Novel multiplex allele-specific PCR assays for the detection of resistance to second-line drugs in Mycobacterium tuberculosis.

OBJECTIVES: The use of rapid molecular assays for the detection of resistance to second-line drugs would significantly decrease the time delay in diagnosing drug-resistant tuberculosis (TB) that is associated with conventional phenotypic drug susceptibility testing. In this study, multiplex allele-specific (MAS)-PCR assays designed to detect the GyrA D94G and rrs A1401G mutations were evaluated for detection of ofloxacin and kanamycin resistance. METHODS: GyrA D94G and rrs A1401G MAS-PCR assays were carried out on 288 Mycobacterium tuberculosis isolates. Phenotypic drug susceptibility testing of ofloxacin and kanamycin was performed on selected multidrug-resistant TB isolates using the indirect proportions method. RESULTS: MAS-PCR assays detected GyrA D94G and rrs A1401G mutations in phenotypically resistant isolates with clinical sensitivities of 54.5% (6 of 11) and 90.0% (9 of 10), respectively, and specificities of 100% were obtained for both assays. A GyrA A90V mutation was identified in 4 of 11 (36.4%) ofloxacin-resistant isolates that did not carry a D94G substitution. CONCLUSIONS: Rapid genotypic assays designed to detect GyrA D94G and A90V mutations and rrs A1401G mutations could detect up to 90.0% of extensively drug-resistant (XDR)-TB in the Western Cape region. The use of these assays in the clinical setting would significantly reduce the time to diagnosis of XDR-TB, enabling the administration of appropriate treatment regimens at the outset of therapy.

Rapid genotypic assays to identify drug-resistant Mycobacterium tuberculosis in South Africa.

OBJECTIVES: Molecular assays to detect drug resistance in Mycobacterium tuberculosis are more rapid than standard drug susceptibility testing. To evaluate the efficacy of such assays in this setting, the GenoType MTBDRplus assay (HAIN Lifescience) and multiplex allele-specific PCR assays were carried out. METHODS: The GenoType MTBDRplus assay was evaluated for the detection of rifampicin and isoniazid resistance in 223 M. tuberculosis isolates of known phenotypic drug sensitivity. The presence of KatG S315T and inhA C-15T mutations that confer isoniazid resistance was determined using multiplex allele-specific PCR assays. The relationship between isolate lineage and resistance determinant was investigated by spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat analysis. RESULTS: The GenoType MTBDRplus assay detected multidrug-resistant, isoniazid-monoresistant and rifampicin-monoresistant isolates with sensitivities of 91.5%, 56.1% and 70%, respectively. Multiplex allele-specific PCR detected isoniazid resistance in 91.5% of the MDR isolates and 53.7% of the isoniazid-monoresistant isolates. The W-Beijing lineage was overrepresented in the MDR subgroup of strains (odds ratio, 3.29; 95% confidence interval, 1.76-6.16). CONCLUSIONS: A proportion of isoniazid resistance, particularly in isoniazid-monoresistant isolates of lineage X3, is due to resistance determinants other than KatG S315T and inhA C-15T. The fact that these isolates will be indicated as drug susceptible highlights the need for determining local patterns of resistance mutations to provide users with information regarding the capabilities of rapid genotypic assays.

Shiga toxin gene-containing Escherichia coli from cattle and diarrheic children in the pastoral systems of southwestern Uganda.

Shiga toxin-producing Escherichia coli (STEC) strains from cattle and diarrheic children in a pastoralist community in Uganda were investigated. The STEC strains belonged to a variety of different serogroups, and 70% of the strains were positive for the intimin gene, eae. STEC strains from two of the children were closely related to bovine strains.

Is IS(ABA-1) customized for Acinetobacter?

An insertion sequence (IS(ABA-1)) was identified in Acinetobacter spp., but not in Enterobacteriacea and Pseudomonas aeruginosa. Numerous copies of the IS were identified in Acinetobacter strains containing the element. In one of the Acinetobacter baumannii strains, IS(ABA-1) was identified adjacent to sulII and transcription of the resistance gene is presumed to be dependent on promoter sequences within the IS. Since the IS is adjacent to ampC and bla(OXA) in this A. baumannii strain, it may be that IS(ABA-1) plays an important role in the expression of antibiotic resistance genes in this genus.

Genetic environment and transcription of ampC in an Acinetobacter baumannii clinical isolate.

An ampC gene was cloned from a clinical isolate of Acinetobacter baumannii (strain RAN). DNA sequencing and primer extension studies showed that ampC is transcribed from a promoter contained within a putative insertion sequence element which has been found to abut several different genes in Acinetobacter spp.

Characterization of class 1 integron resistance gene cassettes and the identification of a novel IS-like element in Acinetobacter baumannii.

Based on hybridization studies, 21/32 multi-resistant clinical isolates of Acinetobacter baumannii contain class 1 integrons. Amplification products were obtained from 20 of the hybridization-positive strains. A single dfrA7 cassette was identified in 18 of the isolates and an integron with two cassettes (aadB-aadA4) was found in only one strain. Amplicons were not obtained from one of the hybridization positive strains. DNA sequence analysis of a 6.080-kb fragment, cloned from this strain, identified the remnant of an integron, following insertion of IS26 into the 5(')-end of intI1. The 6.080-kb sequence carries an aminoglycoside resistance gene, linked to a portion of IS1133, which in turn is linked to a sequence that has properties of IS elements, including sequences that could stimulate transcription, and ORFs encoding amino acid sequences with similarity to a transposase from Deinococcus radiodurans.

Outer membrane protein alterations and blaTEM-1 variants: their role in beta-lactam resistance in Klebsiella pneumoniae.

OBJECTIVES: The aim of the study was to characterize the genetic basis of resistance to selected beta-lactam antibiotics in two clinical isolates of Klebsiella pneumoniae. METHODS AND RESULTS: K. pneumoniae strains were isolated from two hospitalized patients. One of the strains was resistant to amoxicillin, co-amoxiclav, cefuroxime, piperacillin and cefoxitin but susceptible to all the other cephalosporins tested. The second strain displayed a similar phenotype except that it was resistant to piperacillin/tazobactam and susceptible to cefoxitin. PCR assays and DNA sequencing showed that the cefoxitin-susceptible strain contained a novel blaTEM-1 variant downstream of the strong Pa/Pb promoter. SDS-PAGE analysis of the outer membrane proteins (OMPs) did not identify OmpK35 and suggested reduced expression of OmpK36 in this strain. Following passage in non-selective media, expression of OmpK36 was restored with a concomitant increase in cefuroxime susceptibility. A similar experimental approach identified blaTEM-1C in the cefoxitin-resistant K. pneumoniae strain. This strain was deficient in OmpK35 and OmpK36; absence of the latter protein was due to the presence of IS1 in the ompK36 regulatory region. CONCLUSIONS: Resistance to selected beta-lactams in two clinical isolates of K. pneumoniae was due to interplay between the expression of OMPs and TEM-1.

Role of class A penicillin-binding proteins in PBP5-mediated beta-lactam resistance in Enterococcus faecalis.

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.