Determinism of molecular support and epidemiology of resistance to b-lactamines in clinical isolates of gram-negative bacilli in tunisian and libyan hospitals

L’augmentation et la dissémination de la résistance aux β-lactamines chez les bacilles à Gram négatif, particulièrement les Entérobactéries, les bactéries du genre Pseudomonas et Acinetobacter, représentent un problème majeur de santé publique. Les infections nosocomiales causées par ces bactéries multi-résistantes (BMR) ont conduit à une augmentation de la mortalité, de la morbidité et du coût de traitement. L’utilisation abusive et non contrôlée de ces antibiotiques a grandement contribué à la large diffusion de cette résistance. Ainsi, face à cette préoccupation mondiale et suite à de nombreuses recommandations, plusieurs études épidémiologiques et moléculaires ont été rapportées afin de contrôler et de surveiller la diffusion et la dissémination des BMR. Contrairement à de nombreuses régions dans le monde, il existe peu d’informations concernant la caractérisation moléculaire des gènes de résistance aux β-lactamines des bacilles à Gram négatif isolés en Tunisie et surtout en Libye. C’est dans cette optique que ce projet de Thèse de Doctorat s’articule avec comme objectifs: (i) mettre en évidence la prévalence des bacilles à Gram négatifs multi-résistants isolés aux niveaux des hôpitaux tunisiens et libyens (ii) identifier le support génétique de la résistance aux β-lactamines de ces souches cliniques (iii) étudier la diversité clonale des souches multi-résistantes par typage moléculaire.The increase and spread of β-lactam resistance in gram negative bacteria especially Enterobacteriaceae, Pseudomonas and Acinetobacter (E.P.A) species have become a major concern worldwide. The hospital-acquired infections caused by MDR bacteria have led to an increase in mortality, morbidity and cost of treatment. The frequent misuse of antibiotic drug has greatly contributed to worldwide dissemination of antibiotics resistance. Front of this worldwide concern, and various recommendations, several epidemiological and molecular studies have been reported in order to control the spread and the dissemination of these MDR. Unlike many parts of the world, there is little information concerning the molecular characterization of the β-lactam resistance genes of Gram-negative bacilli isolated in Tunisia and especially in Libya. Therefore, it is in this context that the project of this thesis was conducted with essential objectives: (i) highlight the prevalence of multi-resistant Gram negative bacilli isolated in Tunisian and Libyan hospitals (ii) identify the genetic support of resistance to β-lactams of these clinical strains (iii) study the clonal diversity of the multi-resistant strains by molecular typing (iii) study the molecular epidemiology of these BMRs in these countries in order to control the decision-making process of the treatment and the rapid identification of epidemics by implementing appropriate control measures for the spread of infections and especially developing new tools and software for the diagnosis and monitoring of potential MDR bacteria in Mediterranean countries

Prevalence and emergence of carbapenemases-producing Gram-negative bacteria in Mediterranean basin

International audienceThe emergence and the global spread of carbapenemases concern to health services worldwide. Their celestial rise among Gram-negative bacilli has challenged both the scientific and pharmaceutical sectors. Indeed, infections caused by these bacteria have limited treatment options and have been associated with high mortality and morbidity rates. Carbapenemase producers are mainly identified among Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii and still mostly in hospital settings and rarely in the community. They are closely related to KPC, VIM, IMP, NDM, and OXA-48 types. The encoding genes are mostly plasmid located and associated with various mobile genetic elements. The Mediterranean area is of interest due to a great diversity and population mixing. The prevalence of carbapenemases is particularly high and variant among countries, partially depending on the population exchange relationship between the regions and the possible reservoirs of each carbapenemase. This review described the epidemiology of carbapenemases in this region of the world highlighting the worrisome situation and the need to screen and detect these enzymes to prevent and control their dissemination especially as it is clear that very few novel antibiotics will be introduced in the next few years, making the dissemination of carbapenem-resistant Gram-negative bacteria of crucial importance worldwide

Incidence of OXA-23 and OXA-58 Carbapenemases Coexpressed in Clinical Isolates of Acinetobacter baumannii in Tunisia

International audienceAcinetobacter baumannii is an important opportunistic and multidrug-resistant pathogen responsible for nosocomial infections in health facilities. The aim of this study was to characterize the molecular mechanisms of carbapenem resistance in A. baumannii isolates isolated from Mohamed Kassab Orthopedic Institute in Tunis, Tunisia. Twenty-five imipenem-resistant A. baumannii clinical isolates collected between 2013 and 2016 were identified using API 20NE and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Carbapenemase activity was detected using microbiological tests and PCR. The epidemiological relatedness of the isolates was studied using multilocus sequence typing (MLST). The isolates were resistant to all antibiotics tested with increased minimum inhibitory concentration values (>32mg/L). The microbiological tests showed that the 25 A. baumannii were positive for modified Hodge test and for the Carba NP test; however, -lactamase activity was not inhibited by EDTA. All the isolates harbored the naturally occurring bla(OXA-51)-like gene and the bla(OXA-23)-like carbapenemase gene. Among these isolates, one isolate coexpressed the bla(OXA-58) gene. MLST revealed several sequence types (STs) with the predominance of ST2 imipenem-resistant A. baumannii (14/25; 56%). In this study we report the prevalence of ST2 imipenem resistance and for the first time the coexpression of bla(OXA-23) and bla(OXA-58) in clinical isolates of A. baumannii in a Tunisian hospital

Epidemiology, Phenotypic and Genotypic Characterization of Carbapenem-Resistant Gram-Negative Bacteria from a Libyan Hospital

International audienceAntimicrobial resistance, particularly resistance to carbapenems, has become one of the major threats to public health. Seventy-two isolates were collected from patients and hospital environment of Ibn Sina Hospital, Sirte, Libya. Antibiotic susceptibility tests, using the disc diffusion method and E-Test strips, were performed to select carbapenem-resistant strains. The colistin (CT) resistance was also tested by determining the minimum inhibitory concentration (MIC). RT-PCR was conducted to identify the presence of carbapenemase encoding genes and plasmid-mediated mcr CT resistance genes. Standard PCR was performed for positive RT-PCR and the chromosome-mediated CT resistance genes (mgrB, pmrA, pmrB, phoP, phoQ). Gram-negative bacteria showed a low susceptibility to carbapenems. Molecular investigations indicated that the metallo-β-lactamase New Delhi metallo-beta-lactamases-1 was the most prevalent (n = 13), followed by Verona integron-encoded metallo-beta-lactamase (VIM) enzyme (VIM-2 [n = 6], VIM-1 [n = 1], and VIM-4 [n = 1]) that mainly detected among Pseudomonas spp. The oxacillinase enzyme OXA-23 was detected among six Acinetobacter baumannii, and OXA-48 was detected among one Citrobacter freundii and three Klebsiella pneumoniae, in which one coharbored the Klebsiella pneumoniae carbapenemase enzyme and showed resistance to CT (MIC = 64 μg/mL) by modification in pmrB genes. In this study, we report for the first time the emergence of Pseudomonas aeruginosa carrying the blaNDM-1 gene and belonging to sequence type773 in Libya. Our study reported also for the first time CT resistance by mutation in the pmrB gene among Enterobacteriaceae isolates in Libya

Carbapenemases and extended-spectrum beta-lactamases producing ă Enterobacteriaceae isolated from Tunisian and Libyan hospitals

International audienceIntroduction: The aim of the study was to investigate the prevalence of ă extended-spectrum beta-lactamase (ESBL) and carbapenemase production ă among clinical isolates of Enterobacteriaceae recovered from Tunisian ă and Libyan hospitals. ă Methodology: Bacterial isolates were recovered from patients in ă intensive care units and identified by biochemical tests and MALDI-TOF. ă Antibiotic susceptibility testing was performed by disk diffusion and ă the E-test method. ESBL and carbapenemase activities were detected using ă standard microbiological tests. Antibiotic resistance-encoding genes ă were screened by PCR and sequencing. Clonal relationships between ă Klebsiella pneumoniae strains were carried out using multi-locus ă sequence typing (MLST). ă Results: A total of 87 isolates were characterized, with 51 and 36, ă respectively, identified as E. coli and K. pneumoniae. Overall the ă resistance prevalence was high for aminoglycosides (> 60%), ă fluoroquinolones (> 80%), and extended-spectrum cephalosporins (> ă 94%), and was low for imipenem (11.4%). Among this collection, 58 ă strains (66.6%) were ESBL producers and 10 K. pneumoniae strains ă (11.4%) were carbapenemase producers. The antibiotic ă resistance-encoding genes detected were blaC(TX-M-15) (51.7%), ă bla(TEM-1) (35.6%), several variants of bla(SHV) (21.8%), and ă bla(OXA-48) (11.4%). The MLST typing of K. pneumoniae isolates revealed ă the presence of multiple clones and three novel sequence types. Also, ă close relationships between the OXA-48-producing strains from Tunisia ă and Libya were demonstrated. ă Conclusions: This study is the first paper describing the emergence of ă carbapenemase-and ESBL-producing Enterobacteriaceae, sensitive to ă colistin, isolated in Tunisia and Libya. Active surveillance and testing ă for susceptibility to colistin should be implementing because resistance ă to colistin, mainly in Klebsiella, has been recently reported worldwide

Early detection of metallo-beta-lactamase NDM-1-and OXA-23 ă carbapenemase-producing Acinetobacter baumannii in Libyan hospitals

International audienceAcinetobacter baumannii is an opportunistic pathogen causing various ă nosocomial infections. The aim of this study was to characterise the ă molecular support of carbapenem-resistant A. baumannii clinical isolates ă recovered from two Libyan hospitals. Bacterial isolates were identified ă by matrix-assisted laser desorption/ionisation time-of-flight mass ă spectrometry (MALDI-TOF/MS). Antibiotic susceptibility testing was ă performed using disk diffusion and Etest methods, and carbapenem ă resistance determinants were studied by PCR amplification and ă sequencing. Multilocus sequence typing (MLST) was performed for typing ă of the isolates. All 36 imipenem-resistant isolates tested were ă identified as A. baumannii. The bla(OXA-23) gene was detected in 29 ă strains (80.6%). The metallo-beta-lactamase bla(NDM-1) gene was ă detected in eight isolates (22.2%), showing dissemination of ă multidrug-resistant (MDR) A. baumannii in Tripoli Medical Center and ă Burn and Plastic Surgery Hospital in Libya, including one isolate that ă co-expressed the bla(OXA-23) gene. MLST revealed several sequence types ă (STs). Imipenem-resistant A. baumannii ST2 was the predominant clone ă (16/36; 44.4%). This study shows that NDM-1 and OXA-23 contribute to ă antibiotic resistance in Libyan hospitals and represents the first ă incidence of the association of these two carbapenemases in an ă autochthonous MDR A. baumannii isolated from patients in Libya, ă indicating that there is a longstanding infection control problem in ă these hospitals. (C) 2016 Elsevier B.V. and International Society of ă Chemotherapy. All rights reserved