Complete Genome Sequences of Four Mycobacteriophages Involved in Directed Evolution against Undisputed Mycobacterium abscessus Clinical Strains.

Phage therapy is still in its infancy, but it is increasingly promising as a future alternative for treating antibiotic-resistant bacteria. To investigate the effect of phages on Mycobacterium abscessus complex (MABC), we isolated 113 environmental phages, grown them to high titres, and assayed them on MABC clinical strains through the spot test. Of all the phages, only 16 showed killing activity. Their activity was so temperate to MABC that they could not generate any plaque-forming units (PFUs). The Appelmans method of directed evolution was carried out to evolve these 16 phages into more lytic ones. After only 11 of 30 rounds of evolution, every single clinical strain in our collection, including those that were unsusceptible up to this point, could be lysed by at least one phage. The evolved phages were able to form PFUs on the clinical strains tested. Still, they are temperate at best and require further training. The genomes of one random parental phage and three random evolved phages from Round 13 were sequenced, revealing a diversity of clusters and genes of a variety of evolutionary origins, mostly of unknown function. These complete annotated genomes will be key for future molecular characterisations.

Analysis of Phenotypic and Genotypic Susceptibility to Clarithromycin and Amikacin of Mycobacterium abscessus Complex Strains Isolated from Cystic Fibrosis Patients.

Mycobacterium abscessus complex infections are ever on the rise. To curb their increasing evolution, we performed an in-depth study of 43 clinical isolates of cystic fibrosis patients obtained from 2009 to 2020. We identified their subspecies, uncovered their genotypic resistance profiles, characterised their antibiotic-resistant genes, and assessed their phenotypic antibiotic susceptibilities. The phenotypic and genotypic methods showed total agreement in terms of resistance to clarithromycin and amikacin. Of the 43 clinical strains, 28 belonged to M. abscessus subsp. abscessus (65.1%), 13 to M. abscessus subsp. massiliense (30.2%), and 2 to M. abscessus subsp. bolletii (4.6%). The resistant rates for clarithromycin and amikacin, the two main drugs against M. abscessus complex pulmonary infections, were 64.2% and 14.2%, respectively. We found three strains of M. abscessus subsp. abscessus that showed heteroresistance in the rrl and rrs genes, and these strains also presented double-resistance since they were macrolide- and aminoglycoside-resistant. M. abscessus subsp. abscessus showed a high minimum inhibitory concentration (MIC) and a resistant percentage larger than or equal to 88% to cefoxitin, ciprofloxacin, moxifloxacin, doxycycline, imipenem, and trimethoprim-sulfamethoxazole. These results show a panorama of the high resistance of Mycobacterium abscessus complex to current drugs for cystic fibrosis patients. Thus, other treatment methods are urgently needed.