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Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae.

ABSTRACT: Tigecycline resistance has been attributed to ramA overexpression and subsequent acrA upregulation. The ramA locus, originally identified in Klebsiella pneumoniae, has homologues in Enterobacter and Salmonella spp. In this study, we identify in silico that the ramR binding site is also present in Citrobacter spp. and that Enterobacter, Citrobacter and Klebsiella spp. share key regulatory elements in the control of the romA-ramA locus. RACE (rapid amplification of cDNA ends) mapping indicated that there are two promoters from which romA-ramA expression can be regulated in K. pneumoniae. Correspondingly, electrophoretic binding studies clearly showed that purified RamA and RamR proteins bind to both of these promoters. Hence, there appear to be two RamR binding sites within the Klebsiella romA-ramA locus. Like MarA, RamA binds the promoter region, implying that it might be subject to autoregulation. We have identified changes within ramR in geographically distinct clinical isolates of K. pneumoniae. Intriguingly, levels of romA and ramA expression were not uniformly affected by changes within the ramR gene, thereby supporting the dual promoter finding. Furthermore, a subset of strains sustained no changes within the ramR gene but which still overexpressed the romA-ramA genes, strongly suggesting that a secondary regulator may control ramA expression.

SUBMITTER: Rosenblum R

PROVIDER: S-EPMC3117140 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae.

Rosenblum R R Khan E E Gonzalez G G Hasan R R Schneiders T T

International journal of antimicrobial agents 20110422 1

Tigecycline resistance has been attributed to ramA overexpression and subsequent acrA upregulation. The ramA locus, originally identified in Klebsiella pneumoniae, has homologues in Enterobacter and Salmonella spp. In this study, we identify in silico that the ramR binding site is also present in Citrobacter spp. and that Enterobacter, Citrobacter and Klebsiella spp. share key regulatory elements in the control of the romA-ramA locus. RACE (rapid amplification of cDNA ends) mapping indicated tha ...[more]

PMID: 21514798

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Project description:Klebsiella pneumoniae is a major threat to public health, causing significant morbidity and mortality worldwide. The emergence of highly drug-resistant strains is particularly concerning. There has been a recognition and division of Klebsiella pneumoniae into three distinct phylogenetic groups: Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae. K. variicola and K. quasipneumoniae have often been described as opportunistic pathogens that have less virulence in humans than K. pneumoniae does. We recently sequenced the genomes of 1,777 extended-spectrum-beta-lactamase (ESBL)-producing K. pneumoniae isolates recovered from human infections and discovered that 28 strains were phylogenetically related to K. variicola and K. quasipneumoniae. Whole-genome sequencing of 95 additional non-ESBL-producing K. pneumoniae isolates recovered from patients found 12 K. quasipneumoniae strains. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis initially identified all patient isolates as K. pneumoniae, suggesting a potential pitfall in conventional clinical microbiology laboratory identification methods. Whole-genome sequence analysis revealed extensive sharing of core gene content and plasmid replicons among the Klebsiella species. For the first time, strains of both K. variicola and K. quasipneumoniae were found to carry the Klebsiella pneumoniae carbapenemase (KPC) gene, while another K. variicola strain was found to carry the New Delhi metallo-beta-lactamase 1 (NDM-1) gene. K. variicola and K. quasipneumoniae infections were not less virulent than K. pneumoniae infections, as assessed by in-hospital mortality and infection type. We also discovered evidence of homologous recombination in one K. variicola strain, as well as one strain from a novel Klebsiella species, which challenge the current understanding of interrelationships between clades of Klebsiella. IMPORTANCEKlebsiella pneumoniae is a serious human pathogen associated with resistance to multiple antibiotics and high mortality. K. variicola and K. quasipneumoniae are closely related organisms that are generally considered to be less-virulent opportunistic pathogens. We used a large, comprehensive, population-based strain collection and whole-genome sequencing to investigate infections caused by these organisms in our hospital system. We discovered that K. variicola and K. quasipneumoniae isolates are often misidentified as K. pneumoniae by routine clinical microbiology diagnostics and frequently cause severe life-threatening infections similar to K. pneumoniae. The presence of KPC in K. variicola and K. quasipneumoniae strains as well as NDM-1 metallo-beta-lactamase in one K. variicola strain is particularly concerning because these genes confer resistance to many different beta-lactam antibiotics. The sharing of plasmids, as well as evidence of homologous recombination, between these three species of Klebsiella is cause for additional concern.

Project description:The emergence and dissemination of carbapenemases, bacterial enzymes able to inactivate most ?-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.

Dataset Information

Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae.

Publications

Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae.

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