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, �g638 (frameshift), �a64-g73 (frameshift) and NfsB F84S, P45S, W94Stop, E197Stop substitutions, �nfsB 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. © 201

Genotypic characterization of Staphylococcus aureus isolated from a burn centre by using agr, spa and SCCmec typing methods

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. © 201

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

Abstract 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

The role of porins in copper acquisition by mycobacteria

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

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

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. 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. 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. 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

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

ABSTRACT: 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 β 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

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 21 M/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. (C) 2019 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved