Whole-genome sequence analysis of clinically isolated carbapenem resistant Escherichia coli from Iran.

BACKGROUND: The emergence of carbapenem-resistant Enterobacterales (CRE) continues to threaten public health due to limited therapeutic options. In the current study the incidence of carbapenem resistance among the 104 clinical isolates of Escherichia coli and the genomic features of carbapenem resistant isolates were investigated. METHODS: The susceptibility to imipenem, tigecycline and colistin was tested by broth dilution method. Susceptibility to other classes of antimicrobials was examined by disk diffusion test. The presence of blaOXA-48, blaKPC, blaNDM, and blaVIM carbapenemase genes was examined by PCR. Molecular characteristics of carbapenem resistant isolates were further investigated by whole-genome sequencing (WGS) using Illumina and Nanopore platforms. RESULTS: Four isolates (3.8%) revealed imipenem MIC of ≥32 mg/L and positive results for modified carbapenem inactivation method and categorized as carbapenem resistant E. coli (CREC). Colistin, nitrofurantoin, fosfomycin, and tigecycline were the most active agents against all isolates (total susceptibility rate of 99, 99, 96 and 95.2% respectively) with the last three compounds being found as the most active antimicrobials for carbapenem resistant isolates (susceptibility rate of 100%). According to Multilocus Sequence Type (MLST) analysis the 4 CREC isolates belonged to ST167 (n = 2), ST361 (n = 1) and ST648 (n = 1). NDM was detected in all CREC isolates (NDM-1 (n = 1) and NMD-5 (n = 3)) among which one isolate co-harbored NDM-5 and OXA-181 carbapenemases. WGS further detected blaCTX-M-15, blaCMY-145, blaCMY-42 and blaTEM-1 (with different frequencies) among CREC isolates. Co-occurrence of NDM-type carbapenemase and 16S rRNA methyltransferase RmtB and RmtC was found in two isolates belonging to ST167 and ST648. A colistin-carbapenem resistant isolate which was mcr-negative, revealed various amino acid substitutions in PmrB, PmrD and PhoPQ proteins. CONCLUSION: About 1.9% of E. coli isolates studied here were resistant to imipenem, colistin and/or amikacin which raises the concern about the outbreaks of difficult-to-treat infection by these emerging superbugs in the future.

Molecular characterization of quinolone resistance and antimicrobial resistance profiles of Klebsiella pneumoniae and Escherichia coli isolated from human and broiler chickens.

This study characterized quinolone (Q) resistance determinants in a series of Klebsiella pneumoniae (n = 26) and Escherichia coli (n = 19) isolates of human and animal origin. The presence of plasmid-mediated quinolone resistance (PMQR) and carabpenemase genes was examined by PCR. The quinolone resistance-determining regions (QRDRs) of gyrA and parC genes were sequenced. Thirty-three isolates had ciprofloxacin MIC≥8 mg/l. About 34.6% and 10.5% of K. pneumoniae and E. coli isolates were ESBL producers respectively. The PMQR genes were detected in 77% (n = 35) of isolates. The oqxAB was the most prevalent PMQR gene being identified in all K. pneumoniae isolates, followed by aac(6')-Ib-cr (34.6%), qnrS (23%) and qnrB (7.7%). The most frequently detected gene among E. coli isolates was qnrS (36.8%) followed by aac(6')-Ib-cr (10.5%) and qepA (5.2%). All Q resistant isolates harbored amino acid substitutions in both GyrA and ParC QRDRs. High prevalence of PMQR genes among food-producing animal isolates is an issue of great concern.

Evaluating the performance characteristics of different antimicrobial susceptibility testing methodologies for testing susceptibility of gram-negative bacteria to tigecycline.

BACKGROUND: The current emergence of multi-drug resistance among nosocomial pathogens has led to increased use of last-resort agents including Tigecycline (TGC). Availability of reliable methods for testing TGC susceptibility is crucial to accurately predict clinical outcomes. We evaluated the influence of different methodologies and type of media on TGC susceptibility of different gram-negative bacteria of clinical origin. METHODS: The TGC susceptibility of 84 clinical isolates of Klebsiella pneumoniae (n = 29), Escherichia coli (n = 30), and Acinetobacter baumannii (n = 25) was tested by broth microdilution (BMD), Etest, agar dilution (AD) and disk diffusion (DD) methods using Mueller Hinton agar from Difco and Mueller Hinton broth (MHB) from two different manufacturers (Difco and Condalab). FDA TGC susceptibility breakpoints issued for Enterobacteriaceae were used for interpretation of the results. RESULTS: MICs determined by BMD using MHB from two suppliers showed a good correlation with overall essential agreement (EA) and categorical agreement (CA) being 100% and 95% respectively. However, a twofold rise in BMD-Condalab MICs which was detected in 50% of the isolates, resulted in changes in susceptibility categories of few isolates with MICs close to susceptibility breakpoints leading to an overall minor error (MI) rate of 4.7%. Among the tested methods, Etest showed the best correlation with BMD, being characterized with the lowest error rates (only 1% MI) and highest overall EA (100%) and CA (98.8%) for all subsets of isolates. AD yielded the lowest overall agreement (EA 77%, CA 81%) with BMD in a species dependent manner, with the highest apparent discordance being found among the A. baumannii isolates. While the performance of DD for determination of TGC susceptibility among Enterobacteriaceae was excellent, (CA:100% with no errors), the CA was lower (84%) when it was used for A. baumannii where an unacceptably high minor-error rate was noted (16%). No major error or very major error was detected for any of the tested methods. CONCLUSIONS: Etest can be reliably used for TGC susceptibility testing in the three groups of studied bacteria. For the isolates with close-to-breakpoint MICs, testing susceptibility using the reference method is recommended.

Molecular Characterization of Tigecycline Non-Susceptibility among Extensively Drug-Resistant Acinetobacter baumannii Isolates of Clinical Origin.

INTRODUCTION: Tigecycline (TGC) is one of the last-resort therapeutic agents for treating infections caused by extensively drug resistant Acinetobacter baumannii isolates. Although resistance to TGC is not common, non-susceptible A. baumannii (NSAB) isolates have been described. In the current study, we aimed to assess the molecular mechanisms mediating TGC non-susceptibility in 5 clinical isolates of A. baumannii with reduced susceptibility to TGC. METHODS: Susceptibility of isolates to TGC as well as various classes of antibiotics was evaluated by broth dilution and disk diffusion methods, respectively. The presence of tetX and tetX1 genes was investigated by PCR. The nucleotide sequences of adeR and adeS genes were assessed by PCR amplicon sequencing. To evaluate the association between reduced susceptibility to TGC and upregulation of AdeABC efflux pump, transcriptional level of adeB gene was quantified by RT-qPCR analysis. RESULTS: All 5 TGC-NSAB isolates had a TGC MIC of ≥4 mg/L and were resistant to all antimicrobials tested by disk diffusion method except for minocycline and doxycycline for which a susceptibility rate of 40% and 20% was observed, respectively. The tetX/X1 genes were not detected in any isolates. All TGC non-susceptible isolates harbored genetic alterations in the adeRS operon, including AdeS G186V, N268H, and D60N and AdeR A136V and V120I substitutions among, which G186V and D60N were predicted by PROVEAN tool analysis as inactivating alterations. Reduced TGC susceptibility was associated with upregulation of AdeABC efflux pump in all TGC non-susceptible isolates. CONCLUSION: It can be concluded from our results that reduced susceptibility to TGC in the studied isolates was mainly mediated by genetic alterations in the AdeRS system, which resulted in overexpression of AdeABC efflux pump. Emergence of TGC non-susceptibility among isolates that had not been previously exposed to TGC is an issue of great concern.

Molecular characterization of Staphylococcus aureus isolates from southwest of Iran using spa and SCCmec typing methods.

Staphylococcus aureus remains a major cause of nosocomial infection worldwide. Characterization of S. aureus isolates circulating in the southwest of Iran will contribute to understand and control the spread of the strains in this area. spa and SCCmec typing methods were used for genotyping of 125 S. aureus isolates obtained from two teaching hospitals in Ahvaz. Drug susceptibility testing was performed by using disk diffusion method. Frequency of the methicillin resistant S. aureus (MRSA) isolates was 39% (n = 34) and 27% (n = 10) in Emam Khomeini and Golestan hospitals, respectively. Except for Erythromycin, MRSA strains showed high rate of resistance to antimicrobial agents including penicillin (100%), norfloxacine (80%), azitromycin (80%), ciprofloxacin (80%), gentamycin (77%), cotrimoxazole (75%), cephotaxime. All isolates were sensitive to vancomycin. Out of 44 MRSA strains, 39 (88.5%) were SCCmec III, three (7%) were IVc and two (4.5%) of them were nontypeable. spa types t037 (26 isolates; 59%), and t1149 (25 isolates; 31%) were the most dominant types found in MRSA and methicillin sensitive S. aureus (MSSA) strains, respectively. We found SCCmec type III as the most prominent type indicating that most of the studied bacterial population had hospital origin. spa type t037, the most frequent genotype in this study were significantly (100%) associated with MRSA. For the first time we are reporting spa types t692, t706 and t018 from Iran and t342, t704, t2622, t5598, t11270 and t2864 from Asia. Moreover we are reporting types t6871 and t2684 for the second time in the world.

Disulfiram and Copper Ions Kill Mycobacterium tuberculosis in a Synergistic Manner.

Tuberculosis is a severe disease affecting millions worldwide. Unfortunately, treatment strategies are hampered both by the prohibitively long treatment regimen and the rise of drug-resistant strains. Significant effort has been expended in the search for new treatments, but few options have successfully emerged, and new treatment modalities are desperately needed. Recently, there has been growing interest in the synergistic antibacterial effects of copper ions (Cu(II/I)) in combination with certain small molecular compounds, and we have previously reported development of a drug screening strategy to harness the intrinsic bactericidal properties of Cu(II/I). Here, we describe the copper-dependent antimycobacterial properties of disulfiram, an FDA-approved and well-tolerated sobriety aid. Disulfiram was inhibitory to mycobacteria only in the presence of Cu(II/I) and exerted its bactericidal activity well below the active concentration of Cu(II/I) or disulfiram alone. No other physiologically relevant bivalent transition metals (e.g., Fe(II), Ni(II), Mn(II), and Co(II)) exhibited this effect. We demonstrate that the movement of the disulfiram-copper complex across the cell envelope is porin independent and can inhibit intracellular protein functions. Additionally, the complex is able to synergistically induce intracellular copper stress responses significantly more than Cu(II/I) alone. Our data suggest that by complexing with disulfiram, Cu(II/I) is likely allowed unfettered access to vulnerable intracellular components, bypassing the normally sufficient copper homeostatic machinery. Overall, the synergistic antibacterial activity of Cu(II/I) and disulfiram reveals the susceptibility of the copper homeostasis system of Mycobacterium tuberculosis to chemical attacks and establishes compounds that act in concert with copper as a new class of bacterial inhibitors.

Copper complexation screen reveals compounds with potent antibiotic properties against methicillin-resistant Staphylococcus aureus.

Macrophages take advantage of the antibacterial properties of copper ions in the killing of bacterial intruders. However, despite the importance of copper for innate immune functions, coordinated efforts to exploit copper ions for therapeutic interventions against bacterial infections are not yet in place. Here we report a novel high-throughput screening platform specifically developed for the discovery and characterization of compounds with copper-dependent antibacterial properties toward methicillin-resistant Staphylococcus aureus (MRSA). We detail how one of the identified compounds, glyoxal-bis(N4-methylthiosemicarbazone) (GTSM), exerts its potent strictly copper-dependent antibacterial properties on MRSA. Our data indicate that the activity of the GTSM-copper complex goes beyond the general antibacterial effects of accumulated copper ions and suggest that, in contrast to prevailing opinion, copper complexes can indeed exhibit species- and target-specific activities. Based on experimental evidence, we propose that copper ions impose structural changes upon binding to the otherwise inactive GTSM ligand and transfer antibacterial properties to the chelate. In turn, GTSM determines target specificity and utilizes a redox-sensitive release mechanism through which copper ions are deployed at or in close proximity to a putative target. According to our proof-of-concept screen, copper activation is not a rare event and even extends to already established drugs. Thus, copper-activated compounds could define a novel class of anti-MRSA agents that amplify copper-dependent innate immune functions of the host. To this end, we provide a blueprint for a high-throughput drug screening campaign which considers the antibacterial properties of copper ions at the host-pathogen interface.

Genetic Alterations Associated with Colistin Resistance Development in Escherichia coli.

Background: The increased incidence of infections due to multidrug-resistant Gram-negative bacteria has led to the renewed interest in the use of 'forgotten' antibiotics such as colistin. In this work, we studied the chromosomal colistin resistance mechanisms among laboratory-induced colistin-resistant Escherichia coli isolates. Methods: Three colistin-susceptible (ColS) clinical isolates of E. coli assigning to ST131, ST405, and ST361 were exposed to successively increasing concentrations of colistin. The nucleotide sequences of pmrA, pmrB, pmrD, phoP, phoQ, and mgrB genes were determined. The fitness burden associated with colistin resistance acquisition was determined by measuring the in vitro growth rate. Results: Colistin resistance induction resulted in 16-64 times increase in colistin MICs in mutants (n = 8) compared with parental isolates. Analysis of chromosomal genes in colistin-resistant mutants compared with those of ColS ancestors revealed genetic alterations confined to PmrAB two-component system and included PmrA G53R/R81S/L105P and PmrB E121K/E121A/A159P/A159V/G302E changes. The PmrB E121 was found as a critical position for colistin resistance development being altered in three mutants with different ancestors. The acquired colistin-resistance phenotype was stable following 10 consecutive passages in the absence of selective pressure of colistin and it did not alter the susceptibility of mutants to other antimicrobial agents. All mutants exhibited growth rates similar to their respective ColS ancestors, except for one isolate, which revealed a significant growth defect. Conclusion: Our results revealed that colistin resistance in E. coli was more related to PmrAB alterations, which did not impose a fitness cost in most cases.

Assessment of in vitro activity of ceftazidime/avibactam on carbapenemase-producing Enterobacterales from Iran: An experimental study.

Background and Aims: The prevalence of carbapenemase-producing Enterobacterales (CPE) continues to increase worldwide. Combination of ß-lactam and novel ß-lactamase inhibitors introduce a revolutionary treatment option for CPE. Ceftazidime/avibactam (CAZ/AVB) has been recently developed for treatment of severe infections caused by multidrug-resistant bacteria. We aimed to evaluate in vitro activity of CAZ/AVB on a collection of 85 ESBL-producing-carbapenemase negative and CPE from Iran. Methods: ESBL and carbapenemase production was phenotypically confirmed by combined disk test and modified carbapenem inactivation method respectively. The presence of clinically important carbapenemase encoding genes was examined using PCR. Susceptibility of all isolates to CAZ/AVB was determined using discs containing 30 µg ceftazidime +20 µg avibactam (AVB). Minimum inhibitory concentrations (MICs) of CAZ/AVB in 28 CPE (4 Escherichia coli and 24 Klebsiella pneumoniae) was determined by gradient diffusion method using MIC test strips (0.016-256 mg/L ceftazidime +4 mg/L AVB). Results: All phenotypically identified ESBL positive-carbapenemase negative isolates were found to be susceptible to CAZ/AVB. Among the carbapenem resistant isolates, CAZ/AVB showed potent inhibitory activity against all OXA-48-like (MIC ranges 0.125/4-0.75/4 mg/L) and KPC positive isolates (MIC ranges <0.016/4-0.19/4 mg/L). However, AVB could not restore the activity of ceftazdime against isolates producing metallo-ß-lactamases (MLBs) including VIM, NDM (MIC > 256/4 mg/L) and IMP (MIC > 8/4 mg/L). Conclusion: Our data highlighted the excellent in vitro performance of CAZ/AVB against ESBL-producing and CPE suggesting that this combination can efficiently be used as therapeutic option for management of CPE infections particularly in regions with high prevalence of KPC and/or OXA-48-like positive but MBL-negative Enterobacterales.

Porins increase copper susceptibility of Mycobacterium tuberculosis.

Copper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 µM copper by 40% and completely suppressed growth at 15 µM copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis.

High prevalence of OXA-48-like and NDM carbapenemases among carbapenem resistant Klebsiella pneumoniae of clinical origin from Iran.

Background and Objectives: Klebsiella pneumoniae is increasingly developing resistance to last-resort antibiotics such as carbapenems. This study aimed to investigate the dissemination of common carbapenemase encoding genes among 48 clinical isolates of carbapenem-resistant Klebsiella pneumoniae (CRKP). Materials and Methods: Antimicrobial susceptibility testing was performed by broth dilution and disc diffusion methods. The phenotypic evaluation of carbapenemase production was performed by using Modified Carbapenem Inactivation Method. Presence of carbapenemase encoding genes blaKPC, blaNDM, blaOXA-48-like , blaIMP, and blaVIM was screened by PCR. Results: Overall, carbapenemases were produced in all CRKP isolates. The blaOXA-48-like and blaNDM were the most prevalent genes detected among all and 66.6% (n=32) of CRKP isolates respectively. The blaVIM was detected in only one isolate co-harboring NDM and OXA-48-like carbapenemases. The blaKPC and blaIMP genes were not identified in any of the isolates. While tigecycline was the most active agent against CRKP isolates with low resistance rate (4.1%), high rate of resistance was observed to colistin (66.6%), amikacin (79%) and most of other tested antimicrobials. Conclusion: Our results revealed predominant prevalence of OXA-48-like and NDM carbapenemases among CRKP clinical isolates. High rate of resistance to last-resort agents such as colistin among CRKP isolates is a source of great concern.

Genomic features of in vitro selected mutants of Escherichia coli with decreased susceptibility to tigecycline.

OBJECTIVES: The increase in multidrug-resistant bacteria has reached an alarming rate globally, making it necessary to understand the underlying mechanisms mediating resistance in order to discover new therapeutics. Tigecycline (TGC) is a last-resort antimicrobial agent for the treatment of serious infections caused by extensively drug-resistant Enterobacteriaceae. METHODS: The TGC-resistant Escherichia coli mutants were obtained by exposing three different TGC-susceptible isolates belonging to ST131 (n = 2) and ST405 (n = 1) to increasing concentrations of TGC. The genetic alterations associated with reduced susceptibility to TGC were identified using whole genome sequencing. The fitness cost of TGC resistance acquisition, as well as incidence of cross-resistance, was also investigated. RESULTS: The TGC minimum inhibitory concentrations (MICs) of in vitro selected mutants were elevated 8 to 32 times compared with ancestral strains. Inactivating mutations (frameshift and nonsense) or amino acid substitutions were identified in genes encoding proteins with diverse functions, including AcrAB efflux pump or its regulators (lon and marR), Lipopolysaccharides (LPS) inner core biosynthesis enzymes (waaQ and eptB), ribosomal S9 protein (rpsI), and RNA polymerase ß subunit. In most cases (but not all), acquisition of TGC resistance was associated with a fitness cost. While TGC resistance development was associated with cross-resistance to other members of the tetracycline family and chloramphenicol, hypersensitivity to nitrofurantoin was identified among heptose III-less LPS mutants. CONCLUSION: TGC resistance among the studied mutants was found to be multifactorial with extrusion by efflux transports being the most common mechanism. The LPS inner core biosynthesis pathway, as well as ribosomal S9 protein, could be additional targets for TGC resistance.

Characterization of Tigecycline Resistance Among Tigecycline Non-susceptible Klebsiella pneumoniae Isolates From Humans, Food-Producing Animals, and in vitro Selection Assay.

Emergence of extensively drug-resistant isolates of Klebsiella pneumoniae has prompted increased reliance on the last-resort antibiotics such as tigecycline (TGC) for treating infections caused by these pathogens. Consumption of human antibiotics in the food production industry has been found to contribute to the current antibiotic resistance crisis. In the current study, we aimed to investigate the mechanisms of TGC resistance among 18 TGC-non-susceptible (resistant or intermediate) K. pneumoniae (TGC-NSKP) isolates obtained from human (n = 5), food animals (n = 7), and in vitro selection experiment (n = 6). Isolates were genotyped by multilocus sequence typing (MLST). ramR, acrR, rpsJ, tetA, and mgrB (for colistin resistance) genes were sequenced. The presence of tetX, tetX1, and carbapenemase genes was examined by PCR. Susceptibility to different classes of antibiotics was evaluated by disc diffusion and broth macrodilution methods. The expression level of acrB was quantified by RT-qPCR assay. The 12 TGC-NSKP isolates [minimum inhibitory concentrations (MICs) = 4-32 mg/l] belonged to 10 distinct sequence types including ST37 (n = 2), ST11, ST15, ST45, ST1326 (animal isolates); ST147 (n = 2, human and animal isolates); and ST16, ST377, ST893, and ST2935 (human isolates). Co-resistance to TGC and colistin was identified among 57 and 40% of animal and human isolates, respectively. All human TGC-NSKP isolates carried carbapenemase genes (bla OXA - 48, bla NDM - 1, and bla NDM - 5). tetX/X1 genes were not detected in any isolates. About 83% of TGC-NSKP isolates (n = 15) carried ramR and/or acrR alterations including missense/nonsense mutations (A19V, L44Q, I141T, G180D, A28T, R114L, T119S, Y59stop, and Q122stop), insertions (positions +205 and +343), or deletions (position +205) for ramR, and R90G substitution or frameshift mutations for acrR. In one isolate ramR amplicon was not detected using all primers used in this study. Among seven colistin-resistant isolates, five harbored inactivated/mutated MgrB due to premature termination by nonsense mutations, insertion of IS elements, and frameshift mutations. All isolates revealed wild-type RpsJ and TetA (if present). Increased expression of acrB gene was detected among all resistant isolates, with the in vitro selected mutants showing the highest values. A combination of RamR and AcrR alterations was involved in TGC non-susceptibility in the majority of studied isolates.

Evaluation of in vitro activity of ceftaroline on methicillin resistant Staphylococcus aureus blood isolates from Iran.

BACKGROUND AND OBJECTIVES: Ceftaroline (CPT) is a novel cephalosporin with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). Despite its recent introduction, CPT resistance in MRSA has been described worldwide. We aimed in the current study to evaluate the in vitro activity of CPT against 91 clinical MRSA and 3 MSSA isolates. MATERIALS AND METHODS: Susceptibility of isolates to CPT was tested using E-test and disk diffusion (DD) method. The nucleotide sequence of the mecA gene and molecular types of isolates with reduced susceptibility to CPT were further studied to identify resistance conferring mutations in PBP2a and the genetic relatedness of the isolates respectively. RESULTS: Overall, 92.5% of isolates were found to be CPT susceptible (MICs≤1mg/l) and 7 MRSA isolates were characterized with MIC=2mg/l and categorized as susceptible dose dependent. Compared to E-test, DD revealed a categorical agreement rate of 93.6% and the obtained rates for minor, major /very major error were found to be 6.3% and 0% respectively. The MRSA isolates with increased CPT MICs (n=7), belonged to spa types t030 (n=6) and t13927 (n=1) and all carried N146K substitution in PBP2a allosteric domain, except for one isolate which harbored a wild-type PBP2a. CONCLUSION: While resistance to CPT was not detected we found increased CPT MICs in 7.69% of MRSA isolates. Reduced susceptibility to CPT in the absence of mecA mutations is indicative of contribution of secondary chromosomal mutations in resistance development.

Molecular epidemiology and nitrofurantoin resistance determinants of nitrofurantoin-non-susceptible Escherichia coli isolated from urinary tract infections.

OBJECTIVES: The worldwide emergence of multidrug-resistant uropathogens has resulted in the revival of old antibiotics such as nitrofurantoin (NIT) for the treatment of uncomplicated urinary tract infections (UTIs). This study aimed to identify determinants of NIT resistance and to investigate the genetic diversity of NIT-resistant (NIT-R) Escherichia coli isolates. METHODS: Six NIT-R and three NIT-susceptible clinical E. coli isolates from patients with UTI were studied. The susceptibility of the isolates to various classes of antibiotics was evaluated by disk diffusion. The presence of plasmid-encoded efflux pump genes (oqxA and oqxB) was investigated by PCR. Nucleotide sequences of the nfsA, nfsB and ribE genes were determined. The genetic relatedness of the NIT-R isolates was evaluated by multilocus sequence typing (MLST). RESULTS: All six NIT-R isolates were characterised with high-level NIT resistance (MIC ≥ 512 mg/L) and they belonged to five distinct STs including ST131 (n = 2), ST73, ST405, ST10 and ST354 (n = 1 each). Amikacin, carbapenems, minocycline, tigecycline and fosfomycin were the most active agents against the studied uropathogens. The oqxA and oqxB genes were not detected in any isolate. All NIT-R isolates harboured inactivating genetic alterations in nfsA and nfsB [NfsA H11Y, S33N, S38Y, W212R substitutions, Δg638 (frameshift), Δa64-g73 (frameshift) and NfsB F84S, P45S, W94Stop, E197Stop substitutions, ΔnfsB locus]. The ribE gene of most isolates was unaffected, except for one isolate co-harbouring a deleterious RibE G85C substitution and NfsA/B alterations. CONCLUSION: NIT resistance in the studied E. coli isolates was mainly mediated by nfsA and nfsB alterations.

Molecular identification of mutations conferring resistance to rifampin, isoniazid and pyrazinamide among Mycobacterium tuberculosis isolates from Iran.

Here, we aimed to determine the susceptibility of 70 Mycobacterium tuberculosis isolates obtained from different regions of the country to 8 anti-tuberculosis (anti-TB) drugs and possible underlying mechanisms causing resistance to rifampin, isoniazid, and pyrazinamide. The susceptibility of 70 isolates of M. tuberculosis to anti-TB drugs was tested using proportion method. Strains showing resistance to the first line anti-TB drugs were subjected to PCR amplification and sequencing of the rpoB, katG, ahpC, pncA genes, inhA promoter and oxyR-ahpC intergenic regions to detect resistance conferring mutations. Overall, 77.1% and 77.1% of isolates were resistant to at least one of the tested first- and second-line drugs, respectively. Within the rpoB gene the highest rate of mutation was found in codons 531(450) (56.3%), and 533(452) (12.5%). Also, codons 315 (42.4%) of katG, positions -48, -72 and -77 of oxyR-ahpC (total= 3, 9.1%) and -15 of inhA promoter region (33.3%) were the most altered positions in isoniazid resistant isolates. Only a single mutation was detected for pncA among resistant isolates. High prevalence of resistance to essential anti-TB drugs among M. tuberculosis strains isolated from retreated tuberculosis cases is alarming issue necessitating immediate action to prevent the spread of drug resistant isolates in the country.

Prevalence and molecular determinants of colistin resistance among commensal Enterobacteriaceae isolated from poultry in northwest of Iran.

BACKGROUND: The emergence of colistin-resistant Enterobacteriaceae from human and animal sources is a public health concern as this antibiotic is considered to be the last line therapeutic option for infections caused by multidrug-resistant Gram-negative bacteria. Here we aimed to determine the prevalence of colistin resistance, among enterobacteria isolated from poultry and the possible underlying colistin resistance mechanisms. METHODS: A collection of 944 cloacal samples were obtained from poultry and screened for colistin resistance. To uncover the molecular mechanism behind colistin resistance, the presence of plasmid encoded colistin resistance genes mcr-1, mcr-2, mcr-3 and mcr-4 was examined by PCR. The nucleotide sequences of the mgrB, pmrA, pmrB, phoP, phoQ, crrA and crrB genes were determined. The genetic relatedness of the colistin resistant (ColR) isolates was evaluated by Multilocus sequence typing. Three ColR mutants were generated in vitro by repetitive drug exposure. RESULTS: Overall from 931 enteric bacteria isolated from poultry samples obtained from 131 farms, nine ColR bacteria (0.96%) with high level colistin resistance (MICs ≥ 64 mg/L) were detected all being identified as K. pneumoniae. The 9 ColR bacteria originated from different farms and belonged to 7 distinct Sequence types including ST11 (22.2%) and ST726 (22.2%) being the most prevalent STs followed by ST37, ST74, ST485, ST525 and novel sequence type 3380 (11.1% each). mcr-type genes were not detected in any isolate. In 88.8% of the isolates (n = 8), MgrB was inactivated by Insertion of IS elements (IS1-like, IS3-like, IS5-like families, positions + 75, + 113, + 117, + 135) and nonsense mutations at codons 8, 16, 30. All ColR isolates harboured wild type PmrA, PhoP, PhoQ or polymorphic variants of PmrB. Sequence analysis of the CrrB revealed a familiar S195N and 4 novel I27V, T150R, F303S and K325R substitutions. PmrB T93N substitution and mgrB locus deletion were identified in two laboratory induced ColR mutants and one mutant lacked alteration in the studied loci. In one ColR isolate with wild type MgrB an A83V substitution was detected in CrrA. CONCLUSION: It is concluded from our results that colistin resistance in the studied avian K. pneumoniae isolates was mostly linked to alterations identified within the mgrB gene.

Comparison of susceptibility testing methods for determining the activity of colistin against Gram-negative bacilli of clinical origin.

PURPOSE: Despite being in clinical use for decades, colistin susceptibility testing remains challenging because of its inherent cationic properties. We aimed to compare the performance characteristics of different methods for testing susceptibility to colistin in a series of clinical isolates of Gram-negative bacilli. METHODOLOGY: One hundred and nine clinical isolates of Klebsiella pneumoniae (n=34), Escherichia coli (n=20), Acinetobacter baumannii (n=17) and Pseudomonas aeruginosa (n=38) were studied for colistin susceptibility using broth microdilution (BMID), broth macrodilution (BMAD), agar dilution (AD) as well as disc-diffusion (DD) utilizing two different commercial disc sources. RESULTS: By using BMID as reference method, 88 and 21 isolates were found to be colistin susceptible and resistant, respectively. Overall, acceptable essential agreement (EA) and categorical agreement (CA) were observed between BMAD and reference method (100 %). Whereas the AD method revealed the lowest rate of EA (61.7, 11.7, 5.0 and 5.2 % for K. pneumoniae, A. baumannii, E. coli and P. aeruginosa, respectively), it showed acceptable or near acceptable CA for K. pneumoniae (100 %), E. coli (100 %) and A. baumannii (88.2 %) isolates but not for P. aeruginosa (13.1 %). DD failed to detect resistance in colistin-resistant (colR) P. aeruginosa (n=5, very major errors of 100 %) but successfully identified all high-level colistin-resistant A. baumannii and K. pneumoniae isolates. CONCLUSION: We found BMAD to be very reliable for colistin MIC determination. Methods AD and DD should not be used for colistin susceptibility testing in P. aeruginosa isolates as these are associated with false-resistant and -susceptible results, respectively.

Expression analysis of 10 efflux pump genes in multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis clinical isolates.

OBJECTIVES: Active extrusion of antituberculosis drugs via efflux pumps (EPs) has been suggested as contributing to drug resistance in Mycobacterium tuberculosis. This study was conducted to determine the role of 10 drug efflux transporters in the development of drug resistance in a series of clinical M. tuberculosis isolates. METHODS: A total of 31 clinical M. tuberculosis isolates without drug exposure [21 multi/extensively drug-resistant (M/XDR-TB) and 10 drug-susceptible isolates] were studied. The expression profile of 10 EP genes, including efpA, mmr, stp, drrA, drrB, mmpL7, Rv1250, Rv1634, Rv2994 and Rv1258c, was investigated against the H37Rv standard strain by quantitative reverse transcription PCR (RT-qPCR). RESULTS: Among the 21M/XDR-TB isolates, 10 showed significantly increased levels of gene expression (>4-fold) for at least one of the studied EPs. Moreover, of the isolates with overexpressed genes, three and seven lacked genetic alterations in the surveyed regions of the rpoB+katG+inhA and katG+inhA genes, respectively. Whilst no elevation was observed in the expression of mmr, Rv1250, Rv1634 and Rv1258c genes in any of the isolates, drrA, stp and drrB were found to be the most commonly overexpressed, being overexpressed in seven, five and three isolates, respectively. Decreased minimum inhibitory concentrations (MICs) of rifampicin, but not isoniazid, were observed in the presence of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). CONCLUSION: Overexpression of EP genes can contribute to the emergence of a MDR phenotype in M. tuberculosis. Inhibition of EPs may provide a promising strategy for improving tuberculosis treatment outcomes in patients infected with M/XDR-TB isolates.

Molecular Epidemiology and Drug Resistance Pattern of Carbapenem-Resistant Klebsiella pneumoniae Isolates from Iran.

The emergence and dissemination of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates and their involvement in several nosocomial outbreaks are of high concern. This study was conducted to investigate the genetic relatedness and molecular determinants of carbapenem resistance in 100 CRKP isolates. Susceptibility to carbapenems as well as other antibiotics was determined by using disk diffusion method. The Modified Hodge test was performed for detection of carbapenemase production. The minimum inhibitory concentrations of selected antibiotics were determined by broth microdilution method. The presence of blaOXA-48, blaKPC, blaNDM, and blaVIM carbapenemase genes was examined by PCR, and clonal relatedness of CRKP isolates was investigated by pulsed-field gel electrophoresis (PFGE) analysis. blaOXA-48 was the most frequent carbapenemase gene (72%), followed by blaNDM (31%). None of the isolates harbored blaKPC and blaVIM genes. PFGE separated the majority of isolates into 10 clusters, including the major clusters A and B, carrying blaOXA-48, and clusters C and D, carrying blaNDM, and 4 isolates had a unique PFGE pattern. An increased rate of colistin resistance (50%) was detected among the isolates. Tigecycline was found to be the most active agent against CRKP isolates. Our results revealed that high prevalence of blaOXA-48 and blaNDM carbapenamses and resistance to colistin are alarming threats, necessitating an immediate action to prevent the spread of carbapenem-colistin-resistant K. pneumoniae isolates in Iran.