Phage Therapy for Mycobacterium Abscessus and Strategies to Improve Outcomes

Members of Mycobacterium abscessus complex are known for causing severe, chronic infections. Members of M. abscessus are a new “antibiotic nightmare” as one of the most resistant organisms to chemotherapeutic agents. Treatment of these infections is challenging due to the either intrinsic or acquired resistance of the M. abscessus complex to the available antibiotics. Recently, successful phage therapy with a cocktail of three phages (one natural lytic phage and two engineered phages) every 12 h for at least 32 weeks has been reported against a severe case of the disseminated M. abscessus subsp. massiliense infection, which underlines the high value of phages against drug-resistant superbugs. This report also highlighted the limitations of phage therapy, such as the absence of lytic phages with a broad host-range against all strains and subspecies of the M. abscessus complex and also the risk of phage resistant bacteria over treatment. Cutting-edge genomic technologies have facilitated the development of engineered phages for therapeutic purposes by introducing new desirable properties, changing host-range and arming the phages with additional killing genes. Here, we review the available literature and suggest new potential solutions based on the progress in phage engineering that can help to overcome the present limitations of M. abscessus treatment.

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1615. Isolation of Lytic Bacteriophages with Broad Host Range Activity Against Pseudomonas aeruginosa Strains Isolated from Respiratory Samples from Cystic Fibrosis Patients Intended for Therapeutic Application

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1795 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S801-S801

Author(s):

Jose Alexander ◽

Daniel Navas ◽

Marly Flowers ◽

Angela Charles ◽

Amy Carr

Keyword(s):

Cystic Fibrosis ◽

Host Range ◽

Phage Therapy ◽

Clinical Isolates ◽

Clinical Samples ◽

Plaque Morphology ◽

Broad Host Range ◽

Chronic Infections ◽

Clinically Significant ◽

Host Target

Abstract Background With the rise of the antimicrobial resistance between different genera and species of bacteria, Phage Therapy is becoming a more realistic and accessible option for patients with limited or no antimicrobial options. Being able to have rapid access to a collection of clinical active phages is key for rapid implementation of phage therapy. The Microbiology Department at AdventHealth Orlando is performing routine screening of environmental and patient samples for isolation of phages against non-fermenting Gram negative bacteria to develop a Phage Bank. Methods Protocols for phage isolation from environmental sources such as lakes, rivers and sewers and clinical samples were developed. A series of respiratory, throat, stool and urine samples were processed following an internal protocol that includes centrifugation, filtration and enrichment. Clinical samples were centrifugated for 10 minutes, filtered using 0.45µm centrifugation filters, seeded with targeted host bacteria (clinical isolates) and incubated at 35°C for 24 hours. The enriched samples were centrifugated and filtered for a final phage enriched solution. Screening and isolation were performed using the Gracia method over trypticase soybean agar (TSA) for plaque morphology and quantification. Host range screening of other clinical isolates of P. aeruginosa was performed using the new isolated and purified phages. Results 4 lytic phages against clinical strains of P. aeruginosa from patient with diagnosis of cystic fibrosis (CF), were isolated and purified from 4 different respiratory samples, including sputum and bronchial alveolar lavage. All phages showed phenotypical characteristics of lytic activity. 1 phage was active against 4 strains of P. aeruginosa, 1 phage was active against 2 strains of P. aeruginosa and the remaining 2 phages were active only against the initial host target strain. Conclusion With this study we demonstrated the potential use of clinical samples as source for isolating active bacteriophages against clinically significant bacteria strains. Clinical samples from vulnerable population of patients with chronic infections are part of our routine “phage-hunting” process to stock and grow our Phage Bank project for future clinical use. Disclosures All Authors: No reported disclosures

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Alternative and Experimental Therapies of Mycobacterium abscessus Infections

International Journal of Molecular Sciences ◽

10.3390/ijms21186793 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6793

Author(s):

Michal Meir ◽

Daniel Barkan

Keyword(s):

Acquired Resistance ◽

Severe Case ◽

Experimental Models ◽

Mycobacterium Abscessus ◽

Multiple Drug ◽

Beta Lactam ◽

Chronic Infections ◽

Drug Regimens ◽

Human Infections

Mycobacterium abscessus is a non-tuberculous mycobacterium notoriously known for causing severe, chronic infections. Treatment of these infections is challenging due to either intrinsic or acquired resistance of M. abscessus to multiple antibiotics. Despite prolonged poly-antimicrobial therapy, treatment of M. abscessus infections often fails, leading to progressive morbidity and eventual mortality. Great research efforts are invested in finding new therapeutic options for M. abscessus. Clofazimine and rifabutin are known anti-mycobacterial antibiotics, repurposed for use against M. abscessus. Novel antimicrobials active against M. abscessus include delamanid, pretomanid and PIPD1 and the recently approved beta-lactamase inhibitors avibactam, relebactam and vaborbactam. Previously unused antimicrobial combinations, e.g. vancomycin–clarithromycin and dual beta-lactam therapy, have been shown to have synergistic effect against M. abscessus in experimental models, suggesting their possible use in multiple-drug regimens. Finally, engineered phage therapy has been reported to be clinically successful in a severe case of disseminated M. abscessus infection. While many of these experimental therapeutics have shown activity against M. abscessus in vitro, as well as in intracellular and/or animal models, most have little if any evidence of effect in human infections. Clinical studies of M. abscesssus treatments are needed to reliably determine the value of their incorporation in therapeutic regimens.

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Characterisation of bacteriophage JD419, a Staphylococcal phage with an unusual morphology and broad host range

10.1101/2020.11.09.370866 ◽

2020 ◽

Author(s):

Tingting Feng ◽

Sebastian Leptihn ◽

Ke Dong ◽

Belinda Loh ◽

Yan Zhang ◽

...

Keyword(s):

Host Range ◽

Virulence Genes ◽

Phage Therapy ◽

Antibiotic Resistance Genes ◽

Resistant Bacteria ◽

Broad Host Range ◽

Drug Resistant ◽

Ph Values ◽

Drug Resistant Bacteria ◽

Vancomycin Resistant

AbstractAs an antimicrobial therapy, therapeutic phages, also known as “Phage therapy” are able to inactivate multi-drug resistant bacteria such as methicillin and vancomycin resistant S. aureus and thus present a possible treatment for infections that are otherwise incurable. In this paper, we present a novel phage called JD419, which has a remarkably wide host-range. The virulent phage JD419 exhibits an elongated capsid and was able to infect and lyse 83 of all 129 tested clinical strains (64.3%) of multi-drug resistant S. aureus including MRSA. To evaluate the potential as a therapeutic phage, we tested the ability of phage JD419 to remain infectious after treatment exceeding physiological pH or temperature. The lytic activity of the phage was retained at pH values of 6.0-8.0 and at temperatures below 50°C. As phages sometimes contain virulence genes, we sequenced the complete genome of JD419. The 45509 bp genome contains a predicted 65 ORFs, none of which show homology to any known virulence or antibiotic resistance genes. Our study illustrates that Staphylococcus phage JD419 has the potential to be used for diagnostic, prophylaxic and therapeutic purposes.

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Characterization of a Broad-Host-Range Lytic Phage SHWT1 Against Multidrug-Resistant Salmonella and Evaluation of Its Therapeutic Efficacy in vitro and in vivo

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.683853 ◽

2021 ◽

Vol 8 ◽

Author(s):

Chenglin Tao ◽

Zhengfei Yi ◽

Yaodong Zhang ◽

Yao Wang ◽

Hong Zhu ◽

...

Keyword(s):

Antibiotic Resistance ◽

Host Range ◽

Therapeutic Efficacy ◽

Multidrug Resistant ◽

Lytic Activity ◽

Resistant Bacteria ◽

Lytic Phage ◽

Broad Host Range

Inappropriate use of antibiotics has accelerated to the emergence of multidrug-resistant bacteria, becoming a major health threat. Moreover, bacterial biofilms contribute to antibiotic resistance and prolonged infections. Bacteriophage (phage) therapy may provide an alternative strategy for controlling multidrug-resistant bacterial infections. In this study, a broad-host-range phage, SHWT1, with lytic activity against multidrug-resistant Salmonella was isolated, characterized and evaluated for the therapeutic efficacy in vitro and in vivo. Phage SHWT1 exhibited specific lytic activity against the prevalent Salmonella serovars, such as Salmonella Pullorum, Salmonella Gallinarum, Salmonella Enteritidis, and Salmonella Typhimurium. Morphological analysis showed that phage SHWT1 was a member of the family Siphoviridae and the order Caudovirales. Phage SHWT1 had a latent period of 5 min and burst size of ~150 plaque-forming units (PFUs)/cell. The phage was stable from pH 3-12 and 4–65°C. Phage SHWT1 also showed capacity to lyse Salmonella planktonic cells and inhibit the biofilm formation at optimal multiplicity of infection (MOI) of 0.001, 0.01, 0.1, and 100, respectively. In addition, phage SHWT1 was able to lyse intracellular Salmonella within macrophages. Genome sequencing and phylogenetic analyses revealed that SHWT1 was a lytic phage without toxin genes, virulence genes, antibiotic resistance genes, or significant genomic rearrangements. We found that phage SHWT1 could successfully protect mice against S. enteritidis and S. typhimurium infection. Elucidation of the characteristics and genome sequence of phage SHWT1 demonstrates that this phage is a potential therapeutic agent against the salmonellosis caused by multidrug-resistant Salmonella.

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Experimental therapies of Mycobacterium abscessus infections

10.20944/preprints202008.0273.v1 ◽

2020 ◽

Author(s):

Michal Meir ◽

Daniel Barkan

Keyword(s):

Acquired Resistance ◽

Severe Case ◽

Experimental Models ◽

Mycobacterium Abscessus ◽

Multiple Drug ◽

Beta Lactam ◽

Chronic Infections ◽

Drug Regimens ◽

Therapy Treatment

Mycobacterium abscessus is a non-tuberculous mycobacteria notoriously known for causing severe, chronic infections. Treatment of these infections is challenging due to either intrinsic or acquired resistance of M. abscessus to multiple antibiotics. Despite prolonged poly-antimicrobial therapy, treatment of M. abscessus infections often fails, leading to progressive morbidity and eventual mortality. Great research efforts are invested in finding new therapeutic options for M. abscessus. Clofazimine and rifabutin are known anti-mycobacterial antibiotics, repurposed for use against M. abscessus. Novel antimicrobials active against M. abscessus include delamanid, pretomanid and PIPD1 and the recently approved beta-lactamase inhibitors avibactam, relebactam and vaborbactam. Previously unused antimicrobial combinations e.g. vancomycin-clarithromycin and dual beta-lactam therapy have been shown to have synergistic effect against M. abscessus in experimental models, suggesting their possible use in multiple-drug regimens. Finally, engineered phage therapy has been reported to be clinically successful in a severe case of disseminated M. abscessus infection. While many of these experimental therapeutics have shown activity against M. abscessus in vitro, as well as intracellular and/or animal models, most have little if any evidence of effect in humans infections. Clinical studies of M. abscesssus treatments are needed in order to reliably determine the value of their incorporation in therapeutic regimens.

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Pseudomonas aeruginosa PAO 1 In Vitro Time–Kill Kinetics Using Single Phages and Phage Formulations—Modulating Death, Adaptation, and Resistance

Antibiotics ◽

10.3390/antibiotics10070877 ◽

2021 ◽

Vol 10 (7) ◽

pp. 877

Author(s):

Ana Mafalda Pinto ◽

Alberta Faustino ◽

Lorenzo M. Pastrana ◽

Manuel Bañobre-López ◽

Sanna Sillankorva

Keyword(s):

Pseudomonas Aeruginosa ◽

Phage Therapy ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Chronic Infections ◽

Swarming Motility ◽

Resistance Development ◽

Healthcare Settings ◽

Time Kill

Pseudomonas aeruginosa is responsible for nosocomial and chronic infections in healthcare settings. The major challenge in treating P. aeruginosa-related diseases is its remarkable capacity for antibiotic resistance development. Bacteriophage (phage) therapy is regarded as a possible alternative that has, for years, attracted attention for fighting multidrug-resistant infections. In this work, we characterized five phages showing different lytic spectrums towards clinical isolates. Two of these phages were isolated from the Russian Microgen Sextaphage formulation and belong to the Phikmvviruses, while three Pbunaviruses were isolated from sewage. Different phage formulations for the treatment of P. aeruginosa PAO1 resulted in diversified time–kill outcomes. The best result was obtained with a formulation with all phages, prompting a lower frequency of resistant variants and considerable alterations in cell motility, resulting in a loss of 73.7% in swimming motility and a 79% change in swarming motility. These alterations diminished the virulence of the phage-resisting phenotypes but promoted their growth since most became insensitive to a single or even all phages. However, not all combinations drove to enhanced cell killings due to the competition and loss of receptors. This study highlights that more caution is needed when developing cocktail formulations to maximize phage therapy efficacy. Selecting phages for formulations should consider the emergence of phage-resistant bacteria and whether the formulations are intended for short-term or extended antibacterial application.

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How to Train Your Phage: The Recent Efforts in Phage Training

Biologics ◽

10.3390/biologics1020005 ◽

2021 ◽

Vol 1 (2) ◽

pp. 70-88

Author(s):

Abdallah Abdelsattar ◽

Alyaa Dawooud ◽

Nouran Rezk ◽

Salsabil Makky ◽

Anan Safwat ◽

...

Keyword(s):

Host Range ◽

Pathogenic Bacteria ◽

Bacterial Resistance ◽

Phage Therapy ◽

Current Knowledge ◽

Lytic Replication ◽

Resistant Bacteria ◽

Infection Cycles ◽

Different Strains ◽

Specific Species

Control of pathogenic bacteria by deliberate application of predatory phages has potential as a powerful therapy against antibiotic-resistant bacteria. The key advantages of phage biocontrol over antibacterial chemotherapy are: (1) an ability to self-propagate inside host bacteria, (2) targeted predation of specific species or strains of bacteria, (3) adaptive molecular machinery to overcome resistance in target bacteria. However, realizing the potential of phage biocontrol is dependent on harnessing or adapting these responses, as many phage species switch between lytic infection cycles (resulting in lysis) and lysogenic infection cycles (resulting in genomic integration) that increase the likelihood of survival of the phage in response to external stress or host depletion. Similarly, host range will need to be optimized to make phage therapy medically viable whilst avoiding the potential for deleteriously disturbing the commensal microbiota. Phage training is a new approach to produce efficient phages by capitalizing on the evolved response of wild-type phages to bacterial resistance. Here we will review recent studies reporting successful trials of training different strains of phages to switch into lytic replication mode, overcome bacterial resistance, and increase their host range. This review will also highlight the current knowledge of phage training and future implications in phage applications and phage therapy and summarize the recent pipeline of the magistral preparation to produce a customized phage for clinical trials and medical applications.

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Morphologic and genomic characterization of a broad host range Salmonella enterica serovar Pullorum lytic phage vB_SPuM_SP116

Microbial Pathogenesis ◽

10.1016/j.micpath.2019.103659 ◽

2019 ◽

Vol 136 ◽

pp. 103659 ◽

Cited By ~ 2

Author(s):

Hongduo Bao ◽

Khashayar Shahin ◽

Qiaoyan Zhang ◽

Hui Zhang ◽

Zhen Wang ◽

...

Keyword(s):

Host Range ◽

Salmonella Enterica ◽

Lytic Phage ◽

Broad Host Range ◽

Salmonella Enterica Serovar Pullorum ◽

Genomic Characterization

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JD419, a Staphylococcus aureus Phage With a Unique Morphology and Broad Host Range

Frontiers in Microbiology ◽

10.3389/fmicb.2021.602902 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tingting Feng ◽

Sebastian Leptihn ◽

Ke Dong ◽

Belinda Loh ◽

Yan Zhang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Host Range ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Complete Characterization ◽

Resistant Bacteria ◽

Broad Host Range ◽

Gene Engineering ◽

Mrsa Strains

Phage therapy represents a possible treatment option to cure infections caused by multidrug-resistant bacteria, including methicillin and vancomycin-resistant Staphylococcus aureus, to which most antibiotics have become ineffective. In the present study, we report the isolation and complete characterization of a novel phage named JD219 exhibiting a broad host range able to infect 61 of 138 clinical strains of S. aureus tested, which included MRSA strains as well. The phage JD419 exhibits a unique morphology with an elongated capsid and a flexible tail. To evaluate the potential of JD419 to be used as a therapeutic phage, we tested the ability of the phage particles to remain infectious after treatment exceeding physiological pH or temperature. The activity was retained at pH values of 6.0–8.0 and below 50°C. As phages can contain virulence genes, JD419’s complete genome was sequenced. The 45509 bp genome is predicted to contain 65 ORFs, none of which show homology to any known virulence or antibiotic resistance genes. Genome analysis indicates that JD419 is a temperate phage, despite observing rapid replication and lysis of host strains. Following the recent advances in synthetic biology, JD419 can be modified by gene engineering to remove prophage-related genes, preventing potential lysogeny, in order to be deployed as a therapeutic phage.

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