pmrB Genes Research Articles

A nosocomial outbreak of colistin and carbapenem-resistant hypervirulent Klebsiella pneumoniae in a large teaching hospital.

Recently, colistin and carbapenem-resistant hypervirulent Klebsiella pneumoniae (CCR-hvKP) has been observed sporadically. The aim of this study was to report a nosocomial outbreak due to CCR-hvKP, so as to control the transmission of CCR-hvKP and prevent future outbreaks. The clinical characteristics of five involved cases were analyzed and infection prevention and control measures were documented. Five CCR-hvKP isolates were discovered from the five involved cases. Molecular features of the isolates including sequence type, capsule locus, antimicrobial resistance genes, virulence factors and phylogenetic relationship were analyzed by whole-genome sequencing. Validation of the role of the deleterious amino acid mutations to colistin resistance was examined by complementation assays. PCR was performed to identify insertion sequences within the mgrB gene. Mouse intraperitoneal infection models were used to assess virulence phenotype. Five cases infected with CCR-hvKP were identified with a high attributable mortality rate of 60% in the patients. The five outbreak isolates belonged to the high-risk ST11-KL64 clone and were closely clustered. They were highly resistant to commonly used antibiotics and showed hypervirulent in vivo. WGS revealed multiple antimicrobial resistance genes such as blaKPC-2 and blaCTX-M-65 and important virulence factors. Concerning colistin resistance, amino acid mutations G53S in pmrA gene, and T157P, T246A and R256G in pmrB gene were indentified. Among them, the deleterious mutation T157P in pmrB gene was validated to be responsible for the resistance phenotype of isolate KP01, KP03 and KP05. In addition, disruption of mgrB gene by insertion sequences of ISKpn26 and IS903B was indentified in isolate KP02 and KP04, respectively. This is the first report of an outbreak caused by CCR-hvKP. The study highlights infection prevention and control measures are key to successfully fight against CCR-hvKP dissemination and nosocomial infections. Continuous surveillance should be performed to limit the spread of these isolates.

Conventionally Reared Wallon Meat Lambs Carry Transiently Multi-Drug-Resistant Escherichia coli with Reduced Sensitivity to Colistin Before Slaughter.

Major efforts have been made to reduce the use of colistin in livestock since the discovery of the plasmid-borne mobile colistin resistance (mcr) gene in E. coli a decade ago, to curb the burden of its potential transmission to other bacterial species, spread between animals, humans and the environment. This study explored the longitudinal prevalence and characteristics of colistin-resistant and extended-spectrum beta-lactamase-producing (ESBL) E. coli via in vivo fecal and ex vivo carcass swabs from two batches of conventional indoor and organic outdoor Wallon meat sheep from birth to slaughter in 2020 and 2021. Antimicrobial susceptibility testing via broth microdilution revealed that n = 16/109 (15%) E. coli isolates from conventional meat lamb fecal samples had a reduced colistin sensitivity (MIC = 0.5 μg/mL) and thereof, n = 9/109 (8%) were multi-drug-resistant E. coli, while no resistant isolates were recovered from their carcasses. Sequencing revealed causative pmrB genes, indicating that the reduced sensitivity to colistin was not plasmid-borne. While the sample size was small (n = 32), no colistin-resistant and ESBL-producing E. coli were isolated from the organic meat sheep and their carcasses, potentially due to the different husbandry conditions. Prudent and judicious antimicrobial use and strict slaughter hygiene remain imperative for effective risk management to protect consumers in a sustainable One Health approach.

Characterization of colistin-resistant carbapenemase producing Klebsiella pneumoniae in a river receiving wastewater treatment plant effluent.

Genes conferring antibiotic resistance phenotype, particularly to last resort antibiotics, pose a significant concern globally. Wastewater treatment plant (WWTP) effluent substantially contributes to antibiotic resistance in receiving rivers, threatening human health. Globally, colistin- and carbapenem-resistant Klebsiella pneumoniae infections cause high morbidity and mortality. We investigated colistin-resistant carbapenemase-producing K. pneumoniae (Co-CRKP) isolates in Kathajodi river receiving WWTP effluent, their resistance genes, and pathogenic potential. Four isolates (Co-CRKP-7, Co-CRKP-8, Co-CRKP-10, and Co-CRKP-15) exhibited extensively drug-resistant (XDR) phenotype, harbouring blaTEM-1, blaCTX-M-15, blaNDM-5, and blaOXA-48 genes. Colistin resistance was attributed to mutations in the pmrA and pmrB genes. Virulence genes (fimH, mrkD, entB, iucA, iutA, and irp1), capsular serotypes (K1, K2) and biofilm formation in the isolates explicated their pathogenicity. Furthermore, Inc plasmid replicons (Y, FrepB, P, K/B, L/M, N, FIA, A/C, and FIB) indicated the dissemination potential of the resistance genes in Co-CRKP isolates. The multi-locus sequence typing showed that Co-CRKP-7 and Co-CRKP-8 belonged to ST42, while Co-CRKP-10 and Co-CRKP-15 were ST16 and ST231, respectively. These high-risk clones carrying multidrug resistance and virulence genes, implicated in numerous outbreaks, have spread worldwide. Our findings emphasize the necessity for effective treatment of hospital wastes to restrict the spread of clinical isolates into aquatic environments.

Resensitization to colistin results in rapid and stable recovery of adherence, serum resistance and ompW in Acinetobacter baumannii.

Colistin resistance in Acinetobacter baumannii is an emerging problem that limits antimicrobial therapy options. We isolated two pairs of colistin susceptible and colistin-resistant A. baumannii (K1007/K1006 and K408/K409) from two patients diagnosed with carbapenem-resistant A. baumannii infection. Colistin susceptible isolates were exposed to in vitro colistin induction for 50 generations. The selected cell populations were subjected to DNA and RNA sequencing and phenotypic assays. In the in vitro induction assay, K408 gained colistin resistance on the corresponding day of clinical resistance (K408-G25) and got resensitized to colistin in the consecutive generation (K408-G26). A significant upregulation of ompW, ata, adeFGH genes on K408-G25 was followed by a downregulation upon resensitization to colistin (G26). Despite the upregulation of the ompW gene in transcriptomic analysis, the ompW protein disappeared on K408-G25 and recovered in the resensitized generation (G26). In parallel, disrupted cell membrane integrity recovered in K408-G26. In the K408-G25, downregulation of pbpG and upregulation of pbp1a/pbp3 genes decreased serum-resistance which was reversed in the resensitized generation (G26). The K1007 did not gain colistin resistance amongst 50-generations, however, the generation corresponding to clinical resistance day (K1007-G9) had a similar trend with K408-G25. The clinical colistin-resistant K409 and K1006 had SNPs on pmrA and pmrB genes. In this study, we observed that A. baumannii regulates adhesion, efflux pumps and serum-resistance associated genes as an early response to colistin stress. Besides, the ompW protein disappears in the cell membrane of colistin resistant cells which recovers after resensitization to colistin. The lack of ompW protein in colistin-resistant cells should be taken into consideration for escape mutants in development of antivirulence vaccination or treatment options.

Genetic Alterations Associated with Colistin Resistance Development in Escherichia coli.

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

Mutations in the pmrB gene constitute the major mechanism underlying chromosomally encoded colistin resistance in clinical Escherichia coli

ObjectivesThe mechanisms underlying chromosomally encoded colistin resistance in Escherichia coli remain insufficiently investigated. In this study, we investigated the contribution of various pmrB mutations from E. coli clinical isolates to colistin resistance. MethodsThe resistance mechanisms in eight mcr-negative colistin-resistant E. coli isolates obtained from a nationwide surveillance program in Taiwan using recombinant DNA techniques and complementary experiments were investigated. The minimal inhibitory concentrations (MICs) of colistin in the recombinant strains were compared with those in the parental strains. The expression levels of pmrA and pmrK (which are part of the pmrCAB and pmrHFIJKLM operons associated with colistin resistance) were measured using reverse transcription-quantitative real-time polymerase chain reaction. ResultsIn the complementation experiments, various mutated pmrB alleles from the eight mcr-negative colistin-resistant E. coli strains were introduced into an ATCC25922 mutant with a PmrB deletion, which resulted in colistin resistance. The MIC levels of colistin in the most complemented strains were comparable to those of the parental colistin-resistant strains. Increased expression levels of pmrA and pmrK were consistently detected in most complemented strains. The impact for colistin resistance was confirmed for various novel amino acid substitutions, P94L, G19E, L194P, L98R and R27L in PmrB from the parental clinical strains. The detected amino acid substitutions are distributed in the different functional domains of PmrB. ConclusionsColistin resistance mediated by amino acid substitutions in PmrB is a major chromosomally encoded mechanism in E. coli of clinical origin.

Genetic alterations in the pmrAB two-component system and lipid A biosynthesis genes of polymyxin-resistant Acinetobacter baumannii isolates.

The rate of pandrug-resistant Acinetobacter baumannii strains is on the rise in all continents. This bacterium can acquire resistance to all antibiotics, even to colistin. Alterations in the lipid A or/and the two-component pmrAB were earlier detected in colistin resistance. We investigated and analyzed two strains of A. baumannii (ABRC1 and ABRC2) isolated from two patients admitted to intensive care unit with a septic shock. Both strains were resistant to all tested antibiotics including colistin with a MIC >256 mg L-1. Colistin resistance genes (pmrA, pmrB, lpxA, lpxC, lpxD, and lpsB) of two strains (ABRC1 and ABRC2) were investigated by PCR and sequencing. Obtained nucleic acid sequences were aligned with reference sequences of ATCC 19606 and 17987. In this study two amino acid mutations, N287D in the lpxC gene and E117K in the lpxD gene, were detected in both ABRC1 and ABRC2 strains. ABRC1 had an additional H200L mutation in the pmrA gene. Both colistin resistant strains harbored the same A138T mutation in the pmrB gene. The ABRC2 strain also had an alteration in the kinase domain, specifically an R263S substitution of the histidine kinase domain. Three identical mutations were found in the lpsB gene of both A. baumannii strains: Q216K + H218G + S219E. As a result, a newly deduced protein sequence in both ABRC1 and ABRC2 strains differed from those described in ATCC 17978 and 19606 strains was determined. Colistin resistance is multifactorial in A. baumannii. In our study we detected novel mutations in colistin resistant A. baumannii clinical isolates.

Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. Coli strains

BackgroundThe current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales primarily involves the disruption of the upstream PmrAB and PhoPQ two-component system (TCS) control caused by mutations in the regulatory genes. Interestingly, previous studies have yielded conflicting results regarding the interaction of regulatory genes related to colistin resistance in Escherichia coli, specifically those surrounding PhoPQ and PmrAB TCS.ResultsIn our study, we focused on two clinical non-mcr colistin-resistant strains of E. coli, TSAREC02 and TSAREC03, to gain a better understanding of their resistance mechanisms. Upon analysis, we discovered that TSAREC02 had a deletion (Δ27–45) in MgrB, as well as substitutions (G206R, Y222H) in PmrB. On the other hand, TSAREC03 exhibited a long deletion (Δ84–224) in PhoP, along with substitutions (M1I, L14P, P178S, T235N) in PmrB. We employed recombinant DNA techniques to explore the interaction between the PhoPQ and PmrAB two-component systems (TCSs) and examine the impact of the mutated phoPQ and pmrB genes on the minimum inhibitory concentrations (MICs) of colistin. We observed significant changes in the expression of the pmrD gene, which encodes a connector protein regulated by the PhoPQ TCS, in the TSAREC02 wild-type (WT)-mgrB replacement mutant and the TSAREC03 WT-phoP replacement mutant, compared to their respective parental strains. However, the expressions of pmrB/pmrA, which reflect PmrAB TCS activity, and the colistin MICs remained unchanged. In contrast, the colistin MICs and pmrB/pmrA expression levels were significantly reduced in the pmrB deletion mutants from both TSAREC02 and TSAREC03, compared to their parental strains. Moreover, we were able to restore colistin resistance and the expressions of pmrB/pmrA by transforming a plasmid containing the parental mutated pmrB back into the TSAREC02 and TSAREC03 mutants, respectively.ConclusionWhile additional data from clinical E. coli isolates are necessary to validate whether our findings could be broadly applied to the E. coli population, our study illuminates distinct regulatory pathway interactions involving colistin resistance in E. coli compared to other species of Enterobacterales. The added information provided by our study contribute to a deeper understanding of the complex pathway interactions within Enterobacterales.

Upregulation of pmrA, pmrB, pmrC, phoQ, phoP, and arnT genes contributing to resistance to colistin in superbug Klebsiella pneumoniae isolates from human clinical samples in Tehran, Iran

BackgroundAntibiotic resistance in Klebsiella pneumoniae isolates, particularly resistance to colistin, has become a growing concern. This study seeks to investigate the upregulation of specific genes (pmrA, pmrB, pmrC, phoQ, phoP, and arnT) that contribute to colistin resistance in K. pneumoniae isolates collected from human clinical samples in Tehran, Iran. MethodsThirty eight K. pneumoniae isolates were obtained and subjected to antibiotic susceptibility testing, as well as evaluation for phenotypic AmpC and ESBL production according to CLSI guidelines. The investigation of antibiotic resistance genes was conducted using polymerase chain reaction (PCR), whereas the quantification of colistin resistance related genes expressions was performed via Real-Time PCR. ResultsThe highest and lowest antibiotics resistance were observed for cefotaxime 33 (86.8%) and minocycline 8 (21.1%), respectively. Twenty-four (63.2%) and 31 (81.6%) isolates carried AmpC and ESBLs, respectively. Also, antibiotic resistance genes containing blaNDM, blaIMP, blaVIM, blaSHV, blaTEM, blaCTXM, qnrA, qnrB, qnrS, and aac(6')-Ib were detected in K. pneumoniae isolates. Only 5 (13.1%) isolates were resistant to colistin and the MIC range of these isolates was between 4 and 64 μg ml−1. Upregulation of the pmrA, pmrB, pmrC, phoQ, phoP, and arnT genes was observed in colistin-resistant isolates. The colistin-resistant isolates were found to possess a simultaneous presence of ESBLs, AmpC, fluoroquinolone, aminoglycoside, and carbapenem resistant genes. ConclusionsThis study reveals escalating antibiotic resistance in K. pneumoniae, with notable coexistence of various resistance traits, emphasizing the need for vigilant surveillance and innovative interventions.

Phenotypic and molecular characterization of multi-drug resistant Klebsiella spp. isolates recovered from clinical settings

Klebsiella pneumoniae is a Gram-negative bacterium that colonizes the gastrointestinal tract and nasopharynx with many being linked to nosocomial infections. Extended-spectrum β-lactamases (ESBL)-producing and carbapenem-resistant K. pneumoniae is recognized by the World Health Organization (WHO) as a critical public health concern. In this study, whole-genome sequencing (WGS) – based analysis was performed to understand the molecular epidemiology of multi-drug resistant Klebsiella spp. clinical isolates. Genome comparison, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and whole-genome-SNP-based phylogenetic analysis (wg-SNP) were used for in-depth molecular characterization. in silico typing was used to determine the resistance genes, virulence factors, Inc. groups, and capsular types. All except one isolate were non-susceptible to meropenem and 89% were non-susceptible to ertapenem and imipenem. blaNDM, blaOXA-48, and blaKPC were the detected carbapenemases with blaNDM-1 found in half of the sequenced genomes. Resistance to colistin was detected in one isolate and was linked to several genetic alterations in crrB, pmrB, and pmrC genes. The most common plasmid type was IncFIB followed by IncR, and the Type 3 fimbriae, encoded by the mrkABCDF operon, was conserved among all isolates. The most common sequence- (ST) and K-type detected were ST147 and K64. The prevelance and the genomic relatedness of ST147 isolates, as shown by the data from SNP-based phylogenetic analysis, PFGE, and genomic clustering, may be an outbreak marker. However, this can only be validated through a more comprehensive study encompassing a wider sampling scheme and over an extended timeframe.

Determining Susceptibility and Potential Mediators of Resistance for the Novel Polymyxin Derivative, SPR206, in Acinetobacter baumannii.

With the increase in carbapenem-resistant A. baumannii (CRAB) infections, there has been a resurgence in the use of polymyxins, specifically colistin (COL). Since the reintroduction of COL-based regimens in treating CRAB infections, several COL-resistant A. baumannii isolates have been identified, with the mechanism of resistance heavily linked with the loss of the lipopolysaccharide (LPS) layer of the bacterial outer membrane through mutations in lpxACD genes or the pmrCAB operon. SPR206, a novel polymyxin derivative, has exhibited robust activity against multidrug-resistant (MDR) A. baumannii. However, there is a dearth of knowledge regarding its efficacy in comparison with other A. baumannii-active therapeutics and whether traditional polymyxin (COL) mediators of A. baumannii resistance also translate to reduced SPR206 activity. Here, we conducted susceptibility testing using broth microdilution on 30 A. baumannii isolates (17 COL-resistant and 27 CRAB), selected 14 COL-resistant isolates for genomic sequencing analysis, and performed time-kill analyses on four COL-resistant isolates. In susceptibility testing, SPR206 demonstrated a lower range of minimum inhibitory concentrations (MICs) compared with COL, with a four-fold difference observed in MIC50 values. Mutations in lpxACD and/or pmrA and pmrB genes were detected in each of the 14 COL-resistant isolates; however, SPR206 maintained MICs ≤ 2 mg/L for 9/14 (64%) of the isolates. Finally, SPR206-based combination regimens exhibited increased synergistic and bactericidal activity compared with COL-based combination regimens irrespective of the multiple resistance genes detected. The results of this study highlight the potential utility of SPR206 in the treatment of COL-resistant A. baumannii infections.

Expression of transcriptional regulator mgrB is enhanced in colistin-resistant Klebsiella pneumoniae: A report from India

Background: Colistin is regarded as the final option for treating Gram-negative bacteria that are resistant to several drugs. A common resistance mechanism for colistin in Klebsiella pneumoniae is mgrB gene inactivation. Inactivation of mgrB activates the PhoPQ which upregulates the PmrAB leading to resistance by lipopolysaccharide modification. Colistin resistance has been reported in Klebsiella pneumoniae, still, studies related to molecular mechanisms of this resistance are lacking. Aim: Role of mgrB in colistin resistance Methods: In this study, eight colistin-resistant clinical Klebsiella pneumoniae were selected, and performed broth microdilution followed by Rapid polymyxin NP test for screening of colistin resistance. PCR was performed for different types of mcr genes. MgrB was amplified and sequenced. Quantitative real-time PCR was performed for mgrB, PmrA, PmrB, PhoP, and PhoQ genes. Further, clonal analysis by ERIC PCR was done. Results: Colistin MIC of 4μg/ml has been shown by the isolates. No mcr gene was detected. Positive amplification for the mgrB gene was found in all the isolates. Sequence analysis of the mgrB gene found no mutation however, higher expression in the mgrB, PmrA, PmrB, PhoP, and PhoQ genes of all the eight colistin-resistant isolates could be observed when the isolates were exposed to colistin and Mg2+ pressure. Clonal analysis exhibited seven different haplotypes of Klebsiella pneumoniae. Conclusion: This study provides firsthand knowledge about molecular mechanisms of colistin resistance in Klebsiella pneumoniae in this geographical area. Enhancement in mgrB expression in the presence of colistin and Mg2+ pressure regardless of any mutation has a remarkable impact in the context of colistin resistance mechanisms in clinical Klebsiella pneumoniae.

Chromosome-Mediated Colistin Resistance in Clinical Isolates of Klebsiella pneumoniae and Escherichia coli: Mutation Analysis in the Light of Genetic Background.

Colistin resistance mechanisms involving mutations in chromosomal genes associated with LPS modification are not completely understood. Mutations in genes coding for the MgrB regulator frequently account for colistin resistance in Klebsiella pneumoniae, whereas mutations in genes coding for PhoPQ and PmrAB are frequent in E. coli. Our aim was to perform a genetic analysis of chromosomal mutations in colistin-resistant (MIC ≥4 µg/mL) clinical isolates of K. pneumoniae (n = 8) and E. coli (n = 7) of different STs. Isolates were obtained in a 3-year period in a university hospital in Santiago, Chile. Susceptibility to colistin, aminoglycosides, cephalosporins, carbapenems and ciprofloxacin was determined through broth microdilution. Whole genome sequencing was performed for all isolates and chromosomal gene sequences were compared with sequences of colistin-susceptible isolates of the same sequence types. None of the isolates carried mcr genes. Most of the isolates were susceptible to all the antibiotics analyzed. E. coli isolates were ST69, ST127, ST59, ST131 and ST14, and K. pneumoniae isolates were ST454, ST45, ST6293, ST380 and ST25. All the isolates had mutations in chromosomal genes analyzed. K. pneumoniae had mutations mainly in mgrB gene, whereas E. coli had mutations in pmrA, pmrB and pmrE genes. Most of the amino acid changes in LPS-modifying enzymes of colistin-resistant isolates were found in colistin-susceptible isolates of the same and/or different ST. Eleven of them were found only in colistin-resistant isolates. Colistin resistance mechanisms depend on genetic background, and are due to chromosomal mutations, which implies a lower risk of transmission than plasmid-mediated genes. Colistin resistance is not associated with multidrug-resistance, nor to high-risk sequence types.

Stability and genetic insights of the co-existence of blaCTX-M-65, blaOXA-1, and mcr-1.1 harboring conjugative IncI2 plasmid isolated from a clinical extensively-drug resistant Escherichia coli ST744 in Shanghai.

Co-existence of colistin, β-lactam and carbapenem in multidrug-resistant Enterobacteriaceae isolates poses a serious threat to public health. In this study, we investigated and characterized the co-occurrence of blaCTX-M-65, blaOXA-1, and mcr-1.1 strain isolated from a clinical extensively-drug-resistant Escherichia coli ST744 in Shanghai. Antimicrobial susceptibility test was carried out by agar dilution methods. Whole genome sequencing was conducted, and resistance genes, and sequence types of colistin in E. coli isolates were analyzed. Plasmid stability and amino acid mutations were assessed in E. coli isolates. A colistin resistant E. coli ST744, named ECPX221, was identified out of 145 fecal samples collected. The strain carries a 60,168 IncI2 plasmid with the mcr-1.1 gene. The strain also has blaCTX-M-65, blaOXA-1, dfrA14, qnrS1, cmlA5, arr2, ampC, aph(4)-Ia, sul1, and aadA5 resistance genes. The plasmid pECPX221 was capable of conjugation with an efficiency of 2.6 × 10-2. Notably, 45% of the transconjugants were determined as mcr-1.1-harboring in the colistin-free environment after 60 generation of passage. No mutations occurred in pmrB, mgrB, and phoPQ gene in the mcr-1.1-harboring transconjugants. Bioinformatic analysis indicated pECPX221 shared highly similar backbone with the previously reported mcr-1.1-harboring pAH62-1, pMFDS1339.1, pSCZE4, and p2018-10-2CC. Furthermore, sequencing and phylogenetic analyses revealed a similarity between other MCR-1-homolog proteins, indicating that ECPX221 was colistin resistant. The stable transferable mcr-1.1-harboring plasmid found in the E. coli ST744 strain indicated the high risk to disseminate the extensively-drug-resistance phenotype among Enterobacteriaceae.

Epidemiology, Phenotypic and Genotypic Characterization of Carbapenem-Resistant Gram-Negative Bacteria from a Libyan Hospital.

Antimicrobial resistance, particularly resistance to carbapenems, has become one of the major threats to public health. Seventy-two isolates were collected from patients and hospital environment of Ibn Sina Hospital, Sirte, Libya. Antibiotic susceptibility tests, using the disc diffusion method and E-Test strips, were performed to select carbapenem-resistant strains. The colistin (CT) resistance was also tested by determining the minimum inhibitory concentration (MIC). RT-PCR was conducted to identify the presence of carbapenemase encoding genes and plasmid-mediated mcr CT resistance genes. Standard PCR was performed for positive RT-PCR and the chromosome-mediated CT resistance genes (mgrB, pmrA, pmrB, phoP, phoQ). Gram-negative bacteria showed a low susceptibility to carbapenems. Molecular investigations indicated that the metallo-β-lactamase New Delhi metallo-beta-lactamases-1 was the most prevalent (n = 13), followed by Verona integron-encoded metallo-beta-lactamase (VIM) enzyme (VIM-2 [n = 6], VIM-1 [n = 1], and VIM-4 [n = 1]) that mainly detected among Pseudomonas spp. The oxacillinase enzyme OXA-23 was detected among six Acinetobacter baumannii, and OXA-48 was detected among one Citrobacter freundii and three Klebsiella pneumoniae, in which one coharbored the Klebsiella pneumoniae carbapenemase enzyme and showed resistance to CT (MIC = 64 μg/mL) by modification in pmrB genes. In this study, we report for the first time the emergence of Pseudomonas aeruginosa carrying the blaNDM-1 gene and belonging to sequence type773 in Libya. Our study reported also for the first time CT resistance by mutation in the pmrB gene among Enterobacteriaceae isolates in Libya.

Molecular characterisation of colistin and carbapenem-resistant clinical isolates of Acinetobacter baumannii from Southeast Europe

ObjectivesTo characterise 11 colistin- and carbapenem-resistant Acinetobacter baumannii isolates recently emerging in hospital settings. MethodsA. baumannii isolates were collected from hospitalised patients under colistin treatment in three countries of Southeast Europe: Turkey, Croatia, and Bosnia and Herzegovina. Isolates were identified using molecular methods. ResultsIsolates from Turkey and Croatia belong to the sequence types ST195 or ST281 of the clone lineage 2, while the single isolate from Bosnia and Herzegovina belongs to the ST231 of clone lineage 1. All isolates turned out to be highly resistant to colistin (MIC ≥ 16 mg/L) and have point mutations in pmrCAB operon genes. The colistin-resistant isolate from Bosnia and Herzegovina had a unique P170L point mutation in the pmrB gene and the R125H point mutation in the pmrC gene. The L20S mutation in the pmrA gene was detected only in isolates from Croatia and has never been reported before in isolates from this country. ConclusionColistin resistance in A. baumannii in hospitalised patients receiving colistin treatment is a result of chromosomal mutations. The pattern of point mutations in pmrCAB genes suggests a spread of specific colistin-resistant isolates within the hospital.

Molecular characterization of colistin resistance genes in a high-risk ST101/KPC-2 clone of Klebsiella pneumoniae in a University Hospital of Split, Croatia.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has become a major concern worldwide due to multidrug resistance and the ability to spread locally and globally. Infections caused by KPC-KP are great challenge in the healthcare systems because these are associated with longer hospitalization and high mortality. The emergence of colistin resistance has significantly reduced already limited treatment options. This study describes the molecular background of colistin-resistant KPC-KP isolates in the largest hospital in southern Croatia. Thirty-four non-duplicate colistin-resistant KPC-KP isolates were collected during routine work from April 2019 to January 2020 and from February to May 2021. Antimicrobial susceptibility was determined using disk diffusion, broth microdilution, and the gradient strip method. Carbapenemase was detected with an immunochromatographic test. Identification of blaKPC and mcr genes or mutations in pmrA, pmrB, mgrB, phoP, and phoQ genes were performed by polymerase chain reaction (PCR) and positive products were sequenced. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. All isolates were multidrug-resistant, with colistin minimum inhibitory concentrations (MICs) from 4 to >16 mg/L, and all harbored blaKPC-2 and had a single point mutation in the mgrB gene resulting in a premature stop codon, with the exception of one isolate with four point mutations corresponding to stop codons. All isolates were negative for mcr genes. PFGE analysis identified a single genetic cluster, and MLST revealed that all isolates belonged to sequence type 101 (ST101). These results show emergence of the high-risk ST101/KPC-2 clone of K. pneumoniae in Croatia as well as appearance of colistin resistance due to mutations in the mgrB gene. Molecular analysis of epidemiology and possible resistance mechanisms are important to develop further strategies to combat such threats.

Genomic characterization of colistin resistance in Klebsiella spp. under the pressure of colistin.

Introduction. Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a serious threat to global public health. Colistin is regarded as the last-resort antibiotic for CRKP infections, but colistin resistance among CRKP is increasingly being reported, making clinical treatment for CRKP infections more difficult.Hypothesis/Gap Statement. The molecular mechanisms of colistin resistance in Klebsiella spp. under the pressure of colistin have not been fully investigated.Aim. We aimed to investigate the phenotypic and genetic variation in two colistin-susceptible Klebsiella spp. strains under selective pressure of colistin.Methodology. One hundred microlitres of overnight cultures of the CRKP clinical strain CRKP12-130 and of ATCC 700603 was spread on five Mueller-Hinton Agar (MHA) plates with colistin concentrations of 2, 4, 8, 16 and 32 µg ml-1, and growth of colonies was observed for five consecutive days. Colonies collected from plates were passaged daily for 10 days on MHA plates without colistin and susceptibility testing of colistin was performed by broth microdilution. Thirty-four colistin-resistant strains randomly selected were submitted to whole genome sequencing (WGS). Transcriptional levels of genes involved in colistin resistance (mgrB, phoP, phoQ, pmrA, pmrB, pmrD, pmrE and pmrK) were measured by quantitative real-time PCR.Results. A total of 114 and 119 colistin-resistant colonies were obtained from CRKP12-130 and ATCC 700603 in this study, among which 16 and 18 colonies were submitted to WGS, respectively. Among these 34 sequenced isolates, mutation in phoQ (13/16, 81.25 %) was the main genetic factor mediating colistin resistance in strains from CRKP12-130, while for strains from ATCC 700603, mutation associated with mgrB (8/18, 44.44 %) was found to be the commonest. Mutation of mgrB led to a significant increase in the MIC for colistin (from 64 to >128 µg ml-1), and a novel mutation C28R in mgrB was first reported in this study.Conclusion. Colistin-resistant Klebsiella spp. could be easily selected under pressure of different concentrations of colistin. Mutations of mgrB, phoP, phoQ and pmrB genes were the main mechanisms leading to chromosomally mediated colistin resistance in Klebsiella spp.

The role of pmrCAB genes in colistin-resistant Acinetobacter baumannii

The progressively increasing antimicrobial-resistant Acinetobacter baumannii infections have enforced the use of colistin as the last option for therapy, resulting in the colistin resistance evolution. This work aimed to study the pmrCAB expression in A. baumannii isolates as well as the presence of the mcr-1 gene. Colistin MICs of 100 A. baumannii isolates were measured using the broth microdilution assay. In four colistin-susceptible and four colistin-resistant isolates, the relative expression of the pmrA, pmrB, and pmrC genes was determined using reverse transcription PCR, and then selected isolates were sequenced using the Sanger technique. Finally, the mcr-1 gene was detected using conventional PCR. The colistin resistance rate among the studied isolates was 49%. The expression levels of pmrA and pmrB were statistically significantly higher in colistin-resistant isolates than in colistin-susceptible ones, while the pmrC expression had no statistically significant change. There was a weak positive correlation between colistin MICs and the expression levels of each of the pmrA and pmrB genes. By sequencing, two colistin-resistant strains with low pmrCAB expression showed insertion mutations 3277188_3277189T in pmrB and 1185149_1185150T in pmrC. Only one isolate (1%) was positive for the presence of mcr-1. We concluded that pmrCAB increased expression and/or mutations may cause colistin resistance in A. baumannii. However, increased pmrC expression may not necessarily result in colistin resistance. In Egypt, this is the first study to reveal the existence of mcr-1 in A. baumanni. This should attract attention in clinical settings due to the ultimate tendency of spreading colistin resistance.

Prevalence of Mutated Colistin-Resistant Klebsiella pneumoniae: A Systematic Review and Meta-Analysis.

The emergence of genetic mutations in chromosomal genes and the transmissible plasmid-mediated colistin resistance gene may have helped in the spread of colistin resistance among various Klebsiella pneumoniae (K. pneumoniae) isolates and other different bacteria. In this study, the prevalence of mutated colistin-resistant K. pneumoniae isolates was studied globally using a systematic review and meta-analysis approach. A systematic search was conducted in databases including PubMed, ScienceDirect, Scopus and Google Scholar. The pooled prevalence of mutated colistin resistance in K. pneumoniae isolates was analyzed using Comprehensive Meta-Analysis Software (CMA). A total of 50 articles were included in this study. The pooled prevalence of mutated colistin resistance in K. pneumoniae was estimated at 75.4% (95% CI = 67.2-82.1) at high heterogeneity (I2 = 81.742%, p-value < 0.001). Meanwhile, the results of the subgroup analysis demonstrated the highest prevalence in Saudi Arabia with 97.9% (95% CI = 74.1-99.9%) and Egypt, with 4.5% (95% CI = 0.6-26.1%), had the lowest. The majority of mutations could be observed in the mgrB gene (88%), pmrB gene (54%) and phoQ gene (44%). The current study showed a high prevalence of the mutation of colistin resistance genes in K. pneumoniae. Therefore, it is recommended that regular monitoring be performed to control the spread of colistin resistance.