tract infection

Abstract INTRODUCTION : Escherichia coli ranks among the most common sources of urinary tract infections (UTI). METHODS: Between November 2015 and August 2016, 90 isolates of E. coli were isolated from patients at Rize Education and Research Hospital in Turkey. Antibiotic susceptibility was determined for all isolates using the Kirby-Bauer disk diffusion method. These E. coli isolates were also screened for virulence genes, β-lactamase coding genes, quinolone resistance genes, and class 1 integrons by PCR. RESULTS: With respect to the antibiotic resistance profile, imipenem and meropenem were effective against 98% and 90% of isolates, respectively. A high percentage of the isolates showed resistance against β lactam/β lactamase inhibitor combinations, quinolones, and cephalosporins. PCR results revealed that 63% (57/90) of the strains carried class 1 integrons. In addition, a high predominance of extended-spectrum β-lactamases (ESBLs) was observed. The qnrA, qnrB, and qnrS genes were found in 24 (26.6%), 6 (6.6%), and 3 (3.3%), isolates, respectively. The most common virulence gene was fim (82.2%).The afa, hly, and cnf1 genes were detected in 16.6%, 16.6%, and 3.3% of isolates, respectively. Moreover, we observed eleven different virulence patterns in the 90 E. coli isolates. The most prevalent pattern was fım, while hly-fım, afa-aer-cnf-fım, aer-cnf, afa-aer, and afa-cnf-fım patterns were less common. CONCLUSIONS: Most of the E. coli virulence genes investigated in this study were observed in E. coli isolates from UTI patients. Virulence genes are very important for the establishment and maintenance of infection.

Effect of mazEF, higBA and relBE toxin-antitoxin systems on antibiotic resistance in Pseudomonas aeruginosa and Staphylococcus isolates.

Background: A toxin-antitoxin (TA) system is a set of two or more closely linked genes that are encoded as a poison and a corresponding antidote on a protein. In typical bacterial physiology, an antitoxin binds to a toxin and neutralizes it, which prevents the bacterium from killing itself. We aimed to determine whether P.aeruginosa and Staphylococcus isolates have TA genes and to investigate whether there is a relationship between the expression levels of TA genes and resistance to antibiotics. Methods: This study included 92 P. aeruginosa and 148 Staphylococcus isolates. RelBE, higBA genes were investigated in P.aeruginosa by multiplex polymerase chain reaction (PCR). The mazEF gene and the all TA genes expression were detected by real time PCR. Results: RelBE and higBA genes were detected in 100% of P. aeruginosa. It was found that the level of relBE TA gene expression is increased in isolates sensitive to aztreonam compared to resistant isolates (p<0.05). The mazEF gene was detected in 89.1% of Staphylococcus isolates. In terms of MazEF gene expression level there was no significant difference between methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates (p>0.05) whereas there was a significant difference between MSSA and coagulase-negative Staphylococcus (CNS) isolates, MRSA and CNS isolates (p<0.05). The levels of mazEF gene expression were found to be higher in isolates sensitive to gentamicin, ciprofloxacin, levofloxacin, clindamycin, phosphomycine, nitrofurantoin, fusidic acid, cefoxitin compared to resistant isolates (p<0.05). Conclusion: Studies on the prevalence and functionality of TA systems emphasize that it may be possible to have new sensitive regions in bacteria by activating TA systems. The results of this study lead to the idea that resistance to antibiotics can be reduced by increasing TA gene expression levels. But there is need for further studies to support and develop this issue.

High prevalence of NDM metallo-ß-lactamase among ESBL-producing Escherichia coli Isolates.

Resistance to ß-lactams in Enterobacteriaceae has been increasing worldwide. This study aimed to determine the frequency of ß-lactamase genes and antibiotic resistance rates of 140 extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli isolates obtained from urinary tract infection in Ordu Province, Turkey. Isolates were identified by classic methods and by automated system. ESBL production was confirmed by double disk synergy test and antimicrobial susceptibility was investigated by disk diffusion method. All isolates were screened for ß-lactamase coding genes from three groups (A, B, and D) by polymerase chain reaction. The highest rate of susceptible isolates was observed for imipenem (IPM, 99.3%) and ertapenem (ETP, 97.9%), and the highest rate of resistant isolates was observed for cefuroxime (97.9%), ceftriaxone (97.2%), and cefazolin (90.7%). In our study, blaCTX-M1-like group was the most prevalent ß-lactamase (n = 109), followed by blaTEM (n = 68), blaCTX-M2 (n = 22), and blaSHV (n = 2). By contrast to low resistance rate to IPM and ETP, we determined blaNDM in 31 isolates (22.1%). In co-prevalence of blaNDM-1 and ESBL-coding genes, a low carbapenem resistance was determined. We can confirm that blaCTX-M1-types are still the most frequent ß-lactamase coding gene in Turkey. Our study showed the highest prevalence of blaNDM-1 metallo-ß-lactamase coding gene in ESBL-producing E. coli.

Evaluation of the Carbapenem Inactivation Method for Detection of Carbapenemase-Producing Gram-Negative Bacteria in Comparison with the RAPIDEC CARBA NP.

Timely detection of carbapenemases by both phenotypic and genotypic methods is essential for developing strategies to control the spread of infections by carbapenem-resistant isolates and related morbidity and mortality. The aim of this study was to compare the performance of a commercial kit, the RAPIDEC® CARBA NP, and an in-house technique, the carbapenem inactivation method (CIM), against a panel of 136 carbapenemase- and noncarbapenemase-producing Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa isolates. RAPIDEC CARBA NP displayed 99% sensitivity and 100% specificity, whereas the sensitivity and specificity were 78% and 100% for the CIM test, respectively. A slight modification of the CIM test, a prolonged incubation time of 4 hours instead of two, increased the sensitivity of the test to 90% by diminishing false negativity particularly for A. baumannii. In conclusion, both tests possess a high performance and are practical for the detection of carbapenemases. Although RAPIDEC CARBA NP is a more rapid and reliable method, the CIM test may represent a useful tool for microbiology laboratories due to its simplicity and availability at any laboratory with low cost.

Kinetic characterization of GES-22 ß-lactamase harboring the M169L clinical mutation.

The class A ß-lactamase GES-22 has been identified in Acinetobacter baumannii isolates in Turkey, and subsequently shown to differ from GES-11 by a single substitution (M169L). Because M169 is part of the omega loop, a structure that is known to have major effects on substrate selectivity in class A ß-lactamases, we expressed, purified and kinetically characterized this novel variant. Our results show that compared with GES-116 × His, GES-226 × His displays more efficient hydrolysis of penicillins, and aztreonam, but a loss of efficiency against ceftazidime. In addition, the M169L substitution confers on GES-22 more efficient hydrolysis of the mechanistic inhibitors clavulanic acid and sulbactam. These effects are highly similar to other mutations at the homologous position in other class A ß-lactamases, suggesting that this methionine has a key structural role in aligning active site residues and in substrate selectivity across the class.

[Characterization of class 1 and class 2 integron gene cassettes in Escherichia coli strains isolated from urine cultures: a multicenter study]. / Idrar kültürlerinden izole edilen Escherichia coli suslarinda sinif 1 ve sinif 2 integron gen kasetlerinin karakterizasyonu: Çok merkezli bir çalisma.

Escherichia coli is the most common pathogen isolated from both nosocomial and community acquired urinary tract infections. Although there are many studies from different centers concerning the antibiotic susceptibility of E.coli isolates in Turkey, the studies are quite few about class 1 and class 2 integron cassettes in clinical E.coli isolates from urinary samples. The aim of the study was to investigate the antibiotic susceptibility and the carriage of integron gene cassettes in E.coli strains isolated from urinary samples. A total of 626 E.coli strains isolated from urine cultures in microbiology laboratories located at 10 provinces from different regions of Turkey (Denizli, Ankara, Kayseri, Nigde, Sanliurfa, Kahramanmaras, Tokat, Malatya, Konya and Trabzon) between June 2011-June 2012 were included in the study. The identification and antibiotic susceptibility testing of the isolates were studied by conventional methods as well as Vitek® 2 Compact (bioMérieux, France) and BD Phoenix™ 100 (Becton Dickinson, USA) systems. The antibiotic susceptibilities of all the isolates were retested by Kirby-Bauer disk diffusion method according to CLSI recommendations in the main center of the study in order to achive the standardization. The presence of integrons was detected with polymerase chain reaction (PCR) method by using specific primers targeting class 1 (intI1) and class 2 (intI2) integrase gene regions. After integron amplification the samples were cloned and subjected to DNA sequencing. When the antibiotic susceptibility of the isolates were evaluated, the highest resistance was observed against most commonly used empirical antibiotics namely ampicillin and trimethoprim-sulfamethoxazole (SXT) with the mean rate of 58.6% (range: 43.8%-73.2%) and 41.2% (range: 35.4%-45.8%), respectively. The most effective antibiotics detected against the isolates were imipenem and amikacin with the lowest resistance rates of 0.2% (range: 0%-1.1%) and 0.6% (range: 0%-3.2%), respectively. The frequency of positive IntI1 gene and class 1 integron gene cassettes were found as 25.8% (162/626) and 16.6% (104/626), respectively, whereas the frequency of positive intI2 gene II and class 2 integron gene cassettes were 5.1% (32/626) and 3% (19/626), respectively. The lowest intI1 gene frequency was detected in the isolates from Kayseri (16.6%) and the highest in the isolates from Kahramanmaras (35.4%) provinces. While there was no intI2 gene in the isolates from Denizli and Kayseri, the highest frequency was 12.1% in the isolates from Sanliurfa province. dfrA1 gene, the most frequent gene among integron gene cassettes was positive in 31 class 1 integron gene cassette alone, and positive with aadA1 gene in 18 class 1 integron gene cassettes. dfrA1 gene was positive with aadA1a just in one isolate. dfrA17 allele was positive in one isolate alone, in 28 isolates with aadA1, and in 15 isolates with aadA5. aadA1 gene was detected in four isolates. dfrA17-sat-aadA5 co-existence was detected among class 2 integron gene cassette in isolates from six provinces. dfrA1-sat-aadA1 was detected in one isolate from Ankara province and dfrA1 was detected in one isolate in Nigde province only. As a result, dfrA1 and aadA1 genes are the most common types of genes among class 1 and class 2 integron gene cassettes in E.coli isolated from urine cultures. It was concluded that high resistance against streptomycin (31.2%) and SXT (41.2%) supported the dissemination of integron-mediated genes dfr, sul1 and aad in the isolates.

A Multicenter Evaluation of Blood Culture Practices, Contamination Rates, and the Distribution of Causative Bacteria.

BACKGROUND: The prognostic value of blood culture testing in the diagnosis of bacteremia is limited by contamination. OBJECTIVES: In this multicenter study, the aim was to evaluate the contamination rates of blood cultures as well as the parameters that affect the culture results. MATERIALS AND METHODS: Sample collection practices and culture data obtained from 16 university/research hospitals were retrospectively evaluated. A total of 214,340 blood samples from 43,254 patients admitted to the centers in 2013 were included in this study. The blood culture results were evaluated based on the three phases of laboratory testing: the pre-analytic, the analytic, and the post-analytic phase. RESULTS: Blood samples were obtained from the patients through either the peripheral venous route (64%) or an intravascular catheter (36%). Povidone-iodine (60%) or alcohol (40%) was applied to disinfect the skin. Of the 16 centers, 62.5% have no dedicated phlebotomy team, 68.7% employed a blood culture system, 86.7% conducted additional studies with pediatric bottles, and 43.7% with anaerobic bottles. One center maintained a blood culture quality control study. The average growth rate in the bottles of blood cultures during the defined period (1259 - 26,400/year) was 32.3%. Of the growing microorganisms, 67% were causative agents, while 33% were contaminants. The contamination rates of the centers ranged from 1% to 17%. The average growth time for the causative bacteria was 21.4 hours, while it was 36.3 hours for the contaminant bacteria. The most commonly isolated pathogens were Escherichia coli (22.45%) and coagulase-negative staphylococci (CoNS) (20.11%). Further, the most frequently identified contaminant bacteria were CoNS (44.04%). CONCLUSIONS: The high contamination rates were remarkable in this study. We suggest that the hospitals' staff should be better trained in blood sample collection and processing. Sterile glove usage, alcohol usage for disinfection, the presence of a phlebotomy team, and quality control studies may all contribute to decreasing the contamination rates. Health policy makers should therefore provide the necessary financial support to obtain the required materials and equipment.

[Investigation of the frequency and distribution of beta-lactamase genes in the clinical isolates of Acinetobacter baumannii collected from different regions of Turkey: a multicenter study]. / Türkiye'nin farkli bölgelerinden toplanan klinik Acinetobacter baumannii izolatlarinda beta-laktamaz gen sikligi ve dagiliminin arastirilmasi: Çok merkezli bir çalisma.

The diversity and distribution of TEM, SHV and CTX-M type of extended-spectrum beta-lactamases (ESBLs) are important for the treatment and control of infections. Determination of ESBL genes in clinical isolates by polymerase chain reaction (PCR) and DNA sequencing can obtain useful data for their molecular epidemiology and risk. The aim of this study was to investigate the frequency of beta-lactamase genes in Acinetobacter baumannii strains isolated from different regions of Turkey. A total of 519 A.baumannii strains collected from hospitals located at 12 different provinces of Turkey (Bolu (n= 67), Tokat (n= 47), Trabzon (n= 25), Ordu (n= 27), Diyarbakir (n= 47), Nigde (n=31), Kayseri (n= 36), Ankara (n= 41), Kirikkale (n= 26), Kahramanmaras (n= 25), Mersin (n= 40), Istanbul (n= 107)] between 2011-2012 period were included in the study. Identification of the isolates were performed by both conventional methods and automated systems, VITEK2 Compact (BioMerieux, France) and API 32GN (BioMerieux, France). Disc diffusion method was used for the detection of antibiotic susceptibilities of the isolates and the results were evaluated according to CLSI (Clinical and Laboratory Standards Institute) criteria. Tigecycline and colistin sensitivities of the isolates were evaluated according to BSAC (British Society for Antimicrobial Chemotherapy) criteria. The presence of beta-lactamase genes, namely blaoxa-51, blaTEM, blaSHV, blaCTX-M1, blaCTX-M2, blaGES and blaVIM were detected by PCR. In our study, the resistance rates against colistin, tigecycline, ampicillin-sulbactam, amoxicillin-clavulanic acid, cefoperazone/sulbactam, tobramycin, ceftriaxone, piperacillin-tazobactam, gentamicin, ampicillin, tetracycline, cefepime, piperacillin, amikacin, trimethoprim-sulfamethoxazole, meropenem, levofloxacin, ciprofloxacin, imipenem and ceftazidime were detected as; 0.6%, 2.7%, 11.9%, 15.2%, 21%, 22.9%, 23.9%, 48.6%, 59.5%, 61.8%, 66.3%, 67.8%, 69.2%, 71.1%, 77.5%, 78.6%, 81.1%, 82.9%, 87.5% and 89.4%, respectively. All of the isolates (100%) were OXA-51 positive, while 443 (85.4%) out of 519 strains harbored other beta-lactamase genes searched in the study. When the distribution of the genes were evaluated, blaTEM-1 was found as the predominant one with a frequency rate of 55.7% (n=289/519), followed by blaCTX-M2 (63/519, 12.1%), blaCTX-M1 (42/519, 8.1%), blaSHV (40/519, 7.7%), blaGES (8/519, 1.5%) and blaVIM (1/519, 0.2%). Cooccurence of ESBL genes was detected in 16.3% (72/443) of the strains, being mostly TEM+CTX-M2 (20/72, 27.8%), TEM+SHV (11/72, 15.3%) and TEM+CTX-M1 (10/72, 13.9%). In addition, it was noted that the distribution of ESBL genes between isolates showed differences according to the provinces. Accordingly, none of the strains isolated from four provinces (Bolu, Nigde, Mersin, Kahramanmaras) and from three provinces (Bolu, Kahramanmaras, Diyarbakir) harbored blaCTX-M1/M2 and blaSHV genes, respectively. The blaTEM gene was detected in isolates collected from all of the provinces, with a highest frequency in Nigde (28/31, 90.3%) and lowest in Trabzon (1/25, 4%). The presence of GES-11 type ESBLs was found only in the isolates sent from Nigde province (8/31; 25.8%). Screening of metallo-beta-lactamase VIM gene also yielded a single positive result amongst only Nigde isolates (1/31; 3.2%), and this gene was identified as VIM-5 type by DNA sequencing. This study which is the first comprehensive national research to characterize ESBLs in A.baumannii isolates by molecular methods, showed that the most prevalent ESBL type is TEM (289/519, 55.7%) amongst A.baumannii strains isolated from different regions of our country. The data of our study is parallel to the results of previous studies carried out from Turkey.

[Antimicrobial susceptibility and molecular characterization of multidrug-resistant Acinetobacter baumannii isolated in an university hospital]. / Bir üniversite hastanesinde izole edilen çok ilaca dirençli Acinetobacter baumannii izolatlarinin antimikrobiyal duyarliligi ve moleküler karakterizasyonu.

Acinetobacter baumannii, an aerobic, non-motile, gram-negative bacterium is an important nosocomial pathogen which shows resistance to the most antibiotics. Carbapenems are the most commonly used antibiotics for the treatment of infections caused by this pathogen. However the emergence of resistance against carbapenems in an increasing rate generates serious problems for antimicrobial therapy. The aims of this study were to detect the antibiotic susceptibility, and the presence of blaOXA resistance genes of clinical A.baumannii isolates and to determine the clonal relationship between these isolates. A total of 79 A.baumannii strains isolated from various clinical specimens (37 respiratory tract samples, 11 wound, 10 blood, 8 catheters, 6 tissue, 5 urine, 2 abscess) of the patients admitted to Mersin University Medical School Hospital between May 2012-January 2013, were included in the study. The isolates were identified by conventional methods and Vitek®2 Compact automated system. Antibiotic susceptibilities of the isolates were determined by Kirby-Bauer disk diffusion method and evaluated according to CLSI criteria. The presence of blaOXA-51, blaOXA-23, blaOXA-24, blaOXA-48 and blaOXA-58 genes were detected by an in-house polymerase chain reaction (PCR), and the clonal relationship between the isolates were identified by pulsed-field gel electroforesis (PFGE) using the ApaI restriction enzyme. In our study, all of the isolates were susceptible to colistin, while the resistance rates against piperacillin-tazobactam, ciprofloxacin, imipenem, meropenem, cefoperazone/sulbactam, trimethoprim-sulfamethoxazole, ceftazidime, levofloxacin, gentamicin, tetracycline, ampicillin-sulbactam, amikacin, netilmicin and tigecycline were 97.5%, 96.2%, 94.9%, 94.9%, 93.6%, 91.1%, 88.6%, 86%, 83.6%, 77.2%, 69.6%, 55.7%, 27.8% and 3.8%, respectively. All the isolates were identified as A.baumannii with the OXA-specific PCR and OXA16S rDNA sequence analysis. All of the isolates (100%) harboured blaOXA-51 and 71 (89.9%) harboured blaOXA-23 gene, however they were all negative for blaOXA-24, blaOXA-48 and blaOXA-58 genes. According to PFGE results 10 pulsotypes were identified, of these eight pulsotypes formed 77 (97.5%) similar strains with indistinguishable PFGE profiles ranging between 3-30 [A (n= 30), B (n= 20), C (n= 9), D (n= 5), E (n= 4), F (n= 3), G (n= 3), H (n= 3)]. When compared with the other clones, clones A and B were dominant among the samples and they have exhibited high level of antibiotic resistance. The rest two pulsotypes [I (n= 1), J (n= 1)] were in close relation with the main cluster. No common outbreak isolate was detected, but the relationship between the majority of the strains pointed out that there was a cross contamination problem in our hospital. In conclusion blaOXA-51 and blaOXA-23 were detected as predominant genes responsible from carbapenem resistance in our clinical A.baumannii strains, and it was considered that the high prevalence of clones A and B may constitute a threat in terms of hospitalized patients.