[Application of mass spectrometry in mycobacteria]. / Aplicación de la espectrometría de masas en micobacterias.

To date, more than 170 species of mycobacteria have been described, of which more than one third may be pathogenic to humans, representing a significant workload for microbiology laboratories. These species must be identified in clinical practice, which has long been a major problem due to the shortcomings of conventional (phenotypic) methods and the limitations and complexity of modern methods largely based on molecular biology techniques. The aim of this review was to briefly describe different aspects related to the use of MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry (MS) for the identification of mycobacteria. Several difficulties are encountered with the use of this methodology in these microorganisms mainly due to the high pathogenicity of some mycobacteria and the peculiar structure of their cell wall, requiring inactivation and special protein extraction protocols. We also analysed other relevant aspects such as culture media, the reference methods employed (gold standard) in the final identification of the different species, the cut-off used to accept data as valid, and the databases of the different mass spectrometry systems available. MS has revolutionized diagnosis in modern microbiology; however, specific improvements are needed to consolidate the use of this technology in mycobacteriology.

Oleoyl coenzyme A regulates interaction of transcriptional regulator RaaS (Rv1219c) with DNA in mycobacteria.

We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria.

Antimicrobial treatment improves mycobacterial survival in nonpermissive growth conditions.

Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically, we found that under nonpermissive growth conditions, exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, RaaS (for regulator of antimicrobial-assisted survival), encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death under nonpermissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases, such as tuberculosis.

Characterization of the embB gene in Mycobacterium tuberculosis isolates from Barcelona and rapid detection of main mutations related to ethambutol resistance using a low-density DNA array.

OBJECTIVES: Ethambutol resistance has mostly been related to mutations in the embB gene. The objective of the present study was to characterize the embB gene in a collection of ethambutol-resistant and ethambutol-susceptible isolates of Mycobacterium tuberculosis complex (MTBC) from Barcelona, and to develop a DNA microarray for the rapid detection of embB mutations in our area. METHODS: Fifty-three ethambutol-resistant and 702 ethambutol-susceptible isolates of MTBC were sequenced in internal 982-1495 bp fragments of the embB gene. In addition, a low-cost, low-density array was designed to include the embB codons identified as being most frequently mutated in our area (LD-EMB array). RESULTS: The global prevalence of embB mutations found among the ethambutol-resistant isolates was 77.4% (41/53). Substitutions in embB306 were the most common [53.7% (22/41)], followed by substitutions in embB406 [26.8% (11/41)]. The presence of mutations in embB406 was related to higher levels of ethambutol resistance and to multidrug resistance. Among unrelated isolates (from 24-locus MIRU-VNTR genotyping), the percentage of embB-mutated isolates was 72.9% (27/37)--59.3% (16/27) in embB306 and 25.9% (7/27) in embB406. None of the ethambutol-susceptible isolates studied showed a mutation in codon 306 or 406. The LD-EMB array showed 100% sensitivity and specificity in identifying the main embB substitutions in our area. CONCLUSIONS: Mutations at codons 306 and 406 of embB have a relevant role in resistance to ethambutol in our area. The LD-EMB array developed in this study would appear to be a good molecular test for rapid detection of ethambutol resistance.

In Vitro Effect of Three-Antibiotic Combinations plus Potential Antibiofilm Agents against Biofilm-Producing Mycobacterium avium and Mycobacterium intracellulare Clinical Isolates.

Patients with chronic pulmonary diseases infected by Mycobacterium avium complex (MAC) often develop complications and suffer from treatment failure due to biofilm formation. There is a lack of correlation between in vitro susceptibility tests and the treatment of clinical isolates producing biofilm. We performed susceptibility tests of 10 different three-drug combinations, including two recommended in the guidelines, in biofilm forms of eight MAC clinical isolates. Biofilm developed in the eight isolates following incubation of the inoculum for 3 weeks. Then, the biofilm was treated with three-drug combinations with and without the addition of potential antibiofilm agents (PAAs). Biofilm bactericidal concentrations (BBCs) were determined using the Vizion lector system. All selected drug combinations showed synergistic activity, reducing BBC values compared to those treated with single drugs, but BBC values remained high enough to treat patients. However, with the addition of PAAs, the BBCs steadily decreased, achieving similar values to the combinations in planktonic forms and showing synergistic activity in all the combinations and in both species. In conclusion, three-drug combinations with PAAs showed synergistic activity in biofilm forms of MAC isolates. Our results suggest the need for clinical studies introducing PAAs combined with antibiotics for the treatment of patients with pulmonary diseases infected by MAC.

Activity of Antibiotics and Potential Antibiofilm Agents against Biofilm-Producing Mycobacterium avium-intracellulare Complex Causing Chronic Pulmonary Infections.

Nontuberculous mycobacteria (NTM) cause lung infections in patients with underlying pulmonary diseases (PD). The Mycobacteriumavium-intracellulare complex (MAC) is the most frequently involved NTM. The MAC-PD treatment is based on the administration of several antibiotics for long periods of time. Nonetheless, treatment outcomes remain very poor. Among the factors involved is the ability of MAC isolates to form biofilm. The aim of the study was to assess the in vitro activity of different antibiotics and potential antibiofilm agents (PAAs) against MAC biofilm. Four antibiotics and six PAAs, alone and/or in combination, were tested against planktonic forms of 11 MAC clinical isolates. Biofilm was produced after 4 weeks of incubation and analyzed with the crystal violet assay. The antibiotics and PAAs were tested by measuring the absorbance (minimum biofilm inhibition concentrations, MBICs) and by performing subcultures (minimum biofilm eradication concentrations, MBECs). The clarithromycin/amikacin and clarithromycin/ethambutol combinations were synergistic, decreasing the MBECs values compared to the individual antibiotics. The amikacin/moxifloxacin combination showed indifference. The MBIC values decreased significantly when PAAs were added to the antibiotic combinations. These results suggest that antibiotic combinations should be further studied to establish their antibiofilm activity. Moreover, PAAs could act against the biofilm matrix, facilitating the activity of antibiotics.

Differences in Drug-Susceptibility Patterns between Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium chimaera Clinical Isolates: Prospective 8.5-Year Analysis by Three Laboratories.

Background: It has been suggested that Mycobacterium avium, Mycobacterium intracellulare, and M. chimaera have differential drug susceptibility patterns. We prospectively analyzed and compared the drug susceptibility patterns among these species over an 8.5-year period. Methods: A microdilution method (Slomyco®) was performed for drug susceptibility testing of 402 M. avium, 273 M. intracellulare, and 139 M. chimaera clinical isolates. Results: M. avium showed significantly higher resistance to moxifloxacin, ciprofloxacin, rifampicin, ethambutol, streptomycin, linezolid, cotrimoxazole, and clarithromycin. M. avium also showed higher minimum inhibitory concentrations (MIC) than M. intracellulare and M. chimaera against all drugs except ethionamide, to which M. intracellulare and M. chimaera showed greater resistance. Conclusions: Our series demonstrated differential drug resistance patterns among the most frequent M. avium complex species. M. avium was more resistant than M. intracellulare and M. chimaera versus eight antibiotics and showed greater MIC values to most of the antibiotics studied. These data suggest that knowledge of the local distribution and susceptibility profiles of these pathogens is essential for adequate clinical management.

Multicentre study on the reproducibility of MALDI-TOF MS for nontuberculous mycobacteria identification.

The ability of MALDI-TOF for the identification of nontuberculous mycobacteria (NTM) has improved recently thanks to updated databases and optimized protein extraction procedures. Few multicentre studies on the reproducibility of MALDI-TOF have been performed so far, none on mycobacteria. The aim of this study was to evaluate the reproducibility of MALDI-TOF for the identification of NTM in 15 laboratories in 9 European countries. A total of 98 NTM clinical isolates were grown on Löwenstein-Jensen. Biomass was collected in tubes with water and ethanol, anonymized and sent out to the 15 participating laboratories. Isolates were identified using MALDI Biotyper (Bruker Daltonics). Up to 1330 MALDI-TOF identifications were collected in the study. A score ≥ 1.6 was obtained for 100% of isolates in 5 laboratories (68.2-98.6% in the other). Species-level identification provided by MALDI-TOF was 100% correct in 8 centres and 100% correct to complex-level in 12 laboratories. In most cases, the misidentifications obtained were associated with closely related species. The variability observed for a few isolates could be due to variations in the protein extraction procedure or to MALDI-TOF system status in each centre. In conclusion, MALDI-TOF showed to be a highly reproducible method and suitable for its implementation for NTM identification.

Impaired fitness of Mycobacterium tuberculosis resistant isolates in a cell culture model of murine macrophages.

OBJECTIVES: We analysed the ability of Mycobacterium tuberculosis clinical isolates to penetrate and grow inside murine macrophages as a surrogate of fitness. METHODS: Thirty-five drug-resistant and 10 drug-susceptible M. tuberculosis isolates were studied in a murine macrophage model from the J774.2 cell line in a 6 day protocol, performing semi-quantitative counts in Middlebrook 7H11 medium. The mycobacterial penetration index (MPI) after infection and the mycobacterial growth ratio (MGR) inside the macrophages were determined to evaluate the fitness of isolates. RESULTS: Isolates with the katG S315T mutation and multidrug-resistant (MDR) isolates had a significantly lower MGR compared with drug-susceptible isolates. The MPI of the isolates with the katG S315T mutation showed a significant decrease compared with the MPI of those without this mutation. A trend to significantly lower values was also observed on comparing the MPI of the MDR isolates with that of the drug-susceptible isolates and the isolates resistant to isoniazid. CONCLUSIONS: The isoniazid-resistant and MDR isolates with mutations in the katG gene showed decreased multiplication inside murine macrophages, suggesting a lower fitness of M. tuberculosis with these resistance patterns.

Comparison of two-drug combinations, amikacin/tigecycline/imipenem and amikacin/tigecycline/clarithromycin against Mycobacteroides abscessus subsp. abscessus using the in vitro time-kill assay.

Nontuberculous mycobacteria include 198 mycobacterial species. Among these, Mycobacteroides abscessus is a rapidly growing mycobacteria that causes lung and skin infections. M. abscessus lung infections are difficult to treat due to the high levels of resistance to several classes of antibiotics. The current treatment is based on combining at least two or three antibiotics. However, treatment outcomes remain very poor. The objective was to compare the in vitro activity of amikacin, tigecycline, imipenem, and clarithromycin, alone and in two different three-drug combinations (amikacin/tigecycline/imipenem and amikacin/tigecycline/clarithromycin) against seven M. abscessus subsp. abscessus clinical isolates using the time-kill assay. The two combinations showed greater activity than the antibiotics tested individually. Even though both combinations showed similar activity as well as no antagonistic activity, the combination including imipenem could not be an alternative treatment against M. abscessus subsp. abscessus lung infections caused by clarithromycin susceptible isolates. However, this combination could be considered against clarithromycin resistant isolates. Future studies are necessary to confirm this hypothesis.