Increasing antibiotic resistance in preservative-tolerant bacterial strains isolated from cosmetic products.

To ensure the microbiological quality, consumer safety and organoleptic properties of cosmetic products, manufacturers need to comply with defined standards using several preservatives and disinfectants. A drawback regarding the use of these preservatives is the possibility of generating cross-insusceptibility to other disinfectants or preservatives, as well as cross resistance to antibiotics. Therefore, the objective of this study was to understand the adaptive mechanisms of Enterobacter gergoviae, Pseudomonas putida and Burkholderia cepacia that are involved in recurrent contamination in cosmetic products containing preservatives. Diminished susceptibility to formaldehyde-donors was detected in isolates but not to other preservatives commonly used in the cosmetics industry, although increasing resistance to different antibiotics (ß-lactams, quinolones, rifampicin, and tetracycline) was demonstrated in these strains when compared with the wild-type strain. The outer membrane protein modifications and efflux mechanism activities responsible for the resistance trait were evaluated. The development of antibiotic-resistant microorganisms due to the selective pressure from preservatives included in cosmetic products could be a risk for the emergence and spread of bacterial resistance in the environment. Nevertheless, the large contribution of disinfection and preservation cannot be denied in cosmetic products.

Molecular analysis of isoniazid and rifampin resistance in Mycobacterium tuberculosis isolates recovered from Barcelona.

We studied the presence of mutations in the whole katG gene and specific regions of the oxyR-ahpC and mabA-inhA regulatory region in 61 Mycobacterium tuberculosis isoniazid-resistant isolates. An 81-bp region of the rpoB gene was also sequenced in 17 rifampin-resistant strains. Alterations in the katG gene were detected in 55% of the isolates. Mutation in codon 315 was the most prevalent (32%). Strains showed a high level of resistance, and most maintained a substantial catalase-peroxidase activity. Three strains with an isoniazid MIC of >or=32 microg/ml lacked catalase-peroxidase activity. Two of them had deletions in the catalytic domain of the KatG protein. One strain with deletion and three strains with mutations in the C-terminal domain showed low-level resistance and conserved the catalase-peroxidase activity. Mutations in the mabA-inhA regulatory region were identified in 32% of the isolates. All had low-level resistance, and the vast majority conserved catalase-peroxidase activity. Seventeen percent of the isoniazid-resistant isolates had no detectable alterations at the studied loci. Resistance to rifampin was associated with mutations in the 81-bp of the rpoB gene in all cases. IS6110 analysis indicated that recent transmission contributed substantially to the emergence of isoniazid- resistant tuberculosis in Barcelona through short transmission chains. A rapid genotypic assay, including the 315-katG codon and the -15 nucleotide of the mabA-inhA regulatory region, may cover 62% of isoniazid- resistant strains in Barcelona. In contrast, the targeting of the 81-bp region of rpoB would detect all our rifampin-resistant isolates.

Direct detection in clinical samples of multiple gene mutations causing resistance of Mycobacterium tuberculosis to isoniazid and rifampicin using fluorogenic probes.

BACKGROUND: This study evaluates a method based on real-time PCR for direct detection in clinical samples of the common mutations responsible for isoniazid and rifampicin resistance of Mycobacterium tuberculosis. METHODS: Six pairs of fluorogenic 5' exonuclease probes (Taqman), mutated and wild-type, were designed for six targets: codon 315 of katG, substitution C209T in the regulatory region of inhA, and codons 513, 516, 526 and 531 of rpoB. RESULTS: A total of 98 clinical samples harbouring resistant bacilli from 55 patients and 126 samples harbouring susceptible bacilli from 126 patients were processed. The isolates from samples were tested for drug susceptibility with the radiometric method and sequenced for the same genetic targets. Among the samples, 93 harboured isoniazid-resistant bacilli. According to the sequencing results, 30 had mutations in katG, 30 in inhA and 33 (35.4%) had no mutations in these targets. All 27 clinical specimens harbouring rifampicin-resistant bacilli showed mutations in rpoB. The detection threshold of this method in detecting target genes in serial dilutions of artificial samples was 1.5 x 10(3) cfu/mL. In clinical samples, the sensitivity ranged from 30.4 to 35.3% for smear-negative samples and from 95.1 to 99.2% for smear-positive samples, with a specificity of 100%. In this study, the overall sensitivity in detecting patients having the target mutations was 74.3%. CONCLUSIONS: The main advantage of the described method is the possibility of detecting rifampicin and isoniazid resistance within 48-72 h after sample collection, with a sensitivity of nearly 100% in smear-positive samples if the chosen target is responsible for the resistance.

Usefulness of a new mycobacteriophage-based technique for rapid diagnosis of pulmonary tuberculosis.

A new mycobacteriophage-based technique (PhageTek MB) was compared with standard culture and staining techniques for diagnosis of pulmonary tuberculosis. A total of 2,048 respiratory specimens from 1,466 patients collected from February 2000 to March 2001 were studied by both (i) conventional methods (direct microscopic examination [auramine-rhodamine fluorochrome], and culture in BacT/ALERT 3D and solid media) and (ii) the PhageTek MB assay. This phenotypic test utilizes specific mycobacteriophages to detect the presence of live Mycobacterium tuberculosis complex organisms within a decontaminated clinical sample. Overall, 205 (10%) specimens were positive for mycobacteria (134 patients): 144 (70.2%) M. tuberculosis isolates and 61 (29.8%) nontuberculous mycobacterium isolates (30 Mycobacterium kansasii, 12 Mycobacterium xenopi, 9 Mycobacterium gordonae, 7 Mycobacterium avium complex, 2 Mycobacterium chelonae, and 1 Mycobacterium fortuitum isolate). PhageTek MB was more likely to give a positive result with specimens in which high numbers of acid-fast bacilli were observed on the smear. The sensitivity, specificity, and positive and negative predictive values of this mycobacteriophage-based technique versus culture for M. tuberculosis were 58.3, 99.1, 83.2, and 96.9%, respectively. PhageTek MB is a rapid (48-h), specific, safe, and easy-to-perform test. According to the prevalence of the disease in the population studied, the test would require improved sensitivity in order to be used as a screening test for routine diagnosis of respiratory tuberculosis in our setting.