Mechanisms of carbapenem resistance in non-metallo-beta-lactamase-producing clinical isolates of Pseudomonas aeruginosa from a Tunisian hospital.

AIM OF THE STUDY: 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. MATERIALS AND 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. CONCLUSION: 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.

[Bacteriologic profile of bacteremia due to multi-drug resistant bacteria at Charles-Nicolle Hospital of Tunis]. / Profil bactériologique des bactériémies à germes multirésistants à l'hôpital Charles-Nicolle de Tunis.

OBJECTIVE: The authors had for aim to evaluate the place of multi-drug resistant bacteria (MDR) in nosocomial bacteremia. MATERIALS AND METHODS: A retrospective study was carried out at the Microbiology laboratory of Charles Nicolle hospital of Tunis (2001-2003). One hundred and ninety-five isolated MDR [third generation cephalosporin resistant enterobacteria, methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii and Pseudomonas aeruginosa resistant to ceftazidime and imipenem]. An automated system was used to detect bloodstream infections. Microorganism identification was performed by conventional methods and antibiotic susceptibilities were determined by the disk diffusion method. RESULTS: MDR bacteria were resistant to third generation cephalosporins (29%), A. baumannii (24%), P. aeruginosa (24%), and MRSA (10%). ERC3G were resistant to aminosides and fluorquinolones. A. baumannii and P. aeruginosa had high resistance rates. Associated resistance rates in MRSA were moderate. CONCLUSION: MDR bacteria are of great concern in our hospital. This situation emphasizes the importance to maintain rigorous measures of hygiene as well as adapted antibiotic prescriptions.

Evolution of acquired resistance to third-generation cephalosporins in Enterobacteriaceae in a Tunisian hospital 1993-2001.

Between January 1993 and December 2001, the overall frequency of resistance to third-generation cephalosporins in isolates of Enterobacteriaceae from Charles Nicolle Hospital, Tunis, rose from 2.4% to 7.4%. Klebsiella pneumoniae was the most prevalent species (56%), followed by Escherichia coli (15%) and Proteus mirabilis (9%). A rate of 49% was observed among isolates from paediatric patients in 1999, caused mostly by outbreaks in the neonatal intensive care unit of K. pneumoniae and P. mirabilis isolates that produced extended-spectrum beta-lactamases.

[Infectious endocarditis caused by a glycopeptide-resistant enterococus]. / Endocardite infectieuse a entérocoque résistant aux glycopeptides.

Enterococci are an important cause of infective endocarditis. Their resistance to most of the antibiotics involve real therapeutic problems. We report the first clinical isolate of glycopeptide resistant enterococcus from blood culture of patient with a prosthetic valve endocarditis. The strain is an E. faecium with a high level of resistance to vancomycin and teicoplanin (MIC > 256 mg/l), a low level of resistance to gentamycin (MIC = 6 mg/l) and susceptible to ampicillin (MIC = 1.5 mg/l). Therapeutic failure was observed leading to a surgical treatment. Therapy of such infection caused by multiresistant Enterococcus must be based on the study of bactericidal activity of antibiotic associations. In order to control the spread of this emerging resistance, the implementation of control measures is necessary.

Nosocomial outbreak of imipenem-resistant Pseudomonas aeruginosa producing VIM-2 metallo-ß-lactamase in a kidney transplantation unit.

BACKGROUND: Twenty four non replicate imipenem resistant P. aeruginosa were isolated between January and November 2008, in the kidney transplantation unit of Charles Nicolle Hospital of Tunis (Tunisia). This study was conducted in order to establish epidemiological relationship among them and to identify the enzymatic mechanism involved in imipenem resistance. METHODS: Analysis included antimicrobial susceptibility profile, phenotypic (imipenem-EDTA synergy test) and genotypic detection of metallo-ß-lactamase (MBL) (PCR), O-serotyping and pulsed-field gel electrophoresis. RESULTS: All strains showed a high level of resistance to all antimicrobials tested except to colistin. The presence of MBL showed concordance between phenotypic and genotypic methods. Sixteen isolates were identified as VIM-2 MBL-producers and 13 of them were serotype O4 and belonged to a single pulsotype (A). CONCLUSIONS: This study describes an outbreak of VIM-2-producing P. aeruginosa in a kidney transplantation unit. Clinical spread of blaVIM-2 gene is a matter of great concern for carbapenem resistance in Tunisia.

Diversity in VIM-2-encoding class 1 integrons and occasional blaSHV2a carriage in isolates of a persistent, multidrug-resistant Pseudomonas aeruginosa clone from Tunis.

From 2002 to 2006, 35 of 73 multidrug-resistant Pseudomonas aeruginosa isolates from different wards at Charles Nicolle hospital of Tunis were positive for class B carbapenemase (using the imipenem-EDTA test), owing to a bla(VIM-2) gene cassette in a class 1 integron. Twenty-three isolates additionally produced the extended-spectrum beta-lactamase SHV2a. DNA sequences immediately surrounding bla(SHV2a) shared extensive identity with a Klebsiella pneumoniae plasmid sequence. Despite belonging to the same chromosomal type, as shown by pulsed-field gel electrophoresis (PFGE), the VIM-2 producing P. aeruginosa isolates prevalent at Charles Nicolle hospital displayed a diversity of VIM-2-carrying integrons.

Capillary electrophoresis-single-strand conformation polymorphism analysis for rapid identification of Pseudomonas aeruginosa and other gram-negative nonfermenting bacilli recovered from patients with cystic fibrosis.

We used capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis of PCR-amplified 16S rRNA gene fragments for rapid identification of Pseudomonas aeruginosa and other gram-negative nonfermenting bacilli isolated from patients with cystic fibrosis (CF). Target sequences were amplified by using forward and reverse primers labeled with various fluorescent dyes. The labeled PCR products were denatured by heating and separated by capillary gel electrophoresis with an automated DNA sequencer. Data were analyzed with GeneScan 672 software. This program made it possible to control lane-to-lane variability by standardizing the peak positions relative to internal DNA size markers. Thirty-four reference strains belonging to the genera Pseudomonas, Brevundimonas, Burkholderia, Comamonas, Ralstonia, Stenotrophomonas, and Alcaligenes were tested with primer sets spanning 16S rRNA gene regions with various degrees of polymorphism. The best results were obtained with the primer set P11P-P13P, which spans a moderately polymorphic region (Escherichia coli 16S rRNA positions 1173 to 1389 [M. N. Widjojoatmodjo, A. C. Fluit, and J. Verhoef, J. Clin. Microbiol. 32:3002-3007, 1994]). This primer set differentiated the main CF pathogens from closely related species but did not distinguish P. aeruginosa from Pseudomonas alcaligenes-Pseudomonas pseudoalcaligenes and Alcaligenes xylosoxidans from Alcaligenes denitrificans. Two hundred seven CF clinical isolates (153 of P. aeruginosa, 26 of Stenotrophomonas maltophilia, 15 of Burkholderia spp., and 13 of A. xylosoxidans) were tested with P11P-P13P. The CE-SSCP patterns obtained were identical to those for the corresponding reference strains. Fluorescence-based CE-SSCP analysis is simple to use, gives highly reproducible results, and makes it possible to analyze a large number of strains. This approach is suited for the rapid identification of the main gram-negative nonfermenting bacilli encountered in CF.