Characterization of Cephalosporinases Produced by Clinical Isolates of Enterobacteriacae in North Lebanon

Background: The problem of Enterobacteriacae resistance to β-Lactamase drugsis of growing concern in hospitals. Enterobacteria have developed multiple mechanismsof resistance to antibiotics, the main one is the enzymatic resistance mediatedby the beta-lactamases. This study aims to characterize the occurrence ofcephalosporinases in clinical isolates of Enterobacteriacae isolates in North Lebanon.Methods. Twenty two strains of Enterobacteriacae producing high level of cephalosporinaseshave been studied. The antibiotic susceptibility of each strain wastested on Mueller Hinton agar contains cloxacilline (250 mg/L) and by using E-testaccording the guidelines of the Antibiogram Committee of the French Society forMicrobiology. The search for plasmid-mediated cephalosporinases was performedusing PCR and primers for plasmid-mediated cephalosporinases genes (CMY-2,DHA-1, ACT-1, ACC-1, FOX-1 and MOX-1).Results: Thirteen positive strains were detected, of these 9 strains produced theplasmid-mediated cephalosporinase (CMY-2) and one strain produced the plasmidmediatedcephalosporinase (DHA-1). The remaining 9 strains were high-level chromosomalcephalosporinase producers since they belong to group-three Enterobacteria.They did neither produce plasmid-mediated cephalosporinase, nor did theyhave resistance to third generation cephalosporins except for cefepim. Two strains(CMUL E. coli 021) and CMUL E. coli 255) which were not susceptible for cefepim byE-test produced plasmid-mediated cephalosporinase The sequencing result of these2 E.coli strains did not show any mutation in the promoter that is responsible forhigh expression level of the chromosomal cephalosporinase. All examined strainsproducing plasmid-mediated cephalosporinase CMY-2 were analyzed by ERIC-PCRtechnique. The results showed that two of these strains had the same pattern (C4and C5) and three others had another pattern (C10, C12 and C13).Conclusion: This study shows the variations of cephalosporinases produced byclinical isolates of Enterobacteriacae in North Lebanon

Study of the molecular mechanism of antibiotic resistance in the mediterranean basin

La détection, la surveillance et la diffusion de la résistance des bactéries aux antibiotiques est un enjeu majeur au niveau mondial depuis la découverte et la diffusion de bactéries multi résistantes, en particulier la résistance aux carbapénèmes, spécifiquement chez les Entérobactéries et les bactéries du genre Pseudomonas et Acinetobacter. L’émergence et la dissémination des pathogènes Gram- résistants aux carbapénèmes est un contributeur significatif de la morbidité et la mortalité du patient. Malgré les efforts radicaux dans le contrôle de l’infection et les améliorations dans le diagnostique moléculaire, les bacilles Gram- résistants aux carbapénèmes demeurent une formidable menace vue que quelques agents antimicrobiens sont actifs et très peu devraient être disponibles dans le futur proche.L’origine et la source des gènes de résistance dans le monde sont mal connues et des travaux récents suggèrent que les animaux domestiques et sauvages, l’environnement mais également le tube digestif des mammifères et des humains pourraient représenter un réservoir et une source importante de gènes de résistance susceptibles d’être transmissibles à l’homme. C’est dans cette optique que ce projet de thèse s’articule avec comme objectifs : (i) la réalisation d’études épidémiologiques moléculaires d’isolats cliniques et animales résistants aux carbapénèmes isolés dans le basin Méditerranéen et la caractérisation des supports moléculaires de cette résistance ; (ii) la description de nouveaux mécanismes de résistance a l’imipenème ; et enfin (iii) le séquençage de génomes d’isolats cliniques résistants aux carbapénèmes et l’analyse de ces derniers.The detection, monitoring and dissemination of bacterial resistance to antibiotics are a major issue worldwide since the discovery and spread of multi-resistant bacteria, in particular resistance to carbapenems, specifically among Enterobacteriaceae and bacteria of the genus Pseudomonas and Acinetobacter.The emergence and dissemination of carbapenem-resistant Gram-negative pathogens is a significant contributor to patient morbidity and mortality. Despite radical efforts in infection control and improvements in molecular diagnostics, carbapenem-resistant Gram-negative bacilli remain a formidable threat as few antimicrobial agents are reliably active and very little is expected to be available in the near future.The origin and source of resistance genes in the world are not well known and recent works suggest that domestic and wild animals, the environment (soil, water, rivers ..) but also the digestive tract of mammals and humans could represent a reservoir and an important source of resistance genes that may be transmissible to humans.It is in this context that this thesis project articulates with the following objectives: (i) The achievement of molecular epidemiological studies on carbapenem-resistant clinical and animal isolates collected from countries in the Mediterranean basin (Lebanon, Libya, France) and the characterization of the genetic determinants of this resistance; (ii) the description of new resistance mechanisms to imipenem; and finally (iii) The genome sequencing of clinical isolates resistant to carbapenems, the analysis of these genomes and the identification of mechanisms and genetic supports of the resistance to carbapenems and other antibiotics

Molecular characterisation of extended-spectrum beta-lactamase- and plasmid AmpC-producing Escherichia coli strains isolated from broilers in Bejaia, Algeria

International audienceThis study aimed to characterise the molecular support of antibiotic resistance in expanded-spectrum cephalosporin (ESC)-resistant Escherichia coli isolates recovered from healthy broilers in Bejaia, northeast Algeria. A total of 61 intestinal swabs from slaughtered broilers from four regions in Bejaia locality, Algeria, were collected between February and April 2014, from which 20 ESC-resistant E. coli strains were isolated. Escherichia coli isolates were identified by classical biochemical and MALDI-TOF methods. Antibiotic susceptibility testing was performed using disk diffusion and Etest methods. Screening for beta-lactamases, aminoglycoside-modifying enzyme (AME)-encoding genes and qnr determinants was performed by PCR and sequencing. Clonal relatedness was determined using molecular typing by multilocus sequence typing (MLST). Antibiotic susceptibility testing revealed that the isolates showed high rates of resistance (>90%) to amoxicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, aztreonam, ceftazidime, streptomycin, tobramycin, nalidixic acid and ciprofloxacin. Low rates of resistance were observed for kanamycin (35%), amikacin (30%), cefoxitin (20%) and cefotaxime (15%). Molecular characterisation revealed that all of the isolates expressed the bla(TEM-1) gene. Fourteen of them harboured the bla(SHV-12) gene, two harboured the bla(CTX-M-1) gene and four isolates harboured bla(CMY-2). Screening for AME-encoding genes demonstrated that all isolates contained the aadA gene. In addition, qnrA was detected as the quinolone resistance determinant in 13 isolates. MLST revealed four known sequence types (STs), including ST744, ST38, ST1011 and ST2179, as well as one new sequence type (ST5086). Here we report the first study describing the clonal diversity of extended-spectrum beta-lactamase (ESBL)- and plasmid AmpC-producing E. coli isolated from healthy broilers in Algeria. (C) 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved

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

First Detection of Colistin-Resistant Klebsiella pneumoniae in Association with NDM-5 Carbapenemase Isolated from Clinical Lebanese Patients

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Bacterial infection during wars, conflicts and post-natural disasters in Asia and the Middle East: a narrative review

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clbP Gene, a Potential New Member of the β-Lactamase Family

The colibactin island (pks) of Escherichia coli formed by 19 genes (55-Kb), encodes non-ribosomal peptide (NRP) and polyketide (PK) synthases, which allow the synthesis of colibactin, a suspected hybrid PK-NRP compound that causes damage to DNA in eukaryotic cells. The clbP, an unusual essential gene, is found in the operon structure with the clbS gene in the pks-encoded machinery. Interestingly, the clbP gene has been annotated as a β-lactamase but no previous study has reported its β-lactamase characteristics. In this study, we (i) investigated the β-lactamase properties of the clbP gene in silico by analysing its phylogenetic relationship with bacterial β-lactamase and peptidase enzymes, (ii) compared its three-dimensional (3D) protein structure with those of bacterial β-lactamase proteins using the Phyr2 database and PyMOL software, and (iii) evaluated in vitro its putative enzymatic activities, including β-lactamase, nuclease, and ribonuclease using protein expression and purification from an E. coli BL21 strain. In this study, we reveal a structural configuration of toxin/antitoxin systems in this island. Thus, similar to the toxin/antitoxin systems, the role of the clbP gene within the pks-island gene group appears as an antitoxin, insofar as it is responsible for the activation of the toxin, which is colibactin. In silico, our analyses revealed that ClbP belonged to the superfamily of β-lactamase, class C. Furthermore, in vitro we were unable to demonstrate its β-lactamase activity, likely due to the fact that the clbP gene requires co-expression with other genes, such as the genes present in the pks-island (19 genes). More research is needed to better understand its actions, particularly with regards to antibiotics, and to discover whether it has any additional functions due to the importance of this gene and its toxicity

Clonal Dissemination of Plasmid-Mediated Carbapenem and Colistin Resistance in Refugees Living in Overcrowded Camps in North Lebanon

International audienceCarbapenem and colistin-resistant bacteria represent a global public health problem. Refugees carrying these bacteria and living in inadequate shelters can spread these microorganisms. The aim of this study was to investigate the intestinal carriage of these bacteria in Syrian refugees in Lebanon. Between June and July 2019, 250 rectal swabs were collected from two refugee camps in North Lebanon. Swabs were cultured on different selective media. Antibiotic susceptibility testing was performed using the disk diffusion method. Carbapenemase-encoding genes and mcr genes were investigated using real-time polymerase chain reaction (RT-PCR) and standard polymerase chain reaction (PCR). Epidemiological relatedness was studied using multilocus sequence typing (MLST). From 250 rectal swabs, 16 carbapenem-resistant, 5 colistin-resistant, and 4 colistin and carbapenem-resistant Enterobacteriaceae were isolated. The isolates exhibited multidrug-resistant phenotypes. Seven Klebsiella pneumoniae isolates harboured the blaOXA-48 gene, and in addition four K. pneumoniae had mutations in the two component systems pmrA/pmrB, phoP/phoQ and co-harboured the blaNDM-1 gene. Moreover, the blaNDM-1 gene was detected in six Escherichia coli and three Enterobacter cloacae isolates. The remaining five E. coli isolates harboured the mcr-1 gene. MLST results showed several sequence types, with a remarkable clonal dissemination. An urgent strategy needs to be adopted in order to avoid the spread of such resistance in highly crowded underserved communities

Molecular epidemiology of environmental and clinical ă carbapenemase-producing Gram-negative bacilli from hospitals in Guelma, ă Algeria: Multiple genetic lineages and first report of OXA-48 in ă Enterobacter cloacae

International audienceThis study was designed to investigate environmental colonisation in ă Algerian hospitals by carbapenem-resistant Gram-negative bacilli (GNB), ă including molecular characterisation of their resistance, and to perform ă a comparative molecular analysis between clinical and environmental ă strains. GNB isolated from hospitalised patients and the hospital ă environment were identified using microbiological methods and ă matrix-assisted laser desorption/ionisation time-of-flight mass ă spectrometry (MALDI-TOF/MS). Antibiotic susceptibility testing was ă performed by disk diffusion and Etest methods. Carbapenemase- and ă extended-spectrum beta-lactamase (ESBL)-encoding genes were searched for ă using PCR and sequencing. Clonality of the environmental and clinical ă strains was assessed by multilocus sequencing typing (MLST). A total of ă 32 carbapenem-resistant GNB were isolated, including 16 (29%) of 56 ă multidrug-resistant (MDR) GNB from clinical specimens and 16 (48%) of ă 33 MDR-GNB from inanimate surfaces. Of the 32 carbapenem-resistant ă isolates, 14 produced a carbapenemase. The bla(OXA-48) gene was detected ă both in clinical and surface isolates of Klebsiella pneumoniae (n =3) ă and Enterobacter cloacae (n = 2). Clinical and surface isolates of ă Acinetobacter baumannii were found to produce the carbapenemases NDM-1 ă (7 isolates) and OXA-23 (2 isolates). MLST revealed clonal diversity and ă a relationship between environmental and clinical strains with identical ă sequence types. Here we report the first description of an ă OXA-48-producing E. cloacae isolate in Algeria. We also highlight the ă important role of inanimate surfaces in the spread of ă carbapenem-resistant bacteria and the emergence of nosocomial ă infections. (C) 2016 International Society for Chemotherapy of Infection ă and Cancer. Published by Elsevier Ltd. All rights reserved

Carbapenem and colistin-resistant bacteria in North Lebanon: Coexistence of mcr-1 and NDM-4 genes in Escherichia coli

International audienceIntroduction: The increasing incidence of infections caused by multidrug-resistant bacteria is considered a global health problem. This study aimed to investigate this resistance in Gram-negative bacteria isolated from patients hospitalized in North-Lebanon. Methodology: All isolates were identified using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antibiotic susceptibility testing was achieved using disk diffusion, E-test and Broth microdilution methods. Phenotypic detection of carbapenemase was carried out using the CarbaNP test. RT-PCR, standard-PCR and sequencing were performed to detect resistance genes and oprD gene. Conjugal transfer was carried out between our isolates and Escherichia coli J53 to detect the genetic localization of resistance genes. MLST was conducted to determine the genotype of each isolate. Results: Twenty-three carbapenem-resistant Enterobacterales of which eight colistin-resistant Escherichia coli, and Twenty carbapenem-resistant Pseudomonas aeruginosa were isolated. All isolates showed an imipenem MIC greater than 32 mg/mL with MICs for colistin greater than 2 mg/L for E. coli isolates. All the Enterobacterales isolates had at least one carbapenemase-encoding gene, with E. coli isolates coharboring blaNDM-4 and mcr-1 genes. Moreover, 16/20 Pseudomonas aeruginosa harbored the blaVIM-2 gene and 18/20 had mutations in the oprD gene. MLST revealed that the isolates belonged to several clones. Conclusions: We report here the first description in the world of clinical E. coli isolates coharboring blaNDM-4 and mcr-1 genes, and K. pneumoniae isolates producing NDM-6 and OXA-48 carbapenemases. Also, we describe the emergence of NDM-1-producing E. cloacae in Lebanon. Screening for these isolates is necessary to limit the spread of resistant microorganisms in hospitals