Griselda Tudó

Background: Drug susceptibility testing in Mycobacterium species typically requires prolonged incubation periods during which the chemical integrity of antibiotics may not be maintained, potentially compromising the reliability and accuracy of minimum inhibitory concentration (MIC) determinations. Objectives: This study evaluated the in vitro stability of several antibiotics, including recently introduced agents (bedaquiline [BDQ], pretomanid, delamanid and clofazimine) used for treating multidrug-resistant mycobacteriosis (linezolid and moxifloxacin), and those commonly included in combination regimens (rifampicin, isoniazid, ethambutol and clarithromycin). Methods: Antibiotics were pre-incubated at 37 °C before MIC determination and those exhibiting two or more dilutions in MIC were further tested in combination with ascorbic acid (AA) and pyruvate (P). Results: All antibiotics demonstrated stability except BDQ, which showed significant MIC variation after pre-incubation, which was prevented when BDQ was combined with AA and P. Conclusions: These findings suggest that the combined use of AA and P may serve as an effective stabilizing strategy for BDQ during MIC determination.

Objective: To analyze trends in resistance to antituberculous drugs over a 31-year period (1991–2022) at a hospital in Barcelona and to identify associated epidemiological determinants. Methods: This study included culture-confirmed tuberculosis cases diagnosed between 1991 and 2022. Drug susceptibility testing was conducted with clinical data from hospital records and epidemiological data from the Barcelona Public Health Agency. The primary outcome was resistance to first-line drugs. A subset of isolates was tested for second-line drugs. Trends were compared between the periods 1991–2000 and 2001–2022, aligning with increased immigration. Factors associated with resistance were examined using multivariate regression analysis. Results: Among the 2448 patients included, tuberculosis cases peaked in the 1990s and subsequently declined, while drug resistance increased. Overall, 12.2% of isolates showed resistance to at least one drug: 8.5% were monoresistant, 2.3% multiresistant, and 1.4% polyresistant. The 2001–2022 period had a higher resistance rate (OR 1.63; 95%CI 1.28–2.09) but lower multiresistance (OR 0.40; 95%CI 0.23–0.69). Resistance among new cases doubled from 6.4% to 12.8%, while rates among previously treated cases remained stable. The predictors of resistance were foreign-born (OR 1.52; 95%CI 1.21–1.91) and previous tuberculosis treatment (OR 2.88; 95%CI 2.17–3.81). A total of 90% of isolates remained susceptible to fluoroquinolones and aminoglycosides. Conclusions: Although tuberculosis incidence has declined over the past three decades, antibiotic resistance has increased, driven by foreign-born and retreatment cases. Ongoing drug susceptibility testing, access to second-line therapies, and targeted public health interventions for high-risk populations are essential to maintain control in low-incidence settings.

Spanish regulations and World Health Organization (WHO) guidelines recommend not using surgical masks for more than four hours. The high demand and low availability of surgical masks during the COVID-19 pandemic highlighted this as a major limitation. The objective of this study was to compare the bacterial filtration efficiency (BFE) between used surgical masks (eight hours) and unused masks (zero hours). 208 surgical masks worn for eight hours in a row were studied. They were placed on a culture plate containing blood agar (Agar Columbia with sheep blood plus, Thermo Scientific) and sprayed a suspension of Staphylococcus aureus (ATCC 29313) at a distance of thirty centimeters. Culture plates were incubated at 37 °C for twenty-four hours. A count of colony forming units (CFU) was carried out. As a control for BFE, new non-worn masks were included. A t-test and a z-test were used at a 95% confidence level to compare the means of the two groups. To establish the BFE, the CFU of the cultures from the used masks were compared with the control cultures without a mask. Likewise, the BFE of used masks was compared to that of new masks. The mean, median and mode values of the BFE of both masks, used and new ones, were greater than 99%. No statistically significant differences were detected between eight hours used masks and not used ones (zero hours). Surgical masks maintain a BFE equivalent to that new, unused surgical masks at eight hours.

Introduction: Mycobaterium avium complex (MAC) and Mycobacterium abscessus complex are the primary agents of non-tuberculous mycobacteria infection. However, other species within the slow-growing group can also be potentially pathogenic, although information on these species is limited.Objectives: We conducted a prospective analysis of slow-growing species other than MAC, aimed at the identification and microbiological profiles of clinical samples from a tertiary hospital. The Microbiology Department of the Hospital Clinic of Barcelona, the Microbiology Laboratory of SYNLAB Laboratories, and the Microbiology Laboratory of Hospital Sant Joan de Deu participated in the study.Methods: Species identification was conducted by MALDI-TOF MS and/or 16S rRNA and rpoB gene sequencing. Drug susceptibility tests (DST) were performed using the microdilution method. The results of the susceptibility profiles were compared with treatment guidelines, or the most recent literature related to each species.Results: Twenty-five different species belonging to the slow-growing group were identified. The most frequently observed were M. xenopi, M. kansasii, M. gordonae, and M. marinum. In this series, M. lentiflavum presented the highest susceptibility profile, while M. simiae demonstrated the highest level of resistance. Clarithromycin, rifabutin, and amikacin demonstrated high levels of effectiveness across all species. The species most associated with infection, presented a high correlation with the clinical treatment guidelines.Conclusion: A specific susceptibility profile was observed among all the species. The in vitro profiles of the most frequent species correlated with the clinical treatment guidelines, reinforcing the supporting role of DST in the design of individualized treatment for each patient.

Background/Objectives: Nontuberculous mycobacteria (NTM) infections are rising, particularly those by Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MAB). Treating NTM infections is challenging due to their poor response to antibiotics. This study aimed to optimize the treatment of NTM infection by selecting antibiotics with bactericidal activity for combination therapy. To do this, we used the minimum bactericidal concentration (MBC) determination approach to define bactericidal or bacteriostatic activity. We developed three main objectives: validate a new method to determine MBC based on a reincubation method, determine MBC values of 229 NTM clinical isolates using the reincubation method, and evaluate antibiotic stability in preincubated microtiter plates. Methods: First, we assessed the stability of the antibiotics included in SLOWMYCOI Sensititre™ microtiter plates. Five strains of MAC were studied comparing the minimum inhibitory concentrations (MICs) of those preincubated for seven days vs. non-incubated plates. Then, we evaluated the percentage of reproducibility of MBC values using two methods, reincubation and subculturing (standard or traditional method) in 30 MAC isolates. Finally, we validated the reincubation method and prospectively determined the MBC values of the 229 NTM clinical strains. Results: Antibiotic stability: The MIC was equivalent after 7 and 14 days for all the antibiotics, except rifampicin, for which the MIC increased by 2- to 3-fold after preincubation. Reincubation method: The percentage of reproducibility of the MBC values between the two methods was 95.2% (range 76.6% to 100%). Prospective validation: MBC/MIC ratios revealed differential bactericidal activity for most antibiotics according to the different species, being bactericidal in M. avium and Mycobacterium xenopi, and predominantly bacteriostatic in MAB. Conclusions: Preincubation of Sensititre™ microtiter plates did not alter the MIC values of the antibiotics included except for rifampicin, suggesting a loss of activity. MBC determination can be easily performed by the Reincubation method presented. MBC values provide useful additional information regarding MIC values since the MBC/MIC ratio reveals whether antibiotics have bactericidal or bacteriostatic activity according to the species, which is pivotal for selecting the most adequate antibiotic combination to ensure efficient treatment management.

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.

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.

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.