We determined the antibiotic susceptibility and genetic mechanisms of resistance in clinical strains of Acinetobacter baumannii from Istanbul, Turkey. A total of 101 clinical strains were collected between November 2011 and July 2012. Antimicrobial susceptibility was performed using the Vitek 2 Compact system and E-test. Multiplex PCR was used for detecting bla(OXA -51-like), bla(OXA -23-like), bla(OXA -40-like) and bla(OXA -58-like) genes. ISAba1, bla(IMP -like), bla(VIM -like), bla(GES), bla(VEB), bla(PER -2), aac-3-Ia and aac-6'-Ib and NDM-1 genes were detected by PCR and sequencing. By multiplex PCR, all strains were positive for bla(OXA -51), 79 strains carried bla(OXA -23) and one strain carried bla(OXA -40). bla(OXA -51) and bla(OXA -23) were found together in 79 strains. ISAba1 element was detected in 81 strains, and in all cases it was found upstream of blaOXA -51 . GES-type carbapenemases were found in 24 strains (GES-11 in 16 strains and GES-22 in 8 strains) while bla(PER -2), bla(VEB -1), bla(NDM -1), blaIMP - and blaVIM -type carbapenemases were not observed. Aminoglycoside modifying enzyme (aac-3-Ia and aac-6'Ib) genes were detected in 13 and 15 strains, respectively. Ninety-seven (96%) A. baumannii strains were defined as MDR and of these, 98% were extensively drug resistant (sensitive only to colistin). Colistin remains the only active compound against all clinical strains. As seen in other regions, OXA-type carbapenemases, with or without an upstream ISAba1, predominate but GES-type carbapenemases also appear to have a significant presence. REP-PCR analysis was performed for molecular typing and all strains were collected into 12 different groups. To our knowledge, this is the first report of GES-11 and OXA-40 in A. baumannii from Turkey.

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.

BackgroundThe 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.MethodsThirty-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.ResultsAll 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.ConclusionsCombinations 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.

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

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).

Pseudomonas aeruginosa is one of the leading nosocomial pathogens worldwide, and their infections are difficult to treat due to acquired resistance to many antibiotics. This study aimed to detect class 1 and 2 integrons and antibiotic susceptibility of clinical isolates of P. aeruginosa. Two hundred and five P. aeruginosa strains were collected from the seven general state hospitals in Turkey. They were characterized by antimicrobial susceptibility testing, screened for class 1 and class 2 integrons, and evaluated for the association between antibiotic resistance phenotypes and the presence of integrons. intI gene was amplified in 10 isolates (4.87%) by PCR and in seven isolates of them (70%) were found different gene cassettes. The aadA gene integrated into the class 1 integrons was most frequently found and it was followed by aac genes and bla OXA family genes. Sequence analysis of variable regions of the class 1 integrons showed five gene cassette arrays;

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.