Multicenter Evaluation of an MIC-Based Aztreonam and Ceftazidime-Avibactam Broth Disk Elution Test.

Due to limited therapeutic options, there is a clinical need to assess the in vitro activity of the combination of aztreonam (ATM) and ceftazidime-avibactam (CZA) to guide the therapeutic management of multidrug-resistant (MDR) Gram-negative organism infections. We set out to develop a practical MIC-based broth disk elution (BDE) method to determine the in vitro activity of the combination ATM-CZA using readily available supplies and compare it to reference broth microdilution (BMD). For the BDE method, a 30-µg ATM disk, a 30/20-µg CZA disk, both disks in combination, and no disks were added to 4 separate 5-mL cation-adjusted Mueller-Hinton broth (CA-MHB) tubes, using various manufacturers. Three testing sites performed both BDE and reference BMD testing of bacterial isolates in parallel from a single 0.5 McFarland standard inoculum and after overnight incubation, assessed them for growth (not susceptible) or no growth (susceptible) at a final concentration of 6/6/4 µg/mL ATM-CZA. During the first phase, the precision and accuracy of the BDE were analyzed by testing 61 Enterobacterales isolates at all sites. This testing yielded 98.3% precision between sites, with 98.3% categorical agreement and 1.8% major errors (ME). During the second phase, at each site, we evaluated unique, clinical isolates of metallo-ß-lactamase (MBL)-producing Enterobacterales (n = 75), carbapenem-resistant Pseudomonas aeruginosa (n = 25), Stenotrophomonas maltophilia (n = 46), and Myroides sp. (n = 1). This testing resulted in 97.9% categorical agreement, with 2.4% ME. Different results were observed for different disk and CA-MHB manufacturers, requiring a supplemental ATM-CZA-not-susceptible quality control organism to ensure the accuracy of results. The BDE is a precise and effective methodology for determining susceptibility to the combination ATM-CZA.

Functional whole genome screen of nutrient-starved Mycobacterium tuberculosis identifies genes involved in antibiotic tolerance.

Mycobacterium tuberculosis ( Mtb ), the causative agent of tuberculosis (TB), poses a global health challenge and is responsible for over a million deaths each year. Current treatment is lengthy and complex, and new, abbreviated regimens are urgently needed. Mtb adapts to nutrient starvation, a condition experienced during host infection, by shifting its metabolism and becoming tolerant to the killing activity of bactericidal antibiotics. An improved understanding of the mechanisms mediating antibiotic tolerance in Mtb can serve as the basis for developing more effective therapies. We performed a forward genetic screen to identify candidate Mtb genes involved in tolerance to the two key first-line antibiotics, rifampin and isoniazid, under nutrient-rich and nutrient-starved conditions. In nutrient-rich conditions, we found 220 mutants with differential antibiotic susceptibility (218 in the rifampin screen and 2 in the isoniazid screen). Following Mtb adaptation to nutrient starvation, 82 mutants showed differential antibiotic susceptibility (80 in the rifampin screen and 2 in the isoniazid screen). Using targeted mutagenesis, we validated the rifampin-hypersusceptible phenotype under nutrient starvation in Mtb mutants lacking the following genes: ercc3 , moeA1 , rv0049 , and rv2179c . These findings shed light on potential therapeutic targets, which could help shorten the duration and complexity of antitubercular regimens. Importance: Treatment of Mtb infection requires a long course of combination antibiotics, likely due to subpopulations of tolerant bacteria exhibiting decreased susceptibility to antibiotics. Identifying and characterizing the genetic pathways involved in antibiotic tolerance is expected to yield therapeutic targets for the development of novel TB treatment-shortening regimens.

Functional Whole Genome Screen of Nutrient-Starved Mycobacterium tuberculosis Identifies Genes Involved in Rifampin Tolerance.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), poses a global health challenge and is responsible for over a million deaths each year. Current treatment is lengthy and complex, and new, abbreviated regimens are urgently needed. Mtb adapts to nutrient starvation, a condition experienced during host infection, by shifting its metabolism and becoming tolerant to the killing activity of bactericidal antibiotics. An improved understanding of the mechanisms mediating antibiotic tolerance in Mtb can serve as the basis for developing more effective therapies. We performed a forward genetic screen to identify candidate Mtb genes involved in tolerance to the two key first-line antibiotics, rifampin and isoniazid, under nutrient-rich and nutrient-starved conditions. In nutrient-rich conditions, we found 220 mutants with differential antibiotic susceptibility (218 in the rifampin screen and 2 in the isoniazid screen). Following Mtb adaptation to nutrient starvation, 82 mutants showed differential antibiotic susceptibility (80 in the rifampin screen and 2 in the isoniazid screen). Using targeted mutagenesis, we validated the rifampin-hypersusceptible phenotype under nutrient starvation in Mtb mutants lacking the following genes: ercc3, moeA1, rv0049, and rv2179c. These findings shed light on potential therapeutic targets, which could help shorten the duration and complexity of antitubercular regimens.