• bacteriophage
• cystic fibrosis
• Mycobacterium abscessus
• phage
• phage therapy
• terapia fagica

Mycobacterium abscessus is a rapidly growing mycobacterium and an opportunistic pathogen mainly found in the environment. It consists of three subspecies, namely M. abscessus subsp. abscessus, subsp. bolletii, and subsp. massiliense. M. abscessus is associated with several diseases in humans, of which lung infection is the most common, particularly in patients with underlying lung disease such as bronchiec-tasis, chronic obstructive pulmonary disease and cystic fibrosis. During the last years, M. abscessus has appeared as an emerging pathogen, with an increasing number of reported cases worldwide. Soil and water are the main source of exposure, however person-to-person transmission has been attested among cystic fibrosis patients in hospital settings. M. abscessus among the most challenging species of non-tuberculous mycobacteria to treat due to intrinsic and acquired antibiotic resistance. Infections caused by it are often difficult to treat since therapy, involving the use of a combination drug regimen, is long and prone to failure, thus encouraging an urgent need for new and more effective treatment options.
Phage therapy is based on the use of bacteriophages for the treatment of bacterial infection, as an alternative or adjuvant option to antibiotics. The renewed interest towards phage therapy is mostly due to the ability of phages to also infect and kill drug resistant bacteria; other advantages include the selectivity of phages versus a specific bacterial strain, coevolution and self-dosing in their host, low toxicity, and the ability to infect biofilm-embedded cells. Mycobacteriophages, which specifically target species of the Mycobacterium genus, belong to families Siphoviridae and Myoviridae, of double-stranded DNA, tailed viruses. Their use in the treatment of M. abscessus infections is in its infancy but rapidly growing, especially for the treatment of lung infection in cystic fibrosis patients.
This work aims to isolate mycobacteriophages using Mycobacterium smegmatis as host, characterize their general traits and particularly their capacity to infect and kill M. abscessus strains. More than 40 environmental samples (both liquid and solid) were collected from Tuscany, Liguria and Sardinia. Fourteen phage clones with an apparent different plaque morphology were selected from two soil samples and propagated in the M. smegmatis mc2 155 laboratory strain. The lytic activity against their host and M. abscessus clinical isolates provided by the University Hospital of Pisa, including strains belonging to all three subspecies, were tested by spot assay and classified through the observation of lysis plaque morphology, their host range, one-step growth curve and ability to lysogenize. Different phages were found able to infect and lyse drug-resistant M. abscessus strains exhibiting either rough or smooth morphotype.
Genome was extracted from six phage clones using a commercial kit and sequenced by Illumina technology in collaboration with the group of Prof. Lavigne from KU Leuven University, Belgium. Three genetically distinct phages belonging to two novel species were found. From their genome analysis, they were found to carry genes encoding for integrases, transcription repressors and excisionases, all involved in lysogeny. The ability of phages to carry out a lysogenic cycle was also confirmed by an in vitro assay, which results consistent with the genome annotation. Moreover, two of the phages carried a gene of the WhiB family, which also includes a transcription regulator associated with resistance to macrolides and aminoglycosides in M. abscessus. The three novel species identified, though temperate, do not carry any other gene that may be reason to discard them for phage therapy, and their quick replication cycle makes them adequate candidates to be employed after engineering or directly in compassionate use.