Evolution of Mycobacterium abscessus in the human lung: Cumulative mutations and genomic rearrangement of porin genes in patient isolates.

BACKGROUND: Mycobacterium abscessus subspecies massiliense (M. massiliense) is increasingly recognized as an emerging bacterial pathogen, particularly in cystic fibrosis (CF) patients and CF centres' respiratory outbreaks. We characterized genomic and phenotypic changes in 15 serial isolates from two CF patients (1S and 2B) with chronic pulmonary M. massiliense infection leading to death, as well as four isolates from a CF centre outbreak in which patient 2B was the index case. RESULTS: Comparative genomic analysis revealed the mutations affecting growth rate, metabolism, transport, lipids (loss of glycopeptidolipids), antibiotic susceptibility (macrolides and aminoglycosides resistance), and virulence factors. Mutations in 23S rRNA, mmpL4, porin locus and tetR genes occurred in isolates from both CF patients. Interestingly, we identified two different spontaneous mutation events at the mycobacterial porin locus: a fusion of two tandem porin paralogs in patient 1S and a partial deletion of the first porin paralog in patient 2B. These genomic changes correlated with reduced porin protein expression, diminished 14C-glucose uptake, slower bacterial growth rates, and enhanced TNF-α induction in mycobacteria-infected THP-1 human cells. Porin gene complementation of porin mutants partly restored 14C-glucose uptake, growth rate and TNF-α levels to those of intact porin strains. CONCLUSIONS: We hypothesize that specific mutations accumulated and maintained over time in M. massiliense, including mutations shared among transmissible strains, collectively lead to more virulent, host adapted lineages in CF patients and other susceptible hosts.

Disulfiram Is Effective against Drug-Resistant Mycobacterium abscessus in a Zebrafish Embryo Infection Model.

Mycobacterium abscessus is an emerging nontuberculous mycobacterium (NTM) pathogen infecting susceptible people with cystic fibrosis (CF) and non-CF bronchiectasis. Here, we demonstrated the activity of an FDA-approved drug, disulfiram, against drug-susceptible and drug-resistant M. abscessus strains utilizing in vitro and intracellular macrophage assays and a zebrafish embryo infection model. These data demonstrate effective antimicrobial activity of disulfiram against M. abscessus infection in vivo and strongly support further study of disulfiram in human NTM infections.

Detection of Mixed Populations of Clarithromycin-Susceptible and -Resistant Mycobacterium abscessus Strains.

Clarithromycin resistance in Mycobacterium abscessus subsp. abscessus, massiliense, and bolletii occurs through induction of erm(41) or mutations in rrl (23S rRNA) genes. Phenotypic detection of clarithromycin resistance is hindered by the need for extended incubation as well as co-occurrence of mixed populations of M. abscessus with different susceptibility profiles. We developed a quantitative EvaGreen-based droplet digital PCR (ddPCR) scheme for rapid detection of full-length or truncated erm(41) and a probe based ddPCR screening assay for assessment of 23S rRNA rrl mutational resistance. We tested 100 M. abscessus strains, synthetic mixes with different susceptibility profiles, and 13 positive MGIT samples. Truncated and full-length erm(41) genes were detected in 27/100 and 73/100 strains and 4/13 and 9/13 MGIT samples, respectively yielding a sensitivity and specificity of 100%. Clarithromycin resistance mutations in rrl were detected in 26/100 isolates, i.e., A2058G (18/100), A2058C (7/100), and A2059G (1/100), and in 3/13 MGIT samples, i.e., A2058G (2/13) and A2059G (1/13). A screening assay of rrl ddPCR (A2058A/A2058G probes) showed 100% sensitivity in detecting the wild type or A2058G mutation as well as identifying samples requiring further testing. Upon inclusion of additional ddPCR assays, we were able to detect A2058C and A2059G clarithromycin resistance-conferring mutations in the rrl gene. Our ddPCR scheme can differentiate between full-length and truncated erm(41) and identify clarithromycin resistance-conferring mutations in the rrl gene from clinical isolates and positive MGIT samples as well as deconvolute and quantitate mixed populations of M. abscessus with different clarithromycin resistance traits.

Tissue specific diversification, virulence and immune response to Mycobacterium bovis BCG in a patient with an IFN-γ R1 deficiency.

Summary: We characterized Mycobacterium bovis BCG isolates found in lung and brain samples from a previously vaccinated patient with IFNγR1 deficiency. The isolates collected displayed distinct genomic and phenotypic features consistent with host adaptation and associated changes in antibiotic susceptibility and virulence traits. Background: We report a case of a patient with partial recessive IFNγR1 deficiency who developed disseminated BCG infection after neonatal vaccination (BCG-vaccine). Distinct M. bovis BCG-vaccine derived clinical strains were recovered from the patient's lungs and brain. Methods: BCG strains were phenotypically (growth, antibiotic susceptibility, lipid) and genetically (whole genome sequencing) characterized. Mycobacteria cell infection models were used to assess apoptosis, necrosis, cytokine release, autophagy, and JAK-STAT signaling. Results: Clinical isolates BCG-brain and BCG-lung showed distinct Rv0667 rpoB mutations conferring high- and low-level rifampin resistance; the latter displayed clofazimine resistance through Rv0678 gene (MarR-like transcriptional regulator) mutations. BCG-brain and BCG-lung showed mutations in fadA2, fadE5, and mymA operon genes, respectively. Lipid profiles revealed reduced levels of PDIM in BCG-brain and BCG-lung and increased TAGs and Mycolic acid components in BCG-lung, compared to parent BCG-vaccine. In vitro infected cells showed that the BCG-lung induced a higher cytokine release, necrosis, and cell-associated bacterial load effect when compared to BCG-brain; conversely, both strains inhibited apoptosis and altered JAK-STAT signaling. Conclusions: During a chronic-disseminated BCG infection, BCG strains can evolve independently at different sites likely due to particular microenvironment features leading to differential antibiotic resistance, virulence traits resulting in dissimilar responses in different host tissues.

Patients with Idiopathic Pulmonary Nontuberculous Mycobacterial Disease Have Normal Th1/Th2 Cytokine Responses but Diminished Th17 Cytokine and Enhanced Granulocyte-Macrophage Colony-Stimulating Factor Production.

OBJECTIVE: Although disseminated nontuberculous mycobacterial infection is attributed to defects in the interleukin (IL)-12/interferon-γ circuit, the immunophenotype of idiopathic pulmonary nontuberculous mycobacterial (PNTM) disease is not well defined. METHOD: We phenotyped Th1, Th2, Th17, and Treg cytokines and colony-stimulating factor production from patients with idiopathic PNTM disease. Data were compared with healthy donors, cystic fibrosis (CF), and primary ciliary dyskinesia (PCD) patients with PNTM disease. Both supernatant cytokine production and intracellular cytokines expressed by various leukocyte subpopulations following mitogen and antigen stimulation were assayed by electrochemiluminescence-based multiplex immunoassay and flow cytometry, respectively. RESULTS: Regardless of antigen or mitogen stimulation, neither intracellular nor extracellular Th1, Th2, and Treg cytokine levels differed between patients and controls. Th17 cells and IL-17A levels were lower in idiopathic PNTM patients, whereas monocyte granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in response to NTM stimulation was higher compared with healthy donors. Besides, distinct cytokine responses following stimulation by Mycobacterium abscessus and Mycobacterium avium were observed consistently within each group. CONCLUSIONS: The IL-12/IFN-γ circuit appeared intact in patients with idiopathic PNTM disease. However, idiopathic PNTM patients had reduced Th17 response and higher mycobacteria-induced monocyte GM-CSF expression.

Amikacin exposure and susceptibility of macrolide-resistant Mycobacterium abscessus.

Mycobacterium abscessus is associated with antibiotic resistance and poor treatment outcomes. We described within-patient changes in M. abscessus resistance to clarithromycin and amikacin. Patients with amikacin exposure and a >50-month interval between M. abscessus isolates were identified. Antimicrobial susceptibility testing was performed on the first and last isolates by broth microdilution, and genetic markers of resistance were identified. 16 patients were identified with a median amikacin exposure of 2.3 years (range 0.6-8.6 years). 15 patients also received macrolides (median 7.2 years, range 1.3-10.7 years). All initial isolates were resistant to clarithromycin (minimum inhibitory concentration (MIC) ≥8 µg·mL-1). Two patients had later susceptible isolates, which were of a different subspecies (M. abscessus subsp. massiliense) than the initial isolates (M. abscessus subsp. abscessus). All initial isolates were susceptible or intermediately resistant to amikacin, and only one patient had a resistant final isolate (MIC >64 µg·mL-1), accompanied by an A→G mutation at position 1408 of the 16S ribosomal RNA. Forced expiratory volume in 1 s decreased significantly over the study period, while smear quantity and the proportions of patients with elevated C-reactive protein or cavitary lesions all increased significantly. Despite prolonged, mostly inhaled amikacin exposure, development of amikacin resistance was uncommon in this patient population; however, disease progression continued.

Transcriptional Response of Respiratory Epithelium to Nontuberculous Mycobacteria.

The incidence of pulmonary nontuberculous mycobacteria (NTM) disease is increasing, but host responses in respiratory epithelium infected with NTM are not fully understood. In this work, we aimed to identify infection-relevant gene expression signatures of NTM infection of the respiratory epithelium. We infected air-liquid interface (ALI) primary respiratory epithelial cell cultures with Mycobacterium avium subsp. avium (MAC) or Mycobacterium abscessus subsp. abscessus (MAB). We used cells from four different donors to obtain generalizable data. Differentiated respiratory epithelial cells at the ALI were infected with MAC or MAB at a multiplicity of infection of 100:1 or 1,000:1, and RNA sequencing was performed at Days 1 and 3 after infection. In response to infection, we found down-regulation of ciliary genes but upregulation of genes associated with cytokines/chemokines, such as IL-32, and cholesterol biosynthesis. Inflammatory response genes tended to be more upregulated by MAB than by MAC infection. Primary respiratory epithelial cell infection with NTM at the ALI identified ciliary function, cholesterol biosynthesis, and cytokine/chemokine production as major host responses to infection. Some of these pathways may be amenable to therapeutic manipulation.

New Real-Time PCR Assays for Detection of Inducible and Acquired Clarithromycin Resistance in the Mycobacterium abscessus Group.

Members of the Mycobacterium abscessus group (MAG) cause lung, soft tissue, and disseminated infections. The oral macrolides clarithromycin and azithromycin are commonly used for treatment. MAG can display clarithromycin resistance through the inducible erm(41) gene or via acquired mutations in the rrl (23S rRNA) gene. Strains harboring a truncation or a T28C substitution in erm(41) lose the inducible resistance trait. Phenotypic detection of clarithromycin resistance requires extended incubation (14 days), highlighting the need for faster methods to detect resistance. Two real-time PCR-based assays were developed to assess inducible and acquired clarithromycin resistance and tested on a total of 90 clinical and reference strains. A SYBR green assay was designed to distinguish between a full-length and truncated erm(41) gene by temperature shift in melting curve analysis. Single nucleotide polymorphism (SNP) allele discrimination assays were developed to distinguish T or C at position 28 of erm(41) and 23S rRNA rrl gene mutations at position 2058 and/or 2059. Truncated and full-size erm(41) genes were detected in 21/90 and 69/90 strains, respectively, with 64/69 displaying T at nucleotide position 28 and 5/69 containing C at that position. Fifteen isolates showed rrl mutations conferring clarithromycin resistance, including A2058G (11 isolates), A2058C (3 isolates), and A2059G (1 isolate). Targeted sequencing and phenotypic assessment of resistance concurred with molecular assay results. Interestingly, we also noted cooccurring strains harboring an active erm(41), inactive erm(41), and/or acquired mutational resistance, as well as slowly growing MAG strains and also strains displaying an inducible resistance phenotype within 5 days, long before the recommended 14-day extended incubation.

Clonal Diversification and Changes in Lipid Traits and Colony Morphology in Mycobacterium abscessus Clinical Isolates.

The smooth-to-rough colony morphology shift in Mycobacterium abscessus has been implicated in loss of glycopeptidolipid (GPL), increased pathogenicity, and clinical decline in cystic fibrosis (CF) patients. However, the evolutionary phenotypic and genetic changes remain obscure. Serial isolates from nine non-CF patients with persistent M. abscessus infection were characterized by colony morphology, lipid profile via thin-layer chromatography and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), sequencing of eight genes in the GPL locus, and expression level of fadD23, a key gene involved in the biosynthesis of complex lipids. All 50 isolates were typed as M. abscessus subspecies abscessus and were clonally related within each patient. Rough isolates, all lacking GPL, predominated at later disease stages, some showing variation within rough morphology. While most (77%) rough isolates harbored detrimental mutations in mps1 and mps2, 13% displayed previously unreported mutations in mmpL4a and mmpS4, the latter yielding a putative GPL precursor. Two isolates showed no deleterious mutations in any of the eight genes sequenced. Mixed populations harboring different GPL locus mutations were detected in 5 patients, demonstrating clonal diversification, which was likely overlooked by conventional acid-fast bacillus (AFB) culture methods. Our work highlights applications of MALDI-TOF MS beyond identification, focusing on mycobacterial lipids relevant in virulence and adaptation. Later isolates displayed accumulation of triacylglycerol and reduced expression of fadD23, sometimes preceding rough colony onset. Our results indicate that clonal diversification and a shift in lipid metabolism, including the loss of GPL, occur during chronic lung infection with M. abscessus. GPL loss alone may not account for all traits associated with rough morphology.

High-level relatedness among Mycobacterium abscessus subsp. massiliense strains from widely separated outbreaks.

Three recently sequenced strains isolated from patients during an outbreak of Mycobacterium abscessus subsp. massiliense infections at a cystic fibrosis center in the United States were compared with 6 strains from an outbreak at a cystic fibrosis center in the United Kingdom and worldwide strains. Strains from the 2 cystic fibrosis outbreaks showed high-level relatedness with each other and major-level relatedness with strains that caused soft tissue infections during an epidemic in Brazil. We identified unique single-nucleotide polymorphisms in cystic fibrosis and soft tissue outbreak strains, separate single-nucleotide polymorphisms only in cystic fibrosis outbreak strains, and unique genomic traits for each subset of isolates. Our findings highlight the necessity of identifying M. abscessus to the subspecies level and screening all cystic fibrosis isolates for relatedness to these outbreak strains. We propose 2 diagnostic strategies that use partial sequencing of rpoB and secA1 genes and a multilocus sequence typing protocol.

Inhaled amikacin for treatment of refractory pulmonary nontuberculous mycobacterial disease.

RATIONALE: Treatment of pulmonary nontuberculous mycobacteria, especially Mycobacterium abscessus, requires prolonged, multidrug regimens with high toxicity and suboptimal efficacy. Options for refractory disease are limited. OBJECTIVES: We reviewed the efficacy and toxicity of inhaled amikacin in patients with treatment-refractory nontuberculous mycobacterial lung disease. METHODS: Records were queried to identify patients who had inhaled amikacin added to failing regimens. Lower airway microbiology, symptoms, and computed tomography scan changes were assessed together with reported toxicity. MEASUREMENTS AND MAIN RESULTS: The majority (80%) of the 20 patients who met entry criteria were women; all had bronchiectasis, two had cystic fibrosis and one had primary ciliary dyskinesia. At initiation of inhaled amikacin, 15 were culture positive for M. abscessus and 5 for Mycobacterium avium complex and had received a median (range) of 60 (6, 190) months of mycobacterial treatment. Patients were followed for a median of 19 (1, 50) months. Eight (40%) patients had at least one negative culture and 5 (25%) had persistently negative cultures. A decrease in smear quantity was noted in 9 of 20 (45%) and in mycobacterial culture growth for 10 of 19 (53%). Symptom scores improved in nine (45%), were unchanged in seven (35%), and worsened in four (20%). Improvement on computed tomography scans was noted in 6 (30%), unchanged in 3 (15%), and worsened in 11 (55%). Seven (35%) stopped amikacin due to: ototoxicity in two (10%), hemoptysis in two (10%), and nephrotoxicity, persistent dysphonia, and vertigo in one each. CONCLUSIONS: In some patients with treatment-refractory pulmonary nontuberculous mycobacterial disease, the addition of inhaled amikacin was associated with microbiologic and/or symptomatic improvement; however, toxicity was common. Prospective evaluation of inhaled amikacin for mycobacterial disease is warranted.

New rapid scheme for distinguishing the subspecies of the Mycobacterium abscessus group and identifying Mycobacterium massiliense isolates with inducible clarithromycin resistance.

Mycobacterium abscessus (M. abscessus sensu lato, or the M. abscessus group) comprises three closely related taxa whose taxonomic statuses are under revision, i.e., M. abscessus sensu stricto, Mycobacterium bolletii, and Mycobacterium massiliense. We describe here a simple, robust, and cost-effective PCR-based method for distinguishing among M. abscessus, M. massiliense, and M. bolletii. Based on the M. abscessus ATCC 19977(T) genome, regions that discriminated between M. abscessus and M. massiliense were identified through array-based comparative genomic hybridization. A typing scheme using PCR primers designed for four of these locations was applied to 46 well-characterized clinical isolates comprising 29 M. abscessus, 15 M. massiliense, and 2 M. bolletii isolates previously identified by multitarget sequencing. Interestingly, 2 isolates unequivocally identified as M. massiliense were shown to have a full-length erm(41) gene instead of the expected gene deletion and showed inducible clarithromycin resistance after 14 days. We propose using this PCR-based typing scheme combined with erm(41) PCR for straightforward identification of M. abscessus, M. massiliense, and M. bolletii and the assessment of inducible clarithromycin resistance. This method can be easily integrated into a routine workflow to provide subspecies-level identification within 24 h after isolation of the M. abscessus group.

Improved variation calling via an iterative backbone remapping and local assembly method for bacterial genomes.

Sequencing data analysis remains limiting and problematic, especially for low complexity repeat sequences and transposon elements due to inherent sequencing errors and short sequence read lengths. We have developed a program, ReviSeq, which uses a hybrid method composed of iterative remapping and local assembly upon a bacterial sequence backbone. Application of this method to six Brucella suis field isolates compared to the newly revised B. suis 1330 reference genome identified on average 13, 15, 19 and 9 more variants per sample than STAMPY/SAMtools, BWA/SAMtools, iCORN and BWA/PINDEL pipelines, and excluded on average 4, 2, 3 and 19 variants per sample, respectively. In total, using this iterative approach, we identified on average 87 variants including SNVs, short INDELs and long INDELs per strain when compared to the reference. Our program outperforms other methods especially for long INDEL calling. The program is available at http://reviseq.sourceforge.net.

Comparison of genome diversity of Brucella spp. field isolates using Universal Bio-signature Detection Array and whole genome sequencing reveals limitations of current diagnostic methods.

The detection and identification of bio-threat agents and the study of host-pathogen interactions require a high-resolution detection platform capable of discerning closely related species. Diverse analysis methods are used to identify pathogens, specifically Brucella species or biovars. In this study, we compared four diagnostic approaches including serology-based biochemical test, PCR assay, microarray analysis using a Universal Bio-signature Detection Array (UBDA) and whole genome "deep" sequencing for Brucella organisms including a number of field isolates. We found that although there was frequent agreement among the different tests, some tests gave compound/contradictory results that were a consequence of species diversity due to mixed infections or minor contaminants as measured by UBDA and validated from whole genome sequence. By comparing these analysis techniques, we demonstrate that standard diagnostics used in the field are limited in their ability to identify genomic DNA contaminants in field isolates while UBDA and sequencing analysis are highly sensitive in tracing genomic differences among the isolates.

Complete genome sequence of Brucella suis VBI22, isolated from bovine milk.

Brucella suis is the causative agent of swine brucellosis and is known to be able to infect several different hosts, including cattle, dogs, and horses, without causing disease symptoms. Here we report the complete genome sequence of Brucella suis VBI22, which was isolated from raw milk from an infected cow.

Revised genome sequence of Brucella suis 1330.

Brucella suis is a causative agent of porcine brucellosis. We report the resequencing of the original sample upon which the published sequence of Brucella suis 1330 is based and describe the differences between the published assembly and our assembly at 12 loci.

A species independent universal bio-detection microarray for pathogen forensics and phylogenetic classification of unknown microorganisms.

BACKGROUND: The ability to differentiate a bioterrorist attack or an accidental release of a research pathogen from a naturally occurring pandemic or disease event is crucial to the safety and security of this nation by enabling an appropriate and rapid response. It is critical in samples from an infected patient, the environment, or a laboratory to quickly and accurately identify the precise pathogen including natural or engineered variants and to classify new pathogens in relation to those that are known. Current approaches for pathogen detection rely on prior genomic sequence information. Given the enormous spectrum of genetic possibilities, a field deployable, robust technology, such as a universal (any species) microarray has near-term potential to address these needs. RESULTS: A new and comprehensive sequence-independent array (Universal Bio-Signature Detection Array) was designed with approximately 373,000 probes. The main feature of this array is that the probes are computationally derived and sequence independent. There is one probe for each possible 9-mer sequence, thus 49 (262,144) probes. Each genome hybridized on this array has a unique pattern of signal intensities corresponding to each of these probes. These signal intensities were used to generate an un-biased cluster analysis of signal intensity hybridization patterns that can easily distinguish species into accepted and known phylogenomic relationships. Within limits, the array is highly sensitive and is able to detect synthetically mixed pathogens. Examples of unique hybridization signal intensity patterns are presented for different Brucella species as well as relevant host species and other pathogens. These results demonstrate the utility of the UBDA array as a diagnostic tool in pathogen forensics. CONCLUSIONS: This pathogen detection system is fast, accurate and can be applied to any species. Hybridization patterns are unique to a specific genome and these can be used to decipher the identity of a mixed pathogen sample and can separate hosts and pathogens into their respective phylogenomic relationships. This technology can also differentiate between different species and classify genomes into their known clades. The development of this technology will result in the creation of an integrated biomarker-specific bio-signature, multiple select agent specific detection system.

Investigating the genome diversity of B. cereus and evolutionary aspects of B. anthracis emergence.

Here we report the use of a multi-genome DNA microarray to investigate the genome diversity of Bacillus cereus group members and elucidate the events associated with the emergence of Bacillus anthracis the causative agent of anthrax-a lethal zoonotic disease. We initially performed directed genome sequencing of seven diverse B. cereus strains to identify novel sequences encoded in those genomes. The novel genes identified, combined with those publicly available, allowed the design of a "species" DNA microarray. Comparative genomic hybridization analyses of 41 strains indicate that substantial heterogeneity exists with respect to the genes comprising functional role categories. While the acquisition of the plasmid-encoded pathogenicity island (pXO1) and capsule genes (pXO2) represents a crucial landmark dictating the emergence of B. anthracis, the evolution of this species and its close relatives was associated with an overall shift in the fraction of genes devoted to energy metabolism, cellular processes, transport, as well as virulence.

A correlation analysis of protein characteristics associated with genome-wide high throughput expression and solubility of Streptococcus pneumoniae proteins.

We have developed and evaluated a highly parallel protein expression and purification system using ORFs derived from the pathogenic bacterium Streptococcus pneumoniae as a representative test case in conjunction with the Gateway cloning technology. Establishing high throughput protein production capability is essential for genome-wide characterization of protein function. In this study, we focused on protein expression and purification outcomes generated from an expression vector which encodes an NH(2)-terminal hexa-histidine tag and a COOH-terminal S-tag. Purified recombinant proteins were validated by SDS-PAGE, followed by in-gel digestion and identification by MALDI-TOF/TOF analysis. Starting with 1360 sequence-validated destination clones we examined correlation analyses of expression and solubility of a wide variety of recombinant proteins. In total, 428 purified proteins (31%) were recovered in soluble form. We describe a semi-quantitative scoring method using an S-tag assay to improve the throughput and efficiency of expression and solubility studies for recombinant proteins. Given a relatively large dataset derived from proteins representing all functional groups in a microbial genome we correlated various protein characteristics as they relate to protein expression outcomes.

Genome sequence of Theileria parva, a bovine pathogen that transforms lymphocytes.

We report the genome sequence of Theileria parva, an apicomplexan pathogen causing economic losses to smallholder farmers in Africa. The parasite chromosomes exhibit limited conservation of gene synteny with Plasmodium falciparum, and its plastid-like genome represents the first example where all apicoplast genes are encoded on one DNA strand. We tentatively identify proteins that facilitate parasite segregation during host cell cytokinesis and contribute to persistent infection of transformed host cells. Several biosynthetic pathways are incomplete or absent, suggesting substantial metabolic dependence on the host cell. One protein family that may generate parasite antigenic diversity is not telomere-associated.