Development of a nucleic acid chromatography assay for the detection of commonly isolated rapidly growing mycobacteria

Introduction. Non-tuberculosis mycobacterium infections are increasing worldwide, including those caused by rapidly growing mycobacteria (RGM). Gap Statement. The identification of the aetiological agent in the context of infections is essential for the adoption of an adequate therapeutic approach. However, the methods for the rapid distinction of different RGM species are less than optimal. Aim. To develop a nucleic acid chromatography kit to identify clinically common RGM. Methodology. We tried to develop a nucleic acid chromatography kit designed to detect four RGM species (including three subspecies) i.e. Mycobacterium abscessus subsp. abscessus , Mycobacterium abscessus subsp. bolletii (detected as M. abscessus/bolletii) Mycobacterium abscessus subsp. massiliense , Mycobacterium fortuitum , Mycobacterium chelonae and Mycobacterium peregrinum . The amplified target genes for each species/subspecies using multiplex PCR were analysed using a nucleic acid chromatography assay. Results. Among the 159 mycobacterial type strains and 70 RGM clinical isolates tested, the developed assay correctly identified all relevant RGM without any cross-reactivity or false-negatives. The limits of detection for each species were approximately 0.2 pg µl-1. Conclusion. The rapid and simple nucleic acid chromatography method developed here, which does not involve heat denaturation, may contribute to the rapid identification and treatment of RGM infections.

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In Vitro Activities of Omadacycline against Rapidly Growing Mycobacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02522-18 ◽

2019 ◽

Vol 63 (5) ◽

Cited By ~ 15

Author(s):

Carolyn Shoen ◽

David Benaroch ◽

Mary Sklaney ◽

Michael Cynamon

Keyword(s):

Mycobacterium Abscessus ◽

Mycobacterium Fortuitum ◽

Mycobacterium Chelonae ◽

Content Type ◽

Rapidly Growing Mycobacteria ◽

Dilution Assay ◽

Tetracycline Derivative

ABSTRACT The in vitro activity of omadacycline, a new tetracycline derivative, was evaluated against isolates of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum using a broth microtiter dilution assay. Omadacycline had MIC90 values of 2 μg/ml, 0.25 μg/ml, and 0.5 μg/ml, respectively. The in vitro activity of omadacycline against rapidly growing mycobacteria indicates that it may have the potential to improve therapy for infections caused by these organisms.

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Environment in the lung of cystic fibrosis patients stimulates the expression of biofilm phenotype in Mycobacterium abscessus

Journal of Medical Microbiology ◽

10.1099/jmm.0.001467 ◽

2022 ◽

Vol 71 (1) ◽

Author(s):

Bailey F. Keefe ◽

Luiz E. Bermudez

Keyword(s):

Cystic Fibrosis ◽

Host Cell ◽

Biofilm Formation ◽

Type Species ◽

Divalent Cations ◽

Mycobacterium Abscessus ◽

Content Type ◽

Link Type ◽

Populations At Risk

Introduction. Pulmonary infections caused by organisms of the Mycobacterium abscessus complex are increasingly prevalent in populations at risk, such as patients with cystic fibrosis, bronchiectasis and emphysema. Hypothesis. M. abscessus infection of the lung is not observed in immunocompetent individuals, which raises the possibility that the compromised lung environment is a suitable niche for the pathogen to thrive in due to the overproduction of mucus and high amounts of host cell lysis. Aim. Evaluate the ability of M. abscessus to form biofilm and grow utilizing in vitro conditions as seen in immunocompromised lungs of patients. Methodology. We compared biofilm formation and protein composition in the presence and absence of synthetic cystic fibrosis medium (SCFM) and evaluated the bacterial growth when exposed to human DNA. Results. M. abscessus is capable of forming biofilm in SCFM. By eliminating single components found in the medium, it became clear that magnesium works as a signal for the biofilm formation, and chelation of the divalent cations resulted in the suppression of biofilm formation. Investigation of the specific proteins expressed in the presence of SCFM and in the presence of SCFM lacking magnesium revealed many different proteins between the conditions. M. abscessus also exhibited growth in SCFM and in the presence of host cell DNA, although the mechanism of DNA utilization remains unclear. Conclusions. In vitro conditions mimicking the airways of patients with cystic fibrosis appear to facilitate M. abscessus establishment of infection, and elimination of magnesium from the environment may affect the ability of the pathogen to establish infection.

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Activities of the glycylcyclines N,N-dimethylglycylamido-minocycline and N,N-dimethylglycylamido-6-demethyl-6-deoxytetracycline against Nocardia spp. and tetracycline-resistant isolates of rapidly growing mycobacteria.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.4.874 ◽

1996 ◽

Vol 40 (4) ◽

pp. 874-878 ◽

Cited By ~ 15

Author(s):

B A Brown ◽

R J Wallace ◽

G Onyi

Keyword(s):

Therapeutic Potential ◽

Sensu Stricto ◽

Mycobacterium Abscessus ◽

Mycobacterium Fortuitum ◽

New Taxon ◽

Mycobacterium Chelonae ◽

New Agents ◽

Rapidly Growing Mycobacteria ◽

Nocardia Brasiliensis ◽

Mycobacterium Mucogenicum

Susceptibilities to the new semisynthetic tetracycline (Tet) compounds N,N-dimethylglycylamido-minocycline (DMG-MINO) and N,N-dimethylglycylamido-6-demethyl-6-deoxytetracycline (DMG-DMDOT) were compared with those to doxycycline, minocycline, and Tet for 198 Tet-resistant (Tetr) and 33 Tet-susceptible (Tets) clinical isolates of rapidly growing mycobacteria (RGM) including the Mycobacterium fortuitum group, Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium mucogenicum and 68 isolates belonging to six taxa of Nocardia spp. All Tetr RGM were highly susceptible to the glycylcyclines. The MICs at which 50 and 90% of isolates are inhibited were < or = 0.125 and < or = 0.25 microgram/ml, respectively, for DMG-DMDOT and < or = 0.25 and 1 microgram/ml, respectively, for DMG-MINO. The MIC of DMG-DMDOT at which 50% of Tetr strains are inhibited was the same as that for Tets strains for each of the four taxa of RGM. The new agents were less active against Nocardia spp. MICs of DMG-DMDOT were comparable to those of minocycline except for the MICs for Nocardia brasiliensis sensu stricto, the new taxon Nocardia pseudobrasiliensis, and some isolates of Nocardia nova, against which they were four- to eightfold more active. The MICs of DMG-DMDOT were consistently lower than those of DMG-MINO for RGM. This class of drugs offers exciting therapeutic potential for RGM and for selected species of Nocardia.

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A genuine mycobacterial thermophile: Mycobacterium hassiacum growth, survival and GpgS stability at near-pasteurization temperatures

Microbiology ◽

10.1099/mic.0.000898 ◽

2020 ◽

Vol 166 (5) ◽

pp. 474-483 ◽

Cited By ~ 2

Author(s):

Susana Alarico ◽

Daniela Nunes-Costa ◽

Alexandra Silva ◽

Mafalda Costa ◽

Sandra Macedo-Ribeiro ◽

...

Keyword(s):

Drug Targets ◽

Type Species ◽

Mycobacterium Abscessus ◽

Water Supply Systems ◽

Content Type ◽

Link Type ◽

Food And Drink ◽

Supply Systems ◽

Potential Drug Targets ◽

Resistance To Heat

Mycobacterium hassiacum is so far the most thermophilic among mycobacteria as it grows optimally at 50 °C and up to 65 °C in a glycerol-based medium, as verified in this study. Since this and other nontuberculous mycobacteria (NTM) thrive in diverse natural and artificial environments, from where they may access and infect humans, we deemed essential to probe M. hassiacum resistance to heat, a strategy routinely used to control microbial growth in water-supply systems, as well as in the food and drink industries. In addition to possibly being a threat in its own right in rare occasions, M. hassiacum is also a good surrogate for studying other NTM species more often associated with opportunistic infection, namely Mycobacterium avium and Mycobacterium abscessus as well as their strictly pathogenic counterparts Mycobacterium tuberculosis and Mycobacterium leprae . In this regard, this thermophilic species is likely to be useful as a source of stable proteins that may provide more detailed structures of potential drug targets. Here, we investigate M. hassiacum growth at near-pasteurization temperatures and at different pHs and also characterize its thermostable glucosyl-3-phosphoglycerate synthase (GpgS), an enzyme considered essential for M. tuberculosis growth and associated with both nitrogen starvation and thermal stress in different NTM species.

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Molecular epidemiology and phylogenomic analysis of Mycobacterium abscessus clinical isolates in an Asian population

Microbial Genomics ◽

10.1099/mgen.0.000708 ◽

2021 ◽

Vol 7 (11) ◽

Author(s):

Ka Lip Chew ◽

Sophie Octavia ◽

Roland Jureen ◽

Oon Tek Ng ◽

Kalisvar Marimuthu ◽

...

Keyword(s):

Virulence Factors ◽

Type Species ◽

Asian Population ◽

Bioinformatic Analysis ◽

Mycobacterium Abscessus ◽

Phylogenomic Analysis ◽

Pathogenic Potential ◽

Content Type ◽

Link Type ◽

Sequence Types

Mycobacterium abscessus comprises three subspecies: M. abscessus subsp. abscessus , M. abscessus subsp. bolletii , and M. abscessus subsp. massiliense . These closely related strains are typically multi-drug-resistant and can cause difficult-to-treat infections. Dominant clusters of isolates with increased pathogenic potential have been demonstrated in pulmonary infections in the global cystic fibrosis (CF) population. An investigation was performed on isolates cultured from an Asian, predominantly non-CF population to explore the phylogenomic relationships within our population and compare it to global M. abscessus isolates. Whole-genome-sequencing was performed on M. abscessus isolates between 2017 and 2019. Bioinformatic analysis was performed to determine multi-locus-sequence-type, to establish the phylogenetic relationships between isolates, and to identify virulence and resistance determinants in these isolates. A total of 210 isolates were included, of which 68.5 % (144/210) were respiratory samples. These isolates consisted of 140 (66.6 %) M . abscessus subsp. massiliense , 67 (31.9 %) M . abscessus subsp. abscessus, and three (1.4 %) M . abscessus subsp. bolletii . Dominant sequence-types in our population were similar to those of global CF isolates, but SNP differences in our population were comparatively wider despite the isolates being from the same geographical region. ESX (ESAT-6 secretory) cluster three appeared to occur most commonly in ST4 and ST6 M. abscessus subsp. massiliense , but other virulence factors did not demonstrate an association with isolate subspecies or sample source. We demonstrate that although similar predominant sequence-types are seen in our patient population, cross-transmission is absent. The risk of patient-to-patient transmission appears to be largely limited to the vulnerable CF population, indicating infection from environmental sources remains more common than human-to-human transmission. Resistance and virulence factors are largely consistent across the subspecies with the exception of clarithromycin susceptibility and ESX-3.

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Antibacterial activity of high-dose nitric oxide against pulmonary Mycobacterium abscessus disease

Access Microbiology ◽

10.1099/acmi.0.000154 ◽

2020 ◽

Vol 2 (9) ◽

Cited By ~ 1

Author(s):

Kristijan Bogdanovski ◽

Trisha Chau ◽

Chevalia J. Robinson ◽

Sandra D. MacDonald ◽

Ann M. Peterson ◽

...

Keyword(s):

Nitric Oxide ◽

Antibacterial Activity ◽

Type Species ◽

Mycobacterium Abscessus ◽

Clinical Isolates ◽

Content Type ◽

Link Type ◽

Susceptibility Tests ◽

Inhaled No

Introduction. Mycobacterium abscessus is an emerging pulmonary pathogen with limited treatment options. Nitric oxide (NO) demonstrates antibacterial activity against various bacterial species, including mycobacteria. In this study, we evaluated the effect of adjunctive inhaled NO therapy, using a novel NO generator, in a CF patient with pulmonary M. abscessus disease, and examined heterogeneity of response to NO in vitro. Methods. In the compassionate-use treatment, a 24-year-old CF patient with pulmonary M. abscessus was treated with two courses of adjunctive intermittent NO, first at 160 p.p.m. for 21 days and subsequently by escalating the dose up to 240 p.p.m. for 8 days. Methemoglobin, pulmonary function, 6 min walk distance (6MWD), qualify of life and sputum microbiology were assessed. In vitro susceptibility tests were performed against patient’s isolate and comparison clinical isolates and quantified by Hill’s slopes calculated from time–kill curves. Results. M. abscessus lung infection eradication was not achieved, but improvements in selected qualify of life domains, lung function and 6MWD were observed during the study. Inhaled NO was well tolerated at 160 p.p.m. Dosing at 240 p.p.m. was stopped due to adverse symptoms, although methemoglobin levels remained within safety thresholds. In vitro susceptibility tests showed a dose-dependent NO effect on M. abscessus susceptibility and significant heterogeneity in response between M. abscessus clinical isolates. The patient’s isolate was found to be the least susceptible strain in vitro. Conclusion. These results demonstrate heterogeneity in M. abscessus susceptibility to NO and suggest that longer treatment regimens could be required to see the reduction or eradication of more resistant pulmonary strains.

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Intraspecies plasmid and genomic variation of Mycobacterium kubicae revealed by the complete genome sequences of two clinical isolates

Microbial Genomics ◽

10.1099/mgen.0.000497 ◽

2020 ◽

Author(s):

Jo Hendrix ◽

L. Elaine Epperson ◽

David Durbin ◽

Jennifer R. Honda ◽

Michael Strong

Keyword(s):

Type Species ◽

Mycobacterium Abscessus ◽

Genomic Variation ◽

Diagnostic Methods ◽

Pathogenic Potential ◽

Circular Chromosome ◽

Content Type ◽

Link Type ◽

Complete Genomes ◽

Long Read

Mycobacterium kubicae is 1 of nearly 200 species of nontuberculous mycobacteria (NTM), environmental micro-organisms that in some situations can infect humans and cause severe lung, skin and soft tissue infections. Although numerous studies have investigated the genetic variation among prevalent clinical NTM species, including Mycobacterium abscessus and Mycobacterium avium , many of the less common but clinically relevant NTM species, including M. kubicae , still lack complete genomes to serve as a comparative reference. Well-characterized representative genomes for each NTM species are important both for investigating the pathogenic potential of NTM, as well as for use in diagnostic methods, even for species that less frequently cause human disease. Here, we report the complete genomes of two M. kubicae strains, isolated from two unrelated patients. Hybrid short-read and long-read sequencing and assembly, using sequence reads from Illumina and Oxford Nanopore Technologies platforms, were utilized to resolve the chromosome and plasmid sequences of each isolate. The genome of NJH_MKUB1 had 5135 coding sequences (CDSs), a circular chromosome of length 5.3 Mb and two plasmids. The genome of NJH_MKUB2 had 5957 CDSs, a circular chromosome of 6.0 Mb and five plasmids. We compared our completed genomic assemblies to four recently released draft genomes of M. kubicae in order to better understand intraspecies genomic conservation and variability. We also identified genes implicated in drug resistance, virulence and persistence in the M. kubicae chromosome and plasmids. Virulence factors encoded in the genome and in the plasmids of M. kubicae provide a foundation for investigating how opportunistic environmental NTM may cause disease.

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hsp65 Sequencing for Identification of Rapidly Growing Mycobacteria

Journal of Clinical Microbiology ◽

10.1128/jcm.37.3.852-857.1999 ◽

1999 ◽

Vol 37 (3) ◽

pp. 852-857 ◽

Cited By ~ 161

Author(s):

H. Ringuet ◽

C. Akoua-Koffi ◽

S. Honore ◽

A. Varnerot ◽

V. Vincent ◽

...

Keyword(s):

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Mycobacterium Abscessus ◽

Rrna Gene ◽

Mycobacterium Fortuitum ◽

Mycobacterium Chelonae ◽

Rapidly Growing Mycobacteria ◽

Rrna Gene Sequencing ◽

Human Infections ◽

Type Strains

Partial sequencing of the hsp65 gene was used for the identification of rapidly growing mycobacteria (RGM). A 441-bp fragment (A. Telenti, F. Marchesi, M. Balz, F. Bally, E. Böttger, and T. Bodmer, J. Clin. Microbiol. 31:175–178, 1993) was amplified and sequenced by an automated fluorescence-based method involving capillary electrophoresis. Type strains of 10 RGM species were first studied. Each species had a unique nucleotide sequence, distinguishing it clearly from the other species. A panel of strains from the four main RGM species responsible for human infections, Mycobacterium abscessus, Mycobacterium chelonae,Mycobacterium fortuitum, and Mycobacterium peregrinum, was also studied. There were few sequence differences within each of these species (<2% of bases were different from the type strain sequence), and they had no effect on species assignment.hsp65 sequencing unambiguously differentiated M. chelonae and M. abscessus, two species difficult to identify by classical methods and 16S rRNA gene sequencing. The devised procedure is a rapid and reliable tool for the identification of RGM species.

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In Vitro Susceptibilities of Rapidly Growing Mycobacteria to Telithromycin (HMR 3647) and Seven Other Antimicrobials

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.1.181-182.2000 ◽

2000 ◽

Vol 44 (1) ◽

pp. 181-182 ◽

Cited By ~ 31

Author(s):

Ricardo Fernández-Roblas ◽

Jaime Esteban ◽

Froilán Cabria ◽

Juan Carlos López ◽

Maria Soledad Jiménez ◽

...

Keyword(s):

Antimicrobial Activities ◽

Mycobacterium Abscessus ◽

Mycobacterium Fortuitum ◽

Mycobacterium Chelonae ◽

Rapidly Growing Mycobacteria ◽

Mycobacterium Mucogenicum

ABSTRACT The antimicrobial activities of telithromycin (HMR 3647) and seven other antimicrobials against 94 strains of rapidly growing mycobacteria were determined. Telithromycin at a concentration of 1 μg/ml inhibited Mycobacterium peregrinum (100%),Mycobacterium chelonae (80%), Mycobacterium abscessus-Mycobacterium mucogenicum (44.4%), andMycobacterium fortuitum (2.1%). All or most strains ofM. peregrinum, M. fortuitum, and M. mucogenicum were inhibited by 2 μg of quinolones per ml.

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Compromised longevity due to Mycobacterium abscessus pulmonary disease in lungs scarred by tuberculosis

Access Microbiology ◽

10.1099/acmi.0.000003 ◽

2019 ◽

Vol 1 (1) ◽

Author(s):

Urvashi B. Singh ◽

Rojaleen Das ◽

Prajowl Shrestha ◽

Kiran Bala ◽

Pooja Pandey ◽

...

Keyword(s):

Pulmonary Disease ◽

Type Species ◽

Mycobacterium Abscessus ◽

Combination Regimen ◽

Disease Treatment ◽

Content Type ◽

Link Type ◽

Previously Treated ◽

Stopping Treatment

Structural lung diseases or scarring related to prior infections such as tuberculosis (TB) are risk factors for the development of invasive nontuberculous mycobacterial (NTM) pulmonary infections, such as Mycobacterium abscessus . M. abscessus is intrinsically resistant to many antibiotics and in vitro susceptibility correlates poorly with clinical response, especially in pulmonary disease. Treatment is often difﬁcult due to the lack of effective antibiotic regimens. We present a case of a 56-year-old male previously treated for TB, with presumed exacerbation, who was diagnosed after much delay with pulmonary M. abscessus disease and subsequently failed initial treatment with an empirical antibiotic regimen. When placed on a synergistic combination regimen that included amikacin, linezolid, clarithromycin, ethambutol and faropenem, the patient showed a favourable response and was culture-negative for over 12 months when the treatment was stopped as per American Thoracic Society (ATS) recommendations. Unfortunately, he developed recurrent symptoms and died 9 months after stopping treatment, following an acute exacerbation of fever and respiratory failure.

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