Effects of clinical breakpoint changes in CLSI guidelines 2010/2011 and EUCAST guidelines 2011 on antibiotic susceptibility test reporting of Gram-negative bacilli

Objectives The aim of this study was to analyse the effects of clinical breakpoint changes in CLSI 2010 and 2011 guidelines and EUCAST 2011 guidelines on antibiotic susceptibility testing (AST) reports. Methods In total, 3713 non-duplicate clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii were analysed. Inhibition zone diameters were determined for β-lactams, carbapenems, fluoroquinolones, aminoglycosides and trimethoprim/sulfamethoxazole. CLSI 2009-11 and EUCAST 2011 clinical breakpoints were applied. Results Changes in resistance as defined per the guidelines affected individual species and drug classes differently. The cefepime resistance rate in Escherichia coli and Enterobacter cloacae increased from 2.1% and 1.3% to 8.2% and 6.9%, respectively, applying CLSI 2009-11 versus EUCAST 2011 guidelines. Ertapenem resistance rates in E. cloacae increased from 2.6% with CLSI 2009 to 7.2% for CLSI 2010 and 2011, and to 10.1% when applying EUCAST 2011. Cefepime and meropenem resistance rates in P. aeruginosa increased from 12.2% and 20.6% to 19.8% and 27.7%, respectively, comparing CLSI 2009-11 with EUCAST 2011. Tobramycin and gentamicin resistance rates in A. baumannii increased from 15.9% and 25.4% to 34.9% and 44.4% applying CLSI 2009-11 versus EUCAST 2011. Conclusions Higher resistance rates reported due to breakpoint changes in CLSI and EUCAST guidelines will result in increasing numbers of Gram-negative bacilli reported as multidrug resistant. AST reports classifying amoxicillin/clavulanic acid, cefepime or carbapenem resistance will lead clinicians to use alternative agents. Upon implementation of the EUCAST guidelines, laboratories should be aware of the implications of modified drug susceptibility testing reports on antibiotic prescription policie

Tuberculosis vaccine strain Mycobacterium bovis BCG Russia is a natural recA mutant

BACKGROUND: The current tuberculosis vaccine is a live vaccine derived from Mycobacterium bovis and attenuated by serial in vitro passaging. All vaccine substrains in use stem from one source, strain Bacille Calmette-Guérin. However, they differ in regions of genomic deletions, antigen expression levels, immunogenicity, and protective efficacy. RESULTS: As a RecA phenotype increases genetic stability and may contribute restricting the ongoing evolution of the various BCG substrains while maintaining their protective efficacy, we aimed to inactivate recA by allelic replacement in BCG vaccine strains representing different phylogenetic lineages (Pasteur, Frappier, Denmark, Russia). Homologous gene replacement was achieved successfully in three out of four strains. However, only illegitimate recombination was observed in BCG substrain Russia. Sequence analyses of recA revealed that a single nucleotide insertion in the 5' part of recA led to a translational frameshift with an early stop codon making BCG Russia a natural recA mutant. At the protein level BCG Russia failed to express RecA. CONCLUSION: According to phylogenetic analyses BCG Russia is an ancient vaccine strain most closely related to the parental M. bovis. We hypothesize that recA inactivation in BCG Russia occurred early and is in part responsible for its high degree of genomic stability, resulting in a substrain that has less genetic alterations than other vaccine substrains with respect to M. bovis AF2122/97 wild-type

Automated quantitative drug susceptibility testing of non-tuberculous mycobacteria using MGIT 960/EpiCenter TB eXiST

Objectives To assess the predictive value of in vitro drug susceptibility testing (DST) in slow-growing non-tuberculous mycobacteria (NTM), knowledge on quantitative levels of drug susceptibility should be available. The aim of this study was to investigate the suitability of the MGIT 960/TB eXiST system for quantitative DST of NTM. Methods We have assessed quantitative levels of drug susceptibility for clinical isolates of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium kansasii by comparing radiometric Bactec 460TB-based DST with non-radiometric DST using MGIT 960/TB eXiST. Results MGIT 960/TB eXiST gives results comparable to those of Bactec 460TB. Conclusions The MGIT 960/TB eXiST appears suitable for quantitative DST of NT

Evaluation of the AID ESBL line probe assay for rapid detection of extended-spectrum β-lactamase (ESBL) and KPC carbapenemase genes in Enterobacteriaceae

Objectives This study aimed at evaluating the AID ESBL line probe assay for the detection of extended-spectrum β-lactamase (ESBL) and KPC carbapenemase genes in Enterobacteriaceae. Methods The AID ESBL line probe assay was verified for accuracy of its probes using PCR products from clinical ESBL Enterobacteriaceae strains harbouring TEM, SHV and CTX-M ESBL genes and KPC genes and mutant fusion PCR products generated from Enterobacteriaceae strains containing wild-type (wt) TEM and wt SHV. Sensitivity and specificity was determined testing a set of 424 clinical Enterobacteriaceae strains (including 170 strains negative for TEM, SHV, CTX-M and KPC to evaluate the possibility of false positive signals). Results The line probe assay was shown to detect with 100% accuracy ESBL genes for which oligonucleotide probes are present in the assay. Testing a set of 424 clinical Enterobacteriaceae strains showed 100% sensitivity and specificity for the detection and differentiation of TEM, SHV and CTX-M ESBL genes present in that group. In addition, the line probe assay detected KPC genes accurately. Conclusions The AID ESBL line probe assay is an accurate and easy-to-use test for the detection of ESBL and KPC genes, which can readily be implemented in the diagnostic laborator

Erm(41)-dependent inducible resistance to azithromycin and clarithromycin in clinical isolates of Mycobacterium abscessus

Objectives The ribosomal methylase Erm(41) confers inducible resistance to macrolides in Mycobacterium abscessus. The aim of this work was to systematically study and compare drug susceptibility to clarithromycin and azithromycin in M. abscessus and Mycobacterium chelonae clinical isolates with a particular focus on inducible drug resistance. Methods Clinical isolates of M. abscessus subsp. abscessus (n = 21), M. abscessus subsp. bolletii (n = 16), M. abscessus subsp. massiliense (n = 10) and M. chelonae (n = 22) were characterized regarding their erm(41) and rrl genotypes and subjected to drug susceptibility testing (DST) for clarithromycin and azithromycin. Microdilution DST was performed in cation-adjusted Mueller-Hinton broth (pH 7.4) with readings at days 3, 7 and 12 and with pre-incubation at subinhibitory macrolide concentrations for erm(41) induction. In addition, the influence of variations in pH and growth medium on DST results was examined. Results MICs of azithromycin were consistently higher than those of clarithromycin. In strains with an inducible erm(41) gene, high median MICs of ≥256 mg/L on day 12 were observed for both clarithromycin and azithromycin. Inducible resistance was at least as pronounced for azithromycin as for clarithromycin. Conclusions Our findings do not support the suggestion of a preferential use of azithromycin over clarithromycin in order to limit inducible macrolide resistance. Both compounds provoked a comparable resistance phenotype in M. abscessus. Caution is needed when using either azithromycin or clarithromycin for treatment of M. abscessus infection

Stabilization of the genome of the mismatch repair deficient Mycobacterium tuberculosis by context-dependent codon choice

Background: The rate at which a stretch of DNA mutates is determined by the cellular systems for DNA replication and repair, and by the nucleotide sequence of the stretch itself. One sequence feature with a particularly strong influence on the mutation rate are nucleotide repeats. Some microbial pathogens use nucleotide repeats in their genome to stochastically vary phenotypic traits and thereby evade host defense. However, such unstable sequences also come at a cost, as mutations are often deleterious. Here, we analyzed how these opposing forces shaped genome stability in the human pathogen Mycobacterium tuberculosis. M. tuberculosis lacks a mismatch repair system, and this renders nucleotide repeats particularly unstable. Results: We found that proteins of M. tuberculosis are encoded by using codons in a context-dependent manner that prevents the emergence of nucleotide repeats. This context-dependent codon choice leads to a strong decrease in the estimated frame-shift mutation rate and thus to an increase in genome stability. Conclusion: These results indicate that a context-specific codon choice can partially compensate for the lack of a mismatch repair system, and helps to maintain genome integrity in this pathogen

Acquisition of clarithromycin resistance mutations in the 23S rRNA gene of Mycobacterium abscessus in the presence of inducible erm(41)

Objectives Antibiotic therapy of pulmonary Mycobacterium abscessus infection is based on a combination treatment including clarithromycin. Recent data demonstrated that M. abscessus may carry a chromosomal, inducible erm gene coding for the ribosomal methylase Erm(41). The purpose of this study was to investigate whether in patients with chronic M. abscessus infection undergoing clarithromycin therapy, M. abscessus acquires clarithromycin resistance mutations in the rrl gene in addition to the presence of an inducible Erm(41) methylase. Methods We determined clarithromycin MICs, erm(41) and rrl sequences for 29 clinical M. abscessus subsp. abscessus isolates of five different patients. The isolates were obtained between 2007 and 2011 covering a longitudinal observation period of 2-4 years for the individual patients. Results In three out of five patients with an initial rrl wild-type isolate, follow-up isolates demonstrated acquisition of resistance mutations in the rrl gene in addition to the presence of an inducible Erm methylase. Conclusions Our results show that in M. abscessus, clarithromycin resistance mutations in the 23S rRNA peptidyltransferase region provide an additional selective advantage independent of a functional erm(41) gen

Broad-Range 16S rRNA Gene Polymerase Chain Reaction for Diagnosis of Culture-Negative Bacterial Infections

This study defines the role of 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) for diagnosis of culture-negative bacterial infections. Our data show that 16S rRNA PCR is particularly valuable for identification of pathogens in patients pretreated with antibiotic

gyrA mutations and phenotypic susceptibility levels to ofloxacin and moxifloxacin in clinical isolates of Mycobacterium tuberculosis

Objectives To compare mutations in the quinolone resistance-determining region of the gyrA gene and flanking sequences with the MICs of ofloxacin and moxifloxacin for Mycobacterium tuberculosis. Methods The presence of mutations in 177 drug-resistant M. tuberculosis isolates was determined by DNA sequencing and the MICs quantified by MGIT 960. Results Single nucleotide polymorphisms were detected at codons 94 (n = 30), 90 (n = 12), 91 (n = 3), 89 (n = 1), 88 (n = 1) and 80 (n = 1). Four isolates with double mutations D94G plus A90V (n = 2) and D94G plus D94N (n = 2) reflect mixed populations. Agreement between genotypic and phenotypic susceptibility was high (≥97%) for both drugs. Mutant isolates had an MIC50 of 8.0 mg/L and an MIC90 of >10 mg/L for ofloxacin compared with an MIC50 and MIC90 of 2.0 mg/L for moxifloxacin. Codons 94 and 88 were linked to higher levels of fluoroquinolone resistance compared with codons 90, 91 and 89. The MIC distributions for the wild-type isolates ranged from ≤0.5 to 2.0 mg/L for ofloxacin and from ≤0.125 to 0.25 mg/L for moxifloxacin. However, 96% of the isolates with genetic alterations had MICs ≤2.0 mg/L for moxifloxacin, which is within its achievable serum levels. Conclusions This study provides quantitative evidence that the addition of moxifloxacin to extensively drug-resistant tuberculosis (XDR-TB) regimens based on a clinical breakpoint of 2.0 mg/L has merit. The use of moxifloxacin in the treatment of multidrug-resistant tuberculosis may prevent the acquisition of additional mutations and development of XDR-T