George F. Araj

Evaluation of a Multilocus Variable-Number Tandem-Repeat Analysis Scheme for Typing Human Brucella Isolates in a Region of Brucellosis Endemicity

ABSTRACT Brucellosis remains an important anthropozoonosis worldwide. Brucella species are genetically homogeneous, and thus, the typing of Brucella species for epidemiological purposes by conventional molecular typing methods has remained elusive. Although many methods could segregate isolates into the phylogenetically recognized taxa, limited within-species genetic diversity has been identified. Recently, multilocus variable-number tandem-repeat analysis (MLVA) was found to have a high degree of resolution when it was applied to collections of Brucella isolates from geographically widespread locations, and an assay comprising 16 such loci (MLVA-16) was proposed. This scheme includes eight minisatellite loci (panel 1) and eight microsatellites (panel 2, which is subdivided into panels 2A and 2B). The utility of MLVA-16 for the subtyping of human Brucella isolates from geographically restricted regions needs to be further evaluated, and genotyping databases with worldwide coverage must be progressively established. In the present study, MLVA-16 was applied to the typing of 42 human Brucella isolates obtained from 41 patients recovered from 2002 to 2006 at a tertiary-care center in Lebanon. All isolates were identified as Brucella melitensis by MLVA-16 and were found to be closely related to B. melitensis isolates from neighboring countries in the Middle East when their genotypes were queried against those in the web-based Brucella2007 MLVA database (http://mlva.u-psud.fr/ ). Panel 2B, which comprised the most variable loci, displayed a very high discriminatory power, while panels 1 and 2A showed limited diversity. The most frequent genotype comprised seven isolates obtained over 7 weeks in 2002, demonstrating an outbreak from a common source. Two isolates obtained from one patient 5 months apart comprised another genotype, indicating relapsing disease. These findings confirm that MLVA-16 has a good discriminatory power for species determination, typing of B. melitensis isolates, and inferring their geographical origin. Abbreviated panel 2B could be used as a short-term epidemiological tool in a small region of endemicity.

Underlying mechanisms of carbapenem resistance in extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Escherichia coli isolates at a tertiary care centre in Lebanon: role of OXA-48 and NDM-1 carbapenemases

A recent increase in carbapenem resistance among extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae and Escherichia coli isolates at a major tertiary care centre in Lebanon prompted the initiation of this study. Consecutive ESBL-producing isolates were tested for resistance to carbapenems, with initial screening by disk diffusion and Etest using ertapenem. The modified Hodge test was also performed. PCR of β-lactamase-encoding genes, including bla(NDM-1), bla(KPC), bla(OXA-48), bla(CTX-M), bla(TEM), bla(SHV), bla(CMY-2) and bla(OXA-1), as well as outer membrane porin genes (ompC and ompF) was performed. Sequencing, efflux pump inhibitor tests and random amplified polymorphic DNA (RAPD) analysis were performed. In total, 14 (2.45%) of 572 K. pneumoniae and 24 (1.07%) of 2243 E. coli were ertapenem-non-susceptible [minimum inhibitory concentration (MIC) ≥0.25 μg/mL]. Resistance to other carbapenems was variable. PCR and sequencing analysis revealed that isolates harboured different β-lactamase genes, including bla(OXA-1), bla(CTX-M-15), bla(TEM-1), bla(CMY-2), bla(OXA-48) and bla(NDM-1). In addition, K. pneumoniae lacked the outer membrane porin-encoding genes, whilst E. coli harboured them with detected mutations. CTX-M-15 was carried on a 90 kb plasmid, whilst OXA-48 was carried on a 70 kb plasmid. Efflux pump inhibition significantly decreased MICs in E. coli. RAPD analysis demonstrated genomic variability. In conclusion, carbapenem resistance in ESBL-producing K. pneumoniae and E. coli is due to the combined effect of β-lactamases with porin impermeability and/or efflux pump activity observed in these organisms, and in a number of isolates is due to the production of the carbapenemase-encoding genes bla(OXA-48) and the newly emerging bla(NDM-1).

Recent developments in β lactamases and extended spectrum β lactamases

Resistance to β lactam antibiotics is an increasing problem worldwide. This review describes the classification and mechanism of action of βlactamases and the options available for detecting, treating, and controlling extended spectrum β lactamases βlactam antimicrobial agents are the most common treatment for bacterial infections (table 1).1 Rates of bacterial resistance to antimicrobial agents are increasing worldwide, including in Lebanon.2 Production of β lactamases is the most common mechanism of bacterial resistance (table 2).1 3 These enzymes are numerous, and they mutate continuously in response to the heavy pressure of antibiotic use, leading to the development of extended spectrum β lactamases (ESBLs).4 Examples are the mutated TEM and SHV genes, mainly found in strains of Escherichia coli and Klebsiella pneumoniae respectively. Infections with ESBL producing bacterial strains are encountered singly or in outbreaks, especially in critical care units in hospitals, resulting in increasing costs of treatment and prolonged hospital stays. We aim to present a simplified review of this highly complex subject, in the hope that it will guide the practising physician in appropriate decisions relating to the use of β lactams in patient care. View this table: Table 1 Groups and examples of β lactam antimicrobial agents View this table: Table 2 Antimicrobial agents, their modes of action, and the corresponding mechanisms of bacterial resistance We examined new information from the most recent relevant literature retrieved from PubMed and the internet. The β lactams are a family of antimicrobial agents consisting of four major groups: penicillins, cephalosporins, monobactams, and carbapenems (table 1). They all have a β lactam ring, which can be hydrolysed by β lactamases. The groups differ from each other by additional rings (thiazolidine ring for penicillins, cephem nucleus for cephalosporins, none for monobactams, double ring structure for carbapenems). The various antibiotics in each group differ by the nature of one …

DISCREPANCIES BETWEEN MECA PCR AND CONVENTIONAL TESTS USED FOR DETECTION OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS

Conventional and molecular techniques are being used in the detection of methicillin resistance in Staphylococcus aureus but they do not always show concordant results. In this study, a mecA PCR-based amplification was compared with the 1 microg oxacillin disk diffusion test and the Epsilometer test (E-test) for detection of MICs. Among 31 isolates initially characterized as MRSA by the disk diffusion test, mecA was detected in only 13 (42%) isolates. The E-test showed a wide range of oxacillin MICs (0.5 - > 256 microg/ml) among these 31 MRSA isolates: seven isolates had an MIC of > 256 microg/ml, one had 64 microg/ml, two had 4 microg/ml, two had 3 microg/ml, one had 2.5 microg/ml, nine had 2 microg/ml, three had 1.5 microg/ml, five had 1 microg/ml and one had 0.5 microg/ml. Comparing the mecA PCR results with the E-test oxacillin MIC findings revealed that mecA was detected in seven of eight isolates (87.5%) with an MIC of > or = 64 microg/ml, in three of 14 isolates (21.4%) with an MIC of 2-4 microg/ml and in three of nine isolates (33.3%) with an MIC of < 2 microg/ml. Beta-lactamase production was positive in 28/31 isolates (90.3%). Because of this variation between tests and since several resistance mechanisms are known to mediate methicillin resistance in S. aureus, the reliable detection of MRSA cannot be solely based on detection of mecA gene in S. aureus. At this stage and until new guidelines are introduced by an official body, such as NCCLS, a combination of conventional methods alone or together with a molecular method should be used every time S. aureus is tested for detection of methicillin resistance.

First detection of oxacillinase-mediated resistance to carbapenems in Klebsiella pneumoniae from Morocco

Abstract The frequency of carbapenem resistance due to class-D β-lactamases (i.e. oxacillinases) among the worlds Enterobacteriaceae is increasing. Recently, in Morocco, two isolates of carbapenem-resistant Klebsiella pneumoniae were recovered from the same patient, one harbouring plasmid-encoded bla- OXA-48 and the other the bla- OXA-1 gene. This represents the first evidence of bla OXA-48-mediated carbapenem-resistance in Enterobacteriaceae in Morocco.

Characterization of rpoB mutations in rifampin-resistant Mycobacterium tuberculosis isolates from the Middle East

The nature and frequency of mutations in the rpoB gene of rifampin-resistant clinical Mycobacterium tuberculosis isolates vary considerably according to geographical locations. There is no information on the prevalence of specific mutations in clinical M. tuberculosis strains isolated from patients in Middle-Eastern countries. In this study, 13 rifampin-resistant and 6 susceptible clinical M. tuberculosis isolates were tested for identification and characterization of mutations in the rpoB gene by INNO-LiPA Rif. TB kit and DNA sequencing of the PCR amplified target DNA. The kit identified all six susceptible strains as rifampin-sensitive and the DNA sequence of the amplified rpoB gene in the target region matched perfectly with the wild-type sequence. The kit identified 12 resistant isolates as rifampin-resistant with specific detection of mutations in 8 isolates while one of the rifampin-resistant strain was identified as rifampin-susceptible. DNA sequencing confirmed these results and, in addition, led to the specific detection of mutations in 4 rifampin-resistant isolates in which specific base changes within the target region could not be determined by the INNO-LiPA Rif. TB kit. The majority (8 of 13) of resistant isolates involved base changes at codon 531 of the rpoB gene. Mutations at codon position 531 within the rpoB gene have also been reported in majority of rifampin-resistant strains from Greece and St. Petersburg, Russia but not from other geographical locations.

Oxacillinase-mediated resistance to carbapenems in Klebsiella pneumoniae from Lebanon

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Non-pyogenic infections of the spine.

Subacute and chronic spondylodiscitis can be caused by a wide spectrum of infectious aetiologies including Mycobacterium tuberculosis, Brucella spp. and a variety of fungi including Aspergillus spp., Candida spp. and Cryptococcus neoformans. Knowledge of the local epidemiology and prior exposure might suggest the aetiology. Non-invasive diagnostic approaches, such as blood culture or antibody titres in the case of Brucella or antigen detection in the case of fungal infections, can be helpful in reaching the diagnosis. However, direct aspiration or tissue biopsy is usually necessary to identify the causative organism. Specimens are usually sent for pathology, special stains, cultures and, when indicated, molecular analysis. To minimise morbidity and mortality, antibiotic treatment should be initiated promptly directed against the suspected organism, and later adjusted according to the confirmed aetiology. Surgical treatment is reserved for recurrent infection, unstable spinal segment or marked kyphosis in the face of any neurological deficits and uncontrollable pain. Surgical approaches are dictated by the anatomic location of the offending lesion. Once medical treatment fails and surgery becomes warranted, we advocate the use of a two-stage surgical treatment for non-fixed kyphosis and a three-stage operation for fixed kyphosis.

Antimicrobial resistance of Enterococci in Lebanon

There is little information on the types of Enterococcus spp and their antibiotic resistance patterns in Lebanon. One hundred and fifty-three consecutive clinical enterococcal isolates collected between 1998 and 1999 were tested against 11 antimicrobial agents using disk diffusion and the Etest. The isolates were identified by conventional methods and API-Strep and were found to consist of Enterococcus faecalis (72.5%), Enterococcus faecium (22.9%), Enterococcus avium (3.2%) and Enterococcus gallinarum (1.3%). The percent of resistant strains of E. faecalis and E. faecium respectively were, ampicillin 0.9 and 14%, erythromycin 59% and 40%, tetracycline 72% and 34%, chloramphenicol 32 and 11%, rifampin 36% and 57%, ciprofloxacin, 23% and 34%, norfloxacin 22 and 8%. High level aminoglycoside (HLA) resistance was found in 19% E. faecalis and 9% E. faecium for gentamicin and 36% and 26% for streptomycin. Excellent correlation was observed between the high level disk tests and the Etest in the detection of HLA resistance but not with the regular disks. None of the isolates showed resistance to vancomycin or teicoplanin except for one E. gallinarum isolate which showed intermediate resistance (MIC 16 mg/l) to vancomycin. These variable antimicrobial rates of resistance suggest a surveillance programme for antimicrobial resistance in this country would be helpful to help control infection, guide empirical antibiotic therapy and implement a policy of antibiotic usage.

Spread of OXA-48-mediated resistance to carbapenems in Lebanese Klebsiella pneumoniae and Escherichia coli that produce extended spectrum β-lactamase

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