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The emergence of multidrug (MDR) isolates of Escherichia coli has made the treatment of urinary tract infections (UTI) very challenging. Integrons are considered to be one of the most important mechanisms in the dissemination of resistance genes. In the current study, we sought to investigate the antibiotic resistance patterns, class 1–3 integrons and corresponding gene cassettes in urinary isolates of E. coli in the southwest of Iran. A total of 144 E. coli isolates were collected from the patients with UTIs. Antibiotic susceptibility test conducted by the disc diffusion method using 12 different antibiotics. The polymerase chain reaction (PCR) was used for the detection of intl1, intI2, and intI3 genes. To analyze the gene cassettes, variable regions of class 1 integron were amplified by PCR and subjected to sequencing. The highest rates of resistance were exhibited to amoxicillin/clavulanic (73.6%), trimethoprim-sulfamethoxazole (63.9%) and tetracycline (63.2%). Totally, intI1 and intI2 genes were identified in 62.5 and 9% of isolates. One-hundred and nine (75.7%) E. coli isolates were multidrug resistance (MDR), among which 78% harbored intI1 gene. Also, all of the intI2 positive E. coli isolates were MDR. Ten different gene cassette arrays were found in intl1 positive E. coli including dfrA5, dfrA25, dfrA7, aacA4, aadA1, aadB-catB3, dfrA17-aadA5, dfrA12-orfF-aadA2, aadA1-blaOXA-30, and dfrA5-catB3-aacA4. The dfrA17-aadA5 was the most prominent gene cassette array among E. coli isolates. Characterization of resistance gene cassettes located in integrons in this study showed a high frequency of dfrA and aad genes which confer resistance to trimethoprim and aminoglycosides. The obtained result from this study could be useful in planning to prevent the rising rate of antibiotic resistance.

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 plasm...

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.

Background: Staphylococcus aureus is one of the most important food-borne pathogens. The objective of this study was to determine the prevalence, molecular types and drug resistance pattern of S. aureus isolated from retail meat in Tabriz city. Materials & Methods: 60 raw meat samples (chicken and beef) were taken from different markets and were inoculated in selective Mueller Hinton broth media supplemented with 10% NaCl. Identification of S. aureus isolates was performed using conventional biochemical tests. Susceptibility to different antibiotics and genotypes of isolates were determined by disc diffusion and spa typing methods respectively. Results: Fifteen S. aureus strains were isolated from 60 different meat samples which belonged to spa types t14870, t3802, t1814, t491, t386, t3424 and spa type t14870 with the frequency of 33.3% was the most prevalent genotype among S. aureus isolates. spa types of three isolates were not found in Ridom Spa Server data base and were considered as novel types. About 46.6% of isolates were resistant to more than one antibiotic and 13.3% of isolates were identified as methicillin resistant S. aureus (MRSA). Tigecycline, imipenem and ceftaroline were found to be the most effective agents against S. aureus isolates. Conclusion: Oure results revealed a 25% contamination rate with S. aureus. Most of the molecular types of isolates were found to be linked to human infections. High rate of antibiotic resistance was observed among the isolates which poses a great threat to public health.

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.

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 nu- cleotide 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 character- ized with MIC=2mg/l and categorized as susceptible dose dependent. Compared to E-test, DD revealed a categorical agree- ment 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.

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 (blaOXA–48, blaNDM–1, and blaNDM–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.

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.

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.

This study characterized quinolone (Q) resistance determinants in a series of

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.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.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.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.

Here, we aimed to determine the susceptibility of 70

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.

Colistin is considered a last-hope antibiotic against extensively drug-resistant isolates of Acinetobacter baumannii. Resistance to colistin has been rarely reported for A. baumannii. Genetic alterations in the PmrA-PmrB two-component system and lipid A biosynthesis genes may be associated with colistin resistance. We investigated molecular mechanisms of colistin resistance in three clinical colistin-resistant (ColR) and two colistin-susceptible (ColS) A. baumannii isolates. A ColR mutant was generated in vitro by repetitive drug exposure. The pmrA, pmrB, lpxA, lpxC, and lpxD genes were amplified and sequenced. To evaluate association between colistin resistance and upregulation of pmrCAB operon, transcriptional level of the pmrC gene encoding for lipid A phosphoethanolamine (PEtN) transferase was quantified by reverse transcription quantitative PCR (RT-qPCR) analysis. All clinical and in vitro-selected ColR isolates harbored at least one point mutation in the pmrB gene, including A142V, P233S, T235I, and A227V substitutions as well as duplication of H325. No alteration was found in the pmrA and other amino acid substitutions identified in the pmrB as well as lpx genes did not seem to be involved in colistin resistance as they were found in both ColS and ColR isolates. RT-qPCR analysis revealed a correlation between colistin resistance and pmrC overexpression. Specific alterations in the PmrB, linked to overproduction of PEtN transferase, triggered colistin resistance in the studied A. baumannii isolates.

Infections caused by Staphylococcus aureus remain a major global healthcare problem. We aimed to find the common lineages of S. aureus strains circulating in a burn hospital in Tehran. A total of 167 isolates of S. aureus obtained from patients, healthcare workers (HCWs) and environment in Shahid Motahari burn hospital were genotyped by using spa, agr and staphylococcal cassette chromosome mec (SCCmec) typing methods. Antimicrobial susceptibility testing was performed by using the disc diffusion method. The frequency of methicillin-resistant S. aureus (MRSA) was 64.7% (n = 108), with distribution frequencies among patient, HCW and surface isolates of 64.2% (n = 79), 50% (n = 7) and 73.3% (n = 22), respectively. SCCmec type III (75%, n = 81) was found to be the most frequent SCCmec type among MRSA isolates, followed by SCCmec type I (20.4%, n = 22) and SCCmec type IV (1.8%, n = 2). The remaining MRSA isolates (2.8%, n = 3) were nontypeable by this method. About 78.4% (n = 131), 10.2% (n = 17) and 4.8% (n = 8) of all isolates were characterized as agr types I, II and III, respectively, and the other isolates (6.6%) were nontypeable. spa types t030 and t037 constituted the first and second most predominant spa types found in 56.4% (n = 57) and 25.6% (n = 26) of isolates, respectively. We also report here a novel spa type, t16471. The most prevalent genotypes of the isolates found among patient, surface and HCW samples were SCCmec type III/t030, t037/agr type I. Continuous tracking of epidemic isolates and better hospital infection control policies are recommended to efficiently prevent the spread of bacteria to inpatients. Keywords: agr typing, Iran, MRSA, SCCmec typing, spa typing

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.

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.

Background. Considering that Hormozgan province in Iran (southern part of Iran on the Persian Gulf) is among the areas with high prevalence of MDR-MTB and attracts so many sailors and tourists, genetic diversity of MTB isolates circulating in this part of Iran was evaluated. Pattern of TB transmission was also examined. Methods and Material. A total of 38 isolates of MTB were cultured from TB patients from Hormozgan province of Iran and standard MIRU-VNTR typing and spoligotyping were applied to genotype these isolates. Drug susceptibility testing was performed using proportion method. Results. There were 28 VNTR profiles comprising 5 clusters and 23 unique isolates compared to 21 spoligotyping profiles, which contained 9 clusters and 12 unique isolates. Latin American-Mediterranean (n=9, 23.6%) was found to be the most predominant lineage. MIRU-VNTR analysis, with an HGDI of 0.975, was more discriminating than spoligotyping, which had an HGDI of 0.955. The estimated proportion of TB cases due to recent transmission was 26.3% and 44.7% by MIRU-VNTR and spoligotyping, respectively. The rates of monodrug resistance and MDR were 15.8% and 7.9%, respectively. Two of 3 MDR strains were found to be related to MIRU-VNTR and belonged to the same spoligotyping cluster characterized with T1/SIT53 genotype. Conclusions. The high genetic diversity among MTB isolates suggests that transmission occurred from different sources to this area. Reactivation of a priori, latent MTB infection was found to contribute mainly to TB cases in this geographic region.

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.

Absence of mutations within the genes encoding drug targets in some phenotypically drug resistant strains of Mycobacterium tuberculosis suggests possible involvement of alternative mechanisms such as over-expression of efflux pumps. We investigated the expression level of Rv1410c, Rv2459, Rv1218c and Rv1273c efflux pumps gene by real-time quantitative reverse transcription PCR (qRT-PCR) in 31 clinical isolates of M. tuberculosis. Susceptibility to first-line drugs was performed using the proportion method. Twenty one isolates were characterized with drug resistance (DR), and among them 12 showed a significantly elevated level of expression (>4 fold) for at least one of the studied genes encoding for efflux pumps. Point mutations in the katG (codons 315 or 335) and rpoB (codons 456 and 441) genes were found in 42.85% and 66.6% of drug resistant isolates, respectively. Only one isolate showed mutation at position -15 of the inhA promoter region. Among the 7 isolates (33.33%) which had no mutation in the studied regions of drug target genes, 5 isolates showed over-expression for efflux pumps. Our results demonstrated that over-expression of efflux pumps can contribute to drug resistance in M. tuberculosis.

Copper, cobalt and zinc-salophen complexes 3–8 were prepared from the reaction of N,N-bis(salicylidene)4-chloro-1,2-phenylendiamine (1) or N,N-bis(salicylidene)4-bromo-1,2-phenylendiamine (2) with their respective acetate salts. All compounds were characterized with common techniques. In additional, optical, electrochemical, thermal and antibacterial properties of ligands and their complexes were studied. Schiff base ligands 1 and 2 showed the intense emission band at 476 nm in the fluorescence spectra. The emission process of these ligands is related to the excited state intramolecular proton transfer phenomenon (and also π→π* transitions). In general, after the incorporation of copper, cobalt and zinc metal ions into the structure of ligands 1 and 2, the emission intensity of the ligands totally quenched. It can be attributed to the changes in the conformational rigidity of the structure of ligands upon complexation. Thermal stability of complexes was proved by TGA analysis. The cyclic voltammogrames of Cu (II) complexes displayed quasi-reversible one electron Cu(II)/Cu(I) redox process, while those of their cobalt counterparts exhibited two reversible redox couples of Co(II)/Co(I) and Co(III)/Co(II). The investigation of antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and carbapenem &colistin resistant Klebsiella pneumoniae showed both Co(II) complexes 4 and 7 have remarkable antibacterial activity. The antibacterial effect of cobalt complexes against K. pneumoniae is of high interest as the bacterium, is resistant to two most important therapeutic options for treatment of multi-drug resistant Gram-negative bacteria, including polymyxins and carbapenems. Moreover, DFT investigations were performed to obtain a better understanding of structure of compounds 1–8. The values of band gap energies revealed ability of all compounds for using in optical devices. Finally, the electronic spectra of compounds are analyzed by TD-DFT calculations at the B3LYP/6-311G level for Schiff base ligands and the B3LYP/LANL2DZ level for complexes.

Development of molecular techniques for rapid detection of drug resistant tuberculosis allows for the prompt initiation of appropriate anti-TB treatment. We aimed to assess high-resolution melting (HRM) analysis for the detection of rpsL, rrs and embB mutations to identify streptomycin and ethambutol resistance in Mycobacterium tuberculosis.A total of 76 clinical isolates of M. tuberculosis including 25 SM-R, 21 EB-R and 30 drug susceptible - determined by the proportion method of drug susceptibility testing (DST) - were analyzed by HRM analysis, and the results were confirmed using DNA sequencing.The sensitivity and specificity of the HRMA compared to phenotypic DST were 88% and 100.0%, respectively for the detection of streptomycin resistance (SM-R), and 90.4% and 96.6%, respectively for ethambutol resistance (EB-R). Three SM-R and two EB-R isolates had no mutations in the studied regions of rpsL, rrs and embB genes determined by DNA sequencing and therefore were not identified as resistant by HRM assay. Interestingly, one phenotypic EM-S isolate was found by sequencing to have a mutation at codon 423 (Met-Ilu) of embB gene and was clustered as resistant by HRM as well.The sensitivity and specificity of HRM curve assay was consistent with DNA sequencing, which is the gold standard method for genotypic DST. This assay can be utilized as a screening method for detection of drug-resistant tuberculosis, offering the advantages of a high throughput, single step, cost effectiveness, and rapid work flow method.

Objectives Fluoroquinolones (FQs) are recommended as the drugs of choice for the empirical treatment of urinary tract infections (UTIs). This study investigated the molecular determinants of FQ resistance in Escherichia coli and Klebsiella pneumoniae isolates in Iran. Methods A total of 364 clinical isolates of E. coli (n = 144) and K. pneumoniae (n = 220) were collected from patients with UTI. Susceptibility of the isolates to ciprofloxacin, levofloxacin, gatifloxacin and nalidixic acid was evaluated by disk diffusion. The presence of qnrA, qnrB and qnrS genes was assessed by PCR. Nucleotide sequences of the gyrA and parC genes were determined. Results Eighty-seven (60.4%) and 15 (6.8%) E. coli and K. pneumoniae isolates, respectively, were resistant to at least one of the tested FQs. Plasmid-mediated quinolone resistance (PMQR) genes were detected in 12.6% and 60.0% of FQ-resistant E. coli and K. pneumoniae, respectively. Whilst qnrB predominated in K. pneumoniae, qnrS was the most prevalent PMQR gene in E. coli. S83L (98.9%) and D87N (59.8%) were the most frequent mutations identified in GyrA of E. coli, and 55.2% (n = 48) of FQ-resistant E. coli isolates had mutation in ParC harbouring S80I and E84V substitutions. The GyrAS83L substitution was found in only one FQ-resistant K. pneumoniae isolate. Conclusions FQ resistance was much more common in E. coli isolates than in K. pneumoniae. Whilst mutations in the drug target-encoding genes gyrA and parC were the major mechanisms involved in FQ resistance in E. coli, PMQR determinants commonly mediated FQ resistance in K. pneumoniae.

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.

Tuberculosis (TB) persists as a public health problem in Iran. Characterization of Mycobacterium tuberculosis isolates circulating in this area will contribute to understand and control the spread of the strains. The aims of this study were to understand the genetic diversity and drug susceptibility of M. tuberculosis isolates circulating in Iran and to analyze the relationship between genotype and drug resistance. A total of 291 M. tuberculosis isolates collected from TB patients were genotyped by spoligotyping. Drug susceptibility testing was performed using proportion method. Spoligotyping resulted in 75 distinct patterns. 86.2% of isolates were grouped in 35 clusters while the remaining isolates were unique. Ural was found to be the most predominant lineage (34.3%) followed by Central Asian strain (CAS) (24%), T (18.2%), Manu2 (7.5%) and Latin American-Mediterranean (LAM) (6.1%). The five largest clusters were Ural/Spoligotype International Type (SIT)127 (15.8%), CAS1/SIT26 (9.2%), T1/SIT53 (6.1%), T1/SIT284 (5.4%), and CAS1/SIT25 (4.4%). About 5% of isolates had multidrug resistance (MDR) and 10% had other resistance. MDR was significantly associated with Beijing strains, but not with Ural family. This study highlights dominance of Ural, CAS, and T families in Iran. Biogeographic specificity of CAS and T families to border provinces of Iran including Sistan-Baluchestan and Kermanshah, respectively, suggested that this family strains might be transmitted from these regions to other provinces of the country.

Background. Considering that Hormozgan province in Iran (southern part of Iran on the Persian Gulf) is among the areas with high prevalence of MDR-MTB and attracts so many sailors and tourists, genetic diversity of MTB isolates circulating in this part of Iran was evaluated. Pattern of TB transmission was also examined. Methods and Material. A total of 38 isolates of MTB were cultured from TB patients from Hormozgan province of Iran and standard MIRU-VNTR typing and spoligotyping were applied to genotype these isolates. Drug susceptibility testing was performed using proportion method. Results. There were 28 VNTR profiles comprising 5 clusters and 23 unique isolates compared to 21 spoligotyping profiles, which contained 9 clusters and 12 unique isolates. Latin American-Mediterranean (n = 9, 23.6%) was found to be the most predominant lineage. MIRU-VNTR analysis, with an HGDI of 0.975, was more discriminating than spoligotyping, which had an HGDI of 0.955. The estimated proportion of TB cases due to recent transmission was 26.3% and 44.7% by MIRU-VNTR and spoligotyping, respectively. The rates of monodrug resistance and MDR were 15.8% and 7.9%, respectively. Two of 3 MDR strains were found to be related to MIRU-VNTR and belonged to the same spoligotyping cluster characterized with T1/SIT53 genotype. Conclusions. The high genetic diversity among MTB isolates suggests that transmission occurred from different sources to this area. Reactivation of a priori, latent MTB infection was found to contribute mainly to TB cases in this geographic region.

Introduction: Early detection of drug resistant tuberculosis is one of the main priorities of TB control program. Ethambutol (EMB) is a first-line anti-TB drug that is effective for preventing treatment failures caused by Mycobacterium tuberculosis strains that are resistant to other drugs. The aim of this study was to sequence the embB gene to characterize the mutations causing resistance to EMB and to analyze the relationship between bacterial genotype and EMB resistance among M. tuberculosis isolates in Iran. Methodology: A total of 20 M. tuberculosis isolates comprising 10 multidrug-resistant (MDR) and 10 non-MDR isolates, recovered from TB patients in four regions: Tehran, Isfahan, Zahedan, Khorasan, were analyzed. Mutational profiling was performed by amplifying and sequencing the embB gene. Spoligotyping was carried out to characterize the bacterial genotype. Results: Phenotypic EMB resistance was found in 13 strains. Mutations affecting ethambutol resistance-determining region (ERDR) of the embB were identified in 6 of 13 EMB-resistant isolates. The majority of these mutations resulted in amino acid substitution at position 306 (M306V). A novel mutation at codon 366 was identified (S366L) in one isolate. Ural was the most predominant genotype in the studied population. Beijing genotype was associated with both MDR and EMB resistance in which all mutations occurred at codon 306 of the embB gene. Conclusion: A significant association between Beijing genotype and EMB resistance was found, mainly due to mutations at embB306. Results of this study can be used as a basis to develop or improve rapid molecular tests to monitor drug-resistant strains in this country.

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.

Background: Early detection of multidrug-resistant tuberculosis (MDR-TB) is essential to prevent its transmission in the community and initiate effective anti-TB treatment regimen. Materials and Methods: High-resolution melting curve (HRM) analysis was evaluated for rapid detection of resistance conferring mutations in rpoB and katG genes. We screened 95 Mycobacterium tuberculosis clinical isolates including 20 rifampin resistant (RIF-R), 21 isoniazid resistant (INH-R) and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. Nineteen M. tuberculosis isolates with known drug susceptibility genotypes were used as references for the assay validation. The nucleotide sequences of the target regions rpoB and katG genes were determined to investigate the frequency and type of mutations and to confirm HRM results. Results: HRM analysis of a 129-bp fragment of rpoB allowed correct identification of 19 of the 20 phenotypically RIF-R and all RIF-S isolates. All INH-S isolates generated wild-type HRM curves and 18 out of 21 INH-R isolates harboured any mutation in 109-bp fragment of katG exhibited mutant type HRM curves. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible which were confirmed for having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates) and katG315 (85.7% of INH-R isolates), respectively. Conclusion: HRM was found to be a reliable, rapid and low cost method to characterise drug susceptibility of clinical TB isolates in resource-limited settings.

Aims and objectives Copper poisoning in macrophages plays an important role in immunity against invading pathogens. Many bacteria, including Mycobacterium tuberculosis ( Mtb ), have evolved mechanisms to combat copper-derived innate immune responses. Copper homeostasis in Mtb consists of several components, including a multi-copper oxidase, copper efflux pump, cytoplasmic metallothionein and copper-sensing transcriptional regulators. However, components involved in copper uptake are unknown, which prompted this study to investigate the possible role of porins in copper uptake in mycobacteria. Methods Mycobacterium smegmatis porin mutants were created and tested for their ability to grow under copper-reduced or copper-rich conditions. The M. smegmatis porin gene mspA was expressed in Mtb , and its copper susceptibility profile was investigated in the presence of different copper concentrations. The expression level of a copper detoxifying protein, mycobacterial multi-copper oxidase (MmcO), was monitored by western blot to assess intracellular copper content. Results Deletion of porin genes from M. smegmatis caused a severe growth defect on trace copper medium. Copper supplementation alleviated this phenotype. The inability to acquire copper in sufficient amounts due to lack of porins can explain this phenomenon. Moreover, porin mutants showed elevated tolerance to copper at concentrations that were toxic for wild-type strains, indicating that the lack of porins protects these strains from copper poisoning. On the other hand, heterologous expression of mspA in Mtb significantly impaired growth at 2.5 μM copper and eliminated growth at 15 μM, while wild-type Mtb eventually reached its normal cell density at this copper concentration. Consistent with a role of porins in copper uptake, expression levels of MmcO in Mtb expressing the M. smegmatis porin mspA was above wild-type levels, indicating that cytoplasmic copper-sensing transcriptional regulators respond by derepressing the expression of copper resistance genes. Moreover, the polyamine spermine, a known inhibitor of porin activity in gram-negative bacteria, increased the tolerance of wild-type Mtb for copper suggesting that endogenous outer membrane proteins with channel-forming activity exist and contribute to copper acquisition and toxicity in Mtb . Conclusions It was concluded from these results that porins are involved in copper uptake in mycobacteria. Moreover, the outer membrane of Mtb was found to be an important barrier against copper intoxication so that permeabilization of this barrier ( e.g. , by porins) renders Mtb extremely vulnerable to copper. Consequently, copper homeostasis of Mtb provides a promising drug target for the development of a new class of anti-tuberculosis compounds that can induce a copper hypersensitivity phenotype in Mtb .

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 .

Introduction: Nosocomial pneumonia remains an important cause of mortality and morbidity worldwide. Surveillance programs play an important role in the identification of common etiologic agents and local patterns of antimicrobial resistance. Methodology: In this study we determined the frequency and antimicrobial susceptibility of pathogens isolated from patients with nosocomial pneumonia during 2009 to 2011. Results: A total of 642 bacteria were isolated from 516 suspected samples. Acinetobacter baumannii (21.1%, n = 136), was the commonest isolated pathogen followed by Pseudomonas aeruginosa (17.4%, n = 112) , Staphylococcus aureus (15.8%, n = 102) and enterococci (8.4% n = 54). The most effective therapeutic agents against A. baumannii were polymyxin B (95.5% susceptible), ceftriaxone/tazobactam (72% susceptible) and levofloxacin (52.9% susceptible) . Polymixin B (89.2% susceptible), ceftriaxone/tazobactam (89.2% susceptible) and piperacillin-tazobactam (80.3% susceptible) were found to be the most active agents against P. aeruginosa. Extended-spectrum beta-lactamases were detected among isolates of K. pneumoniae (45.4%) and E. coli (20.3%). Overall, the prevalence of methicillin-resistant S. aureus and vancomycin resistant enterococci were 80.4% and 40.7% respectively. Linezolid was found to be the most active antibiotic against these pathogens. The etiology of 50% of the nosocomial infection cases was polymicrobial. Conclusions: The combination of ceftriaxone/tazobactam seems to be beneficial agent against multidrug-resistant Gram-negative bacilli isolated form respiratory tract infections. The results of our study can be used for guiding appropriate empiric therapy in this geographic region.

Aims and objectives Tuberculosis (TB) is considered one of the most important pathogens in the world. Iran has a moderate TB incidence, but borders two high-burden TB countries to the east and one high-burden multidrug-resistant (MDR)-TB country to the north. Limited information is available on the genetic diversity and transmission dynamics of MTB in Iran. To determine the MTB genotypes and their transmission patterns among patients, a collection of isolates from different parts of Iran were genotyped. Methods Genotypes were generated by means of standard 15-locus variable number tandem repeat (VNTR) for 121 MTB clinical isolates collected from three provinces of Iran, including Sistan–Baluchestan (southeast province of Iran, with the highest rate of TB), Kermanshah (western part of Iran with high TB/human immunodeficiency virus [HIV] cases) and Tehran (the capital of Iran). Results Sixty-six distinct mycobacterial interspersed repetitive unit (MIRU)-VNTR patterns were detected among 121 isolates. Seventy-five strains were grouped into 20 clusters, and 46 isolates were unique. The genetic diversity of strains from Sistan–Baluchestan was higher than that in the other province. All isolates from Tehran or Kermanshah that were grouped into clusters shared more identical patterns with Sistan–Baluchestan. The Hunter-Gaston discriminatory index (HGDI) was 0.972, indicating a high power of discrimination for MIRU-VNTR typing. The MIRU 16, ETR-A, ETR-E, MTUB04 loci were designated as highly discriminative. Conclusions MIRU-VNTR typing showed a high genetic diversity and suggests the possibility of transmission from Sistan–Baluchestan to other provinces of Iran. This method could be considered a suitable tool for studying the transmission routes of TB and leading to more appropriate measures for TB control. MIRU-VNTR typing leads to a much better understanding of the bacterial population structure and phylogenetic relationships between strains of MTB in different regions of Iran.

Aims and objectives Early detection of multidrug-resistant tuberculosis (MDR-TB) is essential to prevent its transmission in the community and to initiate an effective anti-TB treatment regimen. The objective of this study is to evaluate molecular technique high resolution melting curve (HRM) assay to rapidly detect resistance-conferring mutations in rpoB and katG genes. Methods HRM analysis was used to screen 95 Mycobacterium tuberculosis (MTB) clinical isolates including 20 rifampin resistant (RIF-R), 21 isoniazid resistant (INH-R), and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. 19 MTB isolates with known drug susceptibility genotypes were used as control strains for initial validation of the assay. Polymerase chain reaction (PCR) amplicons from rpoB and katG genes were sequenced to investigate the frequency and type of mutations and to confirm HRM results. Results All RIF-S and INH-S isolates generated wild-type HRM curves and were accurately identified as susceptible by this method. Similarly 19 out of 20 RIF-R and 18 out of 21 INH-R isolates correctly exhibited mutant-type HRM curves. These results were similar to those obtained by direct sequencing. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible by HRM assay. These strains were confirmed as having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates) and katG315 (85.7% of INH-R isolates), respectively. Conclusions HRM was found to be a reliable, rapid and low-cost method to characterize drug susceptibility of clinical TB isolates in resource-limited settings.