oprD Mutations Research Articles

Pseudomonas aeruginosa carbapenem resistance mechanisms in Spain: impact on the activity of imipenem, meropenem and doripenem

To investigate the mechanisms of carbapenem resistance in the 175 Pseudomonas aeruginosa isolates (39%; 175/448) showing non-susceptibility (European Committee on Antimicrobial Susceptibility Testing breakpoints) to imipenem (35%), meropenem (33%) and/or doripenem (33%) recovered in 2008-09 from 16 Spanish hospitals during the Comparative Activity of Carbapenem Testing (COMPACT) surveillance study. MICs (Etest), clonal relatedness (PFGE) and metallo-β-lactamase (MBL) production (Etest-MBL, PCR and sequencing) were determined. Mutation-driven resistance was studied in 60 non-MBL producers according to the doripenem MICs (15 isolates from each of four MIC groups: ≤ 1, 2-4, 8-16 and ≥ 32 mg/L). The expression of ampC, mexB, mexY, mexD and mexF was determined by real-time reverse transcription-PCR and the presence of mutations in oprD by PCR and sequencing. Isogenic mutants expressing combinations of mutation-driven carbapenem resistance were constructed. Twelve (6.9%) isolates were MBL (VIM-20, VIM-2 or VIM-13) producers and all showed high-level resistance (MIC 32 mg/L) to all three carbapenems. Regarding mutation-driven resistance, all but 1 of the 60 isolates were non-susceptible (MIC >32 mg/L) to imipenem, linked to oprD inactivation. In addition, 50% of the isolates overexpressed ampC, 33% mexY, 32% mexB and 15% mexF, while none overexpressed mexD. Increasing prevalence of ampC overexpression correlated with increasing doripenem MICs (≤ 1, 13%; 2-4, 53%; 8-16, 60%; and ≥ 32, 73%) while overexpression of efflux pumps correlated only with moderate resistance. Doripenem showed slightly higher activity than meropenem against isolates overexpressing ampC, especially mexB or mexY. The analysis of a collection of isogenic laboratory mutants supported this finding. Although the prevalence of MBL producers is increasing, mutation-driven resistance is still more frequent in Spain. Imipenem resistance was driven by OprD inactivation, while additional AmpC and particularly efflux pump hyperproduction had a lower impact on the activity of doripenem compared with meropenem.

Overexpression of AmpC and Efflux Pumps in Pseudomonas aeruginosa Isolates from Bloodstream Infections: Prevalence and Impact on Resistance in a Spanish Multicenter Study

The prevalence and impact of the overexpression of AmpC and efflux pumps were evaluated with a collection of 190 Pseudomonas aeruginosa isolates recovered from bloodstream infections in a 2008 multicenter study (10 hospitals) in Spain. The MICs of a panel of 13 antipseudomonal agents were determined by microdilution, and the expressions of ampC, mexB, mexY, mexD, and mexF were determined by real-time reverse transcription (RT)-PCR. Up to 39% of the isolates overexpressed at least one of the mechanisms. ampC overexpression (24.2%) was the most prevalent mechanism, followed by mexY (13.2%), mexB (12.6%), mexF (4.2%), and mexD (2.2%). The overexpression of mexB plus mexY, documented for 5.3% of the isolates, was the only combination showing a significantly (P=0.02) higher prevalence than expected from the frequencies of the individual mechanisms (1.6%). Additionally, all imipenem-resistant isolates studied (25 representative isolates) showed inactivating mutations in oprD. Most of the isolates nonsusceptible to piperacillin-tazobactam (96%) and ceftazidime (84%) overexpressed ampC, while mexB (25%) and mexY (29%) overexpressions gained relevance among cefepime-nonsusceptible isolates. Nevertheless, the prevalence of mexY overexpression was highest among tobramycin-nonsusceptible isolates (37%), and that of mexB was highest among meropenem-nonsusceptible isolates (33%). Regarding ciprofloxacin-resistant isolates, besides the expected increased prevalence of efflux pump overexpression, a highly significant link to ampC overexpression was documented for the first time: up to 52% of ciprofloxacin-nonsusceptible isolates overexpressed ampC, sharply contrasting with the 24% documented for the complete collection (P<0.001). In summary, mutation-driven resistance was frequent in P. aeruginosa isolates from bloodstream infections, whereas metallo-β-lactamases, detected in 2 isolates (1%) producing VIM-2, although with increasing prevalences, were still uncommon.

Carbapenem resistance mechanisms in Pseudomonas aeruginosa: alterations of porin OprD and efflux proteins do not fully explain resistance patterns observed in clinical isolates

Imipenem resistance in Pseudomonas aeruginosa is considered to be associated with loss of the porin OprD combined with activity of chromosomal beta-lactamase (AmpC), while overexpression of multidrug efflux pumps is considered to confer meropenem resistance. Carbapenem resistance can also result from production of metallo-beta-lactamases. Transcription of oprD and efflux pump genes mexB, mexY and mexF was analysed in 23 clinical isolates of P. aeruginosa by quantitative RT-PCR. oprD was sequenced in all, and mexR, regulator of efflux pump MexAB-OprM, in selected isolates. Four isolates that were imipenem susceptible had significant reduction of oprD mRNA and presence of oprD mutations causing frameshift or translational stop. In strains only resistant to imipenem no significant difference in transcription of oprD was observed between low-level and high-level resistant isolates. The differences could not be explained by either pattern of oprD mutations. Increased transcription of mexB generally correlated well with meropenem resistance. One high-level meropenem-resistant isolate showed no significant change in mexB mRNA, but sequencing confirmed presence of a nalB mutation. Furthermore, one meropenem-susceptible isolate showed significant increase in mexB transcription, but no mexR mutations. In summary, our findings indicate that the resistance patterns observed cannot be fully explained by the currently described carbapenem resistance mechanisms.

Effect of siliconized latex urinary catheters on the activity of carbapenems against Pseudomonas aeruginosa strains with defined mutations in ampC, oprD, and genes coding for efflux systems

The decreased activity of carbapenems against Pseudomonas aeruginosa in the presence of eluates from siliconized latex urinary catheters is related to the loss of OprD and the expression of a new outer membrane protein (OMP). To understand this phenomenon, the activity of carbapenems and quinolones against P. aeruginosa strains, some producing AmpC β-lactamase (BL), OprD, MexA–MexB–OprM and MexE–MexF–OprN, were determined in Mueller–Hinton broth (MH) and in MH-containing eluates. OMP profiles and BL activities were also studied. Resistance to imipenem in P. aeruginosa grown in eluate is due to both the loss of OprD and expression of BL. For meropenem this phenomenon is related to both the loss of OprD and the integrity of MexA–MexB–OprM.

Analysis of the Pseudomonas aeruginosa oprD gene from clinical and environmental isolates.

Genomes are constantly evolving. Our report highlights the wide mutational diversity of clinical as well as environmental isolates, compared with the laboratory strain(s), through the systematic genetic analysis of a chromosomal porin gene (oprD) in relation to a specific antibiotic resistance. Mutational inactivation of the oprD gene is associated with carbapenem resistance in Pseudomonas aeruginosa. The sequence of the oprD gene of 55 Pseudomonas aeruginosa natural isolates obtained from across the world--from sources as diverse as patients and rhizospheres--was analysed. A microscale mosaic structure for this gene--resulting from multiple intra- and possibly interspecies recombinational events--is reported. An array of independent and seemingly fast-occurring defective oprD mutations were found, none of which had been described before. A burn wound isolate demonstrated unusually high overall sequence variability typical of mutator strains. We also present evidence for the existence of OprD homologues in other fluorescent pseudomonads.