A Streptococcus pneumoniae lineage usually associated with pneumococcal conjugate vaccine (PCV) serotypes is the most common cause of serotype 35B invasive disease in South Africa, following routine use of PCV.

Pneumococcal serotype 35B is an important non-conjugate vaccine (non-PCV) serotype. Its continued emergence, post-PCV7 in the USA, was associated with expansion of a pre-existing 35B clone (clonal complex [CC] 558) along with post-PCV13 emergence of a non-35B clone previously associated with PCV serotypes (CC156). This study describes lineages circulating among 35B isolates in South Africa before and after PCV introduction. We also compared 35B isolates belonging to a predominant 35B lineage in South Africa (GPSC5), with isolates belonging to the same lineage in other parts of the world. Serotype 35B isolates that caused invasive pneumococcal disease in South Africa in 2005-2014 were characterized by whole-genome sequencing (WGS). Multi-locus sequence types and global pneumococcal sequence clusters (GPSCs) were derived from WGS data of 63 35B isolates obtained in 2005-2014. A total of 262 isolates that belong to GPSC5 (115 isolates from South Africa and 147 from other countries) that were sequenced as part of the global pneumococcal sequencing (GPS) project were included for comparison. Serotype 35B isolates from South Africa were differentiated into seven GPSCs and GPSC5 was most common (49 %, 31/63). While 35B was the most common serotype among GPSC5/CC172 isolates in South Africa during the PCV13 period (66 %, 29/44), 23F was the most common serotype during both the pre-PCV (80 %, 37/46) and PCV7 period (32 %, 8/25). Serotype 35B represented 15 % (40/262) of GPSC5 isolates within the global GPS database and 75 % (31/40) were from South Africa. The predominance of the GPSC5 lineage within non-vaccine serotype 35B, is possibly unique to South Africa and warrants further molecular surveillance of pneumococci.

In-vitro activity of fosfomycin against Escherichia coli and Klebsiella pneumoniae bloodstream isolates and frequency of OXA-48, NDM, KPC, VIM, IMP types of carbapenemases in the carbapenem-resistant groups.

The aim of this study was to determine the in-vitro activity of fosfomycin against Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) isolates and the frequency of OXA-48, NDM, KPC, VIM, IMP types of carbapenemases in the carbapenem-resistant (CR) groups. A total of 346 isolates (126 E. coli and 220 K. pneumoniae) from nosocomial bloodstream infections were included. Carbapenem and fosfomycin susceptibility were tested by Etest (bioMerieux, France) and agar dilution methods, respectively and evaluated in accordance with EUCAST criteria. The presence of OXA-48, NDM, KPC, VIM, IMP types of carbapenemases were conducted by using PCR method. Of the total 346 isolates, 185 (41 E. coli, 144 K. pneumoniae) were CR. Fosfomycin susceptibility of E. coli was higher than 95% and was not statistically significant between the CR and carbapenem-susceptible (CS) groups. Fosfomycin susceptibility of CS and CR K. pneumoniae was 90.7% and 69.4%, respectively, and statistically significantly lower in CR group. Of the total 185 CR isolates, 163 (32 E. coli, 131 K. pneumoniae) were producing carbapenemases. OXA-48 was the prominent carbapenemase type produced by E. coli (96.8%) and K. pneumoniae (70.9%). The frequency of NDM and KPC types produced by K. pneumoniae was 20.6% and 15.2%, respectively. Fosfomycin has substantial in-vitro activity against nosocomial CS and CR E. coli and CS K. pneumoniae bloodstream isolates. However, due to the risk of emerging resistance with fosfomycin monotherapy, combination therapy should be considered to obtain the possible additive or synergistic activity. Emerging fosfomycin resistance of CR K. pneumoniae isolates is alarming and OXA-48 is still the prominent carbapenemase type in Turkey.

Prevalence of O25b-ST131 clone and fosfomycin resistance in urinary Escherichia coli isolates and their relation to CTX-M determinant.

Escherichia coli ST131 clone and H30-R/H30-Rx subclones are the most common multidrug-resistant high-risk clones in UTIs. Antimicrobial susceptibility of fosfomycin was compared to five other agents in consecutively collected 299 urinary isolates using the agar dilution method. Prevalence of the ST131 clone and the occurrence of blaCTX-M were also investigated. Overall resistance to fosfomycin, cefuroxime, and ceftriaxone were 2.7%, 35.4%, and 30.1% respectively. fosA, fosA3, and fosC2 genes were not detected. In isolates resistant to ciprofloxacin (34.7%), the prevalence of ST131 clone was 31.7%, of which 81.8% belonged to H30-R and 66.7% to H30-Rx subclones. None of the isolates of the ST131 clone were resistant to fosfomycin. However, blaCTX-M occurred in 57.6% of the isolates among this clone, 62.9% in H30-R and 68.2% in H30-Rx subclones. The results of this study suggest that fosfomycin resistance is not prevalent in urinary isolates, however, blaCTX-Mpositive ST131 clone is quite common.

Levels of pro- and anti-inflammatory cytokines in cystic fibrosis patients with or without gingivitis.

BACKGROUND: Inflammatory periodontal diseases are caused by interaction between gram negative, anaerobic bacteria and host response. Persistent infection of Pseudomonas aeruginosa in cystic fibrosis (CF) patients also cause increased pro-inflammatory response and the imbalance of pro- and anti-inflammatory response in brochoalveolar lavage fluid which leads to destruction of lungs. The aim of this study is to evaluate periodontal status of CF patients, to measure level of cytokines and biochemical molecules in gingival crevicular fluid (GCF), and to detect presence of P. aeruginosa in dental plaque samples. MATERIALS AND METHODS: GCF samples were collected from 41 CF patients and 39 healthy (non-CF) subjects. Interleukin (IL)-1ß, IL-17, IL-10, human neutrophil elastase (HNE), cystic fibrosis transmembrane regulator (CFTR) protein, and human ß-defensin-1 (HBD1) in GCF were evaluated by ELISA method. Dental plaque samples were collected from 18 CF patients with history of P. aeruginosa colonization and 15 non-CF subjects. Presence of P. aeruginosa was evaluated by using conventional culture methods and molecular methods. RESULTS: Levels of IL-1ß, HNE, and HBD1 in CF patients were significantly higher than non-CF subjects. However, IL-10 level was significantly lower in CF patients. Increased pro-inflammatory (IL-1ß) and decreased anti-inflammatory (IL-10) cytokine levels were observed in GCF samples from CF patients, irrespective of their periodontal status. P. aeruginosa were detected in four samples of 18 CF patients, and all were negative in non-CF group. CONCLUSIONS: As a result of this study, CF coexists increasing pro-inflammatory and decreasing anti-inflammatory response locally. Due to increasing pro-inflammation, CF patients should be followed-up more often than non-CF children.

A new look at the etiology of interstitial cystitis/bladder pain syndrome: extraordinary cultivations.

PURPOSE: So far, studies have not clearly identified infectious agents as an etiological factor for interstitial cystitis (IC). Specific microbiological diagnosis for detecting the pathogen with higher sensitivity in IC may decrease the treatment costs and increase psychosocial health of the patients. METHODS: A prospective clinical study was performed in 26 IC patients and 20 controls between April and September 2017. All participants were asked to give mid-stream urine sample for routine urine cultures. Followed by the negative results, symptomatic 26 patients were evaluated for L-form pathogen existence by extraordinary cultivation methods. Biopsy samples were taken from 19 patients with ulcerative lesions in the bladder while collecting sterile urine samples from all 26 patients. PG broth, 5% sheep blood agar, EMB, Sabouraud's dextrose, LEM, and GYPA were used. Followed by the 1st day inoculations, all inoculated PG broths were subcultured into the same solid media at the 2nd and 10th days in case of any growth after incubation of 24 h under 35-37 °C. The "O'Leary Sant Symptom and Problem Index" score forms were used to evaluate response to the appropriate treatment for those patients with documented pathogens. RESULTS: Bacterial isolations were yielded from samples of 13 IC patients in PG broth. Eight (61.5%) P. aeruginosa, 2 (15.4%) K. pneumoniae, 2 (15.4%) C. mucifaciens, and 1 (7.7%) E. faecalis were isolated. Antibiotic susceptibility tests were performed. Somehow, the median symptom index and problem scores of those 13 IC patients were lower after the appropriate antibiotic treatment (p < 0.05). CONCLUSIONS: Extraordinary mediums with longer incubation periods may reveal a causative pathogen in the etiology of IC. Future culture techniques may have some value, because still some of IC/BPS patients are describing symptomatic relief by a group of antibiotics.

A prospective multicenter study on the evaluation of antimicrobial resistance and molecular epidemiology of multidrug-resistant Acinetobacter baumannii infections in intensive care units with clinical and environmental features.

BACKGROUND: Multidrug-resistant (MDR) Acinetobacter baumannii infections are considered as emerging nosocomial infections particularly in patients hospitalized in intensive care units (ICUs). Therefore, reliable detection of MDR strains is crucial for management of treatment but also for epidemiological data collections. The purpose of this study was to compare antimicrobial resistance and the clonal distribution of MDR clinical and environmental A. baumannii isolates obtained from the ICUs of 10 different hospitals from five geographical regions of Turkey in the context of the demographic and clinical characteristics of the patients. METHODS: A multicenter-prospective study was conducted in 10 medical centers of Turkey over a 6 month period. A total of 164 clinical and 12 environmental MDR A. baumannii isolates were included in the study. Antimicrobial susceptibility testing was performed for amikacin (AN), ampicillin-sulbactam (SAM), ceftazidime (CAZ), ciprofloxacin (CIP), imipenem (IMP) and colistin (COL) by microdilution method and by antibiotic gradient test for tigecycline (TIG). Pulsed-field gel electrophoresis (PFGE) was performed to determine the clonal relationship between the isolates. The detection of the resistance genes, blaOXA-23, blaOXA-24, blaOXA-51, blaOXA-58, blaIMP, blaNDM, blaKPC, blaOXA-48 and blaPER-1 was carried out using the PCR method. RESULTS: The mortality rate of the 164 patients was 58.5%. The risk factors for mortality included diabetes mellitus, liv1er failure, the use of chemotherapy and previous use of quinolones. Antimicrobial resistance rates for AN, SAM, CAZ, CIP, IMP, COL and TIG were 91.8%, 99.4%, 99.4%, 100%, 99.4%, 1.2% and 1.7% respectively. Colistin showed the highest susceptibility rate. Four isolates did not grow on the culture and were excluded from the analyses. Of 172 isolates, 166 (96.5%) carried blaOXA-23, 5 (2.9%) blaOXA-58 and one isolate (0.6%) was positive for both genes. The frequency of blaPER-1 was found to be 2.9%. None of the isolates had blaIMP, blaKPC, blaNDM and blaOXA-48 genes. PFGE analysis showed 88 pulsotypes. Fifteen isolates were clonally unrelated. One hundred fifty-seven (91.2%) of the isolates were involved in 14 different clusters. CONCLUSIONS: Colistin is still the most effective antibiotic for A. baumannii infections. The gene blaOXA-23 has become the most prevalent carbapenemase in Turkey. The distribution of invasive A. baumannii isolates from different regions of Turkey is not diverse so, infection control measures at medical centers should be revised to decrease the MDR A. baumannii infections across the country. The results of this study are expected to provide an important baseline to assess the future prophylactic and therapeutic options.

Global Distribution of Invasive Serotype 35D Streptococcus pneumoniae Isolates following Introduction of 13-Valent Pneumococcal Conjugate Vaccine.

A newly recognized pneumococcal serotype, 35D, which differs from the 35B polysaccharide in structure and serology by not binding to factor serum 35a, was recently reported. The genetic basis for this distinctive serology is due to the presence of an inactivating mutation in wciG, which encodes an O-acetyltransferase responsible for O-acetylation of a galactofuranose. Here, we assessed the genomic data of a worldwide pneumococcal collection to identify serotype 35D isolates and understand their geographical distribution, genetic background, and invasiveness potential. Of 21,980 pneumococcal isolates, 444 were originally typed as serotype 35B by PneumoCaT. Analysis of the wciG gene revealed 23 isolates from carriage (n = 4) and disease (n = 19) with partial or complete loss-of-function mutations, including mutations resulting in premature stop codons (n = 22) and an in-frame mutation (n = 1). These were selected for further analysis. The putative 35D isolates were geographically widespread, and 65.2% (15/23) of them was recovered after the introduction of pneumococcal conjugate vaccine 13 (PCV13). Compared with serotype 35B isolates, putative serotype 35D isolates have higher invasive disease potentials based on odds ratios (OR) (11.58; 95% confidence interval[CI], 1.42 to 94.19 versus 0.61; 95% CI, 0.40 to 0.92) and a higher prevalence of macrolide resistance mediated by mefA (26.1% versus 7.6%; P = 0.009). Using the Quellung reaction, 50% (10/20) of viable isolates were identified as serotype 35D, 25% (5/20) as serotype 35B, and 25% (5/20) as a mixture of 35B/35D. The discrepancy between phenotype and genotype requires further investigation. These findings illustrated a global distribution of an invasive serotype, 35D, among young children post-PCV13 introduction and underlined the invasive potential conferred by the loss of O-acetylation in the pneumococcal capsule.

Epidemiology of carbapenem-resistant Klebsiella pneumoniae colonization: a surveillance study at a Turkish university hospital from 2009 to 2013.

Between June 2009 and December 2013, 4105 patients were screened for carbapenem-resistant Klebsiella pneumoniae (CR-Kp) colonization in a tertiary care university hospital. The antimicrobial susceptibility and resistance determinants of 279 (6.8%) CR-Kp isolates from single patients were investigated. Additional analysis was performed to evaluate the characteristics and various risk factors for infection in patients with colonization. Of the 279 isolates, 270 harboured OXA-48-like enzymes, and a single isolate harboured IMP-type carbapenemase. A high proportion of isolates were susceptible to carbapenems - except ertapenem. All isolates were susceptible to amikacin and most (94%) were susceptible to colistin and fosfomycin. There was consistent high-level resistance for all isolates to temocillin, piperacillin-tazobactam, amoxicillin-clavulanate and ticarcillin-clavulanate. When colonized and infected patients were compared, only prior carbapenem administration (P = 0.003), was found to be significantly associated with patients with CR-Kp infection.

Antimicrobial Activity of Copper Alloys Against Invasive Multidrug-Resistant Nosocomial Pathogens.

The emergence and spread of antibiotic resistance demanded novel approaches for the prevention of nosocomial infections, and metallic copper surfaces have been suggested as an alternative for the control of multidrug-resistant (MDR) bacteria in surfaces in the hospital environment. This study aimed to evaluate the antimicrobial activity of copper material for invasive MDR nosocomial pathogens isolated over time, in comparison to stainless steel. Clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) (n:4), OXA-23 and OXA-58 positive, MDR Acinetobacter baumannii (n:6) and Pseudomonas aeruginosa (n:4) were evaluated. The antimicrobial activity of coupons containing 99 % copper and a brass alloy containing 63 % copper was assessed against stainless steel. All the materials demonstrated statistically significant differences within each other for the logarithmic reduction of microorganisms. Among the three materials, the highest reduction of microorganisms was seen in 99 % copper and the least in stainless steel. The result was statistically significant especially for 0, 2, and 4 h (P = 0.05). 99 % copper showed a bactericidal effect at less than 1 h for MRSA and at 2 h for P. aeruginosa. 63 % copper showed a bactericidal effect at 24 h for P. aeruginosa strains only. Stainless steel surfaces exhibited a bacteriostatic effect after 6 h for P. aeruginosa strains only. 99 % copper reduced the number of bacteria used significantly, produced a bactericidal effect and was more effective than 63 % copper. The use of metallic copper material could aid in reducing the concentration of bacteria, especially for invasive nosocomial pathogens on hard surfaces in the hospital environment.

Invasive pneumococci before the introduction of pneumococcal conjugate vaccine in Turkey: antimicrobial susceptibility, serotype distribution, and molecular identification of macrolide resistance.

This study evaluates the antimicrobial susceptibilities and serotype distributions of invasive Streptococcus pneumoniae (SP) isolates identified in a Turkish hospital before the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7). The susceptibilities of all isolates were determined by evaluating six antibiotics: penicillin (PEN), ceftriaxone (CRO), levofloxacin (LEV), erythromycin (ERY), clindamycin (CD), and vancomycin (VAN). Serotyping and amplification of macrolide resistance genes were performed. Sixteen (50%) and four (2%) isolates were resistant to PEN and LEV, respectively. No isolates demonstrated VAN resistance. Intermediate resistance to CRO was found in 4% of all invasive isolates. Twenty-three (12.6%) isolates were resistant to ERY. Four (2%) invasive SP isolates demonstrated multidrug resistance. Serogroups 3, 5, 6, 8, 9, and 23 were the most common in both age groups. The potential coverage rates of PCV7 and PCV13 were 44.1 and 66.1% in children and 39.8 and 71.5% in adults, respectively. Continuous surveillance of antimicrobial resistance is required.

[Comparison of GeneXpert® vanA/vanB PCR system and culture methods in the surveillance of vancomycin-resistant enterococci]. / Vankomisine dirençli enterokoklarin sürveyansinda GeneXpert® vanA/vanB PCR sistemi ile kültür yönteminin karsilastirilmasi

Vancomycin-resistant enterococci (VRE) are important agents of hospital infections worldwide. Early recognition of VRE colonization is important in the control of hospital infections. The aim of this study was to compare a real-time PCR (Rt-PCR) system and culture methods in the detection of VRE colonization. A total of 210 perirectal swab samples obtained from the patients (142 were in internal and 68 were in surgical intensive care units) hospitalized at Ankara Training and Research Hospital, Turkey between January-September 2013 were included in the study. The samples were simultaneously evaluated with both Rt-PCR (GeneXpert®vanA/vanB, Cepheid, USA) and the culture methods. The samples were cultivated in enterococcosel agar and incubated at 370C for culture. Culture plates were evaluated for three days on a daily basis. Bacterial identification was done by conventional methods and automated Vitek 2.0 system (BioMérieux, France). Antibiotic susceptibility testing was performed by the E-test. VRE was detected in 76 (36.1%) of the samples by the Rt-PCR method; of them 70 were positive for vanA, two for vanB, and four for vanA + vanB. On the other hand, VRE was detected in 71 (33.8%) of the samples by the culture method. Out of 71 samples, colony growth was observed on the first day in 39 cases, on the second day in 29 cases, and on the third day in three cases. The two strains identified as vancomycin-sensitive enterococci by the Vitek 2 Compact system were determined as vanB positive by PCR. These samples were also confirmed as VRE by E-test. The PCR result of a sample which was found to be invalid, also yielded negative result by culture. Five out of the seven culture-negative samples were positive for vanA, and two for vanB by the GeneXpert® system. In our study, the sensitivity, specificity, positive and negative predictive values of the GeneXpert vanA/vanB PCR system were determined as 97.4%, 98.4%, 97.4%, and 97.4%, respectively. Although the GeneXpert® vanA/vanB RT-PCR method seems to be more attractive regarding the turn around time, it has a higher cost than the culture method. Thus, it was concluded that all laboratories should choose the most appropriate method for screening VRE in the hospital setting according to their own capacities.

Global phylogenomic analysis of nonencapsulated Streptococcus pneumoniae reveals a deep-branching classic lineage that is distinct from multiple sporadic lineages.

The surrounding capsule of Streptococcus pneumoniae has been identified as a major virulence factor and is targeted by pneumococcal conjugate vaccines (PCV). However, nonencapsulated S. pneumoniae (non-Ec-Sp) have also been isolated globally, mainly in carriage studies. It is unknown if non-Ec-Sp evolve sporadically, if they have high antibiotic nonsusceptiblity rates and a unique, specific gene content. Here, whole-genome sequencing of 131 non-Ec-Sp isolates sourced from 17 different locations around the world was performed. Results revealed a deep-branching classic lineage that is distinct from multiple sporadic lineages. The sporadic lineages clustered with a previously sequenced, global collection of encapsulated S. pneumoniae (Ec-Sp) isolates while the classic lineage is comprised mainly of the frequently identified multilocus sequences types (STs) ST344 (n = 39) and ST448 (n = 40). All ST344 and nine ST448 isolates had high nonsusceptiblity rates to ß-lactams and other antimicrobials. Analysis of the accessory genome reveals that the classic non-Ec-Sp contained an increased number of mobile elements, than Ec-Sp and sporadic non-Ec-Sp. Performing adherence assays to human epithelial cells for selected classic and sporadic non-Ec-Sp revealed that the presence of a integrative conjugative element (ICE) results in increased adherence to human epithelial cells (P = 0.005). In contrast, sporadic non-Ec-Sp lacking the ICE had greater growth in vitro possibly resulting in improved fitness. In conclusion, non-Ec-Sp isolates from the classic lineage have evolved separately. They have spread globally, are well adapted to nasopharyngeal carriage and are able to coexist with Ec-Sp. Due to continued use of PCV, non-Ec-Sp may become more prevalent.

[Carbapenem resistance in ESBL positive Enterobacteriaceae isolates causing invasive infections]. / Invazif enfeksiyonlara neden olan GSBL pozitif Enterobacteriaceae izolatlarinda karbapenem direnci.

The aim of this study was to investigate the presence of carbapenem resistance in Enterobacteriaeceae isolates recovered from invasive infections, in Hacettepe University Hospital, Ankara, Turkey, between 2005-2009, by phenotypic and genotypic methods. A total of 210 non-duplicated Escherichia coli (n= 153), Klebsiella pneumoniae (n= 47) and Klebsiella oxytoca (n= 10) isolates which were all determined to be extended-spectrum beta-lactamase (ESBL) positive with the BD Phoenix automated identification and antibiotic susceptibility system (Sparks, USA), were included in the study. The isolates were recovered from patients with bloodstream infections. Susceptibility of the isolates to imipenem, meropenem and ertapenem was detected with microdilution method according to the standards of Clinical and Laboratory Standards Institute (CLSI) minimal inhibitory concentration (MIC) breakpoints. Doripenem susceptibility was detected by the E-test (bioMerieux, Hazelwood, USA). All isolates which were found to be non-susceptible to any of the carbapenem antibiotics tested, were characterized by the phenotypic confirmatory tests and the presence of the resistance genes; blaAmpC, blaCTX-M, blaKPC, blaNDM, blaOXA, blaIMP ve blaVIM were screened by polymerase chain reaction (PCR). Among the 210 ESBL-producing Enterobacteriaceae blood isolates, 23 (11%) were identified as non-susceptible to any of the carbapenems tested. Resistance rates for imipenem, meropenem and ertapenem were 5.7% (n= 12), 1.9% (n= 4) and 2.4% (n= 5), respectively. Doripenem was more active than the other carbapenems, with a resistance rate of 1.0%. Seven of 23 isolates were ESBL negative with cefotaxime/clavulanic acid (CTX/CLA) and ceftazidime/clavulanic acid (CAZ/CLA) combined disk diffusion test, however, six of them were ESBL positive with the addition of boronic acid (BA) to CTX/CLA. Among the three isolates positive for Modifiye Hodge test (MHT) and/or ertapenem-BA tests, blaOXA-48 was detected in one and blaAmpC in the other. Phenotypic pAmpC activity was present in three K.pneumoniae isolates of which one was positive for blaAmpC gene. One K.pneumoniae isolate resistant to all carbapenems with MICs > 256 µg/ml and positive for phenotypic meropenem-BA, MHT, imipenem-EDTA, ceftazidime-CAZ/CLA, cefoxitin-BA production, was found to inhabit blaOXA-48 gene. Five isolates were positive for blaOXA-1 and one for blaOXA-10. Two isolates were positive for blaCTX-M, however blaIMP, blaVIM and blaNDM-1 genes were not detected among the isolates. In conclusion, carbapenem non-susceptibility which was low among the Enterobacteriaceae strains isolated in our center, was mostly attributed to the presence of blaOXA type carbapenemases and no accumulation of blaKPC and blaNDM were detected.

Molecular characterization of oxacillinases and genotyping of invasive Acinetobacter baumannii isolates using repetitive extragenic palindromic sequence-based polymerase chain reaction in Ankara between 2004 and 2010.

BACKGROUND: Multidrug-resistant Acinetobacter baumannii (MDRAB) is an increasing problem worldwide. We aimed to determine the antibiotic susceptibility, diversity of oxacillinases, and molecular types of MDRAB. METHODS: A total of 100 non-duplicate A. baumannii blood culture isolates were evaluated. Antimicrobial susceptibilities of the isolates were determined according to the standard Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. Colistin, doripenem, and tigecycline susceptibilities were analyzed by E-test. The presence of bla(OXA-23-like), bla(OXA-24-like), bla(OXA-51-like), and bla(OXA-58-like) genes was investigated by multiplex polymerase chain reaction (PCR). Typing of A. baumannii isolates was performed using repetitive extragenic palindromic sequence-based PCR (rep-PCR; DiversiLab). RESULTS: Most isolates were susceptible to colistin (98% susceptible) and tigecycline (94% susceptible), whereas fewer isolates were susceptible to imipenem, meropenem, and doripenem (17%, 17%, and 18% susceptible, respectively). Carbapenem resistance was associated with the presence of bla(OXA-23-like) (31% of isolates) and bla(OXA-58-like) (23% of isolates) genes. The occurrence of isolates carrying bla(OXA-58-like) genes increased between y 2004 and 2009, but decreased in 2010. In contrast, isolates with bla(OXA-23-like) genes increased during the 2008-2010 period. Out of 100 isolates, 62 were categorized into 13 major rep-PCR patterns, with the highest prevalence in pattern 1 (10 isolates), followed by patterns 2 and 3 (9 isolates each). CONCLUSIONS: Carbapenem-resistant invasive A. baumannii isolates carrying the bla(OXA-23-like) gene became more prevalent and replaced isolates carrying the bla(OXA-58-like) carbapenemase gene through the 7 y. Rep-PCR genotyping of these strains confirmed that ongoing MDRAB resulted from a long-term persistence and mixture of several clusters.

[Comparison of culture and real-time PCR methods in the detection of Streptococcus pneumoniae and Haemophilus influenzae in acute otitis media effusion specimens]. / Akut Orta Kulak Iltihabi Örneklerinde Streptococcus pneumoniae ve Haemophilus influenzae'nin Saptanmasinda Kültür ve Gerçek Zamanli PCR Yöntemlerinin Karsilastirilmasi

Streptococcus pneumoniae and Haemophilus influenzae are the major etiologic agents of acute otitis media. This study was aimed to compare the detection rate of S.pneumoniae and H.influenzae by culture and real-time polymerase chain reaction (Rt-PCR) in the middle ear effusions of patients diagnosed as acute otitis media. A total of 60 middle ear effusion samples collected from children with acute otitis media were included in the study. The samples were inoculated and incubated in BACTEC Ped Plus blood culture bottles and BACTEC 9120 system (BD Diagnostic Systems, MD), respectively, and the isolates were identified by conventional methods. For the molecular diagnosis of H.influenzae and S.pneumoniae, ply pneumolysin gene and HIB capsule region, respectively were amplified by Rt-PCR (LightCycler, Roche Diagnostics, Germany). H.influenzae and S.pneumoniae were isolated from 5 (8.3%) and 3 (5%) of the patient samples with conventional culture methods, respectively. In addition in 11.6% of the samples other microorganisms (Staphylococcus epidermidis, Streptococcus intermedius, Streptococcus sanguinis, Moraxella catarrhalis, Pseudomonas aeruginosa, Candida albicans) were also isolated. On the other hand H.influenzae and S.pneumoniae were detected in 38 (63.3%) and 24 (40%) of the samples with Rt-PCR, respectively. There was about eight fold increase in the detection frequency of H.influenzae and S.pneumoniae with Rt-PCR compared to culture methods. When culture was accepted as the gold standard method, the sensitivity, specificity and positive predictive value of Rt-PCR in the detection of H.influenzae and S.pneumoniae were estimated as 80%, 51% and 98.2%, respectively. As a result, Rt-PCR was shown to be a sensitive method and could be preferred for the rapid diagnosis of H.influenzae and S.pneumoniae in the etiological diagnosis of acute otitis media, especially in culture negative cases.

Erythromycin and penicillin resistance mechanisms among viridans group streptococci isolated from blood cultures of adult patients with underlying diseases.

The aim of the study was to evaluate the species distribution, antimicrobial susceptibility and erythromycin-penicillin resistance mechanisms of viridans streptococci (VGS) isolates from blood cultures of adult patients with underlying diseases. Fifty VGS blood culture isolates were screened for their antibiotic susceptibilities against penicillin G, erythromycin and tetracycline by E-test. Clindamycin, cefotaxime, chloramphenicol, levofloxacin, linezolid and vancomycin susceptibility were performed by broth microdilution method. Erythromycin and penicillin resistance genotypes, ermB and mefA/E, pbp1a, pbp2b and pbp2x are amplified using PCR method. The clinical isolates included Streptococcus mitis (n. 19), S.oralis (n. 13), S.sanguinis, S.parasanguinis (n. 6, each), S.salivarius, S.vestibularis (n. 2, each), S.constellatus, S.sobrinus (n. 1, each). The percentage resistance against erythromycin and penicillin was 36% and 30%, respectively. The genotypic carriage rate of erythromycin resistance genes were: 56% ermB, 28% mefE, 8% ermB+mefE. Penicillin-resistant isolates carried pbp2b (33.3%) and pbp2x (20%) genes. Twenty-four VGS isolates were recovered from patients with cancer. S.mitis and S.oralis predominated among patients with cancer who had erythromycin and penicillin resistance isolates. The importance of classical antimicrobial agents like penicillin and erythromycin warrants the continuous surveillance of invasive VGS isolates and can guide better treatment options especially in patients with underlying diseases.

[Antimicrobial resistance and existence of metallo-beta-lactamase in Acinetobacter species isolated from adult patients]. / Eriskin hastalardan izole edilen Acinetobacter türlerinde antimikrobiyal direnç ve metallo-beta-laktamaz varligi.

Acinetobacter spp. are the frequent causes of nosocomial infections which are difficult to treat due to multidrug resistance. The aim of this study was to determine the antibiotic susceptibilities and the presence of metallo-beta-lactamases in Acinetobacter spp. isolated from patients admitted to Hacettepe University Adult Hospital. A total of 124 Acinetobacter spp. isolates were included in the study. Antibiotic susceptibilities against imipenem (IMP), meropenem (MER), ceftazidime (CAZ), ciprofloxacin (CIP) and aztreonam (AZT) were studied by microdilution susceptibility testing according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Multidrug-resistant isolates (MDR) were further tested for susceptibility against colistin by microdilution, and against amikacin (AN), piperacillin-tazobactam (PIP-TAZ), cefepime (FEP), ceftriaxone (CRO), tetracycline (TET), trimetoprim-sulfomethoxazole (SXT) and mezlocillin (MEZ) by disk diffusion method according to CLSI guidelines. Each isolate was also tested for metallo-beta-lactamase (MBL) production by using IMP and EDTA combined disk diffusion test and molecular analysis for bla(IMP-1) and bla(VIM-2) genes was done by polymerase chain reaction (PCR). Among 124 non-duplicate isolates, 72 were identified as Acinetobacter baumannii and 52 as Acinetobacter lwoffii. Minimum inhibitor concentration 50 (MIC50) and minimum inhibitor concentration 90 (MIC90) values of the isolates were 32 and 128 microg/ml for IMP, 16 and 32 microg/ml for MER, 128 and 256 microg/ml for CIP, 64 and 256 microg/ml for CAZ, 128 and 256 microg/ml for AZT, respectively. Forty-three (34.7%) isolates were susceptible to IMP. Overall, 51 (41%) Acinetobacter spp. were found to be resistant to > or = 3 antibiotics belonging to different antimicrobial classes and defined as MDR. Colistin MIC50 and MIC90 values were 2 and 8 microg/ml, respectively and the rate of colistin resistance was 27.5% in MDR isolates. The resistance rates for AN, PIP-TAZ, FEP, CRO, TET, SXT and MEZ were 80.4%, 98%, 92.2%, 100%, 100%, 86.3% and 86.3%, respectively. Among 124 isolates, 64 (51.6%) yielded positive result by IMP-EDTA combined disk test, and all the isolates were negative in terms of the tested MBL genes by PCR. These data revealed high level resistance among the Acinetobacter population in our hospital. The high rate of carbapenem resistance and increasing colistin resistance among Acinetobacter isolates should be surveyed cautiously. The increasing incidence of multidrug resistant Acinetobacter spp. emphasizes the need for new and effective treatment options.

[Value of demonstration of pneumococcal surface antigen A and autolysin genes for the identification of Streptococcus pneumoniae clinical isolates]. / Streptococcus pneumoniae klinik izolatlarinin tanimlanmasinda pnömokokal yüzey antijen A ve otolizin genlerinin gösterilmesinin yeri.

Streptococcus pneumoniae is one of the most frequent causative agents of community acquired pneumoniae, meningitis, sinusitis, bronchitis and otitis media both in children and adults. Conventional laboratory methods may sometimes fail to identify S. pneumoniae. The aims of this study were i) to compare the conventional methods and molecular methods which detected pneumococcal surface antigen A (psaA) and autolysin (lytA) genes; ii) to determine the serotype distribution of S. pneumoniae isolated from the respiratory samples. Randomly chosen 62 S. pneumoniae strains isolated from respiratory samples of patients with clinically proven pneumococcal pneumonia (age range: 1-79 years) between years 2000-2006, were included in the study. Classical microbiological analysis for the isolates included Gram staining, optochin sensitivity test performed in 5% CO2 and ambient air and bile solubility test. Capsular serotyping was performed by using latex particles sensitized with mono-specific typing sera (Statens Serum Institut, Denmark). Quellung reaction (Statens Serum Institut, Denmark) was used for serotyping the isolates that gave equivocal results using latex agglutination. Pneumococcal surface antigen A and autolysin genes were detected by in-house polymerase chain reaction (PCR) using psaA1 (5'-CTT TCT GCA ATC ATT CTT G), psaA2 (5'-GCC TTC TTT ACC TTG TTC TGC), lytAF (5'-ACG CAA TCT AGC AGA TGA AGC) and lytAR (5'-TGT TTG GTT GGT TAT TCG TGC) primers. Twenty six different serotypes were detected in 62 S. pneumoniae isolates. The most prevalent capsule serotype was 14 (n= 6), followed by 19A (n= 5). Four isolates could not be typed by the available antisera. All the isolates were optochin sensitive with or without carbondioxide incubation and were bile soluble. All the isolates included in the study have harboured (100%) psaA and lytA genes. No difference was found between the classical and molecular methods for the identification of S. pneumoniae isolates. In conclusion, detection of psaA and/or lytA genes by molecular methods is of value especially in "nonserotypeable strains" when they are performed with conventional methods in clinically proven S. pneumoniae isolates.

CTX-M-3 type beta-lactamase producing Shigella sonnei isolates from pediatric bacillary dysentery cases.

In this study, 153 clinical isolates of Shigella were screened for extended-spectrum beta-lactamase (ESBL) production by double-disk synergy test. After being confirmed by the combined disk-test and inhibitor-combined E-test strips, all positive isolates were tested for the bla gene by PCR and the enzymes by isoelectric focusing. DNA sequencing of PCR products revealed that five Shigella sonnei isolates had CTX-M-3 type ESBL enzymes. All of them were resistant to ampicillin, sulfamethoxazole and cefotaxime but susceptible to ofloxacin and ceftazidime. All isolates displayed identical patterns in pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR. This study reports multidrug-resistant (MDR) S. sonnei isolates producing CTX-M-3 type ESBL from successive pediatric bacillary dysentery patients, indicating widespread and rapid spread of CTX-M type ESBL in Shigella spp. To counter this emerging threat to public health, the surveillance of CTX-M type beta-lactamases should be considered, together with measures designed to prevent outbreaks of MDR Shigella in the community.