Aysegul Saral

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.

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.

Clinical Variants of the Native Class D β-lactamase of Acinetobacter baumannii pose an emerging threat through increased hydrolytic activity against carbapenems

ABSTRACT The threat posed by the chromosomally encoded class D β-lactamase of Acinetobacter baumannii (OXA-51/66) has been unclear, in part because of its relatively low affinity and turnover rate for carbapenems. Several hundred clinical variants of OXA-51/66 have been reported, many with substitutions of active-site residues. We determined the kinetic properties of OXA-66 and five clinical variants with respect to a wide variety of β-lactam substrates. The five variants displayed enhanced activity against carbapenems and in some cases against penicillins, late-generation cephalosporins, and the monobactam aztreonam. Molecular dynamics simulations show that in OXA-66, P130 inhibits the side-chain rotation of I129 and thereby prevents doripenem binding because of steric clash. A single amino acid substitution at this position (P130Q) in the variant OXA-109 greatly enhances the mobility of both I129 and a key active-site tryptophan (W222), thereby facilitating carbapenem binding. This expansion of substrate specificity represents a very worrisome development for the efficacy of β-lactams against this troublesome pathogen.

Determination of a novel integron-located variant (blaOXA -320 ) of Class D β-lactamase in Proteus mirabilis.

Proteus mirabilis (P. mirabilis) is one of Gram‐negative pathogens encountered in clinical specimens. A clinical isolate (TRP41) of P. mirabilis was isolated from a Turkish patient in Turkey. The isolate was identified using the API 32GN system and 16S rRNA gene sequencing and it was found resistant to ampicillin/sulbactam, piperacillin, tetracycline, and trimethoprim/sulfamethoxazole. This isolate was harboring a Class 1 integron gene cassette and its DNA sequence analysis revealed a novel blaOXA variant exhibiting one amino acid substitution (Asn266Ile) from blaOXA‐1. This new variant of OXA was located on Class 1 integron together with aadA1 gene encoding aminoglycoside‐modifying enzymes. According to sequence records, the new variant was named as blaOXA‐320. Cassette array and size of integron were found as blaOXA‐320‐aadA1 and 2086 bp, respectively. The blaOXA‐320 gene is not transferable according to conjugation experiment. In this study, we report the first identification of blaOXA‐320‐aadA1 gene cassette, a novel variant of Class D β‐lactamase, in P. mirabilis from Turkey.

Isolation, identification and characterization of biotechnologically important bacteria from microflora of Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae)

ABSTRACT The chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) is one of the most important insect pests of chestnut. The aim of this study was to isolate and characterize bacteria from D. kuriphilus to obtain new microbial agents for both biological control and other biotechnological applications. D. kuriphilus larvae were collected from chestnut fields located in Bursa and Yalova provinces of Marmara Region of Turkey during May–July 2014. Four bacterial isolates were obtained from D. kuriphilus. According to their morphological, biochemical and molecular properties, these isolates were identified as Staphylococcus saprophyticus (Dk1), Paenibacillus sp. (Dk2), Pseudomonas flourescens (Dk3) and Paenibacillus sp. (Dk4). To the best of our knowledge, this is the first study on the bacterial flora of D. kuriphilus. In our study, the potential of these isolates as a biological control agent against different hazardous pests and other possible biotechnological applications of importance were discussed under the light of literature.

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

The class A β-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 β-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 β-lactamases, suggesting that this methionine has a key structural role in aligning active site residues and in substrate selectivity across the class.