Emergence of Carbapenem Resistance Due to the Novel Insertion Sequence IS<i>Pa</i>8 in <i>Pseudomonas aeruginosa</i>

<div><p>Chronic lung infections due to the persistence of <i>Pseudomonas aeruginosa</i> in cystic fibrosis patients are typically associated with the emergence of antibiotic resistance. The purpose of this study was to investigate the mechanisms responsible for the emergence of carbapenem resistance when a clinical isolate of <i>P. aeruginosa</i> collected from a patient with cystic fibrosis was challenged with meropenem. Nine carbapenem-resistant mutants were selected with subinhibitory concentrations of meropenem from a clinical isolate of <i>P. aeruginosa</i> and characterized for carbapenem resistance. Increased carbapenem MICs were associated with the identification of the novel insertion sequence IS<i>Pa</i>8 within <i>oprD</i> or its promoter region in all the mutants. The position of IS<i>Pa</i>8 was different for each of the mutants evaluated. In addition, Southern blot analyses identified multiple copies of IS<i>Pa</i>8 within the genomes of the mutants and their parent isolate. These data demonstrate that transposition of IS elements within the <i>Pseudomonas</i> genome can influence antibiotic susceptibility. Understanding the selective pressures associated with the emergence of antibiotic resistance is critical for the judicious use of antimicrobial chemotherapy and the successful treatment of bacterial infections.</p></div

Carbapenem susceptibility and gene expression data for <i>P. aeruginosa</i> isolates.

a<p>WT, wild-type isolate; CF, clinical isolate from cystic fibrosis patient; P, parent isolate; M, mutant of PA42.</p>b<p>Subinhibitory concentration (µg/mL) of meropenem used for mutant selection.</p>c<p>IPM, imipenem; MEM, meropenem; DOR, doripenem.</p

Outer membrane analysis of <i>P. aeruginosa</i> PA42 and nine isogenic mutants.

<p>Outer membrane profiles were analyzed for the presence of the porin, OprD. PAO1 and a fully susceptible cystic fibrosis isolate, PA443, were included as positive controls. The locations of OprD and OprF proteins are indicated.</p

PCR amplification of the <i>oprF</i> gene in PAO1, PA42, and nine isogenic mutants.

<p>The expected PCR product size is 1,672(1 kb DNA ladder, Invitrogen), no template control (NTC), or <i>P. aeruginosa</i> isolates.</p

PCR amplification of <i>oprD</i> and IS<i>Pa</i>8 from carbapenem-resistant mutants of <i>P. aeruginosa</i>.

<p>(A) Primers that flanked the <i>oprD</i> gene were used to PCR-amplify the <i>oprD</i> gene giving an expected PCR product size is 1586 bp. Each lane is labeled with its respective DNA ladder (1 kb DNA ladder, Invitrogen), no template control (NTC), or <i>P. aeruginosa</i> isolate. (B) PCR amplification of IS<i>Pa</i>8 within the <i>oprD</i> gene in the nine carbapenem-resistant mutants. Primers ISPa8F1 and OprDRTR3 were used to map the approximate location of the IS<i>Pa</i>8 within the <i>oprD</i> gene. The smaller PCR products indicate IS<i>Pa</i>8 has inserted near the 3′ end of <i>oprD</i>, while larger PCR products indicate IS<i>Pa</i>8 is inserted near the 5′ end. No PCR product was observed for mutant 711M. (C) Primers OprDRTF2 and ISPa8R2 were used to confirm the location of IS<i>Pa</i>8 within the <i>oprD</i> gene in the nine isogenic mutants. Non-specific bands were amplified in PA42, mutant 711M, and mutant 922M suggesting that multiple IS<i>Pa</i>8 elements may be present within the genome. To confirm that IS<i>Pa</i>8 had inserted within the <i>oprD</i> gene or its flanking regions in mutants 711M and 922M, PCR products for these mutants shown in (A) were sequenced using primers OprDRTF2 or ISPa8F1.</p

Pulsed Field Gel Electrophoresis and Southern blot analyses using IS<i>Pa</i>8-specific probe.

<p>(A) PFGE gel of <i>Spe</i>I chromosomal digests of wild-type strain PAO1, parent isolate PA42, and mutant 812M visualized using SYBR gold. (B) Southern blot of the gel depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091299#pone-0091299-g005" target="_blank">Figure 5(A)</a> using an IS<i>Pa</i>8-specific probe. Lane 1, PAO1 (negative control); lane 2, parent isolate PA42; lane 3, mutant 812M.</p

Nontyphoidal Salmonella enterica Nonsusceptible to Both Levofloxacin and Ceftriaxone in Nebraska, United States 2014-2015.

Nontyphoidal Salmonella enterica is a common cause of illness in humans ranging from gastroenteritis to invasive disease. National surveillance programs continually monitor trends in antimicrobial resistance patterns and mechanisms of resistance to identify emerging public health threats. Our study shows the emergence of nonsusceptibility to both levofloxacin and ceftriaxone, a concerning phenotype that threatens first-line antibiotic therapy, in Salmonella isolates recovered between 2014 and 2015. From 2010 to 2013 the rate of resistance increased from 0.0% (0/1181) to 1.5% (9/593) in 2014 and 2015. The isolates with this phenotype were found to be from multiple serotypes, including Typhimurium, Newport, and Enteritidis. Resistance to ceftriaxone was attributed to the presence of either an AmpC or extended-spectrum β-lactamase, and resistance to fluoroquinolones was attributable to the presence of mutations in the quinolone resistance-determining region or the presence of plasmid-mediated quinolone resistance genes. As this resistance pattern was seen in a variety of Salmonella serotypes harboring varied resistance mechanisms, it indicates a worrying trend in the spread of isolates resistant to both first-line treatment options

Evaluation of CTX-M steady-state mRNA, mRNA half-life and protein production in various STs of Escherichia coli.

OBJECTIVES High levels of β-lactamase production can impact treatment with a β-lactam/β-lactamase inhibitor combination. Goals of this study were to: (i) compare the mRNA and protein levels of CTX-M-15- and CTX-M-14-producing Escherichia coli from 18 different STs and 10 different phylotypes; (ii) evaluate the mRNA half-lives and establish a role for chromosomal- and/or plasmid-encoded factors; and (iii) evaluate the zones of inhibition for piperacillin/tazobactam and ceftolozane/tazobactam. METHODS Disc diffusion was used to establish zone size. RNA analysis was accomplished using real-time RT-PCR and CTX-M protein levels were evaluated by immunoblotting. Clinical isolates, transformants and transconjugants were used to evaluate mRNA half-lives. RESULTS mRNA levels of CTX-M-15 were up to 165-fold higher compared with CTX-M-14. CTX-M-15 protein levels were 2-48-fold less than their respective transcript levels, while CTX-M-14 protein production was comparable to the observed transcript levels. Nineteen of 25 E. coli (76%) had extended CTX-M-15 mRNA half-lives of 5-15 min and 16 (100%) CTX-M-14 isolates had mRNA half-lives of <2-3 min. Transformants had mRNA half-lives of <2 min for both CTX-M-type transcripts, while transconjugant mRNA half-lives corresponded to the half-life of the donor. Ceftolozane/tazobactam zone sizes were ≥19 mm, while piperacillin/tazobactam zone sizes were ≥17 mm. CONCLUSIONS CTX-M-15 mRNA and protein production did not correlate. Neither E. coli ST nor phylotype influenced the variability observed for CTX-M-15 mRNA or protein produced. mRNA half-life is controlled by a plasmid-encoded factor and may influence mRNA transcript levels, but not protein levels

Integrative Biology: The challenges of developing a collaborative research environment for heart and cancer modelling

The Integrative Biology project is building a Grid infrastructure which will enable biomedical scientists to develop accurate multi-scale computational models of the heart and of cancer tumours. This infrastructure will remove many of the constraints limiting research progress today by providing access to leading-edge supercomputing and data management resources and by improving facilities to support collaboratio

Integrative Biology - The Challenges of Developing a Collaborative Research Environment for Heart and Cancer Modelling

The Integrative Biology project is building a Grid infrastructure which will enable biomedical scientists to develop accurate multi-scale computational models of the heart and of cancer tumours. This infrastructure will remove many of the constraints limiting research progress today by providing access to leading-edge supercomputing and data management resources and by improving facilities to support collaboratio