Molecular Mechanisms Of Colistin Resistance Research Articles

Molecular mechanisms of colistin resistance mediated by pmrCAB genes in Acinetobacter baumannii isolated from patients hospitalized in Isfahan medical centers

Abstract Introduction In recent years, colistin-resistant Acinetobacter baumannii (A. baumannii) has been found all over the world. In this current study, the main purpose was to examine the occurrence of extensively drug resistant (XDR), resistance to colistin and characterization and mutations in pmrCAB genes among A. baumannii obtained from inpatients. Materials and Methods A total of 108 clinical isolates of A. baumannii were collected from several hospitals located in Isfahan, Iran. The Kirby-Bauer assay was performed to assess the antimicrobial resistance. The Phoenix automated system was utilized to determine the minimum inhibitory concentration (MIC) of colistin for each of the bacterial isolates. Polymerase chain reaction was used to screen for pmrCAB genes that mediate colistin resistance, and sequencing was used to determine the amplicon’s nucleotide sequence. Results The results revealed that all A. baumannii isolates (100%) were resistant to piperacillin tazobactam, meropenem and ciprofloxacin. All isolates were classified as XDR, with seven isolates being pan-drug resistant (PDR). Colistin resistance (CoR) was found in 6.48% (7/108) of studied isolates, all of which were positive for pmrCAB genes. The sequencing results showed a substitution in pmrB and two isolates showed a substitution in pmrC. Conclusions In conclusion, this study is the initial report of the existence and mutations of pmrB and C genes in the clinical isolate of A. baumannii in our region. This outcome highlights the necessity to explore additional mutations in the PMR operon of A. baumannii in forthcoming studies. Moreover, our results highlight the high occurrence of XDR-A. baumannii strain in Isfahan, Iran.

Genomic characterization of colistin-resistant Klebsiella pneumoniae isolated from intensive care unit patients in Egypt

BackgroundEgypt has witnessed elevated incidence rates of multidrug-resistant Klebsiella pneumoniae infections in intensive care units (ICUs). The treatment of these infections is becoming more challenging whilst colistin-carbapenem-resistant K. pneumoniae is upsurging. Due to the insufficiently available data on the genomic features of colistin-resistant K. pneumoniae in Egypt, it was important to fill in the gap and explore the genomic characteristics, as well as the antimicrobial resistance, the virulence determinants, and the molecular mechanisms of colistin resistance in such a lethal pathogen.MethodsSeventeen colistin-resistant clinical K. pneumoniae isolates were collected from ICUs in Alexandria, Egypt in a 6-month period in 2020. Colistin resistance was phenotypically detected by modified rapid polymyxin Nordmann/Poirel and broth microdilution techniques. The isolates susceptibility to 20 antimicrobials was determined using Kirby-Bauer disk diffusion method. Whole genome sequencing and bioinformatic analysis were employed for exploring the virulome, resistome, and the genetic basis of colistin resistance mechanisms.ResultsOut of the tested K. pneumoniae isolates, 82.35% were extensively drug-resistant and 17.65% were multidrug-resistant. Promising susceptibility levels towards tigecycline (88.24%) and doxycycline (52.94%) were detected. Population structure analysis revealed seven sequence types (ST) and K-types: ST383-K30, ST147-K64, ST17-K25, ST111-K63, ST11-K15, ST14-K2, and ST525-K45. Virulome analysis revealed yersiniabactin, aerobactin, and salmochelin siderophore systems in ˃ 50% of the population. Hypervirulence biomarkers, iucA (52.94%) and rmpA/A2 (5.88%) were detected. Extended-spectrum β-lactamase- and carbapenemase-producers accounted for 94.12% of the population, with blaCTX-M-15, blaNDM-5, and blaOXA-48 reaching 64.71%, 82.35%, and 82.35%, respectively. Chromosomal alterations in mgrB (82.35%) were the most prevailing colistin resistance-associated genetic change followed by deleterious mutations in ArnT (23.53%, L54H and G164S), PmrA (11.76%, G53V and D86E), PmrB (11.76%, T89P and T134P), PmrC (11.76%, S257L), PhoQ (5.88%, L322Q and Q435H), and ArnB (5.88%, G47D) along with the acquisition of mcr-1.1 by a single isolate of ST525.ConclusionsIn this study, we present the genotypic colistin resistance mechanisms in K. pneumoniae isolated in Egypt. More effective antibiotic stewardship protocols must be implemented by Egyptian health authorities to restrain this hazard and safeguard the future utility of colistin. This is the first characterization of a complete sequence of mcr-1.1-bearing IncHI2/IncHI2A plasmid recovered from K. pneumoniae clinical isolate belonging to the emerging high-risk clone ST525.

The occurrence and molecular detection of mcr-1 and mcr-5 genes in Enterobacteriaceae isolated from poultry and poultry meats in Malaysia.

The advent of antimicrobials-resistant (AMR), including colistin-resistant bacteria, poses a significant challenge to animal and human health, food safety, socio-economic growth, and the global environment. This study aimed to ascertain the colistin resistance prevalence and molecular mechanisms of colistin resistance in Enterobacteriaceae. The colistin resistance was determined using broth microdilution assay, PCR; and Sanger sequencing of mcr genes responsible for colistin resistance in Enterobacteriaceae (n = 627), including Escherichia coli (436), Salmonella spp. (n = 140), and Klebsiella pneumoniae (n = 51), obtained from chicken and chicken meats. Out of 627 Enterobacteriaceae, 8.6% of isolates exhibited colistin resistance phenotypically. Among these colistin resistant isolates, 9.3% (n = 37) were isolated from chicken meat, 7.2% (n = 11) from the cloacal swab of chicken and 7.9% (n = 6) from the litter samples. Overall, 12.96% of colistin-resistant isolates were positive with mcr genes, in which mcr-1 and mcr-5 genes were determined in 11.11% and 1.85% of colistin-resistant isolates, respectively. The E. coli isolates obtained from chicken meats, cloacal swabs and litter samples were found positive for mcr-1, and Salmonella spp. originated from the chicken meat sample was observed with mcr-5, whereas no mcr genes were observed in K. pneumoniae strains isolated from any of the collected samples. The other colistin resistance genes, including mcr-2, mcr-3, mcr-4, mcr-6, mcr-7, mcr-8, mcr-9, and mcr-10 were not detected in the studied samples. The mcr-1 and mcr-5 genes were sequenced and found to be 100% identical to the mcr-1 and mcr-5 gene sequences available in the NCBI database. This is the first report of colistin resistance mcr-5 gene in Malaysia which could portend the emergence of mcr-5 harboring bacterial strains for infection. Further studies are needed to characterize the mr-5 harbouring bacteria for the determination of plasmid associated with mcr-5 gene.

Prevalence and molecular characteristics of colistin-resistant isolates among clinically isolated carbapenem-resistant Klebsiella pneumoniae in China

Colistin resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) poses health challenges. To investigate the prevalence and molecular characteristics of colistin-resistant CRKP, 708 isolates were collected consecutively from 28 tertiary hospitals in China from 2018 to 2019, and 14 colistin-resistant CRKP were identified. Two-component systems (TCSs) related to colistin resistance (PmrA/B, PhoP/Q, and CrrA/B), the negative regulator mgrB gene and mcr genes, were analysed using genomic sequencing. The relative expression of TCSs genes along with their downstream pmrC and pmrK genes was determined using quantitative real-time PCR (qRT‒PCR). A novel point mutation in PhoQ was confirmed by site-directed mutagenesis, and the subsequent transcriptome changes were analysed by RNA sequencing (RNA-Seq). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect modifications in lipid A. The results showed that only one isolate carried the mcr-8.1 gene, nine exhibited MgrB inactivation or absence, and three exhibited mutations in PmrB. One novel point mutation, L247P, in PhoQ was found to lead to a 64-fold increase in the minimum inhibitory concentration (MIC) of colistin. qRT‒PCR revealed overexpression of phoP/Q and pmrK in isolates with or without MgrB inactivation, and pmrB mutation resulted in overexpression of pmrA and pmrC. Furthermore, transcriptome analysis revealed that the PhoQ L247P novel point mutation caused upregulated expression of phoP/Q and its downstream operon pmrHFIJKLM. Meanwhile, the pmrA/B regulatory pathway did not evolve colistin resistance. Mass spectrometry analysis showed the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A in colistin-resistant isolates with absence of MgrB. These findings illustrate that the molecular mechanisms of colistin resistance in CRKP isolates are complex, and that MgrB inactivation or absence is the predominant molecular mechanism. Interventions should be initiated to monitor and control colistin resistance.

Acinetobacter baumannii under Acidic Conditions Induces Colistin Resistance through PmrAB Activation and Lipid A Modification.

Colistin is a last-resort antimicrobial agent for treating carbapenem-resistant Acinetobacter baumannii infections. The activation of PmrAB by several environmental signals induces colistin resistance in Gram-negative bacteria. This study investigated the molecular mechanisms of colistin resistance in A. baumannii under acidic conditions using wild-type (WT) A. baumannii 17978, ΔpmrA and ΔpmrB mutants, and pmrA-complemented strains. The pmrA or pmrB deletion did not affect the growth of A. baumannii under acidic or aerobic conditions. A. baumannii under acidic (pH 5.5) and high-iron (1 mM) conditions showed 32- and 8-fold increases in the minimum inhibitory concentrations (MICs) of colistin, respectively. The ΔpmrA and ΔpmrB mutants at pH 5.5 showed a significant decrease in colistin MICs compared to the WT strain at pH 5.5. No difference in colistin MICs was observed between WT and mutant strains under high-iron conditions. The pmrCAB expression significantly increased in the WT strain at pH 5.5 compared to the WT strain at pH 7.0. The pmrC expression significantly decreased in two mutant strains at pH 5.5 compared to the WT strain at pH 5.5. The PmrA protein was expressed in the ΔpmrA strain carrying ppmrA_FLAG plasmids at pH 5.5 but not at pH 7.0. Lipid A modification by the addition of phosphoethanolamine was observed in the WT strain at pH 5.5. In conclusion, this study demonstrated that A. baumannii under acidic conditions induces colistin resistance via the activation of pmrCAB operon and subsequent lipid A modification.

Colistin Resistance in Acinetobacter baumannii: Molecular Mechanisms and Epidemiology.

Acinetobacter baumannii is recognized as a clinically significant pathogen causing a wide spectrum of nosocomial infections. Colistin was considered a last-resort antibiotic for the treatment of infections caused by multidrug-resistant A. baumannii. Since the reintroduction of colistin, a number of mechanisms of colistin resistance in A. baumannii have been reported, including complete loss of LPS by inactivation of the biosynthetic pathway, modifications of target LPS driven by the addition of phosphoethanolamine (PEtN) moieties to lipid A mediated by the chromosomal pmrCAB operon and eptA gene-encoded enzymes or plasmid-encoded mcr genes and efflux of colistin from the cell. In addition to resistance to colistin, widespread heteroresistance is another feature of A. baumannii that leads to colistin treatment failure. This review aims to present a critical assessment of relevant published (>50 experimental papers) up-to-date knowledge on the molecular mechanisms of colistin resistance in A. baumannii with a detailed review of implicated mutations and the global distribution of colistin-resistant strains.

Molecular mechanisms of colistin resistance among multi-drug resistant (MDR) Klebsiella pneumoniae and Escherichia coli isolated from ICU patients and their susceptibility towards eravacycline

Molecular mechanisms of colistin resistance among multi-drug resistant (MDR) Klebsiella pneumoniae and Escherichia coli isolated from ICU patients and their susceptibility towards eravacycline

A 12 year experience of colistin resistance in Klebsiella pneumoniae causing neonatal sepsis: two-component systems, efflux pumps, lipopolysaccharide modification and comparative phylogenomics.

Increased use of colistin in healthcare necessitates studies on the trend of colistin resistance and the underlying mechanisms. To understand the susceptibility trend and molecular mechanisms of colistin resistance in neonatal isolates over a 12 year period. Colistin susceptibility, mRNA expression, whole genome sequence and mutational analysis was performed. Phylogenomic comparison with a global collection of colistin-resistant Klebsiella pneumoniae strains (n = 70) was done. Of 319 Enterobacterales (K. pneumoniae and Escherichia coli) studied, colistin resistance was found in 9 K. pneumoniae (2.8%). The transmissible colistin resistance gene, mcr, was absent. Colistin-resistant K. pneumoniae belonged to diverse sequence types (ST14/37/101/147/716) and exhibited multiple mechanisms of colistin resistance including overexpression of the two-component systems (TCS) (phoP/Q, pmrA/B), and AcrAB-TolC pump and its regulators. Mutations in TCS, mgrB, pumps, repressors, and lipopolysaccharide-modifying genes were detected. Phylogenomic comparison revealed that this global collection of colistin-resistant K. pneumoniae was diverse, with the presence of epidemic and international clones. Mutations in mgrB and TCS noted in global strains were comparable to the study strains. Co-occurrence of carbapenem resistance (n = 61, 87%) was observed in global strains. Co-existence of dual carbapenemases (blaNDM-5 with blaOXA-48/181) in multiple lineages within different replicons was found in neonatal colistin-resistant study isolates only. Colistin resistance both in study and global strains is multifaceted and attributed to mutations in chromosomal genes leading to lipopolysaccharide modification or efflux of colistin through pumps. With no transmissible mcr, prevalence of colistin-resistant strains was low in this unit. Colistin-resistant strains with dual carbapenemases causing sepsis are alarming as they are practically untreatable.

Emergence of colistin-resistant hypervirulent Klebsiella pneumoniae (CoR-HvKp) in China

ABSTRACT Colistin is regarded as a last-resort agent to combat infections caused by multidrug-resistant (MDR) Gram-negative bacteria, especially carbapenem-resistant isolates. In recent years, reports of colistin-resistant Klebsiella pneumoniae (CoRKp) are increasing. However, the molecular mechanism and relevance of colistin resistance and virulence remain unclear. Fourteen CoRKp strains were retrospectively screened from 1884 clinical K. pneumoniae isolates during 2017–2018 in China. Six CoRKp strains belonging to ST11 were MDR strains. Plasmid-mediated mobile colistin-resistance genes had a low prevalence in CoRKp. Our results revealed that up-regulated expression of two-component systems, especially phoPQ, contributed more to colistin resistance. mgrB mutation was the most common molecular mechanism of colistin resistance, caused by either nonsense mutations or insertion sequences, which drove the overexpression of phoPQ system. This study also identified three novel point mutations in pmrAB system, in which D313N mutation in pmrB was proved to increase the MIC to colistin by 16-fold. In addition, 6 out of 14 CoRKP strains independently carried hypervirulence genes. All six strains showed medium-to-high virulence phenotype compared with NTUH-K2044 strain in mice intraperitoneal challenge models. We found that 4 strains were biofilm strong producers and transcriptome analysis revealed that three of them significantly up-regulated expression of type III fimbrial shaft gene mrkA. In conclusion, our result revealed the emergence of colistin-resistant and hypervirulent MDR K. pneumoniae, which is a noticeable superbug and could cause a severe challenge to public health.

Molecular and Genomic Insights of mcr-1-Producing Escherichia coli Isolates from Piglets.

The use of colistin in food-producing animals favors the emergence and spread of colistin-resistant strains. Here, we investigated the occurrence and molecular mechanisms of colistin resistance among E. coli isolates from a Mexican piglet farm. A collection of 175 cephalosporin-resistant colonies from swine fecal samples were recovered. The colistin resistance phenotype was identified by rapid polymyxin test and the mcr-type genes were screened by PCR. We assessed the colistin-resistant strains by antimicrobial susceptibility test, pulse-field gel electrophoresis, plasmid profile, and mating experiments. Whole-Genome Sequencing data was used to explore the resistome, virulome, and mobilome of colistin-resistant strains. A total of four colistin-resistant E. coli were identified from the cefotaxime-resistant colonies. All harbored the plasmid-borne mcr-1 gene, which was located on conjugative 170-kb IncHI-2 plasmid co-carrying ESBLs genes. Thus, high antimicrobial resistance rates were observed for several antibiotic families. In the RC2-007 strain, the mcr-1 gene was located as part of a prophage carried on non-conjugative 100-kb-plasmid, which upon being transformed into K. variicola strain increased the polymyxin resistance 2-fold. The genomic analysis showed a broad resistome and virulome. Our findings suggest that colistin resistance followed independent acquisition pathways as clonal and non-genetically related mcr-1-harboring strains were identified. These E. coli isolates represent a reservoir of antibiotic resistance and virulence genes in animals for human consumption which could be potentially propagated into other interfaces.

Molecular Mechanisms of Colistin Resistance in Klebsiella pneumoniae in a Tertiary Care Teaching Hospital.

BackgroundOver the last two decades, the prevalence of colistin resistance among the members of Enterobacteriaceae has been increasing, particularly among Klebsiella pneumoniae isolates; this limits the potential use of colistin and leads to worsened clinical outcomes.MethodsWe investigated the prevalence and genetic characteristics of colistin-resistant K. pneumoniae (COLR-KP) in clinical isolates using genomic sequencing.ResultsIn total, 53 K. pneumoniae isolates (4.5%, 53/1,171) were confirmed as COLR-KP, of which eight isolates carried mobile colistin-resistant (mcr) gene. Although the overall prevalence rate (0.7%, 8/1,171) of mcr-like genes in clinical K. pneumoniae remained relatively low, the presence of mcr (15.1%, 8/53) among the COLR-KP isolates indicated that the mobile resistance gene was already widespread among K. pneumoniae isolates in hospital setting. We randomly selected 13 COLR-KP isolates (four mcr-bearing and nine non-mcr-bearing isolates) for whole-genome sequencing, including two pandrug-resistant and four sequence type 11 (ST11) isolates. Phylogenetic analysis revealed that all COLR-KP isolates were genetically diverse. Among the four mcr-bearing isolates, three (KP4, KP18, and KP30) were positive for mcr-1 and one (KP23) for mcr-8; none of the other mcr genes were detected. The mcr-1 in the KP4 and KP30 isolates were located in an IncX4 plasmid (approximately 33 kb) and could be successfully transferred to Escherichia coli J53AZR. In contrast, for the mcr-8-bearing plasmid in KP23 (IncFII), colistin resistance could not be transferred by conjugation. The mcr-1-producing isolate KP18 coexists a novel plasmid-carried tigecycline resistance gene tmexCD1-toprJ1. The most common chromosomal mutation associated with colistin resistance was a T246A amino acid substitution in PmrB, which was identified in most COLR-KP isolates (11/13, 84.6%). All ST11 isolates additionally had an R256G amino acid substitution. Critical virulence factors associated with hypervirulent K. pneumoniae were detected in four COLR-KP isolates; these virulence factors included aerobactin, salmochelin, and yersiniabactin.ConclusionWe found that mcr-bearing COLR-KP emerged in our hospital and was growing at an increasing rate. Simultaneous emergence of hypervirulence and colistin–tigecycline–carbapenem resistance in the epidemic clone ST11 K. pneumoniae was also observed; this highlights the significance of active and continuous surveillance.

Prevalence and molecular mechanisms of colistin resistance in Acinetobacter baumannii clinical isolates in Tehran, Iran.

Colistin is one of the last remaining active antibiotics against multidrug resistant Gram-negative bacteria. However, several recent studies reported colistin-resistant (ColR) Acinetobacter baumannii from different countries. In the current study, we investigated molecular mechanisms involved in colistin resistance in A. baumannii isolates from different clinical samples.A total of 110 clinical A. baumannii isolates were collected from two hospitals in Tehran. Minimum inhibitory concentrations (MICs) were determined by broth microdilution according to the Clinical and Laboratory Standards Institute. For the ColR isolates, mutation was detected in pmrA, pmrB, lpxA, lpxC, and lpxD genes using the polymerase chain reaction (PCR) and sequencing. Moreover, the relative expression of the pmrC gene was calculated using quantitative reverse transcription PCR. Three colistin resistant isolates were identified with MIC between 8 and 16 μg/mL and were resistant to all the tested antimicrobial agents. All the three isolates had a mutation in the pmrB, pmrA, lpxA, lpxD, and lpxC genes. Moreover, the overexpression of pmrC gene was observed in all isolates. Our results showed that the upregulation of the PmrAB two component system was the primary mechanism linked to colistin resistance among the studied colistin resistant A. baumannii isolates.

Molecular mechanisms of colistin resistance in Africa: A systematic review of literature.

Updated and comprehensive data on the mechanism underlying colistin resistance is lacking in Africa. Herein, we aimed to review available literature on the molecular mechanisms of colistin resistance in Africa. PubMed, Google Scholar, and African Journal online databases were searched on the 15th of January 2020 for original research articles that reported mechanisms of colistin resistance in any of the 54 African countries. Of the 1473 studies identified through initial database search, 36 met the inclusion criteria. Colistin resistance was mostly observed in Escherichia coli isolated from human clinical samples. Plasmid-mediated colistin resistance mechanism (26; 72.2%) was the most frequently reported resistance mechanism. About three-quarters (27; 75.0%) of the 36 studies were done in North Africa. In this zone, the mobilized colistin resistance (mcr) genes were mostly detected in E. coli harboring three plasmid types, IncHI2, IncI2, and IncX4, from animal samples (n=9; 42.8%). Of the six studies performed in Southern Africa, four reported mcr-1 mostly detected from human samples (n=2; 50.0%) in E. coli isolates carrying IncHI2, IncI2, and IncX4 with diverse range of STs. One hitherto unknown mutation, the mutation in the I527N gene was detected in colistin resistant isolates in this region, which was absent in colistin susceptible isolates. In West and Central Africa, two and one studies, respectively, reported mcr-1 gene exclusively in Escherichia coli isolates. Transferable plasmid mediated colistin resistance is rapidly emerging in Africa with mcr-1 as the predominant genetic variant in human, animals, and environmental samples.

Elucidating the mechanisms of colistin resistance and the efficacy of combination therapy in MDR/XDR/PDR Acinetobacter baumannii

Background: The rapid dissemination of carbapenem resistance in Gram-negative bacteria, particularly in Acinetobacter baumannii, has gained focus on the “old” drug colistin. Neverthless, the emergence of colistin resistance, last line of defense, causes a serious global threat. Therefore, antimicrobial treatment for these infections remains an important challenge. In this study, we aimed (1) to understand the involvement of the Two-Component system pmrAB and lipid biosynthesis lpx genes in colistin resistance and their corresponding relative expression levels and (2) implement suitable combination therapy options. Methods and materials: Eighty clinical isolates of A. baumannii were identified phenotypically and confirmed by PCR of GyrA gene. Isolates were subjected to antimicrobial susceptibility testing by BMD. The role of pmrAB and lpx genes on colistin resistance was determined by qRT-PCR. Assessment of the effect of combination therapy was done by the checkerboard analysis, Time kill experiments and the neutropenic pneumonia-induced mouse model. The effect of the electrostatic interaction of colistin action was seen by the Scanning Electron Microscopy. Results: Data have shown that 13 of the 80 (16%) isolates were resistant to colistin. The efficacy of colistin in combination with tigecycline, fosfomycin, zerbaxa, tazocin, teicoplanin, amikacin, levofloxacin, rifampicin and meropenem was assessed against colistin resistant A. baumannii isolates. Checkerboard and Time-kill assays demonstrated a synergestic effect between colistin and rifampicin and between colistin and teicoplanin, with bactericidal activity occuring prior to 4 h of incubation. A preliminary neutropenic pneumonia-induced mouse model, demonstrated improved survival and pathogen clearance from the blood and lung tissue cultures in the rifampicin combination. Up-regulation of pmrAB genes was recorded with a down-regulation of the lpx genes in vitro, nonethless this was not the case in in vivo studies. In addition, qRT-PCR did not show any significant differences between resistant and susceptible isolates upon examining the levels of expression of lpx and pmrAB genes. Conclusion: In conclusion, further understanding of the various molecular mechanisms of colistin resistance in A. baumannii is a must. Our current efforts focused on correlating the results of combination therapy in relation to the different resistance mechanisms to colistin, paving the path for potential treatment options against XDR and PDR A. baumannii infections.

Colistin Resistance in Environmental Isolates of Acinetobacter baumannii.

Although the molecular mechanisms of carbapenem resistance of environmental isolates of Acinetobacter baumannii are well described, data on the mechanisms of colistin resistance are scarce. In this study, we report the molecular mechanisms of colistin resistance in environmental isolates of A. baumannii. Seven clinically relevant isolates of A. baumannii belonging to ST-2Pasteur were recovered from hospital wastewater and wastewater treatment plant. The phenotypic resistance to colistin was confirmed by broth microdilution with minimum inhibitory concentration values ranging from 20 to 160 mg/L. Colistin sulfate and colistimethate sodium showed bactericidal activity against two colistin-heteroresistant isolates in vitro, but substantially recovery of population was observed after prolonged incubation. In silico genome analysis revealed nucleotide variations resulting in amino acid changes in LpxC (N286D), LpxD (E117K), PmrB (A138T, R263S, L267W, Q309P, and A444V), and EptA (F166L, I228V, R348K, A370S, and K531T). According to reverse transcription quantitative PCR, all isolates had increased levels of eptA mRNA and decreased levels of lpxA and lpxD mRNA. Isolates expressed low hydrophobicity, biofilm, and pellicle formation, but showed excellent survival in river water during 50 days of monitoring. Colistin- and pandrug-resistant A. baumannii disseminated in the environment could represent the source for the occurrence of serious community-acquired infections.

A novel plasmid-encoded mcr-4.3 gene in a colistin-resistant Acinetobacter baumannii clinical strain.

To identify the molecular mechanism of colistin resistance in an MDR Acinetobacter baumannii clinical strain isolated in 2008 from a meningitis case in Brazil. Long- and short-read WGS was used to identify colistin resistance genes in A. baumannii strain 597A with a colistin MIC of 64 mg/L. MS was used to analyse lipid A content. mcr was cloned into pET-26b (+) and transformed into Escherichia coli BL21(λDE3)pLysS for analysis. A novel plasmid (pAb-MCR4.3) harbouring mcr-4.3 within a Tn3-like transposon was identified. The A. baumannii 597A lipid A MS spectra showed a main molecular ion peak at m/z=2034, which indicated the addition of phosphoethanolamine to the lipid A structure. E. coli BL21 transformed with pET-26b-mcr-4.3 gained colistin resistance with a colistin MIC of 8 mg/L. Colistin resistance in A. baumannii 597A was correlated with the presence of a novel plasmid-encoded mcr-4.3 gene.

Colistin resistance emerges in pandrug-resistant Klebsiella pneumoniae epidemic clones in Rio de Janeiro, Brazil

Klebsiella pneumoniae is an important human pathogen, able to accumulate and disseminate a variety of antimicrobial resistance genes. Resistance to colistin, one of the last therapeutic options for multi-drug-resistant bacteria, has been reported increasingly. Colistin-resistant K. pneumoniae (ColRKp) emerged in two hospitals in Rio de Janeiro state, Brazil in 2016. The aim of this study was to investigate if these ColRKp isolates were clonally related when compared between hospitals, to identify the molecular mechanisms of colistin resistance, and to describe other antimicrobial resistance genes carried by isolates. Twenty-three isolates were successively recovered, and the whole-genome sequence was analysed for 10, each of a different pulsed-field gel electrophoresis (PFGE) type. Although some PFGE clusters were found, none of them included isolates from both hospitals. Half of the isolates were assigned to CC258, three to ST152 and two to ST15. One isolate was pandrug resistant, one was extensively drug resistant, and the others were multi-drug resistant. Colistin resistance was related to mutations in mgrB, pmrB, phoQ and crrB. Eleven new mutations were found in these genes, including two nucleotide deletions in mgrB. All isolates were carbapenem resistant, and seven were associated with carbapenemase carriage (blaKPC-2 in six isolates and blaOXA-370 in one isolate). All isolates had a blaCTX-M, and two had a 16S ribosomal RNA methyltransferase encoding gene (armA and rmtB). ColRKp were composed of epidemic clones, but cross-dissemination between hospitals was not detected. Colistin resistance emerged with several novel mutations amid highly resistant strains, further restricting the number of drugs available and leading to pandrug resistance.

Multifaceted mechanisms of colistin resistance revealed by genomic analysis of multidrug-resistant Klebsiella pneumoniae isolates from individual patients before and after colistin treatment

Polymyxins (i.e., polymyxin B and colistin) are used as a last-line therapy to combat multidrug-resistant (MDR) Klebsiella pneumoniae. Worryingly, polymyxin resistance in K. pneumoniae is increasingly reported worldwide. This study identified the genetic variations responsible for high-level colistin resistance in MDR K. pneumoniae clinical isolates. Sixteen MDR K. pneumoniae isolates were obtained from stool samples of 8 patients before and after colistin treatment. Their genomes were sequenced on Illumina MiSeq to determine genetic variations. Fifteen of 16 isolates harboured ISKpn26-like element insertion at nucleotide position 75 of mgrB, abolishing its negative regulation on phoPQ; while colistin-susceptible ATH7 contained intact mgrB and phoQ. Interestingly, each of the 7 mgrB-disrupted, colistin-susceptible isolates contained a nonsynonymous substitution in PhoQ (G39S, L239P, N253T or V446G), potentially impairing its function and intergenically suppressing the effect caused by mgrB inactivation. Additionally, three of the 7 corresponding mgrB-disrupted, colistin-resistant isolates harboured a secondary nonsynonymous substitution in PhoQ (N253P, D438H or T439P). This is the first report of phoQ mutations in mgrB-disrupted, colistin-susceptible K. pneumoniae clinical isolates. We also discovered multiple phoQ mutations in mgrB-disrupted, colistin-resistant strains. Our findings highlight the multifaceted molecular mechanisms of colistin resistance in K. pneumoniae.

Molecular mechanism of colistin resistance mediated by mcr-1 and pmrAB genes in Escherichia coli

Objective To investigate the molecular mechanism of colistin resistance mediated by mcr-1and pmrAB genes in clinical Escherichia coli (E.coli) isolates. Methods A total of 1 988 clinical E. coli isolates were collected from the First People′s Hospital of Shangqiu from 2010 to 2017 and screened for colistin-resistant isolates using agar dilution method. The minimum inhibitory concentrations (MICs) of nine common clinical antibiotics were determined using broth microdilution method. PCR and sequencing analysis were performed to detect the colistin resistance genes of mcr-1 and pmrAB. Conjugation experiments were used to test the transferability of the plasmid carrying mcr-1 gene. S1-PFGE and Southern blot were used to locate the plasmid carrying mcr-1. All colistin-resistant E. coli isolates were typed by multilocus sequence typing (MLST). Results Six colistin-resistant E. coli isolates were obtained by agar dilution method. The results of susceptibility testing showed that all of the six isolates were multidrug resistant. PCR and sequencing analysis revealed that four out of the six strains carried mcr-1 gene, and the other two isolates both had an amino acid substitution (L167P) caused by pmrB gene mutation. The results of conjugation experiments, S1-PFGE and Southern hybridization analysis showed that the plasmids of four mcr-1 gene-positive E. coli strains were located on a conjugative plasmid about 60 kb in length. MLST analysis classified the six isolates into six distinct sequence types (STs). Conclusion This study suggested that mcr-1 gene and mutations in pmrAB gene were the main mechanisms mediating the resistance of E. coli to colistin. In clinical practice, the occurrence and spread of colistin-resistant E. coli should be further monitored, and the rational use of antibiotics should be promoted to prevent the spread of colistin-resistant strains. Key words: Escherichia coli; Colistin; mcr-1; pmrAB; Resistance mechanism

Molecular mechanism of colistin resistance in Klebsiella pneumoniae

Objective To investigate the molecular mechanism of colistin resistance in Klebsiella pneumoniae (K.pneumoniae). Methods Three clinical isolates of colistin-resistant K. pneumoniae (FK1149, FK1920 and FK1934) and three colistin-resistant mutants (FK660R, FK713R and FK729R) were investigated. Resistance genes of pmrAB, phoPQ, mgrB, crrAB, mcr-1 and mcr-2 were detected by PCR and then analyzed by sequencing. PROVEAN platform was used to predict changes in the biological functions of proteins related to drug resistance. Expression of pmrH, pmrC, mgrB and phoP genes was measured using quantitative real-time PCR. LPS silver staining and conjugation assay were performed to analyze the three clinical colistin-resistant isolates. Results Amino acid substitutions in PmrA (G53V), PmrB (T157P, R256G), MgrB (F44C) and CrrB (E189K) were detected. ISkpn14 and IS5-like insertion sequences were detected in FK713R and FK729R, respectively. FK1149, FK1920 and FK1934 were negative for mcr genes. Compared with the wild-type strain, expression of pmrH and pmrC genes at the transcriptional level was increased in all investigated isolates. Changes in the expression of phoP and mgrB genes were also observed. A partial deletion of LPS was identified in FK1149. Conclusion LPS modification induced by inactivation of PmrAB or MgrB is the main molecular mechanism of colistin resistance in K. pneumoniae isolates in this study. Mutations in PmrA (G53V), MgrB (F44C) and CrrB (E189K) that might be related to colistin resistance are detected for the first time in clinical isolates of K. pneumoniae. Key words: Colistin; Klebsiella pneumoniae; Drug resistance mechanism; Two-component regulatory system