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Detection of point mutations associated with antibiotic resistance in Pseudomonas aeruginosa.

ABSTRACT: Excessive use of broad-spectrum antibiotics in hospitals has led to the emergence of highly resistant strains of Pseudomonas aeruginosa. To reduce the selection pressure for resistance, it is important to determine the antibiotic susceptibility pattern of bacteria so that hospital patients can be treated with more narrow-spectrum and target-specific antibiotics. This study describes the development of a technique for detecting point muations in the fluoroquinolone resistance-determining region of the gyrA and parC genes as well as the efflux regulatory genes mexR, mexZ and mexOZ that are associated with fluoroquinolone and aminoglycoside resistance. The assay is based on a short DNA sequencing method using multiplex-fast polymerase chain reaction (PCR) and Pyrosequencing for amplification and sequencing of the selected genes. Fifty-nine clinical isolates of P. aeruginosa were examined for mutations in the abovementioned genes. Mutations related to antibiotic resistance were detected in codons 83 and 87 of gyrA and codon 126 of the mexR regulatory gene. Results of this study suggest Pyrosequencing as a substitute for traditional methods as it provides a rapid and reliable technique for determining the antibiotic resistance pattern of a given bacterial strain in <1 h.

SUBMITTER: Gorgani N

PROVIDER: S-EPMC2744841 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Detection of point mutations associated with antibiotic resistance in Pseudomonas aeruginosa.

Gorgani Neda N Ahlbrand Scott S Patterson Andrew A Pourmand Nader N

International journal of antimicrobial agents 20090804 5

Excessive use of broad-spectrum antibiotics in hospitals has led to the emergence of highly resistant strains of Pseudomonas aeruginosa. To reduce the selection pressure for resistance, it is important to determine the antibiotic susceptibility pattern of bacteria so that hospital patients can be treated with more narrow-spectrum and target-specific antibiotics. This study describes the development of a technique for detecting point muations in the fluoroquinolone resistance-determining region o ...[more]

PMID: 19656662

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Project description:BackgroundPseudomonas aeruginosa is a ubiquitous environmental microorganism and also a common cause of infection. Its ability to survive in many different environments and persistently colonize humans is linked to its presence in biofilms formed on indwelling device surfaces. Biofilm promotes adhesion to, and survival on surfaces, protects from desiccation and the actions of antibiotics and disinfectants.ResultsWe examined the genetic basis for biofilm production on polystyrene at room (22 °C) and body temperature (37 °C) within 280 P. aeruginosa. 193 isolates (69 %) produced more biofilm at 22 °C than at 37 °C. Using GWAS and pan-GWAS, we found a number of accessory genes significantly associated with greater biofilm production at 22 °C. Many of these are present on a 165 kb region containing genes for heavy metal resistance (arsenic, copper, mercury and cadmium), transcriptional regulators and methytransferases. We also discovered multiple core genome SNPs in the A-type flagellin gene and Type II secretion system gene xpsD. Analysis of biofilm production of isolates of the MDR ST111 and ST235 lineages on stainless-steel revealed several accessory genes associated with enhanced biofilm production. These include a putative translocase with homology to a Helicobacter pylori type IV secretion system protein, a TA system II toxin gene and the alginate biosynthesis gene algA, several transcriptional regulators and methytransferases as well as core SNPs in genes involved in quorum sensing and protein translocation.ConclusionsUsing genetic association approaches we discovered a number of accessory genes and core-genome SNPs that were associated with enhanced early biofilm formation at 22 °C compared to 37 °C. These included a 165 kb genomic island containing multiple heavy metal resistance genes, transcriptional regulators and methyltransferases. We hypothesize that this genomic island may be associated with overall genotypes that are environmentally adapted to survive at lower temperatures. Further work to examine their importance in, for example gene-knockout studies, are required to confirm their relevance. GWAS and pan-GWAS approaches have great potential as a first step in examining the genetic basis of novel bacterial phenotypes.

Project description:UnlabelledElongation factor P (EF-P) is a ubiquitous bacterial protein that is required for the synthesis of poly-proline motifs during translation. In Escherichia coli and Salmonella enterica, the posttranslational ?-lysylation of Lys34 by the PoxA protein is critical for EF-P activity. PoxA is absent from many bacterial species such as Pseudomonas aeruginosa, prompting a search for alternative EF-P posttranslation modification pathways. Structural analyses of P. aeruginosa EF-P revealed the attachment of a single cyclic rhamnose moiety to an Arg residue at a position equivalent to that at which ?-Lys is attached to E. coli EF-P. Analysis of the genomes of organisms that both lack poxA and encode an Arg32-containing EF-P revealed a highly conserved glycosyltransferase (EarP) encoded at a position adjacent to efp. EF-P proteins isolated from P. aeruginosa ?earP, or from a ?rmlC::acc1 strain deficient in dTDP-L-rhamnose biosynthesis, were unmodified. In vitro assays confirmed the ability of EarP to use dTDP-L-rhamnose as a substrate for the posttranslational glycosylation of EF-P. The role of rhamnosylated EF-P in translational control was investigated in P. aeruginosa using a Pro4-green fluorescent protein (Pro4GFP) in vivo reporter assay, and the fluorescence was significantly reduced in ?efp, ?earP, and ?rmlC::acc1 strains. ?rmlC::acc1, ?earP, and ?efp strains also displayed significant increases in their sensitivities to a range of antibiotics, including ertapenem, polymyxin B, cefotaxim, and piperacillin. Taken together, our findings indicate that posttranslational rhamnosylation of EF-P plays a key role in P. aeruginosa gene expression and survival.ImportanceInfections with pathogenic Salmonella, E. coli, and Pseudomonas isolates can all lead to infectious disease with potentially fatal sequelae. EF-P proteins contribute to the pathogenicity of the causative agents of these and other diseases by controlling the translation of proteins critical for modulating antibiotic resistance, motility, and other traits that play key roles in establishing virulence. In Salmonella spp. and E. coli, the attachment of ?-Lys is required for EF-P activity, but the proteins required for this posttranslational modification pathway are absent from many organisms. Instead, bacteria such as P. aeruginosa activate EF-P by posttranslational modification with rhamnose, revealing a new role for protein glycosylation that may also prove useful as a target for the development of novel antibiotics.

Dataset Information

Detection of point mutations associated with antibiotic resistance in Pseudomonas aeruginosa.

Publications

Detection of point mutations associated with antibiotic resistance in Pseudomonas aeruginosa.

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