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Genomic and transcriptomic analyses of colistin-resistant clinical isolates of Klebsiella pneumoniae reveal multiple pathways of resistance.

ABSTRACT: The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae has resulted in a more frequent reliance on treatment using colistin. However, resistance to colistin (Col(r)) is increasingly reported from clinical settings. The genetic mechanisms that lead to Col(r) in K. pneumoniae are not fully characterized. Using a combination of genome sequencing and transcriptional profiling by RNA sequencing (RNA-Seq) analysis, distinct genetic mechanisms were found among nine Col(r) clinical isolates. Col(r) was related to mutations in three different genes in K. pneumoniae strains, with distinct impacts on gene expression. Upregulation of the pmrH operon encoding 4-amino-4-deoxy-L-arabinose (Ara4N) modification of lipid A was found in all Col(r) strains. Alteration of the mgrB gene was observed in six strains. One strain had a mutation in phoQ. Common among these seven strains was elevated expression of phoPQ and unaltered expression of pmrCAB, which is involved in phosphoethanolamine addition to lipopolysaccharide (LPS). In two strains, separate mutations were found in a previously uncharacterized histidine kinase gene that is part of a two-component regulatory system (TCRS) now designated crrAB. In these strains, expression of pmrCAB, crrAB, and an adjacent glycosyltransferase gene, but not that of phoPQ, was elevated. Complementation with the wild-type allele restored colistin susceptibility in both strains. The crrAB genes are present in most K. pneumoniae genomes, but not in Escherichia coli. Additional upregulated genes in all strains include those involved in cation transport and maintenance of membrane integrity. Because the crrAB genes are present in only some strains, Col(r) mechanisms may be dependent on the genetic background.

SUBMITTER: Wright MS

PROVIDER: S-EPMC4291396 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Genomic and transcriptomic analyses of colistin-resistant clinical isolates of Klebsiella pneumoniae reveal multiple pathways of resistance.

Wright Meredith S MS Suzuki Yo Y Jones Marcus B MB Marshall Steven H SH Rudin Susan D SD van Duin David D Kaye Keith K Jacobs Michael R MR Bonomo Robert A RA Adams Mark D MD

Antimicrobial agents and chemotherapy 20141110 1

The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae has resulted in a more frequent reliance on treatment using colistin. However, resistance to colistin (Col(r)) is increasingly reported from clinical settings. The genetic mechanisms that lead to Col(r) in K. pneumoniae are not fully characterized. Using a combination of genome sequencing and transcriptional profiling by RNA sequencing (RNA-Seq) analysis, distinct genetic mechanisms were found among nine Col(r) clinical isolates. C ...[more]

PMID: 25385117

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Project description:The emergence and dissemination of carbapenemases, bacterial enzymes able to inactivate most β-lactam antibiotics, in Enterobacteriaceae is of increasing concern. The concurrent spread of resistance against colistin, an antibiotic of last resort, further compounds this challenge further. Whole-genome sequencing (WGS) can play a significant role in the rapid and accurate detection/characterization of existing and emergent resistance determinants, an essential aspect of public health surveillance and response activities to combat the spread of antimicrobial resistant bacteria. In the current study, WGS data was used to characterize the genomic content of antimicrobial resistance genes, including those encoding carbapenemases, in 10 multidrug-resistant Klebsiella pneumoniae isolates from Pakistan. These clinical isolates represented five sequence types: ST11 (n = 3 isolates), ST14 (n = 3), ST15 (n = 1), ST101 (n = 2), and ST307 (n = 1). Resistance profiles against 25 clinically-relevant antimicrobials were determined by broth microdilution; resistant phenotypes were observed for at least 15 of the 25 antibiotics tested in all isolates except one. Specifically, 8/10 isolates were carbapenem-resistant and 7/10 isolates were colistin-resistant. The blaNDM-1 and blaOXA-48 carbapenemase genes were present in 7/10 and 5/10 isolates, respectively; including 2 isolates carrying both genes. No plasmid-mediated determinants for colistin resistance (e.g. mcr) were detected, but disruptions and mutations in chromosomal loci (i.e. mgrB and pmrB) previously reported to confer colistin resistance were observed. A blaOXA-48-carrying IncL/M-type plasmid was found in all blaOXA-48-positive isolates. The application of WGS to molecular epidemiology and surveillance studies, as exemplified here, will provide both a more complete understanding of the global distribution of MDR isolates and a robust surveillance tool useful for detecting emerging threats to public health.

Project description:PurposeCarbapenemases-producing Klebsiella pneumoniae are challenging antimicrobial therapy of hospitalised patients, which is further complicated by colistin resistance. This study describes molecular epidemiological insights into colistin-resistant and carbapenemases-producing clinical K. pneumoniae.Patients and methodsCultures collected from 26 hospitalised patients during 2014-2017 in the main hospital in Molise Region, central Italy, were characterized. The minimum inhibitory concentration for 19 antibiotics was determined, including carbapenems and colistin. Prevalence of resistance-associated genes was investigated through PCR, detecting bla KPC, bla GES, bla VIM, bla IMP, bla NDM, bla OXA-48, bla CTX-M, bla TEM, bla SHV, and mcr-1,2,3,4,5,6,7,8. The mgrB gene was also analysed in colistin-resistant strains by PCR and sequencing assays. K. pneumoniae were typed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).ResultsTwenty out of 26 K. pneumoniae were phenotypically resistant to carbapenems and 19 were resistant to colistin. All isolates harbored bla KPC, and bla SHV, bla TEM and bla VIM were further the most common resistance-associated genes. In colistin-resistant strains, mcr-1,2,3,4,5,6,7,8 variants were not detected, while mutations and insertion elements in mgrB were observed in 68.4% (n=13) in 31.6% (n=6) isolates, respectively. PFGE revealed 12 clusters and 18 pulsotypes at 85% and 95% cut-off, while the Sequence Types ST512 (n=13, 50%), ST101 (n=10, 38.5%), ST307 (n=2, 7.7%) plus a novel ST were detected using MLST.ConclusionAll K. pneumoniae showed a multidrug-resistant phenotype, particularly to carbapenems and colistin. According to national data, bla KPC was the prevailing carbapenemase, followed by bla VIM, while bla TEM and bla SHV were among the most frequent beta-lactamases. Consistent with previous reports in Italy, ST512 was the most common clone, particularly during 2014-15, whilst ST101 became dominant in 2016-17. Colistin resistance was mainly associated with deleterious mutations and transposon in the mgrB gene. Improvements of surveillance, compliance with infection prevention procedures and antimicrobial stewardship are essential to limit the spread of resistant K. pneumoniae.

Project description: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.

Dataset Information

Genomic and transcriptomic analyses of colistin-resistant clinical isolates of Klebsiella pneumoniae reveal multiple pathways of resistance.

Publications

Genomic and transcriptomic analyses of colistin-resistant clinical isolates of Klebsiella pneumoniae reveal multiple pathways of resistance.

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