Cemal Sandalli

Class 1 and class 2 integrons and plasmid-mediated antibiotic resistance in coliforms isolated from ten rivers in northern Turkey

We aimed to determine the molecular mechanisms of antibiotic resistance in coliforms isolated from ten rivers in northern region of Turkey. A total of 183 isolates were tested for antimicrobial susceptibility by disk diffusion and agar dilution methods. Resistance to ampicillin, streptomycin, trimethoprim, tetracycline, and chloramphenicol was detected in 58%, 51.9%, 24%, 28.4%, and 12.5%, respectively. Twelve (6.5%) phylogenetically distant organisms were detected to harbor self-transmissible plasmids ranging 52 to >147 kb in sizes. Resistances to ampicillin, tetracycline, trimethoprim, streptomycin, and nalidixic acid were commonly transferable traits. Transferable nalidixic acid-resistant strains harbored qnrS gene, which was the first report of plasmid-mediated quinolone resistance in bacteria of environmental origin in Turkey. Fourteen and five coliforms harbored class 1 and class 2 integrons, respectively, and some of them were located on transferable plasmids. Sequence analyses of variable regions of the class 1 and 2 integrons harbored various gene cassettes, dfrA1, dfr2d, dfrA7, dfrA16, dfrA17, aadA1, aadA5, blaoxA-30, and sat1. A gene cassette array, dfrA16 has been demonstrated for the first time in a Citrobacter koseri isolate. Class 1 and class 2-bearing strains were clustered in different groups by BOX-PCR fingerprinting. Rivers in the northern Turkey may act as receptacle for the multi-drug resistant enterobacteria and can serve as reservoirs of the antimicrobial resistance determinants in the environment. The actual risk to public health is the transfer of resistance genes from the environmental bacteria to human pathogens.

Characterization of novel VIM carbapenemase, VIM-38, and first detection of GES-5 carbapenem-hydrolyzing β-lactamases in Pseudomonas aeruginosa in Turkey.

Pseudomonas aeruginosa isolates were collected form a Turkish hospital. Antimicrobial susceptibility was performed using the Vitek 2 Compact system, and 24 isolates were categorized as multidrug resistant (n = 18), extensively-drug resistant (n = 5), or pan-drug resistant (n = 1). PCR and DNA sequence analysis revealed that 1 strain possessed the blaGES-5 and another carried a novel blaVIM variant, named VIM-38. This new gene exhibited 1 amino acid substitution (Ala265Val) in comparison to its closest variant, VIM-5. Both VIM encoding genes were clones and demonstrated similar susceptibility profile when expressed in identical background. The presence of VIM-38 increases the diversity of carbapenemases in Turkey.

Characterization of a xylanase from a thermophilic strain of Anoxybacillus pushchinoensis A8

A facultatively anaerobic, thermophilic, xylanolytic bacterium was isolated from a sample collected from the Diyadin Hot Springs, Turkey. According to morphological, biochemical and molecular identification, this new strain was suggested to be representative of the Anoxybacillus pushchinoensis and it was designated as Anoxybacillus pushchinoensis strain A8. It exhibited 97% similarity to 16S rRNA gene sequence of A. pushchinoensis and 77% DNA homology by DNA-DNA hybridization studies. Q-sepharose and CM-sepharose chromatography was used to purify an extracellular xylanase to >90% purity from this species. The enzyme had a molecular mass of approximately 83 kDa. The enzyme showed optimum activity at pH 6.5 and it was 96% stable over a broad pH range of 6.5–11 for 24 hours. The enzyme had optimum activity at 55°C and it was 100% stable at temperature between 50–60°C up to 24 hours. Kinetic characterization of the enzyme was performed at temperature optima (55°C) and Vmax and Km were found to be 59.88 U/mg protein and 0.909 mg/mL, respectively. Oat spelt xylan but not xylooligosaccharides was degraded by the enzyme and xylose was the only product detected from oat xylan degradation. This suggested that the enzyme was an exo-acting xylanase.

Cloning, expression, purification and characterisation of a thermostable chitinase fromBacillus licheniformis A1

The chitinase B gene (chiB65) ofBacillus licheniformis A1 (BlicA1) isolated from Diyadin hotspring in Turkey was cloned and sequenced. The gene is 1779 bp long and encodes a protein 592 amino acids with a 35-amino acid signal peptide at N-terminal. The gene has 99% percent similarity tochiB gene ofBacillus licheniformis under the GenBank number AY205293. The gene without signal peptide was overexpressed inEscherichia coli and the recombinant protein purified by nickel affinity chromatography. The activity of enzyme was shown on SDS-PAGE with the flourogenic substrate 4-methylumbelliferyl β-D-N,N′-diacetylchitobioside. Kinetic characterisation of the enzyme was performed at 65 °C by using chromogenic substratep-nitrophenylN,N′-diacetyl-β-D-chitobioside, andKm and Vmax were found to be 0.02 μM and 1017 U/mg protein, respectively. Enzyme has maximal activity at pH 6.0 and was stable over a broad pH range of 5.0–9.0 for 24 h at room temperature and 4 h at 65 °C. Enzyme was 60% stable at 65 °C for 1.5 h. The inhibition or activation of some substances on the activity of enzyme was determined. High kinetic properties of enzyme open the possibility of an extensive structural and enzyme-substrate interaction studies.

Comparison of Verona Integron-Borne Metallo-β-Lactamase (VIM) Variants Reveals Differences in Stability and Inhibition Profiles

ABSTRACT Metallo-β-lactamases (MBLs) are of increasing clinical significance; the development of clinically useful MBL inhibitors is challenged by the rapid evolution of variant MBLs. The Verona integron-borne metallo-β-lactamase (VIM) enzymes are among the most widely distributed MBLs, with >40 VIM variants having been reported. We report on the crystallographic analysis of VIM-5 and comparison of biochemical and biophysical properties of VIM-1, VIM-2, VIM-4, VIM-5, and VIM-38. Recombinant VIM variants were produced and purified, and their secondary structure and thermal stabilities were investigated by circular dichroism analyses. Steady-state kinetic analyses with a representative panel of β-lactam substrates were carried out to compare the catalytic efficiencies of the VIM variants. Furthermore, a set of metalloenzyme inhibitors were screened to compare their effects on the different VIM variants. The results reveal only small variations in the kinetic parameters of the VIM variants but substantial differences in their thermal stabilities and inhibition profiles. Overall, these results support the proposal that protein stability may be a factor in MBL evolution and highlight the importance of screening MBL variants during inhibitor development programs.

Nationwide study of Escherichia coli producing extended-spectrum β-lactamases TEM, SHV and CTX-M in Turkey

Four hundred and forty extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates were collected from 10 different hospitals in Turkey between 2011 and 2012. Clinical specimens consisted of urine (80.45%), blood (6.59%), cerebrospinal fluid (1.13%), pleural fluid (2.95%), wound (4.31%) and sputum (4.54%). ESBL-coding genes (CTX-M1, CTX-M2, TEM, SHV) were detected by PCR. According to the PCR and sequencing results, CTX-M1 was the most prevalent β-lactamase 83.18% (366/440), followed by TEM 44.09% (194/440), CTX-M2 31.81% (140/440) and SHV 1.81% (8/440). Sequencing results showed that TEM and SHV types were TEM-1b and SHV-11, respectively. Rate of the strains harboring only CTX-M1, CTX-M2, TEM-1b and SHV-11 were 30.90%, 3.63%, 2.27% and 0.23%, respectively. Rate of the strains harboring the combinations of CTX-M1-CTX-M2, CTX-M1-CTX-M2-TEM-1b, CTX-M2-TEM-1b, CTX-M1-TEM-1b, CTX-M1-CTX-M2-TEM-1b-SHV-11, CTX-M1-TEM-1b-SHV-11, CTX-M1-SHV-11, CTX-M1-CTX-M2-SHV-11, CTX-M2-SHV-11, CTX-M2-TEM-1b-SHV-11, TEM-1b-SHV-11 were 12.95%, 11.59%, 2.95%, 26.13%, 0.45%, 0.68%, 0.22%, 0.22%, 0%, 0% and 0%, respectively. This is a nationwide study of ESBL-producing E. coli in Turkey. These results shows that CTX-M1 group is the most common type of class A β-lactamases among ESBL-producing E. coli strains in Turkey.

Detection of class 1 integron in Acinetobacter baumannii isolates collected from nine hospitals in Turkey

OBJECTIVE To investigate the antibiotic resistance genes inserted into class 1 and class 2 integrons in Acinetobacter baumannii (A. baumannii) isolates obtained from nine different cities in Turkey. METHODS A collection of 281 A. baumannii clinical isolates were collected from nine diferent state hospitals in Turkey and were confirmed as A. baumannii by conventional biochemical, API testing and bla-OXA-51 specific PCR. The isolates were examined by PCR for existence of class 1 and 2 integron gene cassettes. RESULTS They were characterized by antimicrobial susceptibility testing and the highest resistance rates were determined for piperacillin (90.03%), ciprofloxacin (87.54%), cefepime and trimethoprim/sulfamethoxazole (81.13%). The lowest resistance rates was for cefotaxime (3.55%). class I integrons were detected in 6.4% (18/281) of A. baumannii strains and no class 2 integron was detected. The gene cassettes of class 1 integrons AacC1-AAC(3)I-aadA1, AacC1-aadA1, AAC(3)-I, AAC(3)-I -AAC(3)-I -aadA1, TEM-1, AAC(3)-I-aadA1 - AAC(3)-I -AAC(3)-I, AAC(3)-I -AAC(3)-I -AAC(3)-I -aadA1, AAC(3)-I - aadA1, AAC(3)-I-AAC(3)-I, AAC(3)-I-aadA1- AAC(3)-I-aadA1, AAC(3)-I- AAC(3)-I- aadA1-AAC(3)-I-aadA1 were detected in eighteen strains. The aac genes family were most frequently found integrated into the class 1 integrons and it was followed by aadA genes and TEM-1 genes. CONCLUSIONS This is an extensive study on the distribution of class 1 integron among A. baumannii in Turkey. In addition to these, two new alleles were observed. Their percentage rates of similarity to other cassettes are 95% aadA1 ( TKA18) and 89% aadA1 (ANKA3).

Distribution of β-lactamase genes among carbapenem-resistant Klebsiella pneumoniae strains isolated from patients in Turkey.

Background The emergence of carbapenem-resistant Klebsiella pneumoniae poses a serious problem to antibiotic management. We investigated the β-lactamases in a group of carbapenem-resistant K. pneumoniae clinical isolates from Turkey. Methods Thirty-seven strains of K. pneumoniae isolated from various clinical specimens were analyzed by antimicrobial susceptibility testing, PCR for the detection of β-lactamase genes, DNA sequencing, and repetitive extragenic palindronic (REP)-PCR analysis. Results All 37 isolates were resistant to ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, ceftazidime, cefoperazone/sulbactam, cefepime, imipenem, and meropenem. The lowest resistance rates were observed for colistin (2.7%), tigecycline (11%), and amikacin (19%). According to PCR and sequencing results, 98% (36/37) of strains carried at least one carbapenemase gene, with 32 (86%) carrying OXA-48 and 7 (19%) carrying NDM-1. No other carbapenemase genes were identified. All strains carried a CTX-M-2-like β-lactamase, and some carried SHV- (97%), TEM- (9%), and CTX-M-1-like (62%) β-lactamases. Sequence analysis of blaTEM genes identified a blaTEM-166 with an amino acid change at position 53 (Arg53Gly) from blaTEM-1b, the first report of a mutation in this region. REP-PCR analysis revealed that there were seven different clonal groups, and temporo-spatial links were identified within these groups. Conclusions Combinations of β-lactamases were found in all strains, with the most common being OXA-48, SHV, TEM, and CTX-M-type (76% of strains). We have reported, for the first time, a high prevalence of the NDM-1 (19%) carbapenemase in carbapenem-resistant K. pneumoniae from Turkey. These enzymes often co-exist with other β-lactamases, such as TEM, SHV, and CTX-M β-lactamases.

Antimicrobial Resistance Patterns and Integron Carriage of Escherichia coli Isolates Causing Community-Acquired Infections in Turkey

We aimed to observe antimicrobial resistance patterns and integron carriage of Escherichia coli isolates causing community-acquired infections. Two hundred sixty-eight E. coli strains were obtained from outpatients with various infections at different polyclinics at the 82nd Year of State Hospital in Rize, Turkey. Susceptibility to antimicrobials was tested using a disk diffusion method. The presence of integrons was examined using PCR with specific primers. Positive PCR results were confirmed by sequencing. A broth mating method was used for conjugation assays. Extragenic palindromic-PCR was performed using the oligonucleotide primer BOXA1R. Resistance frequency for ampicillin, trimethoprim/sulfamethoxazole, and tetracycline was determined as 50.6%, 33.5%, and 36.8% respectively. No strains were resistant to amikacin. Seventy isolates were positive for the intI1 gene, of which 49 carried gene cassettes. Eleven isolates were positive for the intI2 gene, eight of which carried gene cassettes. Seven gene cassettes (dfrA1, dfrA5, dfrA7, dfrA17, aadA1, aadA5, and sat2) were predominantly harbored in integrons. We detected conjugative plasmids harboring integrons in two E. coli strains. Four strain clusters were yielded by BOX-PCR fingerprints showing that they were clonally related. No apparent relationship occurred among class 1 and 2 integron-carrying strains. We conclude that integrons are widespread in genetically variable E. coli strains and will continue to mediate dissemination of resistance genes in the community.

Molecular characterisation and control of Acinetobacter baumannii isolates resistant to multi-drugs emerging in inter-intensive care units

BackgroundA nosocomial outbreak of Acinetobacter baumannii (AB) infections occurred among intensive care units (ICU) (surgery, medical, cardiovascular surgery, coronary unit) of Recep Tayyip Erdogan University Medical School (Rize, Turkey) between January 2011 and May 2012. The identification of isolates and clonal relation among them were investigated by molecular techniques.MethodsA total of 109 AB isolates were obtained from 64 clinical materials from 54 ICU patients and 3 from the hands of healthcare workers (HCWs) of 42 environmental samples. The isolates were identified by 16S rDNA sequencing and OXA- specific PCR. The clonal relation between isolates was investigated by PFGE methods using ApaI restriction enzyme.ResultsAll isolates were determined as AB by 16S rDNA sequencing and OXA-spesific PCR. While the blaOXA-51-like gene was amplified in all isolates, the blaOXA-23-like gene was amplified from 103 isolates. The PFGE pattern generated 9 pulsotypes and showed that the isolates from patients, HCWs, and the environment were genetically related. In 7 of these pulsotypes, there were 107 strains (98%) showing similar PFGE profiles that cannot be distinguished from each other, ranging from 2 to 53. The remaining 2 pulsotypes were comprised of strains closely associated with the main cluster. Two major groups were discovered with similarity coefficient of 85% and above. The first group consisted of 97 strains that are similar to each other at 92.7% rate, and the second group consisted of 12 strains that are 100% identical.ConclusionsThe common utilization of the blood gas device among ICU was the reason for the contamination. AB strains can remain stable for a long period of time, although due to the disinfection procedures applied in hospitals, there is a small chance that the same clone might reappear and cause another epidemic. For that reason, the resistance profiles of the strains must be continuously followed with amplification-based methods, and these methods should be used to support the PFGE method in the short term.