Mechanisms of carbapenem resistance in non-metallo-beta-lactamase-producing clinical isolates of Pseudomonas aeruginosa from a Tunisian hospital.
OBJECTIVE
An increasing rate of imipenem-resistant Pseudomonas aeruginosa infections has become an important clinical problem in our hospital. The aim of this study is to determine the mechanisms involved in carbapenem resistance.


METHODS
Ten strains have been randomly selected among 144 clinical isolates of carbapenem-resistant non-metallo-beta-lactamase (MBL)-producing P. aeruginosa. A phenotypic and genotypic study was performed using serotyping, antimicrobial susceptibility, detection of MBL and clonality. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used for the expression of the genes oprD, mexA and mexE and by western blot for the expression of OprM. Sequencing of oprD gene was performed.


RESULTS
Five genotypes have been determined by arbitrary primer polymerase chain reaction and seven strains were selected to study the mechanisms involved. The predominant serotype was O12. All isolates exhibited high minimum inhibitory concentration (MICs) to both imipenem and meropenem (MIC ranged from 16 to more than 32 microg/ml) and did not harbor genes encoding MBL as confirmed by PCR. RT-PCR showed a decline in oprD expression with increased expression of mexA compared to PAO1 wild type strain. None of the isolates overexpressed mexE. Western blot analysis of outer membrane showed overproduction of OprM in all isolates.


CONCLUSIONS
Resistance to both imipenem and meropenem of clinical isolates of P. aeruginosa was due to two combined mechanisms: decreased transcription of oprD gene and overproduction of the MexAB-OprM efflux system.
Hammami
S
S
Laboratoire de recherche résistance aux antimicrobiens, département de microbiologie, faculté de medicine, Tunis, Tunisia.
Ghozzi
R
R
Burghoffer
B
B
Arlet
G
G
Redjeb
S
S
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D015780:Carbapenems / Q000494:pharmacology* / Q000627:therapeutic use; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D005784:Gene Amplification; D005838:Genotype; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D018272:Porins / Q000737:chemistry / Q000235:genetics; D011552:Pseudomonas Infections / Q000188:drug therapy / Q000235:genetics*; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014416:Tunisia; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D015780:Carbapenems; D017931:DNA Primers; D004269:DNA, Bacterial; D026901:Membrane Transport Proteins; C118598:MexA protein, Pseudomonas aeruginosa; D018272:Porins; C077157:OprD protein, Pseudomonas aeruginosa; D001618:beta-Lactamases