Antibiotic resistance in Campylobacter jejuni and Campylobacter coli: significant contribution of an RND type efflux pump in erythromycin resistance.

In this study, we aimed to determine the antibiotic resistance status of Campylobacter spp. isolated from human infections in our region, including the role of mechanisms involved in erythromycin resistance. Standard methods were used for the isolation, identification and antibiotic susceptibility testing of Campylobacter spp. isolates. Erythromycin-resistant mutants were selected from erythromycin-susceptible clinical isolates, and the erythromycin resistance mechanisms were investigated phenotypically by determining the erythromycin MICs of isolates in the presence and absence of the resistance nodulation cell division (RND) type efflux pump inhibitor, phenylalanine-arginine ß-naphthylamide dihydrochloride (PAßN), and genotypically by determining ribosomal and cmeABC alterations using PCR and DNA sequence analysis. Campylobacter spp., including 184 C. jejuni and 20 C. coli in a two-year period, were the most frequently isolated gastrointestinal bacterial pathogens in our region. However, in both C. jejuni and C. coli, resistance to tetracycline and ciprofloxacin were found to be high, erythromycin resistance was especially high (20%) in C. coli. With a ribosomal alteration, A2075G, which was found to be associated with high-level erythromycin resistance in clinical isolates, PAßN significantly reduced the erythromycin MICs in both clinical isolates and mutants. An important finding of this study, while considering cmeABC operon, is the explanation of why erythromycin resistance is more common among C. coli than C. jejuni, bearing in mind the specific deletions and alterations in the intergenic region of the operon in all erythromycin-resistant C. coli isolates. Ultimately, these findings revealed the significant role of RND-type efflux activity in increased erythromycin MICs of the isolates.

One-Year Post-Vaccination Longitudinal Follow-Up of Quantitative SARS-CoV-2 Anti-Spike Total Antibodies in Health Care Professionals and Evaluation of Correlation with Surrogate Neutralization Test.

Numerous vaccines have been generated to decrease the morbidity and mortality of COVID-19. This study aims to evaluate the immunogenicity of the heterologous boosts by BioNTech against homologous boosts by CoronaVac at three-month intervals in two health care worker (HCW) cohorts, with or without prior COVID-19, for one year post-vaccination. This is a prospective cohort study in which the humoral responses of 386 HCWs were followed-up longitudinally in six main groups according to their previous COVID-19 exposure and vaccination status. Anti-SARS-CoV-2 spike-RBD total antibody levels were measured and SARS-CoV-2 neutralization antibody (NAbs) responses against the ancestral Wuhan and the Omicron variant were evaluated comparatively using international standard serum for Wuhan and Omicron, as well as with the aid of a conversion tool. The anti-SARS-CoV-2 spike-RBD total Ab and Nab difference between with and without prior COVID-19, three months after two-dose primary vaccination with CoronaVac, was statistically significant (p = 0.001). In the subsequent follow-ups, this difference was not observed between the groups. Those previously infected (PI) and non-previously infected (NPI) groups receiving BioNTech as the third dose had higher anti-SARS-CoV-2 spike total Ab levels (14.2-fold and 17.4-fold, respectively, p = 0.001) and Nab responses (against Wuhan and Omicron) than those receiving CoronaVac. Ab responses after booster vaccination decreased significantly in all groups at the ninth-month follow-up (p < 0.05); however, Abs were still higher in all booster received groups than that in the primary vaccination. Abs were above the protective level at the twelfth-month measurement in the entire of the second BioNTech received group as the fourth dose of vaccination. In the one-year follow-up period, the increased incidence of COVID-19 in the groups vaccinated with two or three doses of CoronaVac compared with the groups vaccinated with BioNTech as a booster suggested that continuing the heterologous CoronaVac/BioNTech vaccination, revised according to current SARS-CoV-2 variants and with at least a six-month interval booster would be an effective and safe strategy for protection against COVID-19, particularly in health care workers.

Diversity of HIV-1 Subtypes and Transmitted Drug-resistance Mutations Among Minority HIV-1 Variants in a Turkish Cohort.

BACKGROUND: The World Health Organization (WHO) recommends the surveillance of transmitted drug resistance mutations (TDRMs) to ensure the effectiveness and sustainability of HIV treatment programs. OBJECTIVE: Our aim was to determine the TDRMs and evaluate the distribution of HIV-1 subtypes using and compared next-generation sequencing (NGS) and Sanger-based sequencing (SBS) in a cohort of 44 antiretroviral treatment-naïve patients. METHODS: All samples that were referred to the microbiology laboratory for HIV drug resistance analysis between December 2016 and February 2018 were included in the study. After exclusions, 44 treatment-naive adult patients with a viral load of >1000 copies/mL were analyzed. DNA sequencing for reverse transcriptase and protease regions was performed using both DeepChek ABL single round kit and Sanger-based ViroSeq HIV-1 Genotyping System. The mutations and HIV-1 subtypes were analyzed using the Stanford HIVdb version 8.6.1 Genotypic Resistance software, and TDRMs were assessed using the WHO surveillance drug-resistance mutation database. HIV-1 subtypes were confirmed by constructing a maximum-likelihood phylogenetic tree using Los Alamos IQ-Tree software. RESULTS: NGS identified nucleos(t)ide reverse transcriptase inhibitor (NRTI)-TDRMs in 9.1 % of the patients, non-nucleos(t)ide reverse transcriptase inhibitor (NNRTI)-TDRMs in 6.8 % of the patients, and protease inhibitor (PI)-TDRMs in 18.2 % of the patients at a detection threshold of ≥ 1 %. Using SBS, 2.3 % and 6.8 % of the patients were found to have NRTI- and NNRTI-TDRMs, respectively, but no major PI mutations were detected. M41L, L74I, K65R, M184V, and M184I related to NRTI, K103N to NNRTI, and N83D, M46I, I84V, V82A, L24I, L90M, I54V to the PI sites were identified using NGS. Most mutations were found in low-abundance (frequency range: 1.0 % - 4.7 %) HIV-1 variants, except M41L and K103N. The subtypes of the isolates were found as follows; 61.4 % subtype B, 18.2 % subtype B/CRF02_AG recombinant, 13.6 % subtype A, 4.5 % CRF43_ 02G, and 2.3 % CRF02_AG. All TDRMs, except K65R, were detected in HIV-1 subtype B isolates. CONCLUSION: The high diversity of protease site TDRMs in the minority HIV-1 variants and prevalence of CRFs were remarkable in this study. All minority HIV-1 variants were missed by conventional sequencing. TDRM prevalence among minority variants appears to be decreasing over time at our center.

Detection of a New Resistance-Mediating Plasmid Chimera in a blaOXA-48-Positive Klebsiella pneumoniae Strain at a German University Hospital.

Mobile genetic elements, such as plasmids, facilitate the spread of antibiotic resistance genes in Enterobacterales. In line with this, we investigated the plasmid-resistome of seven blaOXA-48 gene-carrying Klebsiella pneumoniae isolates, which were isolated between 2013 and 2014 at the University Medical Center in Göttingen, Germany. All isolates were subjected to complete genome sequencing including the reconstruction of entire plasmid sequences. In addition, phenotypic resistance testing was conducted. The seven isolates comprised both disease-associated isolates and colonizers isolated from five patients. They fell into two clusters of three sequence type (ST)101 and two ST11 isolates, respectively; and ST15 and ST23 singletons. The seven isolates harbored various plasmids of the incompatibility (Inc) groups IncF, IncL/M, IncN, IncR, and a novel plasmid chimera. All blaOXA-48 genes were encoded on the IncL/M plasmids. Of note, distinct phenotypical resistance patterns associated with different sets of resistance genes encoded by IncL/M and IncR plasmids were observed among isolates of the ST101 cluster in spite of high phylogenetic relatedness of the bacterial chromosomes, suggesting nosocomial transmission. This highlights the importance of plasmid uptake and plasmid recombination events for the fast generation of resistance variability after clonal transmission. In conclusion, this study contributes a piece in the puzzle of molecular epidemiology of resistance gene-carrying plasmids in K. pneumoniae in Germany.

Evaluation of phenotypic tests for detection of carbapenemases: New modifications with new interpretation.

INTRODUCTION: The emergence and spread of carbapenemase-producing Enterobacterales (CPE) is a worldwide public health threat. Rapid and accurate detection of CPE is essential to prevent their dissemination within health care settings. The aim of this study was to evaluate the performance of CIM, mCIM and mCIM with ammonium bicarbonate (mCIM-A) methods by using different interpretation criteria for detection of carbapenemases. METHODS: One hundred and fifty-three Klebsiella pneumoniae isolates previously characterized by molecular tests, including 133 carbapenemase producers and 20 non-carbapenemase producers, were collected in this study. CIM and mCIM tests were performed as described previously. mCIM-A by adding 50 mM ammonium bicarbonate to the bacterial suspension prepared in tryptic soy broth. The inhibition zone diameter of around meropenem disc was measured and interpreted as positive according to i) Pierce and colleagues (<19 mm), ii) EUCAST meropenem susceptibility breakpoint (<22). RESULTS: CIM, although seems to be good for carbapenemases other than OXA-48-like and NDM, is not satisfactory (42.3% and 83.4%, respectively) for those enzymes with any of the interpretation criteria. OXA-48-like and NDM were detected with a better performance (88.7% and 92.8, respectively) with mCIM when results were interpreted according to <22 mm zone diameter for OXA-48-like and NDM. The best results were obtained with mCIM-A using <22 mm criteria without any difference in the results of other enzymes and negative strains. CONCLUSIONS: mCIM-A method interpreted with <22 mm meropenem zone diameter seems to be preferable compared to CIM and mCIM. mCIM-A is simple and useful tool for identification of CPEs in clinical microbiology laboratories.

Evaluation of two commercial methods for rapid detection of the carbapenemase-producing Klebsiella pneumoniae.

In this study, we evaluated the performance of the two commercial methods (Rapidec Carba NP and NG-Test Carba-5) for the rapid detection of carbapenemase-producing Klebsiella pneumoniae. A total of 224 Klebsiella pneumoniae strains previously characterized for carbapenemase genes by polymerase chain reaction were included in the study. The strain collection included 30 non-carbapenemase producers, 85 OXA-48-like, 59 NDM, 14 IMP, 7 KPC, 7 VIM, 19 OXA-48-like plus NDM, and 3 KPC plus NDM producers. Rapidec Carba NP and NG-Test Carba 5 was performed according to the manufacturer's instructions. NG-Test Carba 5 correctly detected all carbapenemase-producing K. pneumoniae, however, Rapidec Carba NP failed to detect 41% of OXA-48-like producers even with extended incubation time. The overall sensitivity and specificity were 81,9% and 100% for Rapidec Carba NP, 100% and 100% for NG-Test Carba 5, respectively. Both tests seem to be fast and reliable for the detection of carbapenemase-producing K. pneumoniae especially for microbiology laboratories where molecular tests cannot be performed. However, Rapidec Carba NP with weak hydrolysis activity for OXA-48 like might be used in regions where OXA-48 is not prevalent. The additional advantage of NG-Test Carba 5 is that it specifically detects carbapenemases giving way to threat-related infections with an effective drug such as ceftazidime-avibactam or meropenem- vaborbactam.

Comparative in vitro activity of plazomicin and older aminoglyosides against Enterobacterales isolates; prevalence of aminoglycoside modifying enzymes and 16S rRNA methyltransferases.

Comparative in vitro activity of plazomicin and 4 older aminoglycosides was evaluated with broth microdilution in 714 blood isolates from 14 hospitals in Turkey. Isolates included Escherichia coli (n=320), Klebsiella spp. (n=294), Enterobacter spp. (n=69), Serratia marcescens (n=20), and Citrobacter spp. (n=11). Isolates resistant to older aminoglycosides (n=240) were screened for aminoglycoside modifying enzyme genes: aac(6')-Ib, aac(3)-Ia, aac(3)-IIa, ant(2″)-Ia. Isolates with high MICs for plazomicin (n=41) were screened for 16S rRNA methyltransferase genes (armA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF, rmtG, rmtH, npmA) and 2 carbapenemase genes (blaOXA-48, blaNDM-1). Overall, resistance to plazomicin, amikacin, netilmicin, gentamicin, and tobramycin was 7.7%, 7.4%, 31.5%, 32.9%, and 34.7%, respectively. aac(6')-Ib and aac(3)-IIa were the most common AME genes. Co-occurrence of blaNDM-1 with armA and rmtC and blaOXA-48 with armA was striking. Enterobacter cloacae carrying rmtC+blaNDM-1, S. marcescens with armA+blaOXA-48, and rmtF+ blaOXA-48 in K. pneumoniae were reported for the first time.

Alterations in AdeS and AdeR regulatory proteins in 1-(1-naphthylmethyl)-piperazine responsive colistin resistance of Acinetobacter baumannii.

Colistin resistant Acinetobacter baumannii strains are of great concern worldwide. However, the role of efflux pumps in colistin resistance needs to be elucidated. We investigated the changes in colistin MICs of 29 colistin resistant A. baumannii isolates in response to resistance-nodulation-division (RND)-type efflux pump inhibitor (EPI) and the alterations in AdeR and AdeS two-component regulatory proteins previously associated with the overproduction of AdeAB. The EPI, 1-(1-naphthylmethyl)-piperazine (NMP), led to significant reductions in colistin MICs. At least one of the following amino acid substitutions was found in AdeS proteins from 18 of the isolates: L172P, A94V, V27I, V32I, G186V, and G164A. Besides, A136V and V120I alterations were identified in AdeR from five isolates. Therefore, EPI-responsive colistin resistance in our isolates is most likely due to the action of an RND-type efflux system. The underlying mechanism of resistance might be the result of certain AdeRS alterations, leading to AdeAB overexpression.

Performance of "RESIST-3 O.K.N. K-SeT" immunochromatographic assay for the detection of OXA-48 like, KPC, and NDM carbapenemases in Klebsiella pneumoniae in Turkey

ABSTRACT In this study, the performance of the "RESIST-3 O.K.N. K-SeT" (Coris BioConcept, Gembloux, Belgium) immunochromatographic assay was evaluated in 132 Klebsiella pneumoniae comprising 102 carbapenem resistant and 30 carbapenem susceptible isolates. Genotypically known isolates of Gram negative bacteria (n = 22) including various species were also tested by the assay as controls. The isolates tested by the immunochromatographic assay and also were run PCR for bla KPC, bla IMP, bla VIM, bla NDM, and bla OXA-48. The rates of bla NDM, bla OXA-48, and bla KPC in carbapenem resistant isolates were found at 52.9%, 39.2%, and 2.0%, respectively. Both bla NDM and bla OXA-48 were found in six (5.9%) isolates. The results of the assay showed 100% concordance with those obtained by PCR in 132 K. pneumoniae. The agreement between the two methods was found to be identical at the isolate level. The assay also correctly detected all genotypically known isolates of Escherichia coli, Serratia marcescens, Citrobacter freundii, Enterobacter cloacae, K. pneumoniae carrying bla KPC, bla NDM, and/or bla OXA-48. On the other hand, the assay did not exhibit any cross-reaction in control isolates harboring bla IMP and bla VIM. We conclude that the RESIST-3 O.K.N. K-SeT is a reliable, rapid, and user friendly test and we recommend it for routine diagnostic laboratories.

Assessment of Transmitted HIV-1 Drug Resistance Mutations Using Ultra- Deep Pyrosequencing in a Turkish Cohort.

BACKGROUND: Antiretroviral treatment (ART) reduces morbidity and mortality caused by human immunodeficiency virus (HIV) infection; however, the emergence of drug-resistant strains poses an important obstacle to treatment success. Using conventional sequencing methods to determine antiretroviral resistance, mutations present in ≥20% of quasispecies can be identified, but drug-resistant minority variants can lead to virologic failure. OBJECTIVE: We aimed to assess transmitted drug resistance mutations (TDRMs) within minority variants using ultra-deep pyrosequencing (UDPS). METHOD: Treatment-naive adult patients were included in this observational study. Surveillance TDRMs were classified as ≥20% or at minority variant level (≥2% - <20%). Genotypic sensitivity score calculated by using all pre-treatment drug resistance mutations (PDRMs) was also evaluated. RESULTS: Thirty-six patients were analyzed. Any TDRM at ≥20% level was detected in 8.3% of the patients (n=3). This prevalence increased to 30.6% (n=11) with the inclusion of minority variants. All non-nucleoside reverse transcriptase inhibitor and protease inhibitor-related TDRMs were within minority variants. The genotypic sensitivity score of rilpivirine-based regimens was considerably diminished when minority variants were included in the PDRM analysis. CONCLUSION: UDPS was used for the first time to assess TDRM in a Turkish HIV cohort and uncovered several mutations hidden within minority variants. UDPS may be preferred to detect PDRMs for avoiding virologic failure with rilpivirine-based ART regimens.

Performance of "RESIST-3 O.K.N. K-SeT" immunochromatographic assay for the detection of OXA-48 like, KPC, and NDM carbapenemases in Klebsiella pneumoniae in Turkey.

In this study, the performance of the "RESIST-3 O.K.N. K-SeT" (Coris BioConcept, Gembloux, Belgium) immunochromatographic assay was evaluated in 132 Klebsiella pneumoniae comprising 102 carbapenem resistant and 30 carbapenem susceptible isolates. Genotypically known isolates of Gram negative bacteria (n=22) including various species were also tested by the assay as controls. The isolates tested by the immunochromatographic assay and also were run PCR for blaKPC, blaIMP, blaVIM, blaNDM, and blaOXA-48. The rates of blaNDM, blaOXA-48, and blaKPC in carbapenem resistant isolates were found at 52.9%, 39.2%, and 2.0%, respectively. Both blaNDM and blaOXA-48 were found in six (5.9%) isolates. The results of the assay showed 100% concordance with those obtained by PCR in 132K. pneumoniae. The agreement between the two methods was found to be identical at the isolate level. The assay also correctly detected all genotypically known isolates of Escherichia coli, Serratia marcescens, Citrobacter freundii, Enterobacter cloacae, K. pneumoniae carrying blaKPC, blaNDM, and/or blaOXA-48. On the other hand, the assay did not exhibit any cross-reaction in control isolates harboring blaIMP and blaVIM. We conclude that the RESIST-3 O.K.N. K-SeT is a reliable, rapid, and user friendly test and we recommend it for routine diagnostic laboratories.

The impact of a pneumococcal conjugate vaccination program on the nasopharyngeal carriage, serotype distribution and antimicrobial resistance of Streptococcus pneumoniae among healthy children in Turkey.

BACKGROUND: The 7-valent conjugate pneumococcal vaccine (PCV7) was introduced by the Turkey National Immunization Program in 2008 and replaced by the PCV13 in 2011. We assessed the impact of PCV vaccination on the nasopharyngeal (NP) carriage, serotype distribution and antimicrobial resistance of Streptococcus pneumoniae (SP) among healthy Turkish children. METHODS: A prospective surveillance study was performed between September 2011 and September 2013 in Istanbul, Turkey. NP swabs, demographic data, and vaccination statuses were obtained from 2165 healthy children aged 0-18years. Pneumococcal carriage was defined by a positive culture; serotyping was performed via multiplex conventional PCR, and the antibiotic susceptibilities of the isolates were determined based on the minimum inhibitory concentration (MIC) values of the Clinical Laboratory Standards Institute (CLSI). RESULTS: The prevalence of pneumococcal carriage was 6.4%. The carriage rates were 8%, 7%, and 5% in the following age groups: 0-24months, 25-60months, and >60months, respectively. The carriage rate was significantly higher in the 0-24month age group than in the >60months age group (p=0.03). Sixty percent of the children were not vaccinated with any PCV; 4%, 2%, and 4% received at least 1, 2 or 3 doses and 30% children received the full schedule (4 doses) of either PCV7 or PCV13. Among the isolated S. pneumoniae strains, 45% were of the non-vaccine type (NVT) and 55% were of the vaccine type (VT). The children who received at least a single PCV dose had significantly lower odds of colonization via VT serotypes than the non-vaccinated children [odds ratio: 0.61 (95% confidence interval=0.41-0.91), p=0.01]. The percentages of the serotypes covered by PCV7 and PCV13 were 51% and 56%, respectively. The most frequently isolated serotypes were 6A/B/C (n=22, 16.5%), 19F (n=18, 13.5%), 23F (n=15, 11.2%), serotype 9V/A (n=10, 7.5%), 12F (n=5, 4.5%), 15A/F (n=7, 4.5%) and 22 A/F (n=6, 4.5%). Using the meningitis criteria and the MIC, 62% of the isolates were resistant to penicillin and 13% were non-sensitive to ceftriaxone. Erythromycin and clindamycin resistance were 43% and 31%, respectively. CONCLUSION: We shown that following nation-wide PCV vaccination, S. pneumoniae NP carriage was decreased.

[Investigation of carbapenemases in carbapenem-resistant Escherichia coli and Klebsiella pneumoniae strains isolated in 2014 in Turkey]. / Türkiye'de 2014 yili içinde izole edilen karbapeneme dirençli Escherichia coli ve Klebsiella pneumoniae izolatlarinda karbapenemaz varliginin arastirilmasi.

Carbapenems are the choice of treatment in infections caused by multidrug resistant Enterobacteriaceae. In recent years carbapenem-resistant Enterobacteriaceae isolates due to carbapenemases have been increasingly reported worldwide. Multicenter studies on carbapenemases are scarce in Turkey. The aim of this study was to determine the distribution of carbapenemases from different parts of Turkey as a part of the European Survey of Carbapenemase Producing Enterobacteriaceae (EuSCAPE) project. Beginning in November 2013, carbapenem-resistant isolates resistant to at least one of the agents, namely imipenem, meropenem, and ertapenem were sent to the coordinating center. Minimum inhibitory concentrations for these carbapenems were determined by microdilution tests following EUCAST guidelines. Production of carbapenemase was confirmed by combination disk synergy tests. Types of carbapenemases were investigated using specific primers for VIM, IMP; NDM, KPC and OXA-48 genes by multiplex polymerase chain reaction. In a six month period, 155 suspected carbapenemase-positive isolates were sent to the coordinating center of which 21 (13.5%) were E.coli and 134 (86.5%) were K.pneumoniae. Nineteen (90.5%) strains among E.coli and 124 (92.5%) strains among K.pneumoniae were shown to harbour at least one carbapenemase gene by molecular tests, with a total of 92.3% (143/155). Carbapenemases were determined as a single enzyme in 136 strains (OXA-48: 84.6%; NDM: 6.3%; VIM: 2.8%; IMP: 1.4%) and as a combination in seven isolates (OXA-48 + NDM: 2.1%; OXA-48 + VIM: 2.1%; VIM + NDM: 0.7%). KPC was not detected in any of the isolates. According to the microdilution test results, resistance to imipenem, meropenem and ertapenem in OXA-48 isolates were 59.5%, 52.9% and 100%, respectively. The combination disk synergy test was 100% compatible with the molecular test results. As most of the OXA-48 producing isolates were susceptible to meropenem but all were resistant to ertapenem, ertapenem seems to be the most sensitive agent in screening carbapenemases in areas where OXA-48 is prevalent and phenotypic combination tests can be useful in centers where molecular tests are not available.

[Serotype distribution and antibiotic susceptibilities of Streptococcus pneumoniae causing acute exacerbations and pneumonia in children with chronic respiratory diseases]. / Kronik Akciger Hastaligi Olan, Akut Alevlenme ve Pnömoni Tanisi ile Basvuran Çocuklarda Streptococcus pneumoniae Serotip Dagilimi ve Antimikrobiyal Duyarliliklari

This study aimed to investigate serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates obtained from children with chronic respiratory diseases admitted to hospital with a diagnosis of acute exacerbations between 2008-2010 at Marmara University Hospital, Istanbul, Turkey. Sixty one S.pneumoniae strains isolated from the respiratory samples of patients were studied for erythromycin, clindamycin, tetracyline, trimethoprim-sulphametoxazole (TMP-SMX), vancomycin, levofloxacin susceptibilities by disk diffusion method; MIC values of penicillin and ceftriaxone were determined by E-test (AB Biodisk, Sweden). Results were evaluated according to the CLSI standards. The erythromycin-clindamycin double disc method was applied for the detection of macrolide resistance phenotypes. The presence of macrolide resistance genes, ermB, mef(A)/(E), ermTR were determined by PCR using specific primers for each gene. The serotypes were determined by multiplex PCR using specific primers for 40 different serotypes. According to CLSI criteria, penicillin resistance in S.pneumoniae isolates were found to be 8.2% (5/61) and intermediate resistance rate was 54% (33/61) for oral penicillin. Penicillin resistance were found to be only 1.6% (1/61) for parenteral penicillin. Resistance rates of erythromycin, clindamycin, tetracyline, TMP-SMX were detected as 55.8%, 46%, 47.5% and 67.2%; respectively. No resistance was detected to vancomycin and levofloxacin. Constitutive macrolide-lincosamide-streptogramin B (cMLSB) phenotype and M phenotype were observed in 82.4% (n= 28) and 17.6% (n= 6) of the macrolide resistant isolates, respectively. Inducible macrolide-lincosamide-streptogramin B (iMLSB) phenotype was not detected. The macrolid resistance genotypes, ermB, mef(A)/(E), were positive 50% and 14.7%; respectively. Both ermB and mef(A)/(E) genes were detected 35.3% of the macrolid resistant isolates. None of the isolates were positive for ermTR gene. The most common S.pneumoniae serotypes were determined as serotype 19F, 23F and 6, furthermore penicillin (34%, 15.7% and 18.4%, respectively) and macrolide (38.2%, 20.6% and 14.7%, respectively) resistance rates of those serotypes were found relatively high. Serotype covarage of 7-, 10-, 13-valent conjugated pneumococcal vaccines and 23-valent pneumococcal vaccine were 65%, 67%, 69%, and 78.6%, respectively. In our country, use of the vaccines with these coverage rates has been observed to be effective in children exposed to intensive use of antibiotics with chronic lung disease.

[The role of cell wall organization and active efflux pump systems in multidrug resistance of bacteria]. / Qoklu ilaç direncinde bakteri hücre duvari organizasyonu ve aktif pompa sistemlerinin rolü.

Multiple antibiotic resistance of clinically important bacteria are of major concern worldwide. Alterations of drug targets or enzymatic inactivation of antimicrobial agents are the well known mechanisms of antimicrobial drug resistance. Besides these well known mechanisms, recent studies have shown that a further resistance mechanism, active drug efflux, has become increasingly important in the current threat of multidrug resistance. It involves certain bacterial transport proteins which pump out toxic antimicrobial compounds from the cell. Drug efflux pump proteins in bacteria fall into five distinct protein super families [ATP binding cassette super family (ABC), Major facilitator super family (MFS), Small multidrug resistance super family (SMR), Multidrug and toxic compound extrusion (MATE) super family, Resistance-nodulation-cell division (RND) super family] and are mostly encoded by chromosomal genes. Among them, the members of RND protein super family are widely distrubuted in Gram negative bacteria and play siginificant role in both, intrinsic and acquired multidrug resistance of these bacteria with very wide substrate specificity. RND type multidrug efflux proteins usually function together with an outer membrane canal protein (OMP) and a membrane fusion protein (MFP) to pump out drugs. AcrAB-TolC of Escherichia coli and MexAB-OprM of Pseudomonas aeruginosa are the typical examples of these tripartite systems. They are constitutively expressed in wild type cells and play significant role in intrinsic resistance of these bacteria. However, multidrug resistance which is of major clinical significance, rises as a result of overexpression of these pump systems due to mutations and elevated levels of resistance are recorded to structurally unrelated antimicrobial drugs such as fluoroquinolones, beta-lactams, tetracyclines, chloramphenicol, trimethoprim, aminoglycosides and toxic compunds. Synthesis of RND type pump proteins are regulated by complex genetic mechanisms and global activator proteins (MarA, SoxS, Rob) are significant in the induction of overexpression of these efflux pump systems. Outer membrane of Gram negative bacteria with its unique lipopolysaccharide rich structure also contributes to drug efflux and other antimicrobial resistance mechanisms by reducing the influx rate of toxic antimicrobial compunds. Multidrug efflux pump proteins found in Gram positive bacteria and mycobacteria are usually the members of protein super families other than RND family and their substrate profiles are more limited. However, some of these efflux proteins (NorA, MsrA, QacA in Staphylococcus aureus; PmrA and EmeA in Streptococcus pneumoniae) have clinical significance in the resistance to several antimicrobial agents (fluoroquinolones, macrolids) and toxic substances (quarternery ammonium compounds). In this review article, the role of cell wall organization and active efflux pump systems in multidrug resistance of bacteria have been discussed.

Detection and prevalence of active drug efflux mechanism in various multidrug-resistant Klebsiella pneumoniae strains from Turkey.

The prevalence of active drug efflux pump and porin alterations was investigated in Turkish nosocomial strains of Klebsiella pneumoniae exhibiting a multidrug-resistant phenotype. MICs of various antibiotics, including quinolones, chloramphenicol, tetracycline, and beta-lactams, for those strains were determined either with or without the efflux pump inhibitor phenylalanine arginine beta-naphthylamide (PAbetaN). Thirty-nine percent of the strains exhibited a PAbetaN-modulated resistance for quinolones, chloramphenicol, and tetracycline. In these strains, a significant increase of chloramphenicol accumulation was gained in the presence of the efflux pump inhibitor PAbetaN or with the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone. Moreover, high-level expression of the membrane fusion protein AcrA, which was immunodetected in most of those isolates, suggests that the AcrAB/TolC efflux machinery contributed to their antibiotic resistance. Studies of K. pneumoniae porins indicated that the majority of the strains, including extended-spectrum beta-lactamase producers and efflux-positive ones, presented an alteration in their sorbitol-sensitive porin (OmpK35) expression. This is the first report showing the prominent role of active drug efflux in the antibiotic resistance of nosocomial K. pneumoniae strains from Turkey.

Simple and reliable detection of slime production of Candida spp. directly from blood culture bottles: comparison of visual tube method and transmission electron microscopy.

Early detection of slime production may be useful for clinical decision because of its suggestive property for potential pathogenic capacity of a Candida strain especially in patients with a prosthetic device. In this study we aimed to compare the visual tube method (VTM) with transmission electron microscopy (TEM) in order to confirm the reliability of the former method. In order to demonstrate the reproducibility of the tube method and to determine the correct timing for the test, Candida isolates directly obtained from blood culture (DBC) bottles and their two subsequent subcultures were used. The results of this study showed that VTM is a simple and reliable method which can be used in every clinical mycology laboratory, provided that the test is applied on DBC isolates; as the ability of slime production is decreased or lost even after the first subculturing. We suggest that this simple method can be used and may have some contributions to the ongoing studies on the controversial issue concerning removal of biomaterials in candidemic patients.