In vitro activity of cefoxitin, imipenem, meropenem, and ceftaroline in combination with vaborbactam against Mycobacterium abscessus.

Mycobacterium abscessus (MAB) infections pose a growing public health threat. Here, we assessed the in vitro activity of the boronic acid-based ß-lactamase inhibitor, vaborbactam, with different ß-lactams against 100 clinical MAB isolates. Enhanced activity was observed with meropenem and ceftaroline with vaborbactam (1- and >4-fold MIC50/90 reduction). CRISPRi-mediated blaMAB gene knockdown showed a fourfold MIC reduction to ceftaroline but not the other ß-lactams. Our findings demonstrate vaborbactam's potential in combination therapy against MAB infections.

Prospective healthcare-associated links in transmission of nontuberculous mycobacteria among people with cystic fibrosis (pHALT NTM) study: Rationale and study design.

BACKGROUND: Healthcare-associated acquisition and transmission of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) has been described, and remains a concern for both patients and providers. This report describes the design of a prospective observational study utilizing the standardized epidemiologic investigation toolkit for healthcare-associated links in transmission of NTM among pwCF. METHODS: This is a parallel multi-site study of pwCF who have infections with respiratory NTM isolates and receive healthcare within a common CF Care Center. Participants have a history of one or more NTM positive airway cultures and have been identified as having NTM infections suggestive of a possible outbreak within a single Center, based on NTM isolate genomic analysis. Participants are enrolled in the study over a 3-year period. Primary endpoints are identification of shared healthcare-associated source(s) among pwCF in a Center, identification of healthcare environmental dust and water biofilm NTM isolates that are genetically highly-related to respiratory isolates, and identification of common home of residence watersheds among pwCF infected with clustered isolates. Secondary endpoints include characterization of healthcare-associated transmission and/or acquisition modes and settings as well as description of incidence and prevalence of healthcare-associated environmental NTM species/subspecies by geographical region. DISCUSSION: We hypothesize that genetically highly-related isolates of NTM among pwCF cared for at the same Center may arise from healthcare sources including patient-to-patient transmission and/or acquisition from health-care environmental dust and/or water biofilms. This study design utilizes a published, standardized, evidence-based epidemiologic toolkit to facilitate confidential, independent healthcare-associated NTM outbreak investigations within CF Care Centers. This study will facilitate real-time, rapid detection and mitigation of healthcare-associated NTM outbreaks to reduce NTM risk, inform infection prevention and control guidelines, and characterize the prevalence and origin of NTM outbreaks from healthcare-associated patient-to-patient transmission and/or environmental acquisition. This study will systematically characterize human disease causing NTM isolates from serial collection of healthcare environmental dust and water biofilms and define the most common healthcare environmental sources harboring NTM biofilms. TRIAL REGISTRATION: ClinicalTrials.gov NCT05686837.

In vitro susceptibility patterns for slowly growing non-tuberculous mycobacteria in the USA from 2018 to 2022.

BACKGROUND: Treatment of slowly growing non-tuberculous mycobacteria (SGM) is challenging. In vitro antimicrobial susceptibility testing (AST) is needed to optimize a multidrug regimen but requires weeks to result. Aggregated AST patterns, or an antibiogram, of SGM would be helpful to providers. OBJECTIVES: We aggregated and analysed human SGM isolates sent to our laboratory from across the USA between 2018 and 2022 to describe their in vitro susceptibility patterns and construct an antibiogram. METHODS: SGM isolates' species/subspecies and mutations in rrs or rrl were identified by a line probe assay. AST was done primarily by broth microdilution and interpreted using the latest CLSI guideline. Mutational and AST results for SGM with ≥15 isolates were collated and analysed with descriptive statistics. RESULTS: There were 32 different species/subspecies of SGM from 10 131 isolates between January 2018 and December 2022 from across the USA, 80% of which were from organisms in Mycobacterium avium complex (MAC). Most specimens were sputum and came from Florida (2892). MAC ranged from 94% to 100% susceptible to clarithromycin, 64% to 91% to amikacin, 2% to 31% to linezolid, and 4% to 41% to moxifloxacin. Non-MAC SGM ranged from 82% to 100% susceptible to clarithromycin, 49% to 100% to amikacin, and 76% to 100% to rifabutin, but susceptibilities to other antimicrobials varied widely. WT rrs and rrl predicted >96% of phenotypic non-resistance to amikacin and clarithromycin, respectively, whereas mutant genotypes predicted >90% of phenotypic resistance. CONCLUSIONS: Most SGM are likely to be susceptible to clarithromycin and amikacin, complementing their treatment guidance by mycobacterial experts. Molecular identification of resistant genotypes is accurate and helpful. This antibiogram for SGM will help providers.

Molecular Epidemiologic Investigation of Mycobacterium intracellulare Subspecies chimaera Lung Infections at an Adult Cystic Fibrosis Program.

Rationale: Outbreaks of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) have been reported at CF centers with conflicting conclusions. The occurrence of NTM at the UVMC (University of Vermont Medical Center) adult CF program was investigated. Objectives: Use the HALT NTM (Healthcare-associated Links in Transmission of NTM) toolkit to investigate the healthcare-associated transmission and/or acquisition of NTM among pwCF having genetically similar NTM isolates. Methods: Whole genome sequencing of NTM isolates from 23 pwCF was conducted to identify genetically similar NTM isolate clusters (30 or fewer single-nucleotide polymorphism differences). The epidemiological investigation, comparison of respiratory and healthcare environmental isolates, and home residence watershed mapping were analyzed. Results: Whole genome sequencing analysis revealed two clusters of NTM isolates (Mycobacterium avium and M. intracellulare ssp. chimaera) among pwCF. The epidemiologic investigation demonstrated opportunities for healthcare-associated transmission within both clusters. Healthcare environmental M. avium isolates revealed no genetic similarity to respiratory isolates. However, M. intracellulare ssp. chimaera respiratory isolates revealed greater genetic similarity to a hospital water biofilm isolate than to each other. Neither cluster had all subjects residing in the same watershed. Conclusions: This study suggests the healthcare-associated transmission of M. avium among pwCF is unlikely at UVMC but supports the healthcare-associated environmental acquisition of M. intracellulare ssp. chimaera. The presence of genetically similar isolates alone is insufficient to confirm healthcare-associated transmission and/or acquisition. The HALT NTM toolkit standardizes outbreak investigation with genetic analysis, epidemiologic investigation, healthcare environmental sampling, and home of residence watershed identification to test the frequency and nature of healthcare-associated NTM transmission among pwCF.

Investigating Nontuberculous Mycobacteria Transmission at the Colorado Adult Cystic Fibrosis Program.

Rationale: Healthcare-associated transmission of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) has been investigated at CF centers worldwide, with conflicting conclusions. We investigated transmission at the Colorado Adult CF Program. Objectives: To systematically investigate healthcare-associated transmission and/or acquisition of NTM to determine similarity among respiratory and environmental isolates, and to compare home residence watershed mapping among pwCF having genetically similar NTM isolates. Methods: Whole-genome sequencing of NTM isolates from 80 pwCF was conducted to identify genetically similar isolate clusters (⩽30 SNP differences). Epidemiology, comparison of respiratory and environmental isolates, and home residence watershed mapping were analyzed. Measurements and Main Results: Whole-genome sequencing analysis revealed 11 clusters of NTM [6 Mycobacterium abscessus subspecies (ssp.) abscessus, 1 M. abscessus ssp. massiliense, 2 Mycobacterium avium, and 2 Mycobacterium intracellulare] among pwCF. Epidemiologic investigation demonstrated opportunities for healthcare-associated transmission in two M. abscessus and two M. avium clusters. Respiratory and healthcare environmental isolate comparisons revealed no genetic similarity. Individuals comprising one M. abscessus cluster, with no plausible healthcare-associated transmission, resided in the same watershed. Conclusions: This study suggests healthcare-associated transmission of M. abscessus is rare and includes a report of potential healthcare-associated transmission of M. avium among pwCF. One M. abscessus cluster possibly had common acquisition arising from residing in the same watershed. The presence of genetically similar isolates is insufficient to demonstrate healthcare-associated NTM transmission. Standardizing epidemiologic investigation, combined with environmental sampling and watershed analysis, will improve understanding of the frequency and nature of healthcare-associated NTM transmission among pwCF.

MmpL3 as a Target for the Treatment of Drug-Resistant Nontuberculous Mycobacterial Infections.

Nontuberculous mycobacterial (NTM) pulmonary infections are emerging as a global health problem and pose a threat to susceptible individuals with structural or functional lung conditions such as cystic fibrosis, chronic obstructive pulmonary disease and bronchiectasis. Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC) species account for 70-95% of the pulmonary NTM infections worldwide. Treatment options for these pathogens are limited, involve lengthy multidrug regimens of 12-18 months with parenteral and oral drugs, and their outcome is often suboptimal. Development of new drugs and improved regimens to treat NTM infections are thus greatly needed. In the last 2 years, the screening of compound libraries against M. abscessus in culture has led to the discovery of a number of different chemotypes that target MmpL3, an essential inner membrane transporter involved in the export of the building blocks of the outer membrane of all mycobacteria known as the mycolic acids. This perspective reflects on the therapeutic potential of MmpL3 in Mycobacterium tuberculosis and NTM and the possible reasons underlying the outstanding promiscuity of this target. It further analyzes the physiological and structural factors that may account for the apparent looser structure-activity relationship of some of these compound series against M. tuberculosis compared to NTM.

Design, synthesis and evaluation of indole-2-carboxamides with pan anti-mycobacterial activity.

Current treatment regimens for non-tuberculous mycobacteria (NTM) and tuberculosis (TB) generally require long duration of therapy with multiple drugs, some of which are broad spectrum antibiotics. Despite some advances in antimicrobial compounds, there remains a need in therapy for antibiotics with specific mycobacterial targets. It has been shown that MmpL3 is an essential transporter required for the translocation of mycolic acids to the mycobacterial cell envelope. Here, we synthesized a series of indole-2-carboxamides that inhibit MmpL3 and have potent pan-activity against mycobacterial species. The compounds were tested against several fast and slow-growing Mycobacterium species, including M. abscessus, M. massiliense, M. bolletii, M. chelonae, M. tuberculosis, M. avium, M. xenopi and M. smegmatis. The target of these indole-based compounds makes them selective for mycobacteria, while showing no clinically relevant bactericidal activity against S. aureus or P. aeruginosa. These compounds were tested against THP-1, a human-cell line, and showed minimal in vitro cytotoxicity and good selectivity indices. The data shown and discussed suggest that lead indole-2-carboxamides are strong contenders for further preclinical testing as NTM therapeutics.

Gene replacement in Mycobacterium chelonae: application to the construction of porin knock-out mutants.

Mycobacterium chelonae is a rapidly growing mycobacterial opportunistic pathogen closely related to Mycobacterium abscessus that causes cornea, skin and soft tissue infections in humans. Although M. chelonae and the emerging mycobacterial pathogen M. abscessus have long been considered to belong to the same species, these two microorganisms considerably differ in terms of optimum growth temperature, drug susceptibility, pathogenicity and the types of infection they cause. The whole genome sequencing of clinical isolates of M. chelonae and M. abscessus is opening the way to comparative studies aimed at understanding the biology of these pathogens and elucidating the molecular bases of their pathogenicity and biocide resistance. Key to the validation of the numerous hypotheses that this approach will raise, however, is the availability of genetic tools allowing for the expression and targeted mutagenesis of genes in these species. While homologous recombination systems have recently been described for M. abscessus, genetic tools are lacking for M. chelonae. We here show that two different allelic replacement methods, one based on mycobacteriophage-encoded recombinases and the other on a temperature-sensitive plasmid harboring the counterselectable marker sacB, can be used to efficiently disrupt genes in this species. Knock-out mutants for each of the three porin genes of M. chelonae ATCC 35752 were constructed using both methodologies, one of which displays a significantly reduced glucose uptake rate consistent with decreased porin expression.