The problem of Mycobacterium abscessus complex: multi-drug resistance, bacteriophage susceptibility and potential healthcare transmission.

OBJECTIVES: Mycobacterium abscessus complex is responsible for 2.6-13.0% of all non-tuberculous mycobacterial pulmonary infections and these are notoriously difficult to treat due to the complex regimens required, drug resistance and adverse effects. Hence, bacteriophages have been considered in clinical practice as an additional treatment option. Here, we evaluated antibiotic and phage susceptibility profiles of M. abscessus clinical isolates. Whole-genome sequencing (WGS) revealed the phylogenetic relationships, dominant circulating clones (DCCs), the likelihood of patient-to-patient transmission and the presence of prophages. METHODS: Antibiotic susceptibility testing was performed using CLSI breakpoints (n = 95), and plaque assays were used for phage susceptibility testing (subset of n = 88, 35 rough and 53 smooth morphology). WGS was completed using the Illumina platform and analysed using Snippy/snp-dists and Discovery and Extraction of Phages Tool (DEPhT). RESULTS: Amikacin and Tigecycline were the most active drugs (with 2 strains resistant to amikacin, and one strain with Tigecycline MIC of 4 µg/mL). Most strains were resistant to all other drugs tested, with Linezolid and Imipenem showing the least resistance, at 38% (36/95) and 55% (52/95), respectively. Rough colony morphotype strains were more phage-susceptible than smooth strains (77%-27/35 versus 48%-25/53 in the plaque assays, but smooth strains are not killed efficiently by those phages in liquid infection assay). We have also identified 100 resident prophages, some of which were propagated lytically. DCC1 (20%-18/90) and DCC4 (22%-20/90) were observed to be the major clones and WGS identified 6 events of possible patient-to-patient transmission. DISCUSSION: Many strains of M. abscessus complex are intrinsically resistant to available antibiotics and bacteriophages represent an alternative therapeutic option, but only for strains with rough morphology. Further studies are needed to elucidate the role of hospital-borne M. abscessus transmission.

Unusual prophages in Mycobacterium abscessus genomes and strain variations in phage susceptibilities.

Mycobacterium abscessus infections are relatively common in patients with cystic fibrosis and are clinically challenging, with frequent intrinsic resistance to antibiotics. Therapeutic treatment with bacteriophages offers some promise but faces many challenges including substantial variation in phage susceptibilities among clinical isolates, and the need to personalize therapies for individual patients. Many strains are not susceptible to any phages or are not efficiently killed by lytic phages, including all smooth colony morphotype strains tested to-date. Here, we analyze a set of new M. abscessus isolates for the genomic relationships, prophage content, spontaneous phage release, and phage susceptibilities. We find that prophages are common in these M. abscessus genomes, but some have unusual arrangements, including tandemly integrated prophages, internal duplications, and they participate in active exchange of polymorphic toxin-immunity cassettes secreted by ESX systems. Relatively few strains are efficiently infected by any mycobacteriophages, and the infection patterns do not reflect the overall phylogenetic relationships of the strains. Characterization of these strains and their phage susceptibility profiles will help to advance the broader application of phage therapies for NTM infections.

Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease.

BACKGROUND: Nontuberculous Mycobacterium infections, particularly Mycobacterium abscessus, are increasingly common among patients with cystic fibrosis and chronic bronchiectatic lung diseases. Treatment is challenging due to intrinsic antibiotic resistance. Bacteriophage therapy represents a potentially novel approach. Relatively few active lytic phages are available and there is great variation in phage susceptibilities among M. abscessus isolates, requiring personalized phage identification. METHODS: Mycobacterium isolates from 200 culture-positive patients with symptomatic disease were screened for phage susceptibilities. One or more lytic phages were identified for 55 isolates. Phages were administered intravenously, by aerosolization, or both to 20 patients on a compassionate use basis and patients were monitored for adverse reactions, clinical and microbiologic responses, the emergence of phage resistance, and phage neutralization in serum, sputum, or bronchoalveolar lavage fluid. RESULTS: No adverse reactions attributed to therapy were seen in any patient regardless of the pathogen, phages administered, or the route of delivery. Favorable clinical or microbiological responses were observed in 11 patients. Neutralizing antibodies were identified in serum after initiation of phage delivery intravenously in 8 patients, potentially contributing to lack of treatment response in 4 cases, but were not consistently associated with unfavorable responses in others. Eleven patients were treated with only a single phage, and no phage resistance was observed in any of these. CONCLUSIONS: Phage treatment of Mycobacterium infections is challenging due to the limited repertoire of therapeutically useful phages, but favorable clinical outcomes in patients lacking any other treatment options support continued development of adjunctive phage therapy for some mycobacterial infections.

Nebulized Bacteriophage in a Patient With Refractory Mycobacterium abscessus Lung Disease.

An elderly man with refractory Mycobacterium abscessus lung disease previously developed anti-phage neutralizing antibodies while receiving intravenous phage therapy. Subsequent phage nebulization resulted in transient weight gain, decreased C-reactive protein, and reduced Mycobacterium burden. Weak sputum neutralization may have limited the outcomes, but phage resistance was not a contributing factor.

Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection.

Two mycobacteriophages were administered intravenously to a male with treatment-refractory Mycobacterium abscessus pulmonary infection and severe cystic fibrosis lung disease. The phages were engineered to enhance their capacity to lyse M. abscessus and were selected specifically as the most effective against the subject's bacterial isolate. In the setting of compassionate use, the evidence of phage-induced lysis was observed using molecular and metabolic assays combined with clinical assessments. M. abscessus isolates pre and post-phage treatment demonstrated genetic stability, with a general decline in diversity and no increased resistance to phage or antibiotics. The anti-phage neutralizing antibody titers to one phage increased with time but did not prevent clinical improvement throughout the course of treatment. The subject received lung transplantation on day 379, and systematic culturing of the explanted lung did not detect M. abscessus. This study describes the course and associated markers of a successful phage treatment of M. abscessus in advanced lung disease.

Bacteriophage treatment of disseminated cutaneous Mycobacterium chelonae infection.

Mycobacterium chelonae is a rare cause of chronic disseminated cutaneous infections in immunocompromised patients. Multidrug-resistant M. chelonae infections present a challenge for treatment, and prolonged antimicrobial courses lead to significant toxicities and further antimicrobial resistance. We report a case of refractory cutaneous disseminated M. chelonae infection in a patient with seronegative arthritis on immunotherapy with tofacitinib that was treated with combination antimicrobial, surgical, and single bacteriophage therapy with excellent clinical response. The patient developed neutralizing antibodies against the bacteriophage but continues to have stable improvement of disease with negative biopsies and no evidence of bacterial resistance to the phage.

Phage Therapy for Antibiotic-Resistant Bacterial Infections.

Antibiotic resistance in bacterial pathogens presents a substantial threat to the control of infectious diseases. Development of new classes of antibiotics has slowed in recent years due to pressures of cost and market profitability, and there is a strong need for new antimicrobial therapies. The therapeutic use of bacteriophages has long been considered, with numerous anecdotal reports of success. Interest in phage therapy has been renewed by recent clinical successes in case studies with personalized phage cocktails, and several clinical trials are in progress. We discuss recent progress in the therapeutic use of phages and contemplate the key factors influencing the opportunities and challenges. With strong safety profiles, the main challenges of phage therapeutics involve strain variation among clinical isolates of many pathogens, battling phage resistance, and the potential limitations of host immune responses. However, the opportunities are considerable, with the potential to enhance current antibiotic efficacy, protect newly developed antibiotics, and provide a last resort in response to complete antibiotic failure.

Application of Bacteriophages for Mycobacterial Infections, from Diagnosis to Treatment.

Mycobacterium tuberculosis and other non-tuberculous mycobacteria are responsible for a variety of different infections affecting millions of patients worldwide. Their diagnosis is often problematic and delayed until late in the course of disease, requiring a high index of suspicion and the combined efforts of clinical and laboratory colleagues. Molecular methods, such as PCR platforms, are available, but expensive, and with limited sensitivity in the case of paucibacillary disease. Treatment of mycobacterial infections is also challenging, typically requiring months of multiple and combined antibiotics, with associated side effects and toxicities. The presence of innate and acquired drug resistance further complicates the picture, with dramatic cases without effective treatment options. Bacteriophages (viruses that infect bacteria) have been used for decades in Eastern Europe for the treatment of common bacterial infections, but there is limited clinical experience of their use in mycobacterial infections. More recently, bacteriophages' clinical utility has been re-visited and their use has been successfully demonstrated both as diagnostic and treatment options. This review will focus specifically on how mycobacteriophages have been used recently in the diagnosis and treatment of different mycobacterial infections, as potential emerging technologies, and as an alternative treatment option.

Mycobacteriophage-antibiotic therapy promotes enhanced clearance of drug-resistant Mycobacterium abscessus.

Infection by multidrug-resistant Mycobacterium abscessus is increasingly prevalent in cystic fibrosis (CF) patients, leaving clinicians with few therapeutic options. A compassionate study showed the clinical improvement of a CF patient with a disseminated M. abscessus (GD01) infection, following injection of a phage cocktail, including phage Muddy. Broadening the use of phage therapy in patients as a potential antibacterial alternative necessitates the development of biological models to improve the reliability and successful prediction of phage therapy in the clinic. Herein, we demonstrate that Muddy very efficiently lyses GD01 in vitro, an effect substantially increased with standard drugs. Remarkably, this cooperative activity was retained in an M. abscessus model of infection in CFTR-depleted zebrafish, associated with a striking increase in larval survival and reduction in pathological signs. The activity of Muddy was lost in macrophage-ablated larvae, suggesting that successful phage therapy relies on functional innate immunity. CFTR-depleted zebrafish represent a practical model to rapidly assess phage treatment efficacy against M. abscessus isolates, allowing the identification of drug combinations accompanying phage therapy and treatment prediction in patients. This article has an associated First Person interview with the first author of the paper.

Potent antibody-mediated neutralization limits bacteriophage treatment of a pulmonary Mycobacterium abscessus infection.

An 81-year-old immunocompetent patient with bronchiectasis and refractory Mycobacterium abscessus lung disease was treated for 6 months with a three-phage cocktail active against the strain. In this case study of phage to lower infectious burden, intravenous administration was safe and reduced the M. abscessus sputum load tenfold within one month. However, after two months, M. abscessus counts increased as the patient mounted a robust IgM- and IgG-mediated neutralizing antibody response to the phages, which was associated with limited therapeutic efficacy.

Toward a Phage Cocktail for Tuberculosis: Susceptibility and Tuberculocidal Action of Mycobacteriophages against Diverse Mycobacterium tuberculosis Strains.

The global health burden of human tuberculosis (TB) and the widespread antibiotic resistance of its causative agent Mycobacterium tuberculosis warrant new strategies for TB control. The successful use of a bacteriophage cocktail to treat a Mycobacterium abscessus infection suggests that phages could play a role in tuberculosis therapy. To assemble a phage cocktail with optimal therapeutic potential for tuberculosis, we have explored mycobacteriophage diversity to identify phages that demonstrate tuberculocidal activity and determined the phage infection profiles for a diverse set of strains spanning the major lineages of human-adapted strains of the Mycobacterium tuberculosis complex. Using a combination of genome engineering and bacteriophage genetics, we have assembled a five-phage cocktail that minimizes the emergence of phage resistance and cross-resistance to multiple phages, and which efficiently kills the M. tuberculosis strains tested. Furthermore, these phages function without antagonizing antibiotic effectiveness, and infect both isoniazid-resistant and -sensitive strains.IMPORTANCE Tuberculosis kills 1.5 million people each year, and resistance to commonly used antibiotics contributes to treatment failures. The therapeutic potential of bacteriophages against Mycobacterium tuberculosis offers prospects for shortening antibiotic regimens, provides new tools for treating multiple drug-resistant (MDR)-TB and extensively drug-resistant (XDR)-TB infections, and protects newly developed antibiotics against rapidly emerging resistance to them. Identifying a suitable suite of phages active against diverse M. tuberculosis isolates circumvents many of the barriers to initiating clinical evaluation of phages as part of the arsenal of antituberculosis therapeutics.

CRISPY-BRED and CRISPY-BRIP: efficient bacteriophage engineering.

Genome engineering of bacteriophages provides opportunities for precise genetic dissection and for numerous phage applications including therapy. However, few methods are available for facile construction of unmarked precise deletions, insertions, gene replacements and point mutations in bacteriophages for most bacterial hosts. Here we describe CRISPY-BRED and CRISPY-BRIP, methods for efficient and precise engineering of phages in Mycobacterium species, with applicability to phages of a variety of other hosts. This recombineering approach uses phage-derived recombination proteins and Streptococcus thermophilus CRISPR-Cas9.

Mycobacterium abscessus Strain Morphotype Determines Phage Susceptibility, the Repertoire of Therapeutically Useful Phages, and Phage Resistance.

Mycobacterium abscessus is an opportunistic pathogen whose treatment is confounded by widespread multidrug resistance. The therapeutic use of bacteriophages against Mycobacterium abscessus infections offers a potential alternative approach, although the spectrum of phage susceptibilities among M. abscessus isolates is not known. We determined the phage infection profiles of 82 M. abscessus recent clinical isolates and find that colony morphotype-rough or smooth-is a key indicator of phage susceptibility. None of the smooth strains are efficiently killed by any phages, whereas 80% of rough strains are infected and efficiently killed by at least one phage. The repertoire of phages available for potential therapy of rough morphotype infections includes those with relatively broad host ranges, host range mutants of Mycobacterium smegmatis phages, and lytically propagated viruses derived from integrated prophages. The rough colony morphotype results from indels in the glycopeptidolipid synthesis genes mps1 and mps2, negating reversion to smooth as a common route to phage resistance. Resistance is thus rare, and although mutations in polyketide synthesis, uvrD2, and rpoZ can confer resistance, these likely also impair survival in vivo The expanded therapeutic repertoire and the resistance profiles show that small cocktails or single phages could be suitable for controlling infections with rough strains.IMPORTANCEMycobacterium abscessus infections in cystic fibrosis patients are challenging to treat due to widespread antibiotic resistance. The therapeutic use of lytic bacteriophages presents a new potential strategy, but the great variation among clinical M. abscessus isolates demands determination of phage susceptibility prior to therapy. Elucidation of the variation in phage infection and factors determining it, expansion of the suite of therapeutic phage candidates, and a greater understanding of phage resistance mechanisms substantially advances the potential for broad implementation of new therapeutic options for M. abscessus infections.

The Prophage and Plasmid Mobilome as a Likely Driver of Mycobacterium abscessus Diversity.

Mycobacterium abscessus is an emerging pathogen that is often refractory to antibiotic control. Treatment is further complicated by considerable variation among clinical isolates in both their genetic constitution and their clinical manifestations. Here, we show that the prophage and plasmid mobilome is a likely contributor to this variation. Prophages and plasmids are common, abundant, and highly diverse, and code for large repertoires of genes influencing virulence, antibiotic susceptibility, and defense against viral infection. At least 85% of the strains we describe carry one or more prophages, representing at least 17 distinct and diverse sequence "clusters," integrated at 18 different attB locations. The prophages code for 19 distinct configurations of polymorphic toxin and toxin-immunity systems, each with WXG-100 motifs for export through type VII secretion systems. These are located adjacent to attachment junctions, are lysogenically expressed, and are implicated in promoting growth in infected host cells. Although the plethora of prophages and plasmids confounds the understanding of M. abscessus pathogenicity, they also provide an abundance of tools for M. abscessus engineering.IMPORTANCEMycobacterium abscessus is an important emerging pathogen that is challenging to treat with current antibiotic regimens. There is substantial genomic variation in M. abscessus clinical isolates, but little is known about how this influences pathogenicity and in vivo growth. Much of the genomic variation is likely due to the large and varied mobilome, especially a large and diverse array of prophages and plasmids. The prophages are unrelated to previously characterized phages of mycobacteria and code for a diverse array of genes implicated in both viral defense and in vivo growth. Prophage-encoded polymorphic toxin proteins secreted via the type VII secretion system are common and highly varied and likely contribute to strain-specific pathogenesis.

Genome Sequence of Mycobacterium abscessus Phage phiT45-1.

Mycobacteriophage phiT45-1 is a newly isolated bacteriophage spontaneously released from Mycobacterium abscessus strain Taiwan-45 that lytically infects M. abscessus strain BWH-C; phiT45-1 also infects M. abscessus ATCC 19977 but not Mycobacterium smegmatis Phage phiT45-1 has a 43,407-bp genome and carries a polymorphic toxin-immunity cassette associated with type VII secretion systems.

Genome Sequence of Mycobacterium abscessus Phage phiT46-1.

Mycobacteriophage phiT46-1 is a newly isolated Mycobacterium phage that was isolated by spontaneous release from Mycobacterium abscessus strain Taiwan-46 and infects M. abscessus strain BWH-C. Phage phiT46-1 is unrelated to previously described mycobacteriophages, has a 52,849-bp genome, and includes a polymorphic toxin-immunity cassette associated with type VII secretion systems.

Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus.

A 15-year-old patient with cystic fibrosis with a disseminated Mycobacterium abscessus infection was treated with a three-phage cocktail following bilateral lung transplantation. Effective lytic phage derivatives that efficiently kill the infectious M. abscessus strain were developed by genome engineering and forward genetics. Intravenous phage treatment was well tolerated and associated with objective clinical improvement, including sternal wound closure, improved liver function, and substantial resolution of infected skin nodules.

Mycobacteriophage ZoeJ: A broad host-range close relative of mycobacteriophage TM4.

A collection of over 1600 sequenced bacteriophages isolated on a single host strain, Mycobacterium smegmatis mc2155, can be grouped into over two dozen types that have little or no nucleotide sequence similarity to each other. One group, Cluster K, can be divided into several subclusters, and the well-characterized and much exploited phage TM4 lies in Subcluster K2. Many of the Cluster K phages have broad host ranges and infect both fast- and slow-growing mycobacterial strains. Here we describe phage ZoeJ, a new Subcluster K2 member, which infects a broad spectrum of mycobacterial hosts including M. smegmatis, Mycobacterium tuberculosis, and Mycobacterium avium. ZoeJ has extensive sequence similarity to TM4, and comparative analysis reveals the precise deletion conferring the lytic phenotype of TM4. The ZoeJ immunity repressor was identified as gene 45, which is prophage-expressed, is required for lysogeny, and is sufficient to confer superinfection immunity to ZoeJ. ZoeJ gp45 also confers immunity to Subcluster K2 phage Milly, and Subcluster K1 phages Adephagia and CrimD, but surprisingly not to TM4. RNAseq analysis reveals the temporal pattern of early and late gene expressions in ZoeJ lytic growth and suggests a role for the ESAS motifs for gene regulation.

Yet More Evidence of Collusion: a New Viral Defense System Encoded by Gordonia Phage CarolAnn.

Temperate phages play important roles in the physiology of their bacterial hosts and establish a lysogenic relationship with the host through which prophage-expressed genes confer new phenotypes. A key phenotype is prophage-mediated defense against heterotypic viral attack, in which temperate phages collude with their bacterial host to prevent other phages from attacking, sometimes with exquisite specificity. Such defense systems have been described in Pseudomonas and Mycobacterium phages but are likely widespread throughout the microbial community. Here, we describe a novel prophage-mediated defense system encoded by Gordonia phage CarolAnn, which defends against infection by unrelated phages grouped in cluster CZ. CarolAnn genes 43 and 44 are coexpressed with the repressor and are necessary and sufficient to confer defense against phage Kita and its close relatives. Kita and these relatives are targeted through Kita gene 53, a gene that is of unknown function but which is the location of defense escape mutations that overcome CarolAnn defense. Expression of Kita gene 53 is toxic to Gordonia terrae in the presence of CarolAnn genes 43 and 44, suggesting that defense may be mediated by an abortive infection type of mechanism. CarolAnn genes 43 and 44 are distant relatives of mycobacteriophage Sbash genes 31 and 30, respectively, which also confer viral defense but use a different targeting system.IMPORTANCE Prophage-mediated viral defense systems play a key role in microbial dynamics, as lysogeny is established relatively efficiently, and prophage-expressed genes can strongly inhibit lytic infection of other, unrelated phages. Demonstrating such defense systems in Gordonia terrae suggests that these systems are widespread and that there are a multitude of different systems with different specificities for the attacking phages.

More Evidence of Collusion: a New Prophage-Mediated Viral Defense System Encoded by Mycobacteriophage Sbash.

The arms race between bacteria and their bacteriophages profoundly influences microbial evolution. With an estimated 1023 phage infections occurring per second, there is strong selection for both bacterial survival and phage coevolution for continued propagation. Many phage resistance systems, including restriction-modification systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) systems, a variety of abortive infection systems, and many others that are not yet mechanistically defined, have been described. Temperate bacteriophages are common and form stable lysogens that are immune to superinfection by the same or closely related phages. However, temperate phages collude with their hosts to confer defense against genomically distinct phages, to the mutual benefit of the bacterial host and the prophage. Prophage-mediated viral systems have been described in Mycobacterium phages and Pseudomonas phages but are predicted to be widespread throughout the microbial world. Here we describe a new viral defense system in which the mycobacteriophage Sbash prophage colludes with its Mycobacterium smegmatis host to confer highly specific defense against infection by the unrelated mycobacteriophage Crossroads. Sbash genes 30 and 31 are lysogenically expressed and are necessary and sufficient to confer defense against Crossroads but do not defend against any of the closely related phages grouped in subcluster L2. The mapping of Crossroads defense escape mutants shows that genes 132 and 141 are involved in recognition by the Sbash defense system and are proposed to activate a loss in membrane potential mediated by Sbash gp30 and gp31.IMPORTANCE Viral infection is an ongoing challenge to bacterial survival, and there is strong selection for development or acquisition of defense systems that promote survival when bacteria are attacked by bacteriophages. Temperate phages play central roles in these dynamics through lysogenic expression of genes that defend against phage attack, including those unrelated to the prophage. Few prophage-mediated viral defense systems have been characterized, but they are likely widespread both in phage genomes and in the prophages integrated in bacterial chromosomes.