pmrB Genes Research Articles

Emergence of extensively drug-resistant and colistin-resistant Klebsiella pneumoniae isolated from hospitalized patients in Isfahan, Iran

The current study was performed to investigate the antibiotic resistance pattern, the prevalence of colistin resistance and its molecular mechanisms in clinical isolates of Klebsiella pneumoniae obtained from hospitalized patients in teaching hospitals in Isfahan, Iran. This cross-sectional study was performed during 2019–2020 at several teaching hospitals in Isfahan, Iran. All the K. pneumoniae isolates were screened against 14 antimicrobial agents based on standard disk diffusion method. Moreover, the minimal inhibitory concentration (MIC) of colistin was determined by the E-test strips. Also, PCR assay was carried out to detect genes encoding resistance to colistin, including mcr-1, mcr-2, pmrA, pmrB, and crrB. In the present study, a total of 79 strains of multidrug-resistant K. pneumoniae were isolated. Of these, 35 colistin-resistant clinical K. pneumoniae isolates were obtained. Antibiotic susceptibility pattern showed a high rate of antibiotic resistance to ceftazidime (94.9%), while, the lowest resistance rate was observed against tigecycline (2.5%). All colistin-resistant isolates were classified as extensively drug-resistant (XDR). PCR results showed that mcr-1 and mcr-2 genes were not detected, while, 10.1% contained pmrA and pmrB genes. In addition, the frequency of crrB gene among our isolates was 3.8%. Due to the fact that the last line of treatment for infections associated with K. pneumoniae is colistin, increasing resistance to this antibiotic causes many concerns and problems in the treatment of patients. Detection of colistin-resistant strains and reporting of occurrence of genes associated with this resistance can greatly help in the treatment of diseases.

Characterization of Carbapenemase-Producing Klebsiella pneumoniae Isolates from Two Romanian Hospitals Co-Presenting Resistance and Heteroresistance to Colistin.

Klebsiella pneumoniae is a notorious human pathogen involved in healthcare-associated infections. The worldwide expansion of infections induced by colistin-resistant and carbapenemase-producing Enterobacterales (CPE) isolates has been increasingly reported. This study aims to analyze the phenotypic and molecular profiles of 10 colistin-resistant (CR) isolates and 2 pairs of colistin-heteroresistant (ChR) (parental and the corresponding resistant mutants) isolates of K. pneumoniae CPE sourced from two hospitals. The phenotypes of strains in the selected collection had been previously characterized. Antimicrobial susceptibility testing was performed using a Vitek 2 Compact system (BioMérieux SA, Marcy l’Etoile, France), the disc diffusion method, and broth microdilution (BMD) for colistin. Whole-genome sequencing (WGS) did not uncover evidence of any mobile colistin resistance (mcr) genes, although the mgrB gene of seven isolates appeared to be disrupted by insertion sequences (ISKpn25 or ISKpn26). Possible deleterious missense mutations were found in phoP (L4F), phoQ (Q426L, L26Q, L224Q, Q317K), pmrB (R256G, P95L, T157P, V352E), and crrB (P151S) genes. The identified isolates belonged to the following clonal lineages: ST101 (n = 6), ST147 (n = 5), ST258 (n = 2), and ST307 (n = 1). All strains harbored IncF plasmids. OXA-48 producers carried IncL and IncR plasmids, while one blaNDM-1 genome was found to harbor IncC plasmids. Ceftazidime–avibactam remains a therapeutic option for KPC-2 and OXA-48 producers. Resistance to meropenem–vaborbactam has emerged in some blakPC-2-carrying isolates. Our study demonstrates that the results of WGS can provide essential evidence for the surveillance of antimicrobial resistance.

Polymyxin Resistance in Clinical Isolates of K. pneumoniae in Brazil: Update on Molecular Mechanisms, Clonal Dissemination and Relationship With KPC-Producing Strains.

In Brazil, the production of KPC-type carbapenemases in Enterobacteriales is endemic, leading to widespread use of polymyxins. In the present study, 502 Klebsiella pneumoniae isolates were evaluated for resistance to polymyxins, their genetic determinants and clonality, in addition to the presence of carbapenem resistance genes and evaluation of antimicrobial resistance. Resistance to colistin (polymyxin E) was evaluated through initial selection on EMB agar containing 4% colistin sulfate, followed by Minimal Inhibitory Concentration (MIC) determination by broth microdilution. The susceptibility to 17 antimicrobials was assessed by disk diffusion. The presence of bla KPC, bla NDM and bla OXA-48-like carbapenemases was investigated by phenotypic methods and conventional PCR. Molecular typing was performed by PFGE and MLST. Allelic variants of the mcr gene were screened by PCR and chromosomal mutations in the pmrA, pmrB, phoP, phoQ and mgrB genes were investigated by sequencing. Our work showed a colistin resistance frequency of 29.5% (n = 148/502) in K. pneumoniae isolates. Colistin MICs from 4 to >128 µg/mL were identified (MIC50 = 64 µg/mL; MIC90 >128 µg/mL). All isolates were considered MDR, with the lowest resistance rates observed for amikacin (34.4%), and 19.6% of the isolates were resistant to all tested antimicrobials. The bla KPC gene was identified in 77% of the isolates, in consonance with the high rate of resistance to polymyxins related to its use as a therapeutic alternative. Through XbaI-PFGE, 51 pulsotypes were identified. MLST showed 21 STs, with ST437, ST258 and ST11 (CC11) being the most prevalent, and two new STs were determined: ST4868 and ST4869. The mcr-1 gene was identified in 3 K. pneumoniae isolates. Missense mutations in chromosomal genes were identified, as well as insertion sequences in mgrB. Furthermore, the identification of chromosomal mutations in K. pneumoniae isolates belonging from CC11 ensures its success as a high-risk epidemic clone in Brazil and worldwide.

The molecular characterization of colistin-resistant isolates of Acinetobacter baumannii from patients at intensive care units.

The objective of this study was to determine molecular characterization and genetic diversity of colistin-resistant A. baumannii clinical isolates in Intensive Care Unit hospitalized patients. A total of 127 A. baumannii clinical isolates were evaluated for antimicrobial susceptibility. PCR reaction and sequencing were performed for the detection of mutations in pmrAB and lpx ACD genes. Based on antimicrobial susceptibility testing, 40.94% and 33.85% of the isolates were MDR and XDR respectively whereas 3.93% of them were found to be PDR. Results of agar dilution MIC and E-test indicated that 76% of the isolates were sensitive to colistin. All of the isolates were positive for bla OXA-51 and 50% of them were positive for both bla OXA-23 -like and bla OXA-143 -like genes while only 25% of the isolates were positive for bla OXA-72 . None of them were positive for the bla OXA-58 -like gene. There is no mutation in pmrA. The V162A substitution for pmrB gene was repeated in two isolates, and E394D and Y292H substitutions in lpxA were observed in two isolates; also, C120R and F165L substitutions in lpxC gene was repeated in two isolates. Analysis of phylogenetic tree based on alterations in lpxACD and pmrB genes indicated the appearance of new isolates compared to the reference strain ATCC17978 A. baumannii isolates. The present study indicated the prevalence of MDR and XDR A. baumannii isolates and the emergence of PDR isolates in the northwest portion of Iran. The appearance of colistin-resistant isolates with new mutations in pmrB, lpxACD genes indicates new resistance mechanisms.

FIRST REPORT IN IRAQ : AMINO ACID SUBSTITUTION IN PMRCAB GENES AND THERE CORELLATION WITH COLISTIN RESISTANCE AMONG A.BAUMANNII ISOLATES

In the present study,out of 120 samples were collected from different clinical sites from patients who admitted to different hospitals in Baghdad city,only 65 samples were detected as A. baumannii.The antibiotics susceptibility test showed that the bacteria were resistant to piperacillin (92.31%), cefotaxime (87.69%),trimethoprimsulfamethoxazole(73.85%).While Meropenem (70.77%)and gentamycin(67.69 %).The resistance rate to imipenem and tetracycline were 58.46% and 43.08% respectively. Eventually, just(20%)among all isolates are resistant to levofloxacin. While the percentage of colistin resistance was performed among 23 isolates and the results showed that all isolates were reported resistance in a percentage 100% with MIC values ≥ 16 µg/100µl.The results of pmrCAB genes detection and sequencing among seven A.baumannii isolates, which were choices because they were resistant to all antibiotics even colistin, were showed that the entire region of the pmrC containing fourteen missense mutations. Out of twelve detected mutations in pmrA, only one of them was found to exhibit a missense mutations. While the results of pmrB gene showed three missenses mutations.

Mutation in mgrB is the major colistin resistance mechanism in Klebsiella pneumoniae clinical isolates in Tehran, Iran.

Colistin is considered as one of a last resort antimicrobial agent against multidrug-resistant Gram-negative bacteria including Escherichia coli and Klebsiella pneumoniae. However, the recent emergence of colistin resistance (ColR) worldwide that severely restricts therapeutic options is a serious threat to global public health. In this study we have investigated the molecular determinants in ColR K. pneumoniae isolates collected from clinical specimens. A total of 98 E. coli and 195 K. pneumoniae clinical isolates were collected from two hospitals from August 2018 to December 2019 in Tehran, Iran. Colistin susceptibility and minimum inhibitory concentrations (MIC) were determined according to the Clinical and Laboratory Standards Institute by disk diffusion method, and microdilution method, respectively. For isolates with colistin MIC ≥4 μg mL-1, PCR was performed for the detection of mcr-1 to mcr-4 genes. Moreover, nucleotide sequences of mgrB, phoP, phoQ, pmrA, and pmrB genes were determined by sequencing. Finally, the transcriptional level of pmrK and pmrC genes was evaluated by quantitative reverse transcription PCR (RT-qPCR). None of the E. coli isolates were resistant to colistin while 21 out 195 K. pneumoniae isolates were identified as resistant, 19 of which carried mutation in the mgrB gene. Three different mutations were observed in the pmrB gene in 3 K. pneumoniae isolates. None of the ColR isolates showed alternations in pmrA, phoP, and phoQ genes. Furthermore, none of the plasmid-encoding genes were detected. Transcriptional level of the pmrK gene increased in all ColR isolates meanwhile, pmrC overexpression was detected in 16 out 21 (76.19%) isolates. Eventually, all ColR isolates were susceptible to tigecycline. Our results demonstrated that the alternation of mgrB gene is the main mechanism related to colistin resistance among ColR K. pneumoniae isolates in this study.

Evolved resistance to a novel cationic peptide antibiotic requires high mutation supply.

Background and ObjectivesA key strategy for resolving the antibiotic resistance crisis is the development of new drugs with antimicrobial properties. The engineered cationic antimicrobial peptide WLBU2 (also known as PLG0206) is a promising broad-spectrum antimicrobial compound that has completed Phase I clinical studies. It has activity against Gram-negative and Gram-positive bacteria including infections associated with biofilm. No definitive mechanisms of resistance to WLBU2 have been identified.MethodologyHere, we used experimental evolution under different levels of mutation supply and whole genome sequencing (WGS) to detect the genetic pathways and probable mechanisms of resistance to this peptide. We propagated populations of wild-type and hypermutator Pseudomonas aeruginosa in the presence of WLBU2 and performed WGS of evolved populations and clones.ResultsPopulations that survived WLBU2 treatment acquired a minimum of two mutations, making the acquisition of resistance more difficult than for most antibiotics, which can be tolerated by mutation of a single target. Major targets of resistance to WLBU2 included the orfN and pmrB genes, previously described to confer resistance to other cationic peptides. More surprisingly, mutations that increase aggregation such as the wsp pathway were also selected despite the ability of WLBU2 to kill cells growing in a biofilm.Conclusions and implicationsThe results show how experimental evolution and WGS can identify genetic targets and actions of new antimicrobial compounds and predict pathways to resistance of new antibiotics in clinical practice.

Analysis of colistin resistance in carbapenem-resistant Enterobacterales and XDR Klebsiella pneumoniae.

Introduction:Increasing occurrence of infections caused by multidrug-resistant Gram-negative bacteria resulted in colistin being the last agent for treatment. Apart from plasmid-mediated mcr genes, mutations involving several genes like mgrB, phoP/phoQ, pmrA, pmrB, pmrC, and crrABC genes, are leading causes of colistin resistance. Four colistin susceptibility testing methods were compared against broth microdilution (BMD) and determined the presence of the mcr1-5 gene.Methodology:A total of 100 carbapenem-resistant Enterobacterales isolates were tested for colistin susceptibility by commercial broth microdilution (cBMD), E-test, VITEK-2, and rapid polymyxin NP assay (RPNP) and compared with BMD. The presence of the mcr1-5 gene was determined by modified RPNP and PCR. Two non-mcr colistin-resistant XDR isolates were subjected to whole-genome sequencing using Illumina MiSeq sequencing platform.Results:Among 100 carbapenem-resistant Enterobacterales isolates, 15% were resistant to colistin. Essential agreement, categorical agreement, major error, and very major error for cBMD/E-test/VITEK-2/RPNP were 96%/73%/82%/NA; 99%/86%/88%/91%, 1.2%/9.4%/11.8%/8.2% and 0%/40%/13.3%/13.3%, respectively. Only one Klebsiella pneumoniae isolate harbored the mcr-1 gene, observed by both methods. Whole-genome sequencing of two non-mcr XDR Klebsiella pneumoniae showed multiple mutations in 10 genes responsible for lipopolysaccharide biosynthesis.Conclusions:The performance of cBMD was excellent, whereas the E-test was unacceptable. VITEK-2 and RPNP performed better but remained unreliable due to high error rates. Multiple mutations in the target proteins involving lipopolysaccharide formation, modification, and regulation were seen, resulting in colistin resistance.

Emergence of colistin resistance in Klebsiella pneumoniae ST15 disseminating blaKPC-2 in a novel genetic platform

Isolation of colistin- and carbapenem-resistant Klebsiella pneumoniae (CCR-Kp) is increasing in hospital settings worldwide, which is related to increased morbidity, mortality and healthcare costs. The aim of this work was to perform whole-genome sequencing (WGS), genomic and phylogenetic analysis, and conjugation assays of an extensively drug-resistant (XDR) CCR-Kp isolate from Argentina. WGS of strain KpS26 isolated from a bloodstream infection was performed using Illumina MiSeq-I, and de novo assembly was achieved using SPAdes v.3.11. A maximum likelihood tree was created using MEGA7 based on core genome single nucleotide polymorphisms from whole-genome alignment of K. pneumoniae isolates identified in silico as sequence type 15 (ST15). The resistome, plasmids and integrons were analysed using ResFinder, AMRFinderPlus, ISfinder, plasmidSPAdes, PlasmidFinder and IntegronFinder. Standard conjugation was performed. KpS26 belonged to ST15, which is less common than ST258, ST25 and ST11 that are globally reported as responsible for CCR-Kp outbreaks. Fourteen transferable antimicrobial resistance genes (ARGs), including blaKPC-2 in a novel genetic platform transferable by conjugation, were detected contributing to the XDR phenotype. The amino acid substitution T157P in the protein encoded by the pmrB gene of KpS26, previously reported as being responsible for resistance to colistin in K. pneumoniae lineages globally disseminated, was also identified in this strain. The XDR CCR-Kp isolate analysed here shows that ST15 is also disseminating blaKPC-2 in Argentina alongside other ARGs, evidencing that KPC epidemiology continues to be shaped by intricate and assorted ways of lateral gene transfer.

Colistin resistance in Gram-negative bacteria analysed by five phenotypic assays and inference of the underlying genomic mechanisms

BackgroundColistin is used against multi-drug resistant pathogens, yet resistance emerges through dissemination of plasmid-mediated genes (mcr) or chromosomal mutation of genes involved in lipopolysaccharide synthesis (i.e. mgrB, phoPQ, pmrCAB). Phenotypic susceptibility testing is challenging due to poor diffusion of colistin in agar media, leading to an underestimation of resistance. Performance of five phenotypic approaches was compared in the context of different molecular mechanisms of resistance. We evaluated Vitek 2® (bioMérieux, AST N242), Colistin MIC Test Strip (Liofilchem Diagnostici), UMIC (Biocentric), and Rapid Polymyxin™ NP test (ELITechGroup) against the standard broth microdilution (BMD) method. We used whole genome sequencing (WGS) to infer molecular resistance mechanisms. We analysed 97 Enterobacterales and non-fermenting bacterial isolates, largely clinical isolates collected up to 2018. Data was analysed by comparing susceptibility categories (susceptible or resistant) and minimal inhibitory concentrations (MIC). Susceptibility category concordance is the percentage of test results sharing the same category to BMD. MIC concordance was calculated similarly but considering ±1 MIC titre error range. We determined genomic diversity by core genome multi locus sequencing typing (cgMLST) and identified putative antimicrobial resistance genes using NCBI and CARD databases, and manual annotation.ResultsOf 97 isolates, 54 (56%) were resistant with standard BMD. Highest susceptibility category concordance was achieved by Rapid Polymyxin™ NP (98.8%) followed by UMIC (97.9%), Colistin E-test MIC strip (96.9%) and Vitek 2® (95.6%). Highest MIC concordance was achieved by UMIC (80.4%), followed by Vitek 2® (72.5%) and Colistin E-test MIC strip (62.9%). Among resistant isolates, 23/54 (43%) were intrinsically resistant to colistin, whereas 31/54 (57%) isolates had acquired colistin resistance. Of these, mcr-1 was detected in four isolates and mcr-2 in one isolate. Non-synonymous mutations in mgrB, phoQ, pmrA, pmrB, and pmrC genes were encountered in Klebsiella pneumoniae, Escherichia coli, and Acinetobacter bereziniae resistant isolates. Mutations found in mgrB and pmrB were only identified in isolates exhibiting MICs of ≥16 mg/L.ConclusionsThe Rapid Polymyxin™ NP test showed highest categorical concordance and the UMIC test provided MIC values with high concordance to BMD. We found colistin resistance in diverse species occurred predominantly through spontaneous chromosomal mutation rather than plasmid-mediated resistance.

МОЛЕКУЛЯРНО-ГЕНЕТИЧЕСКИЕ МЕХАНИЗМЫ УСТОЙЧИВОСТИ НОЗОКОМИАЛЬНЫХ ШТАММОВ KLEBSIELLA PNEUMONIAE К ПОЛИМИКСИНАМ И АНТИБИОТИКАМ ДРУГИХ ГРУПП ПО ДАННЫМ ПОЛНОГЕНОМНОГО СЕКВЕНИРОВАНИЯ

Identification of numerous mechanisms of resistance to colistin and other antibiotics is possible using whole genome sequencing. Objectives. To assess the molecular-genetic mechanisms of resistance to polymyxins and antibiotics of other groups in nosocomial Klebsiella pneumoniae strains. Material and methods. For 13 multidrug- and extensively drug-resistant K.pneumoniae strains semiconductor sequencing was performed in the Ion PGM System genomic sequencer (Thermo Fisher Scientific, USA). The assembly of genomic sequences and their annotation were carried out. The PROVEAN software tool was used to predict the influence of nucleotide replacements on the structure of amino acid sequences and functional activity of proteins. The identification of antibiotic resistance genes and the search for efflux mechanisms were performed by the ResFinder v.4.1 and CARD web resources. Results. Several types of β-lactamase genes were detected simultaneously in all strains, as well as genes of resistance to fosfomycin. Genes of resistance to aminoglycosides were identified in 11 strains, to chloramphenicol - in 10, to rifampicin - in 5, to macrolides - in 4. The mcr phosphoethanolamine transferase genes were absent in all strains. Functionally significant substitutions were revealed in the pmrB gene (D150Y, T157P, G207S) comparing the studied samples with the reference K. pneumoniae strain ATCC 700603. Changes in the mgrB gene were also found in colistin-resistant strains (W20R replacement, insertional inactivation of the gene by transposons of the IS1, IS4 and IS5 families). Conclusions. The results of whole genome sequencing represent the significant resistance of nosocomial Klebsiella pneumoniae strains to the majority of antibiotics including β-lactams, aminoglycosides, fluoroquinolones, fosfomycin, chloramphenicol, polymyxins. Genetic determinants of colistin resistance were revealed (insertional inactivation and deletion of the mgrB gene; D150Y, T157P and G207S substitutions in the pmrB gene) in strains with colistin MIC 64-128 mg/l and their absence in colistin-susceptible strains.

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.

Colistin Resistance Among Multiple Sequence Types of Klebsiella pneumoniae Is Associated With Diverse Resistance Mechanisms: A Report From India.

Background: The resistance to colistin and carbapenems in Klebsiella pneumoniae infections have been associated with increased morbidity and mortality worldwide. A retrospective observational study was conducted to determine the prevalence and molecular events contributing to colistin resistance.Methods: Clinical samples were screened for colistin resistance and underlying mechanisms were studied by PCR-based amplification and sequence analysis of genes of two-component regulatory system (phoPQ and pmrAB), regulatory transmembrane protein-coding mgrB, and mobilized colistin resistance genes (mcr-1-8). Gene expression of pmrC and pmrK was analyzed by qRT-PCR, and the genetic relationship was assessed by MLST. The putative effect of amino-acid substitutions was predicted by a combination of bioinformatics tools.Results: Of 335 Klebsiella spp. screened, 11 (3.2%) were identified as colistin-resistant (MIC range, 8 to >128 μg/ml). K. pneumoniae isolates belonged to clonal complex-11 (CC11) with sequence types (STs): 14, 16, 43, 54, 147 and 395, whereby four isolates conferred three novel STs (3986, 3987 and 3988) profiles. Sequence analysis revealed non-synonymous potentially deleterious mutations in phoP (T151A), phoQ (del87–90, del263–264, L30Q, and A351D), pmrA (G53S), pmrB (D150V, T157P, L237R, G250C, A252G, R315P, and Q331H), and mgrB (C28G) genes. The mgrB gene in three strains was disrupted by insertion sequences encoding IS1-like and IS5/IS1182 family-like transposase genes. All 11 isolates showed an elevation in the transcription level of pmrC gene. Mobilized colistin-resistance (mcr) genes were not detected. All but one of the colistin-resistant isolates was also resistant to carbapenems; β-lactamase genes blaNDM-1-like, blaOXA-48-like, and blaCTX-M-like were detected in eight, five, and nine isolates, respectively.Conclusion: All the studied colistin- and carbapenem-resistant K. pneumoniae isolates were genetically distinct, and various mechanisms of colistin resistance were detected, indicating its spontaneous emergence in this bacterial species.

Colistin Resistance Mechanisms in Clinical Escherichia coli and Klebsiella spp. Isolates from the Western Cape of South Africa.

Objectives: Colistin is a last-resort antibiotic for the treatment of carbapenem-resistant Gram-negative infections. Colistin resistance thus poses a threat to human health. Colistin resistance is most commonly encoded by mutations in chromosomal pmrA, pmrB, phoP, phoQ, ccrB, and mgrB genes, and the presence of plasmid-mediated mcr genes. This study describes colistin resistance mechanisms in clinical Enterobacterales isolates from the Western Cape, South Africa. Results: Escherichia coli (n = 22) and Klebsiella spp. (n = 7) isolates, from nine health care facilities, were confirmed to be colistin resistant during 2016 and 2017. mcr-1 was present in 55% (12/22) of E. coli and 71% (5/7) of Klebsiella spp. isolates. Colistin resistance mutations in pmrB were identified in 8/10 mcr-negative E. coli isolates using whole-genome sequencing, with pmrB Pro-94→Gln being the most frequent with presence in 4 isolates. One mcr-negative Klebsiella spp. isolate had a complete deletion of the mgrB and one contained an insertion sequence (IS1) in mgrB. Conclusion: A reduction in the proportion of colistin-resistant isolates harboring mcr-1 from 2016 to 2017 was observed. Colistin-resistant E. coli attributed by chromosomal mutations in pmrB in 2017 were mostly clonal related, which contrasts with the 2016 unrelated mcr-1-positive isolates. The diverse strains, hospitals, and resistance mechanisms may suggest that selective pressure is the main driver of colistin resistance.

Resistance evolution of hypervirulent carbapenem-resistant Klebsiella pneumoniae ST11 during treatment with tigecycline and polymyxin

ABSTRACT Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has recently aroused increasing attention, especially ST11, the predominant CRKP clone in China. Here, we report a case of hv-CRKP-associated infection and reveal the in-host evolution of its mechanism of resistance to tigecycline and polymyxin under clinical therapy. A total of 11 K. pneumoniae carbapenemase (KPC)-producing CRKP strains were consecutively isolated from a male patient who suffered from continuous and multisite infections. String and antimicrobial susceptibility tests identified seven hypermucoviscous strains and three tigecycline-resistant and four colistin-resistant strains. Galleria mellonella larvae infection model confirmed the hypervirulence. Pulsed-field gel electrophoresis (PFGE) separated three PFGE clusters among all strains, and further Southern blotting detected that bla KPC-2 was located on the same-sized plasmid. Whole-genome sequencing showed that all strains belonged to the hv-CRKP ST11-KL64 clone. Diverse hypervirulence factors and resistance genes were identified. Further sequencing with the Nanopore platform was performed on the CRKP-Urine1 strain, which contained one virulence plasmid (pVi-CRKP-Urine1) and two resistance plasmids (pKPC-CRKP-Urine1 and pqnrS1-CRKP-Urine1). The gene mutations responsible for tigecycline or colistin resistance were then amplified with PCR followed by sequencing, which indicated that mutations of ramR and lon were the potential loci for tigecycline resistance and that the pmrB, phoQ and mgrB genes for colistin resistance. A novel frameshift mutation of lon was identified in the high-level tigecycline-resistant strain (MIC, 128 mg/L). The results indicate that the hypervirulent ST11-KL64 clone is a potential threat to antiinfection treatment and is capable of rapid and diverse evolution of resistance during tigecycline and polymyxin treatment.

Genomic Characteristics of Colistin-Resistant Salmonella enterica subsp. enterica Serovar Infantis from Poultry Farms in the Republic of Serbia.

The antimicrobial susceptibility testing was conducted on 174 single isolates from poultry farms in Serbia and it was determined that seven Salmonella spp. were multidrug resistant. Sixteen serotypes were detected, but only serotype Infantis confirmed reduced susceptibility to colistin. Seven colistin resistant Salmonella Infantis were studied in detail using the WGS approach. Three sequence types were identified corresponding to different epizootiology region. The isolate from the Province of Vojvodina 3842 and isolates from Jagodina (92 and 821) are represented by the sequence type ST413 and ST11, respectively. Four isolates from Kraljevo are ST32, a common S. Infantis sequence type in humans, poultry and food. The fosfomycin resistance gene fosA7 in isolate 3842 and the vgaA gene in isolate 8418/2948 encoding resistance to pleuromutilins were reported for the first time in serovar Infantis. The changes in relative expression of the phoP/Q, mgrB and pmrA/B genes were detected. Single nucleotide polymorphisms of the pmrB gene, including transitions Val164Gly or Val164Met, and Arg92Pro are described. Analyses of quinolone resistance determining region revealed substitutions Ser83Tyr in GyrA protein and Thr57Ser and Ser80Arg in ParC protein. Based on WGS data, there are two major clusters among analyzed Salmonella Infantis isolates from central Serbia.

Molecular and genome characterization of colistin-resistant Escherichia coli isolates from wild sea lions (Zalophus californianus).

Using molecular and whole-genome sequencing tools, we investigated colistin-resistant Escherichia coli isolates from wild sea lions. Two unrelated E. coli colistin-resistant isolates, ST8259 and ST4218, were identified, both belonging to the B2 phylogroup and different serotypes. Polymorphisms in PmrA, PmrB, and PhoQ proteins were identified, and the role of PmrB and PhoQ in contributing to colistin resistance was determined by complementation assays. However, the mutations characterized in the present study are not involved in colistin resistance, which have been described in E. coli isolates from clinical settings. Therefore, the acquired mutations in pmrB and phoQ genes in resistance to colistin in bacteria related to marine environment animals are different. This work contributes to the surveillance and characterization of colistin resistance in Escherichia coli obtained from animals from aquatic environments.

Survey on Genetic Diversity, Biofilm Formation, and Detection of Colistin Resistance Genes in Clinical Isolates of Acinetobacter baumannii.

IntroductionAcinetobacter baumannii is an opportunistic pathogen responsible for nosocomial infections. The emergence of colistin-resistant A. baumannii is a significant threat to public health. The aim of this study was to investigate the molecular characterization and genotyping of clinical A. baumannii isolates in Southwestern Iran.MethodsA total of 70 A. baumannii isolates were collected from patients admitted to Imam Khomeini Hospital in Ahvaz, Southwestern Iran. Minimum inhibitory concentration test was conducted by using Vitek 2 system. The presence of biofilm-forming genes and colistin resistance-related genes were evaluated by PCR. The isolates were also examined for their biofilm formation ability and the expression of pmrA and pmrB genes. Finally, multilocus sequence typing (MLST) and PCR-based sequence group were used to determine the genetic relationships of the isolates.ResultsOverall, 61 (87.1%) and 9 (12.8%) isolates were multidrug-resistant (MDR) and extensively drug-resistant (XDR), respectively. Colistin and tigecycline with 2 (2.8%) and 32 (45.7%) resistance rates had the highest effect. Among all the isolates, 55 (78.5%), 7 (10%), and 3 (4.3%) were strong, moderate, and weak biofilm producers, respectively. The frequency rates of biofilm-related genes were 64 (91.4%), 70 (100%), 56 (80%), and 22 (31.42%) for bap, ompA, csuE, and blaPER1, respectively. Overexpression of pmrA and pmrB genes was observed in two colistin-resistance isolates, but the expression of these genes did not change in colistin-sensitive isolates. Additionally, 37 (52.8%) and 8 (11.4%) isolates belonged to groups 1 (ICII) and 2 (IC I), respectively. MLST analysis revealed a total of nine different sequence types that six isolates belonged to clonal complex 92 (corresponding to ST801, ST118, ST138, ST 421, and ST735). Other isolates were belonging to ST133 and ST216, and two colistin-resistant (Ab4 and Ab41) isolates were belonging to ST387 and ST1812.ConclusionThe present study revealed the presence of MDR and XDR A. baumannii isolates harboring biofilm genes and emergence of colistin-resistant isolates in Southwestern Iran. These isolates had high diversity, which was affirmed by typing techniques. The control measures and regular surveillance are urgently needed to preclude the spread of these isolates.

In Vivo Development of Polymyxin B Resistance in Klebsiella pneumoniae owing to a 42 bp Deletion in the Sequence of phoQ.

Polymyxins resistance has emerged worldwide and is threatening the treatment efficacy of multidrug resistant Klebsiella pneumoniae in humans and animals. In this research, we employed whole-genome sequencing (WGS) to investigate the polymyxin B resistance mechanism in selected polymyxin B-susceptible and polymyxin B-resistant K. pneumoniae, isolated from one patient of Huashan Hospital affiliated to Fudan University. The WGS results showed that the two K. pneumoniae all belong to ST11. The average nucleotide identity between the two K. pneumoniae was nearly 100%. No sense mutations of polymyxins resistance associated genes (pmrA, pmrB, phoP, mgrB) were observed in polymyxin B-resistant K. pneumonia (PRKP) compared to the polymyxin B-susceptible isolate. A 42 bp deletion was found in the sequence of phoQ in PRKP. The deletion of amino acid occurred on the periplasmic domain of PhoQ protein. We speculate that this is the domain that MgrB protein interact with the PhoQ protein and negatively regulate the PhoP/PhoQ system. qRT-PCR analysis revealed an overexpression of the pmrA (6.8-fold), pmrB (151.9-fold), pmrC (14.5-fold), pmrK (287.9-fold), phoP (14.5-fold), and phoQ (16.8-fold) genes in the polymyxin B-resistant isolate compared to the expression of the polymyxin B-susceptible K. pneumoniae isolate, suggesting that the phoQ deletion maybe responsible for the increased expression levels of those genes. In conclusion, this study identified a 42 bp deletion in the sequence of phoQ as being responsible for the overexpression of pmrCAB and pmrHFIJKLM operons, leading to resistance to polymyxin B.

Analysis of the Extracellular Proteome of Colistin-Resistant Korean Acinetobacter baumannii Strains.

We analyzed the extracellular proteome of colistin-resistant Korean Acinetobacter baumannii (KAB) strains to identify proteome profiles that can be used to characterize extensively drug-resistant KAB strains. Four colistin-resistant KAB strains with colistin resistance associated with point mutations in pmrB and pmrC genes were analyzed. Analysis of the extracellular proteome of these strains revealed the presence of 506 induced common proteins, which were hence considered as the core extracellular proteome. Class C ADC-30 and class D OXA-23 β-lactamases were abundantly induced in these strains. Porins (CarO and CarO-like porin), outer membrane proteins (OmpH and BamABDE), transport protein (AdeK), receptor (TonB), and several proteins of unknown function were among the specifically induced proteins. Based on the sequence homology analysis of proteins from the core proteome and those of other A. baumannii strains and pathogenic bacterial species as well as further in silico screening, we propose that CarO-like porin is an A. baumannii-specific protein and that two tryptic peptides that originate from CarO-like porin detected by tandem mass spectrometry are peptide makers of this protein.