Genetic and biochemical characterization of a novel metallo-beta-lactamase, TMB-1, from an Achromobacter xylosoxidans strain isolated in Tripoli, Libya

An Achromobacter xylosoxidans strain from the Tripoli central hospital produced a unique metallo-β-lactamase, designated TMB-1, which is related to DIM-1 (62%) and GIM-1 (51%). bla was embedded in a class 1 integron and located on the chromosome. The TMB-1β-lactamase has lower k values than both DIM-1 and GIM-1 with cephalosporins and carbapenems. The K values were more similar to those of GIM-1 than those of DIM-1, with the overall k /K values being lower than those for GIM-1 and DIM-1. Copyrigh

Characterization of IncA/C conjugative plasmid harboring bla (TEM-52) and bla (CTX-M-15) extended-spectrum beta-lactamases in clinical isolates of Escherichia coli in Tunisia

To characterize the extended-spectrum β-lactamases (ESBLs) as well as their genetic environment in different isolates of Escherichia coli from patients with repeated urinary tract infections, large multidrug resistance (MDR) plasmids have been found. Definitive evidence for the presence of an A/C incompatibility complex (IncA/C) plasmid in the MDR isolates was provided by the probing of plasmids extracted from the clinical isolates. Conjugation experiments showed that bla genes were transferred by conjugation from the ten E. coli clinical isolates to E. coli XL1-Blue recipient.A comparative restriction fragment length polymorphism (RFLP) analysis of these plasmids showed that they are genetically similar, while the overall similarity of these plasmids supports the likelihood of recent movements among these E. coli isolates. Polymerase chain reaction (PCR) amplification and sequencing of the amplicons showed that the IncA/C plasmids harbor two ESBLs, identified as TEM-52 and CTX-M-15. Analysis of the plasmid DNA surrounding the blaCTX-M-15 gene in the clinical isolates under study revealed a partially truncated fragment of ISEcp1 tnpA transposase. This result indicates the variety of genetic events that have enabled associations between ISEcp1 sequences and bla genes in these clinical isolates

The diamidine diminazene aceturate is a substrate for the high-affinity pentamidine transporter: implications for the development of high resistance levels in trypanosomes

African trypanosomiasis is a disease of humans and livestock in many areas south of the Sahara. Resistance to the few existing drugs is a major impediment to the control of these diseases, and we investigated how resistance to the main veterinary drug diminazene aceturate correlates with changes in drug transport in resistant strains. The strain tbat1(-/-), lacking the TbAT1/P2 aminopurine transporter implicated previously in diminazene transport, was adapted to higher levels of diminazene resistance. The resulting cell line was designated ABR and was highly cross-resistant to other diamidines and moderately resistant to cymelarsan. Procyclic trypanosomes were shown to be a convenient model to study diamidine uptake in Trypanosoma brucei brucei given the lack of TbAT1/P2 and a 10-fold higher activity of the high-affinity pentamidine transporter (HAPT1). Diminazene could be transported by HAPT1 in procyclic trypanosomes. This drug transport activity was lacking in the ABR line, as reported previously for the pentamidine-adapted line B48. The K(m) for diminazene transport in bloodstream tbat1(-/-) trypanosomes was consistent with uptake by HAPT1. Diminazene transport in ABR and B48 cells was reduced compared with tbat1(-/-), but their resistance phenotype was different: B48 displayed higher levels of resistance to pentamidine and the melaminophenyl arsenicals, whereas ABR displayed higher resistance to diminazene. These results establish a loss of HAPT1 function as a contributing factor to diminazene resistance but equally demonstrate for the first time that adaptations other than those determining the initial rates of drug uptake contribute to diamidine and arsenical resistance in African trypanosome

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

The Diamidine Diminazene Aceturate Is a Substrate for the High-Affinity Pentamidine Transporter: Implications for the Development of High Resistance Levels in TrypanosomesS⃞

African trypanosomiasis is a disease of humans and livestock in many areas south of the Sahara. Resistance to the few existing drugs is a major impediment to the control of these diseases, and we investigated how resistance to the main veterinary drug diminazene aceturate correlates with changes in drug transport in resistant strains. The strain tbat1(−/−), lacking the TbAT1/P2 aminopurine transporter implicated previously in diminazene transport, was adapted to higher levels of diminazene resistance. The resulting cell line was designated ABR and was highly cross-resistant to other diamidines and moderately resistant to cymelarsan. Procyclic trypanosomes were shown to be a convenient model to study diamidine uptake in Trypanosoma brucei brucei given the lack of TbAT1/P2 and a 10-fold higher activity of the high-affinity pentamidine transporter (HAPT1). Diminazene could be transported by HAPT1 in procyclic trypanosomes. This drug transport activity was lacking in the ABR line, as reported previously for the pentamidine-adapted line B48. The Km for diminazene transport in bloodstream tbat1(−/−) trypanosomes was consistent with uptake by HAPT1. Diminazene transport in ABR and B48 cells was reduced compared with tbat1(−/−), but their resistance phenotype was different: B48 displayed higher levels of resistance to pentamidine and the melaminophenyl arsenicals, whereas ABR displayed higher resistance to diminazene. These results establish a loss of HAPT1 function as a contributing factor to diminazene resistance but equally demonstrate for the first time that adaptations other than those determining the initial rates of drug uptake contribute to diamidine and arsenical resistance in African trypanosomes

Symmetrical choline-derived dications display strong anti-kinetoplastid activity

Objectives To investigate the anti-kinetoplastid activity of choline-derived analogues with previously reported antimalarial efficacy. Methods From an existing choline analogue library, seven antimalarial compounds, representative of the first-, second- and third-generation analogues previously developed, were assessed for activity against Trypanosoma and Leishmania spp. Using a variety of techniques, the effects of choline analogue exposure on the parasites were documented and a preliminary investigation of their mode of action was performed. Results The activities of choline-derived compounds against Trypanosoma brucei and Leishmania mexicana were determined. The compounds displayed promising anti-kinetoplastid activity, particularly against T. brucei, to which 4/7 displayed submicromolar EC50 values for the wild-type strain. Low micromolar concentrations of most compounds cleared trypanosome cultures within 24–48 h. The compounds inhibit a choline transporter in Leishmania, but their entry may not depend only on this carrier; T. b. brucei lacks a choline carrier and the mode of uptake remains unclear. The compounds had no effect on the overall lipid composition of the cells, cell cycle progression or cyclic adenosine monophosphate production or short-term effects on intracellular calcium levels. However, several of the compounds, displayed pronounced effects on the mitochondrial membrane potential; this action was not associated with production of reactive oxygen species but rather with a slow rise of intracellular calcium levels and DNA fragmentation. Conclusions The choline analogues displayed strong activity against kinetoplastid parasites, particularly against T. b. brucei. In contrast to their antimalarial activity, they did not act on trypanosomes by disrupting choline salvage or phospholipid metabolism, instead disrupting mitochondrial function, leading to chromosomal fragmentation