Types Of Β-lactamases Research Articles

Prevalence and molecular epidemiology of ESBLs, plasmid-determined AmpC-type β-lactamases and carbapenemases among diarrhoeagenic Escherichia coli isolates from children in Gwangju, Korea: 2007-16.

Young children could act as important carriers of cefotaxime-resistant Enterobacteriaceae. However, most studies on these bacteria have focused on hospitalized adults. Therefore, we determined the prevalence and characteristics of ESBL-, plasmid-determined AmpC-type β-lactamase (PABL)- and carbapenemase-producing diarrhoeagenic Escherichia coli isolates mainly from infants and children in the south-west region of Korea over a 10 year period. Non-duplicate E. coli clinical isolates were recovered from diarrhoeagenic patient specimens at 12 hospitals in Gwangju, Korea, between January 2007 and December 2016. Antimicrobial susceptibilities and molecular features of ESBL- and carbapenemase-producing isolates were determined. A total of 1047 pathogenic E. coli isolates were collected and 58 cefotaxime-resistant E. coli isolates (5.5%) were identified. The prevalence and types of β-lactamase genes increased steadily from 5.7% in 2007 to 11.6% in 2016 with some fluctuations. CTX-M-14 (53.4%) was the predominant CTX-M genotype. PFGE revealed high genetic heterogeneities among diarrhoeagenic E. coli isolates, suggesting horizontal transfer of antibiotic resistance genes, which was also proved by conjugation assay. Progressive increases in carriage rates and the number of β-lactamase types, and the possibility of community outbreaks of these food-borne bacteria in young children, may pose tangible public health threats.

X-ray Crystallography Deciphers the Activity of Broad-Spectrum Boronic Acid β-Lactamase Inhibitors.

Recent decades have witnessed a dramatic increase of multidrug resistant (MDR) bacteria, compromising the efficacy of available antibiotics, and a continual decline in the discovery of novel antibacterials. We recently reported the first library of benzo[b]thiophen-2-ylboronic acid inhibitors sharing broad spectrum activity against β-lactamases (BLs). The ability of these compounds to inhibit structurally and mechanistically different types of β-lactamases has been here structurally investigated. An extensive X-ray crystallographic analysis of boronic acids (BAs) binding to proteins representative of serine BLs (SBLs) and metallo β-lactamases (MBLs) have been conducted to depict the role played by the boronic group in driving molecular recognition, especially in the interaction with MBLs. Our derivatives are the first case of noncyclic boronic acids active against MBLs and represent a productive route toward potent broad-spectrum inhibitors.

Distribution of Extended-spectrum β-lactamase and Metallo-β-lactamase-producing Pseudomonas aeruginosa in Tertiary Care Hospitals of Lahore, Pakistan

Background: Pseudomonas aeruginosa (P. aeruginosa) is an important bacterial pathogen most frequently associated with nosocomial infections, especially in immuno-compromised patients. Early detection of these life threatening, β-lactamase producing bacteria is essential for infection control and to prevent their dissemination. The aim of our study was to detect the presence of Extended-Spectrum β-Lactamase (ESBL) and Metallo-β-Lactamase (MBL) strains of Pseudomonas aeruginosa.Material and Methods: Eighty-eight identified strains of P. aeruginosa were collected from Chughtai Laboratories, Combined Military Hospital and Children Hospital, Lahore. These strains were sub-cultured and after confirming the cultural characteristics by Gram staining and colony morphology, manual biochemical identification was done. Susceptibility to various antibiotics and production of extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) were determined using modified Kirby Bauer disk diffusion method, double disk synergy test, combined disk synergy test (CDST) and inhibitor-potentiated disk diffusion test (IPD) respectively.Results: Out of eighty-eight strains tested, three were ESBL producers (3.4%) and eleven strains (12.5%) were found to be resistant to carbapenems. Of these, eight were MBL producers (72.7%). All these β-lactamase producing strains (14 strains) were multidrug-resistant (MDR). Piperacillin and piperacillin/tazobactam proved to be the most effective antibiotics in both types of β-lactamase producing strains.Conclusion: Our study shows noticeable emergence of β-lactamases (ESBLs & MBLs) in P. aeruginosa. All of these strains were MDR. It reveals a correlation of these β-lactamases with multidrug resistant genes.

Significant role of Asn-247 and Arg-64 residues in close proximity of the active site in maintaining the catalytic function of CTX-M-15 type β-lactamase.

Members of Enterobacteriaceae cause antibiotic-resistant infections worldwide. One such marker, CTX-M-15, expressed by Enterobacteriaceae produces β-lactamases, which hydrolyze the cephalosporin group of antibiotics, such as cefotaxime, used in the treatment of both Gram-positive and negative bacterial infections. Amino acid residues present in close proximity of the active site might also play a major role in the structure and function of CTX-M-15, hence the objective of this study was to investigate the significance of two amino acid residues, Asn-247 and Arg-64, present near to the active site in the hydrolysis of cefotaxime. blaCTX-M-15, cloned from the E. cloacae strain, and using Polymerase Chain Reaction (PCR)-based site-directed mutagenesis, Asn247Val and Arg64Leu mutations were introduced. The minimum inhibitory concentrations of cefotaxime for the CTX-M-15 (N247V) and CTX-M-15 (R64L) mutants were reduced by 512 and 128 fold, respectively. Proteins/enzymes of wild-type CTX-M-15, CTX-M-15 (N247V) and CTX-M-15 (R64L) mutants were expressed and purified. Kinetic studies showed that the catalytic efficiencies of the N247V mutant and R64L mutant enzymes in the hydrolysis of cefotaxime were reduced by 89.66% and 71.11%, respectively. Circular dichroism spectroscopic studies showed considerable changes in the α-helical content of the mutant enzymes. A fluorescence study showed that N247V mutant-cefotaxime and R64L mutant-cefotaxime underwent complex formation with strong interactions. The study provides an understanding of the crucial role of the amino acid residues asparagine 247 and arginine 64 present in close proximity of the active site in the hydrolytic mechanism of CTX-M-15 type β-lactamases. Hence, Asn-247 and Arg-64 can be used as potential target sites for the design of inhibitory molecules against CTX-M-15-producing bacterial strains.

Scaffold hopping computational approach for searching novel β-lactamase inhibitors

We present a novel computational ligand-based virtual screening approach with scaffold hopping capabilities for the identification of novel inhibitors of β-lactamases which confer bacterial resistance to β-lactam antibiotics. The structures of known β-lactamase inhibitors were used as query ligands, and a virtual in silico screening a database of 8 million drug-like compounds was performed in order to select the ligands with similar shape and charge distribution. A set of numerical descriptors was used such as chirality, eigen spectrum of matrices of interatomic distances and connectivity together with higher order moment invariants that showed their efficiency in the field of pattern recognition but have not yet been employed in drug discovery. The developed scaffold-hopping approach was applied for the discovery of analogues of four allosteric inhibitors of serine β-lactamases. After a virtual in silico screening, the effect of two selected ligands on the activity of TEM type β-lactamase was studied experimentally. New non-β-lactam inhibitors were found that showed more effective inhibition of β-lactamases compared to query ligands.

Virulence and resistance determinants of Klebsiella pneumoniae isolated from a Portuguese tertiary university hospital centre over a 31-year period

IntroductionThe rapid and complex evolution of bacterial resistance mechanisms in Klebsiella pneumoniae producing extended-spectrum β-lactamases and carbapenemases in Klebsiella pneumoniae is one of the most significant threats to public health. However, questions and controversies regarding the interactions between resistance and virulence in multidrug-resistant K. pneumoniae isolates remain unclear. MethodsA retrospective cohort study was performed with 100 K. pneumoniae isolates recovered from a tertiary care university hospital centre in Lisbon over a 31-year period. Resistance and virulence determinants were screened using molecular methods (PCR, M13-PCR and MLST). ResultsThe predominant virulence profile (fimH, mrkDv1, khe) was shared by all isolates, indicative of an important role of type 1 and 3 fimbrial adhesins and haemolysin, regardless of the type of β-lactamase produced. However, accumulation of virulence factors was identified in KPC-3-producers, with a higher frequency (p<0.05) of capsular serotype K2 and iucC aerobactin when compared with non-KPC-3 β-lactamases or carbapenemases. Additionally, 9 different virulence profiles were found, indicating that the KPC-3 carbapenemase producers seem to adapt successfully to the host environment and maintain virulence via several pathways. ConclusionThis study describes an overlapping of multidrug-resistance and virulence determinants in ST-14K2 KPC-3 K. pneumoniae clinical isolates that may impose an additional challenge in the treatment of infections caused by this pathogen.

Evaluation of the RAPIDEC CARBA NP, Conventional CarbaNP, and the Modified Carbapenem Inactivation Method (mCIM) Tests for Phenotypic Detection of Carbapenemase-Producing Organisms

Decreased susceptibility to carbapenems is being increasingly reported among Gram-negative organisms worldwide. It is of critical importance for patient management and infection control purposes to detect carbapenemase-producing organisms rapidly and reliably. In this study, the performance of the commercially available RAPIDEC CARBA NP test (bioMérieux, Durham, NC) was evaluated against the performance of the conventional CarbaNP and the modified Carbapenem Inactivation Method (mCIM) for the detection of carbapenemase activity in Enterobacteriaceae and Pseudomonas aeruginosa. A total of 73 Enterobacteriaceae and 32 Pseudomonas aeruginosa isolates were tested for carbapenemase activity using the RAPIDEC CARBA NP, conventional CarbaNP, and mCIM methods. Using available whole-genome sequence information, this collection included 72 isolates with and 33 isolates without carbapenemase genes. The isolates harboring a carbapenemase gene encoded 20 class A, 47 class B, and 5 class D–type β-lactamases. All methods were performed using bacteria from a single overnight blood agar plate. The conventional CarbaNP and mCIM were performed according to guidelines from the Clinical and Laboratory Standards Institute. The RAPIDEC CARBA NP was performed per the manufacturer’s instructions. The RAPIDEC CARBA NP detected 65 of 72 (90%) carbapenemase-producing isolates; the seven isolates yielding false-negative results included five Enterobacteriaceae with blaOXA-48-like and two with blaKPC-4 genes. The sensitivity and specificity were 90.2% and 100%, respectively, for the RAPIDEC test; 94.4% and 100% for conventional CarbaNP; and 100% and 81.8% for mCIM. The conventional CarbaNP detected all carbapenemase producers except four OXA-48-like–producing isolates. The mCIM, which accurately detected all carbapenemase producers, yielded false-positive results in six isolates of P aeruginosa. Overall, the RAPIDEC CARBA NP was easy to use and required less hands-on time than the conventional CarbaNP and mCIM methods. However, the RAPIDEC CARBA NP showed decreased sensitivity for detecting low-level activity carbapenemases such as OXA-48-like enzymes and KPC-4.

Molecular Characterization of Extended-Spectrum β-Lactamase-Producing Multidrug Resistant Escherichia coli From Swine in Northwest China.

Objectives: The aim of the present study was to explore the prevalence and molecular characterization of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli collected from pig farms in Northwest China.Methods: Between May 2015 and June 2017, a total of 456 E. coli isolates were collected from fecal samples of healthy and diarrheal pigs in Northwest China to screen the ESBL producers. The β-lactamases, plasmid-mediated quinolone resistance (PMQR) genes and virulence genes among ESBL producers were corroborated by PCR and sequencing. Finally, ESBL producers were further grouped according to phylogenetic background and genetic relatedness.Results: Forty-four (9.6%) out of the 456 E. coli isolates were identified as ESBL-producing isolates. All ESBL producers exhibited multidrug resistance (MDR) phenotype, and more than 90% of the ESBL producers were resistant to amoxicillin, amoxicillin-clavulanic acid, oxytetracycline, enrofloxacin and sulfamethoxazole/trimethoprim. All ESBL producers harbored at least one type of β-lactamase, with blaCTX−M, blaTEM, blaSHV, blaOXA−48, and blaKPC−2 being detected in forty, thirty, seven, four, two and one isolates, respectively. Sequencing revealed the most common blaCTX−M subtype was blaCTX−M−14 (n = 24), followed by blaCTX−M−15 (n = 14), blaCTX−M−64 (n = 11), blaCTX−M−9 (n = 10) and blaCTX−M−123 (n = 9). qnrS (n = 23) was the predominant PMQR gene, and all PMQR genes were detected in co-existence with β-lactamase genes. estA (n = 18) and F4 (n = 18) were the most prevalent enterotoxin and fimbrial adhesin, respectively, and 27 different virotypes were found with respect to the association of enterotoxins and fimbrial adhesins. Twenty-four different sequence types (STs) were identified among 44 ESBL producers, and clones ST405, ST10 and ST648 were strongly present in more than one-third (34.1%) of ESBL producers.Conclusion: All ESBL-producing E. coli isolates exhibited MDR phenotype, and showed high prevalence of β-lactamase and PMQR genes. Especially, one isolate harbored ESBL genes blaTEM, blaSHV, blaCTX−M−9, blaCTX−M−14, blaCTX−M−64, and carbapenemase gene blaOXA−48 and blaKPC−2, as well as PMQR genes qnrS, qnrB, qnrD, qepA and aac(6')-Ib-cr.

Diversity Determination of CTX-M1 Producing Klebsiella pneumoniae Using Multilocus Variable-Number Tandem Repeat Analysis, Semnan, Iran

Background: CTX-M is the most prevalent and rapidly growing type of the extended-spectrum β-lactamase (ESBL) family and CTX-M1 is the most common type of blaCTX-M. Objectives: The current study aimed at investigating the genetic diversity of CTX-M-1-producing Klebsiella pneumoniae circulating in Semnan, Iran evaluated by multilocus variable-number tandem repeat analysis (MLVA). Methods: A total of 110 isolates of K. pneumoniae were collected from different clinical samples. The antibiotic suceptibility and double disk synergy test were determined according to CLSI (the clinical and laboratory standards institute) guidelines. The polymerase chain reaction (PCR) method was performed to detect CTX-M-1. The eight loci for MLVA genotyping were selected along with the primers previously described. Results: Imipenem, with 84.7% susceptibility, was the most effective antibiotic against K. pneumoniae. Seventy (63.63%) isolates had ESBL positive results and 42 (60 %) of them were positive for CTX-M-1 gene. Totally, 28 MLVA genotypes were discriminated, evaluation of diversity indexes (DIs) for eight loci showed that six different alleles were the most polymorphic and the most DI was 0.807. Conclusions: The findings of the current study demonstrated heterogeneity among CTX-M-1-producing K. pneumonia strains. The presence of CTX-M-1 in different MLVA types demonstrated that a certain clone is not responsible for spreading the isolates.

Identification of CFE-2, a new plasmid-encoded AmpC β-lactamase from a clinical isolate of Citrobacter freundii

A clinical isolate of Citrobacter freundii (JA99) obtained from a bile culture of a Taiwanese patient was found to produce a plasmid-encoded β-lactamase conferring resistance to oxyimino-cephalosporins and cephamycins. Resistance arising from production of the β-lactamase could be transferred by conjugation with an IncW plasmid (pJA99) into Escherichia coli J53. The substrate and inhibition profiles of this enzyme resembled that of an AmpC β-lactamase. The resistance gene of pJA99, cloned and expressed in E. coli DH5α, was shown to contain an open reading frame showing 92% amino acid identity with the plasmid-encoded enzyme CFE-1 of E. coli KU6400. DNA sequence analysis also identified a gene upstream of ampC in pJA99 whose sequence was 95.0% identical to the ampR gene from E. coli KU6400. In addition, orf1, the fumarate operon (frdABCD), blc, lolB and repB surrounding the ampR-ampC genes in C. freundii were identified. This DNA fragment was absent in other Citrobacter spp. Therefore, we describe a new plasmid-encoded AmpC β-lactamase, named CFE-2. This study highlights the emergence of broad-spectrum cephalosporin resistance in C. freundii owing to a new type of AmpC β-lactamase.

Combination of aztreonam and cefotaxime against CTX-M-15 type β-lactamases: A mechanism based effective therapeutic approach

CTX-M-15 type β-lactamases are a class of enzymes which hydrolyzes cefotaxime and aztreonam (a monobactam) antibiotics. The emergence of CTX-M-15 producing Enterobacteriaceae member is a major threat to public health. The objective of the study was to check the potency of aztreonam and cefotaxime in combination against β-lactamase producing strains and to monitor the mechanism behind their interaction. FICI results showed the synergistic effect of aztreonam-cefotaxime against CTX-M-15 producing strain. The expressed and purified CTX-M-15 protein was used as the source of enzyme. Kinetic studies confirmed that the catalytic efficiency of the CTX-M-15 enzyme was decreased to about 78% when it was treated with aztreonam then with cefotaxime in 5 and 10 times molar ratio, respectively, in comparison to the studies where efficiency was enhanced by 33% when cefotaxime was taken alone. Fluorescence study showed that aztreonam binding with CTX-M-15 was an endothermic and spontaneous process with Ka of the order of 104 M−1. CD spectroscopic study showed conformational changes upon aztreonam/aztreonam-cefotaxime binding with CTX-M-15. The study concludes that aztreonam in combination with cefotaxime synergistically inhibits CTX-M-15 efficiency significantly. Hence the combination of a monobactam and cephalosporin can be used as the potential therapeutic candidates against β-lactamase producing CTX-M-15 strains.

Analysis of resistance genes of clinical Pannonibacter phragmitetus strain 31801 by complete genome sequencing.

To clarify the resistance mechanisms of Pannonibacter phragmitetus 31801, isolated from the blood of a liver abscess patient, at the genomic level, we performed whole genomic sequencing using a PacBio RS II single-molecule real-time long-read sequencer. Bioinformatic analysis of the resulting sequence was then carried out to identify any possible resistance genes. Analyses included Basic Local Alignment Search Tool searches against the Antibiotic Resistance Genes Database, ResFinder analysis of the genome sequence, and Resistance Gene Identifier analysis within the Comprehensive Antibiotic Resistance Database. Prophages, clustered regularly interspaced short palindromic repeats (CRISPR), and other putative virulence factors were also identified using PHAST, CRISPRfinder, and the Virulence Factors Database, respectively. The circular chromosome and single plasmid of P. phragmitetus 31801 contained multiple antibiotic resistance genes, including those coding for three different types of β-lactamase [NPS β-lactamase (EC 3.5.2.6), β-lactamase class C, and a metal-dependent hydrolase of β-lactamase superfamily I]. In addition, genes coding for subunits of several multidrug-resistance efflux pumps were identified, including those targeting macrolides (adeJ, cmeB), tetracycline (acrB, adeAB), fluoroquinolones (acrF, ceoB), and aminoglycosides (acrD, amrB, ceoB, mexY, smeB). However, apart from the tripartite macrolide efflux pump macAB-tolC, the genome did not appear to contain the complete complement of subunit genes required for production of most of the major multidrug-resistance efflux pumps.

Increased exposure to extended-spectrum β-lactamase-producing multidrug-resistant Enterobacteriaceae through the consumption of chicken and sushi products

The aim of this study was to determine the occurrence and patterns of resistance of extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae in food products purchased in Navarra, northern Spain. A total of 174 samples of fish and chicken were analyzed from September 2015 to September 2016, including raw and ready-to-eat products: trout (n = 25), salmon (n = 28), panga (n = 13), chicken nuggets and chicken scalopes (n = 32), sushi (n = 31) and sliced cooked poultry (n = 45). Cefpodoxime-resistant strains were isolated on ChromID ESBL agar and further phenotypic (antimicrobial study on MicroScan© NM37 panel) and genotypic characterization (multiplex PCR, sequencing and multi-locus sequence typing, MLST) was performed to confirm and characterize ESBL producers. Raw chicken and sushi have been determined as the most risky products regarding transmission of ESBL-producing Enterobacteriaceae (occurrence 53.1% and 19.4%, respectively), while sliced cooked poultry products appear to be a safe product in this aspect. With regard to raw fish, prevalence in salmon was lower (3.6%) than in trout and panga (16.0%). Ninety-eight per cent of ESBL isolates (n = 50) show multidrug-resistant profiles, highlighting the high resistances against quinolones and tetracyclines observed in chicken isolates, as well as against ertapenem and chloramphenicol in sushi strains. Predominant β-lactamase type was SHV-12 (50.1%), followed by TEM-type (24.5%) and CTX-M (20.8%). In addition, CTX-M type was only detected in chicken products. The phylogenetic study showed the prevalence of groups A (35%), F (25%) and B1 (15%), usually related to nonvirulent strains. MLST E. coli isolates (n = 20) were grouped into 5 clonal complexes (CC) and 15 sequence types (ST), showing high clonal diversity. ST117 was the prevalent sequence type, while the human pathogen ST131 was not detected in this study. The high prevalence of ESBL-producing multidrug-resistant Enterobacteriaceae detected in products of widespread consumption such as chicken and sushi, increases the concern regarding human exposure to superbugs and encourages the need to improve surveillance of this public health issue.

Synergistic Effect of Doripenem and Cefotaxime to Inhibit CTX-M-15 Type β-Lactamases: Biophysical and Microbiological Views

CTX-M-15 type β-lactamase has the ability to hydrolyse cefotaxime, a third generation cephalosporin. The infections caused by multidrug resistant strains, especially CTX-M-15 producing strains are being treated with carbapenem group of β-lactam antibiotics. The objective of the study was to know if cefotaxime in combination with doripenem (carbapemen antibiotic) at very low concentration, inhibits the CTX-M-15 producing bacterial strains. blaCTX−M−15 gene was cloned to express CTX-M-15 enzyme and construct CTX-M-15 producing strain. The clone carrying CTX-M-15 was found susceptible to doripenem. Doripenem and CTX-M-15 binding was an endothermic and spontaneous process leading to change in polarity in the micro-environment and conformational changes of enzyme as shown by fluorescence, UV and CD spectroscopic study. The catalytic efficiency of CTX-M-15 enzyme was reduced to about 15.86% when it was treated with doripenem along with cefotaxime (in 5 times molar ratio each of doripenem and cefotaxime w.r.t CTX-M-15), as compared to the studies where enzyme's efficiency was increased by 33% when treated with cefotaxime alone. Hence, doripenem in combination with cefotaxime reduces enzyme's efficiency to hydrolyse cefotaxime by about 48%. FIC study showed that doripenem paired with cefotaxime showed synergistic effect against CTX-M-15 producing bacterial strain. The study concludes that doripenem at very low concentration (25 nM), induces such a structural changes in CTX-M-15 which reduced enzyme's activity to hydrolyse cefotaxime. Hence, the synergistic use of doripenem and cefotaxime plays a significant role in inhibiting the efficiency of CTX-M-15 type β-lactamase, and may provide an alternative approach to reduce the resistance against the cephalosporin type antibiotics.

Designing of inhibitors against CTX-M-15 type β-lactamase: potential drug candidate against β-lactamases-producing multi-drug-resistant bacteria

CTX-M-15 are the most prevalent types of β-lactamases that hydrolyze almost all antibiotics of β-lactam group lead to multiple-antibiotic resistance in bacteria. Three β-lactam inhibitors are available for use in combination with different antibiotics of cephalosporine group against the CTX-M-15-producing strains. Therefore, strategies to identify novel anti β-lactamase agents with specific mechanisms of action are the need of an hour. In this study, we screened three novel non-β-lactam inhibitors against CTX-M-15 by multi-step virtual screening approach. The potential for virtually screened drugs was estimated through in vitro cell assays. Hence, we proposed a study to understand the binding mode of CTX-M-15 with inhibitors using bioinformatics and experimental approach. We calculated the dissociation constants (Kd), association constant (Ka), stoichiometry (n) and binding energies (ΔG) of compounds with the respective targets. Molecular dynamic simulation carried out for 25 ns, revealed that these complexes were found stable throughout the simulation with relative RMSD in acceptable range. Moreover, microbiological and kinetic studies further confirmed high efficacies of these inhibitors by reducing the minimum inhibitory concentration (MIC) and catalysis of antibiotics by β-lactamases in the presence of inhibitors. Therefore, we conclude that these potential inhibitors may be used as a lead molecule for future drug candidates against β-lactamases-producing bacteria.

Antimicrobial Susceptibility Pattern and Prevalence of Extended-Spectrum β-Lactamase Genotypes among Clinical Isolates of Acinetobacter baumanii in Tabriz, North-West of Iran

Background: Multidrug resistant (MDR) Acinetobacter baumanii strains have emerged as novel nosocomial pathogens threatening patients’ lives, especially in intensive-care units (ICU). Various types of extended-spectrum β-lactamases (ESBLs) are involved in conferring resistance to β-lactam antibiotics, making their genotypic characterization an essential prerequisite to take proper preventative measures. Objectives: The aim of this study was to determine the antimicrobial susceptibility and prevalence of blaTEM, blaSHV, blaCTX-M, blaOXA-2, and blaOXA-10 genes among A. baumanii isolates obtained from patients in Tabriz city, North-west Iran. Methods: The clinical isolates of A. baumanii were collected from patients hospitalized in the Imam Reza hospital of Tabriz. Antimicrobial susceptibility patterns were determined by the disk diffusion method. The frequency of different ESBLS resistance genes were determined by PCR. Results: Antimicrobial susceptibility testing through the disk diffusion method revealed that the lowest resistance rates were against polymyxin B (16%), colistin (23%), and rifampin (27%); whereas the highest resistance rate was observed against ticarcillin (100%), cefixime (100%), and ceftizoxim (100%). Screening by double disk synergy test showed that 60% of the isolates were ESBL producers. PCR technique on ESBL-positive isolates determined blaSHV gene as the most prevalent (31.6%) and blaOXA-10 as the least prevalent (8.3%) among the studied resistance genes. Conclusions: The high prevalence of resistance genes supported the essential role of ESBLs in antibiotic resistance of A. baumanii.

The study of the role of mutations M182T and Q39K in the TEM-72 β-lactamase structure by the molecular dynamics method

Synthesis of β-lactamases is one of the common mechanisms of bacterial resistance to β-lactam antibiotics such as penicillins and cephalosporins. The widespread use of antibiotics resulted in appearance of numerous extended-spectrum β-lactamase variants or inhibitor-resistant β-lactamases. In TEM type β-lactamases mutations of 92 residues have been described. Several mutations are functionally important and they determine the extended substrate specificity. However, roles of the most so-called associated mutations, located far from the active site, remain unknown. We have investigated the role of associated mutations in structure of β-lactamase TEM-72, which contains two key mutations (G238S, E240K) and two associated mutations (Q39K, M182T) by means of molecular dynamics simulation. Appearance of the key mutations (in 238 and 240 positions) caused destabilization of the protein globule, characterized by increased mobility of amino acid residues. Associated mutations (Q39K, M182T) exhibited opposite effect on the protein structure. The mutation M182T stabilized, while the mutation Q39K destabilized the protein. It appears that the latter mutation promoted optimization of the conformational mobility of β-lactamase and may influence the enzyme activity.

CTX-M-15 Type β-lactamases From Clinical Isolates of Escherichia coli by Polymerase Chain Reaction and DNA Sequencing

Background: E. coli is a common cause of urinary tract infection and bacteremia in humans. That is frequently resistant to β-lactam antibiotics. Objectives: The aim of this study was to determine the antibiotic resistance and CTX-M extended-spectrum β-lactamases (ESBL) carrying E. coli isolated in hospitals from two different areas in Iran. Methods: In this descriptive cross-sectional study, 93 clinical isolates of E. coli were collected from urine culture of inpatients in Mashhad and Qom hospitals from August 2013 to February 2014. Bacteria were re-confirmed by Microgen kit. Antimicrobial and ESBL tests were performed using by standard disc diffusion method. Polymerase chain reaction (PCR) was used for detection of plasmid mediated CTX-M-1 and CTX-M-2 ESBLs. Four of the blaCTX-Mgenes were sequenced. Statistical analysis carried out using SPSS (version 16) software and Chi-square test. Results: High prevalent isolates were resistant to ampicillin, cephalothin, nalidixic acid and cotrimoxazole, but sensitive to imipenem, nitrofurantoin and amikacin. The ESBL phenotype was detected in 48 (51.6%) isolates, 42 of which were carrying blaCTX-Mgene. There was no significant difference between the β-lactamase-producing bacteria from the two studied cities. 81% and 85% of blaCTX-Mcarriers from hospitals in Qom and Mashhad were respectively found to have CTX-M-1 ESBL, whereas none of them showed CTX-M-2 ESBL cluster. BlaCTX-M-15was found in four selected bacteria by sequencing. Conclusions: This study showed a high rate of CTX-M-1 cluster and also presence of CTX-M-15 ESBL in two different areas of Iran, which emphasize the need for employing an excellent management program in antibiotic therapy.

WCK 5222 (cefepime/zidebactam) antimicrobial activity tested against Gram-negative organisms producing clinically relevant β-lactamases.

Zidebactam is a β-lactam enhancer antibiotic with a dual mechanism of action involving binding to Gram-negative PBP2 and β-lactamase inhibition. Cefepime combined with zidebactam (WCK 5222) is under clinical development for treatment of Gram-negative infections. To evaluate the in vitro activities of cefepime and zidebactam separately and combined at 1:1 and 2:1 ratios when tested against Gram-negative organisms producing the most clinically relevant β-lactamase types. β-Lactamase-producing (193) and WT (71) isolates were tested for susceptibility by broth microdilution method against cefepime/zidebactam, cefepime and zidebactam. Cefepime/zidebactam (1:1) was very active against Enterobacteriaceae producing CTX-M-15 (21; MIC 50/90 0.25/1 mg/L), SHV (20; MIC 50/90 0.12/0.25 mg/L), other ESBLs (20, including GES-18, OXA-1/30 and OXY-, PER-, TEM- and VEB-like; MIC 50/90 0.25/1 mg/L), plasmidic AmpC (10; MIC 50/90 ≤0.06/≤0.06 mg/L), derepressed AmpC (23; MIC 50/90 0.12/0.5 mg/L), KPC (35; MIC 50/90 0.25/1 mg/L) and metallo-β-lactamases (MBLs; 20 including VIM, IMP and NDM; MIC 50/90 0.5/8 mg/L). Cefepime/zidebactam (1:1) was also active against Pseudomonas aeruginosa with overexpression of AmpC (21; MIC 50/90 4/8 mg/L) and MBLs [12 (VIM and IMP); MIC 50/90 4/8 mg/L]. Zidebactam alone exhibited potent in vitro activity against some Enterobacteriaceae and P. aeruginosa , including β-lactamase-producing strains. Cefepime/zidebactam MIC values were lower than those of each agent tested alone for many β-lactamase-producing strains, indicating synergy. Cefepime/zidebactam showed moderate activity against OXA-23/24/58-producing Acinetobacter baumannii [MIC 50/90 32 mg/L (1:1)]. Cefepime/zidebactam showed potent activities against Enterobacteriaceae and P. aeruginosa producing various clinically relevant β-lactamases, including ESBLs, KPCs, AmpC and MBLs for which limited treatment options are currently available.

Antibiotic Resistance Determinant-Focused Acinetobacter baumannii Vaccine Designed Using Reverse Vaccinology.

As one of the most influential and troublesome human pathogens, Acinetobacter baumannii (A. baumannii) has emerged with many multidrug-resistant strains. After collecting 33 complete A. baumannii genomes and 84 representative antibiotic resistance determinants, we used the Vaxign reverse vaccinology approach to predict classical type vaccine candidates against A. baumannii infections and new type vaccine candidates against antibiotic resistance. Our genome analysis identified 35 outer membrane or extracellular adhesins that are conserved among all 33 genomes, have no human protein homology, and have less than 2 transmembrane helices. These 35 antigens include 11 TonB dependent receptors, 8 porins, 7 efflux pump proteins, and 2 fimbrial proteins (FilF and CAM87009.1). CAM86003.1 was predicted to be an adhesin outer membrane protein absent from 3 antibiotic-sensitive strains and conserved in 21 antibiotic-resistant strains. Feasible anti-resistance vaccine candidates also include one extracellular protein (QnrA), 3 RND type outer membrane efflux pump proteins, and 3 CTX-M type β-lactamases. Among 39 β-lactamases, A. baumannii CTX-M-2, -5, and -43 enzymes are predicted as adhesins and better vaccine candidates than other β-lactamases to induce preventive immunity and enhance antibiotic treatments. This report represents the first reverse vaccinology study to systematically predict vaccine antigen candidates against antibiotic resistance for a microbial pathogen.