Loss of LpqM proteins in Mycobacterium abscessus is associated with impaired intramacrophage survival.

Mycobacterium abscessus, an emerging pathogen responsible for severe pulmonary infections in cystic fibrosis patients, displays either a smooth (S) or a rough (R) morphotype. Infections with M. abscessus R are associated with increased pathogenicity in animal models and humans. While the S-to-R transition correlating with reduced glycopeptidolipid (GPL) production is well-documented, the recent screening of a transposon library revealed additional gene candidates located outside of the GPL locus involved in this transition. These genes include MAB_1470c, encoding the putative lipoprotein peptidase LpqM. However, experimental confirmation of the implication of this gene in the morphotype switch is lacking. Herein, we re-examined the role of MAB_1470c, and its homolog MAB_1466c, in colonial morphotype changes by generating unmarked deletion mutants in M. abscessus S. Our results indicate that the morphotype of these mutants stayed smooth in different media. Unexpectedly, the intracellular growth of ΔMAB_1470c and ΔMAB_1466c in THP-1 macrophages was significantly reduced as compared to the parental S strain, and these defects were rescued upon complementation with their corresponding genes. Strikingly, the intracellular survival defect was further exacerbated in a mutant lacking both MAB_1470c and MAB_1466c genes. This implies that, despite their primary sequence relatedness, the two proteins are not functionally redundant. Collectively, this suggests that these two LpqM-related lipoproteins are unlikely to be involved in the S-to-R transition but are key players for intramacrophage survival of M. abscessus. IMPORTANCE: Mycobacterium abscessus causes persistent infections in patients with underlying pulmonary diseases, resulting in progressive lung function deterioration. The rough (R) morphotype is well-established as associated with chronic and more aggressive infections in patients. In this study, we individually and simultaneously deleted the MAB_1470c and MAB_1466c genes in M. abscessus S, without observing changes in colony morphotypes. However, these mutants exhibited a severe impairment in their ability to survive within human macrophages, highlighting the critical role of these two lipoproteins in M. abscessus virulence.

Efflux pump effects on levofloxacin resistance in Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an important role in mediating drug resistance. The current study examined the potential of efflux pump inhibitors to reverse levofloxacin (LFX) resistance in M. abscessus. The reference strain of M. abscessus (ATCC19977) and 60 clinical isolates, including 41 M. abscessus subsp. abscessus and 19 M. abscessus subsp. massilense, were investigated. The drug sensitivity of M. abscessus against LFX alone or in conjunction with efflux pump inhibitors, including verapamil (VP), reserpine (RSP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or dicyclohexylcarbodiimide (DCC), were determined by AlarmarBlue microplate assay. Drug-resistant regions of the gyrA and gyrB genes from the drug-resistant strains were sequenced. The transcription level of the efflux pump genes was monitored using qRT-PCR. All the tested strains were resistant to LFX. The drug-resistant regions from the gyrA and gyrB genes showed no mutation associated with LFX resistance. CCCP, DCC, VP, and RSP increased the susceptibility of 93.3% (56/60), 91.7% (55/60), 85% (51/60), and 83.3% (50/60) isolates to LFX by 2 to 32-fold, respectively. Elevated transcription of seven efflux pump genes was observed in isolates with a high reduction in LFX MIC values in the presence of efflux pump inhibitors. Efflux pump inhibitors can improve the antibacterial activity of LFX against M. abscessus in vitro. The overexpression of efflux-related genes in LFX-resistant isolates suggests that efflux pumps are associated with the development of LFX resistance in M. abscessus.

Gene knock-out in Mycobacterium abscessus using Streptococcus thermophilus CRISPR/Cas.

The CRISPRi system using dCas9Sth1 from Streptococcus thermophilus developed for Mycobacterium tuberculosis and M. smegmatis was modified to allow gene knock-out in M. abscessus. Efficacy of the knock-out system was evaluated by applying deletions and insertions to the mps1 gene. A comparative genomic analysis of mutants and wild type validated the target specificity.

A dual-plasmid CRISPR/Cas9-based method for rapid and efficient genetic disruption in Mycobacterium abscessus.

Mycobacterium abscessus is increasingly recognized for causing infections that are notoriously difficult to treat, owing to its large arsenal of intrinsic antibiotic resistance mechanisms. Tools for the genetic manipulation of the pathogen are critical for enabling a better understanding of M. abscessus biology, pathogenesis, and antibiotic resistance mechanisms. However, existing methods are largely recombination-based, which are relatively inefficient. Meanwhile, CRISPR/Cas9 has revolutionized the field of genome editing including its recent adaptation for use in mycobacteria. In this study, we report a streamlined and efficient method for rapid genetic disruptions in M. abscessus. Harnessing the CRISPR1 loci from Streptococcus thermophilus, we have developed a dual-plasmid workflow that introduces Cas9 and sgRNA cassettes in separate steps but requires no other additional factors to engineer mutations in single genes or multiple genes simultaneously or sequentially using multiple targeting sgRNAs. Importantly, the efficiency of mutant generation is several orders of magnitude higher than reported for homologous recombination-based methods. This work, thus, reports the first application of CRISPR/Cas9 for gene editing in M. abscessus and is an important tool in the arsenal for the genetic manipulation of this human pathogen. IMPORTANCE: Mycobacterium abscessus is an opportunistic pathogen of increasing clinical importance due to its poor clinical outcomes and limited treatment options. Drug discovery and development in this highly antibiotic-resistant species will require further understanding of M. abscessus biology, pathogenesis, and antibiotic resistance mechanisms. However, existing methods for facile genetic engineering are relatively inefficient. This study reports on the first application of CRISPR/Cas9 for gene editing in M. abscessus using a dual-plasmid workflow. We establish that our method is easily programmable, efficient, and versatile for genetic disruptions in M. abscessus. This is a critical advancement to facilitating targeted gene function studies in this emerging pathogen.

Lsr2, a pleiotropic regulator at the core of the infectious strategy of Mycobacterium abscessus.

Mycobacterium abscessus is a non-tuberculous mycobacterium, causing lung infections in cystic fibrosis patients. During pulmonary infection, M. abscessus switches from smooth (Mabs-S) to rough (Mabs-R) morphotypes, the latter being hyper-virulent. Previously, we isolated the lsr2 gene as differentially expressed during S-to-R transition. lsr2 encodes a pleiotropic transcription factor that falls under the superfamily of nucleoid-associated proteins. Here, we used two functional genomic methods, RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), to elucidate the molecular role of Lsr2 in the pathobiology of M. abscessus. Transcriptomic analysis shows that Lsr2 differentially regulates gene expression across both morphotypes, most of which are involved in several key cellular processes of M. abscessus, including host adaptation and antibiotic resistance. These results were confirmed through quantitative real-time PCR, as well as by minimum inhibitory concentration tests and infection tests on macrophages in the presence of antibiotics. ChIP-seq analysis revealed that Lsr2 extensively binds the M. abscessus genome at AT-rich sequences and appears to form long domains that participate in the repression of its target genes. Unexpectedly, the genomic distribution of Lsr2 revealed no distinctions between Mabs-S and Mabs-R, implying more intricate mechanisms at play for achieving target selectivity.IMPORTANCELsr2 is a crucial transcription factor and chromosome organizer involved in intracellular growth and virulence in the smooth and rough morphotypes of Mycobacterium abscessus. Using RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), we investigated the molecular role of Lsr2 in gene expression regulation along with its distribution on M. abscessus genome. Our study demonstrates the pleiotropic regulatory role of Lsr2, regulating the expression of many genes coordinating essential cellular and molecular processes in both morphotypes. In addition, we have elucidated the role of Lsr2 in antibiotic resistance both in vitro and in vivo, where lsr2 mutant strains display heightened sensitivity to antibiotics. Through ChIP-seq, we reported the widespread distribution of Lsr2 on M. abscessus genome, revealing a direct repressive effect due to its extensive binding on promoters or coding sequences of its targets. This study unveils the significant regulatory role of Lsr2, intricately intertwined with its function in shaping the organization of the M. abscessus genome.

Antibiotic resistance and genomic characterization of Mycobacterium abscessus complex isolates from patients with pulmonary tuberculosis in Iran: A multi-center study (2010-2021).

One hundred and eighteen sputum specimens suspected of Mycobacterium abscessus infection were collected. Species level identification of M. abscessus was performed by rpoB sequencing. Clonality analysis was done by multilocus sequence typing (MLST) for M. abscessus. Antibiotic susceptibility testing was performed for clarithromycin, amikacin, ciprofloxacin and moxifloxacin. Altogether 128 isolates were obtained and were subjected to rpoB gene sequencing for definite identification. Among them 59 were identified as M. abscessus, and these included 22 (37.28%) isolates of M. abscessus subsp. abscessus, 22 (37.28%) isolates of M. abscessus subsp. massiliense, and 15 (25.42%) isolates of M. abscessus subsp. bolletii. All 59 M. abscessus complex isolates were analyzed by MLST in this study. Certain sequence types (STs) were identified among the 59 isolates and were specific for each subspecies. Two STs (ST40 and ST33) were specific to M. abscessus subsp. abscessus, one ST (ST20) was specific to M. abscessus subsp. bolletii, and one ST (ST15) was specific to M. abscessus subsp. massiliense. In antibiotic resistance, clarithromycin susceptibility testing of 22 M. abscessus subsp. abscessus strains detected 15 (68.18%) resistant strains, while among 22 M. abscessus subsp. massiliense strains 5 (22.72%) exhibited resistance, and among 15 M. abscessus subsp. bolletii 8 (53.33%) were resistant. Our study revealed a significant level of antibiotic resistance in isolates of the M. abscessus complex.

Mycothione reductase as a potential target in the fight against Mycobacterium abscessus infections.

While infections caused by Mycobacterium abscessus complex (MABC) are rising worldwide, the current treatment of these infections is far from ideal due to its numerous shortcomings thereby increasing the urge for novel drug targets. In this study, mycothione reductase (Mtr) was evaluated for its potential as a drug target for MABC infections since it is a key enzyme needed in the recycling of mycothiol, the main low-molecular-weight thiol protecting the bacteria against reactive oxygen species and other reactive intermediates. First, a Mab∆mtr mutant strain was generated, lacking mtr expression. Next, the in vitro sensitivity of Mab∆mtr to oxidative stress and antimycobacterial drugs was determined. Finally, we evaluated the intramacrophage survival and the virulence of Mab∆mtr in Galleria mellonella larvae. Mab∆mtr demonstrated a 39.5-fold reduction in IC90 when exposed to bedaquiline in vitro. Furthermore, the Mab∆mtr mutant showed a decreased ability to proliferate inside macrophages and larvae, suggesting that Mtr plays an important role during MABC infection. Altogether, these findings support the assumption of Mtr being a potential target for antimycobacterial drugs.IMPORTANCEMycobacterium abscessus complex (MABC) is a group of bacteria causing a serious public health problem worldwide due to its ability to cause progressive disease, its highly resistant profile against various antibiotics, and its lengthy treatment. Therefore, new drugs are needed to alleviate antibiotic resistance and reduce the length of the current treatment. A potential new target for new antibiotics is mycothione reductase (Mtr), an important enzyme belonging to a pathway that protects the bacteria against harmful conditions. Our research created a bacterium deficient of mtr by using advanced genetic techniques and demonstrated that mtr-deficient bacteria have a decreased ability to multiply during infection. Furthermore, we show evidence that currently used antibiotics combined with mtr deficiency can lead to a better treatment of MABC infection. Altogether, our results validate Mtr as a potential new target and suggest that Mtr plays a role during MABC infection.

Pharmacological validation of dihydrofolate reductase as a drug target in Mycobacterium abscessus.

The Mycobacterium abscessus drug development pipeline is poorly populated, with particularly few validated target-lead couples to initiate de novo drug discovery. Trimethoprim, an inhibitor of dihydrofolate reductase (DHFR) used for the treatment of a range of bacterial infections, is not active against M. abscessus. Thus, evidence that M. abscessus DHFR is vulnerable to pharmacological intervention with a small molecule inhibitor is lacking. Here, we show that the pyrrolo-quinazoline PQD-1, previously identified as a DHFR inhibitor active against Mycobacterium tuberculosis, exerts whole cell activity against M. abscessus. Enzyme inhibition studies showed that PQD-1, in contrast to trimethoprim, is a potent inhibitor of M. abscessus DHFR and over-expression of DHFR causes resistance to PQD-1, providing biochemical and genetic evidence that DHFR is a vulnerable target and mediates PQD-1's growth inhibitory activity in M. abscessus. As observed in M. tuberculosis, PQD-1 resistant mutations mapped to the folate pathway enzyme thymidylate synthase (TYMS) ThyA. Like trimethoprim in other bacteria, PQD-1 synergizes with the dihydropteroate synthase (DHPS) inhibitor sulfamethoxazole (SMX), offering an opportunity to exploit the successful dual inhibition of the folate pathway and develop similarly potent combinations against M. abscessus. PQD-1 is active against subspecies of M. abscessus and a panel of clinical isolates, providing epidemiological validation of the target-lead couple. Leveraging a series of PQD-1 analogs, we have demonstrated a dynamic structure-activity relationship (SAR). Collectively, the results identify M. abscessus DHFR as an attractive target and PQD-1 as a chemical starting point for the discovery of novel drugs and drug combinations that target the folate pathway in M. abscessus.

GlnR activated transcription of nitrogen metabolic pathway genes facilitates biofilm formation by mycobacterium abscessus.

OBJECTIVES: Nitrogen is indispensable for the synthesis of biomacromolecules. The correlation between nitrogen metabolism and Mycobacterium abscessus (M. abscessus) biofilm formation is unclear. This study constructed global nitrogen regulator gene GlnR (Mab_0744) knockout (ΔglnR) and complementation (ΔglnR::glnR) M. abscessus strains. METHODS: Global nitrogen regulator gene glnR (Mab_0744) knockout (ΔglnR) and complementation (ΔglnR::glnR) M. abscessus strains were constructed. Sauton's medium was used to culture M. abscessus pellicle biofilm. To test the antibiotic susceptibility of pellicle biofilm, clarithromycin, amikacin, cefoxitin or imipenem was added to the medium under biofilms after 14 days of incubation. RT-qPCR and ChIP-qPCR were performed to analyse the transcriptional regulatory function of GlnR. RESULTS: GlnR knockout decreased the growth rate of planktonic cells, reduced biofilm mass and wrinkle formation, and diminished the resistance of biofilms to antibiotics. However, the susceptibility of planktonic cells to antibiotics was not changed by glnR knockout. The growth rate of planktonic ΔglnR cells was accelerated by adding nitrogen sources to the medium; the addition of glutamine or sodium glutamate rescued ΔglnR biofilm morphology and resistance to amikacin, cefoxitin, clarithromycin and imipenem. GlnR bound the promoter region and activated the transcription of eight nitrogen metabolic pathway genes (i.e. glnA, amt, ansP, nirB, nirD, glnD, glnK and narK3), which are closely related to glutamine/glutamate biosynthesis and, thus, regulate biofilm formation. CONCLUSION: This study provides insights into the mechanisms of M. abscessus biofilm formation and its resistance to antibiotics.

Genetic stability of Mycobacterium abscessus during antibiotic treatment.

OBJECTIVES: Genetic changes in Mycobacterium abscessus during antibiotic treatment are not fully understood. This study aimed to investigate the genetic changes in M. abscessus in patients receiving antibiotic treatment, and their clinical implications. METHODS: Pretreatment and 12-month post-treatment M. abscessus isolates were obtained from patients with M. abscessus pulmonary disease. Isolates from each time point were separated into six groups based on their distinctive morphological characteristics. Twenty-four isolates, comprising 12 from patient A exhibiting progressive disease and 12 from patient B demonstrating stable disease, underwent sequencing. Subsequently, minimal inhibitory concentrations (MICs) for the administered antibiotics were measured. RESULTS: Persistent infection with a single strain was observed in patients A and B. During 12 months of treatment, MICs for administered drugs did not generally change over time in either patient and single nucleotide variations (SNV) associated with antimicrobial resistance (rrl, rrs, erm(41), gyrA, gyrB, whiB7 and hflX) were not mutated. Although not significant, 47 and 52 non-synonymous SNVs occurred in M. abscessus from patients A and B, respectively, and the accumulation of these SNVs differed in patients A and B, except for five SNVs. The most variable positions were within a probable NADH-dependent glutamate synthase gene and a putative YrbE family protein gene in patients A and B, respectively. CONCLUSIONS: Persistent infections by a single strain of M. abscessus were observed in two patients with different clinical courses. Genetic changes in M. abscessus during antibiotic treatment were relatively stable in these patients. CLINICAL TRIALS IDENTIFIER: NCT01616745 (ClinicalTrials.gov ID).

Hierarchy and interconnected networks in the WhiB7 mediated transcriptional response to antibiotic stress in Mycobacterium abscessus.

Mycobacterium abscessus is intrinsically resistant to antibiotics effective against other pathogenic mycobacteria largely due to the drug-induced expression of genes that confer resistance. WhiB7 is a major hub controlling the induction of resistance to ribosome-targeting antibiotics. It activates the expression of >100 genes, 7 of which are known determinants of drug resistance; the function of most genes within the regulon is however unknown, but some conceivably encode additional mechanisms of resistance. Furthermore, the hierarchy of gene expression within the regulon, if any, is poorly understood. In the present work we have identified 56 WhiB7 binding sites using chromatin immunoprecipitation sequencing (CHIP-Seq) which accounts for the WhiB7-dependent upregulation of 72 genes, and find that M. abscessus WhiB7 functions exclusively as a transcriptional activator at promoters recognized by σA/σB. We have investigated the role of 18 WhiB7 regulated genes in drug resistance. Our results suggest that while some genes within the regulon (eg. erm41, hflX, eis2 and the ABCFs) play a major role in resistance, others make smaller contributions (eg. MAB_4324c and MAB_1409c) and the observed hypersensitivity ΔMabwhiB7 is a cumulative effect of these individual contributions. Moreover, our CHIP-Seq data implicate additional roles of WhiB7 induced genes beyond antibiotic resistance. Finally, we identify a σH-dependent network in aminoglycoside and tigecycline resistance which is induced upon drug exposure and is further activated by WhiB7 demonstrating the existence of a crosstalk between components of the WhiB7-dependent and -independent circuits.

Molecular Identification of Strains within the Mycobacterium abscessus Complex and Determination of Resistance to Macrolides and Aminoglycosides.

One of the most relevant and pathogenic groups among the rapidly growing mycobacteria (RGM) is Mycobacterium abscessus complex (MABC) that includes three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. The aim of this study was the analysis of prevalence of MABC among other non-tuberculous mycobacteria isolated from patients in the Malopolska Region of Poland, between 2018 and 2021, as well as determination of their subspecies and molecular mechanisms of resistance to macrolides and aminoglycosides. The incidence of MABC was 5,4% (12/223). Eight strains were classified as M. abscessus subsp. abscessus, three as M. abscessus subsp. massiliense and one M. abscessus subsp. bolletii. Molecular analysis showed resistance to macrolides for eight strains of M. abscessus subsp. abscessus associated with erm(41)T28 gene mutations. One strain of M. abscessus subsp. abscessus showed resistance to macrolides (two mutations simultaneously: in erm(41)T28 and rrl genes) and aminoglycosides (point mutation in rrs gene). One strain of M. abscessus subs. bolletii was resistant to macrolides (erm(41)T28 mutation), whereas presented no mutations for aminoglycosides. M. abscessus subsp. massiliense reveal no mutations. High clarithromycin resistance of M. abscessus, determines the urgent need for susceptibility-based treatment. Molecular determination of resistance mechanisms to aminoglycosides and macrolides enables fast and accurate targeted treatment implementation.

Mycobacterium abscessus Complex-Associated Chronic Meningitis: Time to Think Beyond Tuberculosis.

Background: Mycobacterium abscessus complex (MabC) has emerged as an important cause of human infections, including meningitis. In the absence of correct microbiological identification, cases of MabC meningitis are treated with conventional anti-tubercular therapy, thereby worsening the outcome. Objective: The current study was conducted to determine the clinical features, antimicrobial susceptibility, and outcome of patients with MabC meningitis. Material and Methods: Cerebrospinal fluid specimens processed between 2011 and 2021 were subjected to smear, culture, MALDI TOF identification, hsp65 gene sequencing, and susceptibility testing using Sensititre™ RAPMYCOI plates along with a literature review. Results: 12 cases of MabC meningitis were identified between 2011 and 2021, 11 of which were M. abscessus subspecies abscessus on hsp65 gene sequencing. A pioneer case of meningitis with M. abscessus subspecies bolletii was also identified. The common predispositions were TB elsewhere, HIV positivity, and head injury. Two patients had dual infections, both MabC and TB. Ten patients succumbed to infection with a mean survival of 11 months. All isolates were susceptible to amikacin and tigecycline and subspecies bolletii had a higher minimum inhibitory concentration (MIC) than subspecies abscessus. A combined analysis with the available literature, reporting 19 more cases, revealed that the overall mortality of MabC meningitis was 61.3% (19/31) and that of shunt-associated/neurosurgical intervention-related MabC meningitis was 66.7% (12/20). To date, out of 20 MabC meningitis isolates in which subspecies identification was carried, 13 were M. abscessus, six were M. massiliense, and one was M. bolletii. Conclusion: MabC is an important differential diagnosis of chronic meningitis. Prompt identification and speciation are imperative for targeted therapy, thus improving the overall patient outcome.

Aortic endograft infection by Mycobacterium abscessus subsp. massiliense with acquired clarithromycin resistance: a case report.

BACKGROUND: Mycobacterium abscessus subsp. massiliense (MMA) comprises a group of non-tuberculous, rapidly growing mycobacteria. Although MMA can cause pulmonary diseases, surgical site infections, and disseminated diseases, aortic endograft infection has not been reported. Here, we describe the first case of aortic endograft infection caused by MMA. CASE PRESENTATION: Two months after stent-graft insertion for an abdominal aortic aneurysm, an 85-year-old man was admitted with fever and abdominal pain and was diagnosed with aortic endograft infection. Despite 14 days of meropenem and vancomycin intravenous administration, periaortic fluid pooling increased as compared to that before antibiotic administration. The abscess was drained, and fluorescent acid-fast staining of the abscess fluid revealed bacilli. We conducted genetic tests on the genes hsp65, rpoB, and sodA, performed Whole Genome Sequencing (WGS), and identified the organism as MMA. Intravenous imipenem-cilastatin (IPM/CS), amikacin (AMK), and oral clarithromycin (CAM) were administered. After 2 months, oral CAM and sitafloxacin were administered because the abscess had decreased in size. However, after 6 weeks, the abscess increased in size again. Antimicrobial susceptibility testing of the drainage fluid from the abscess resulted in the isolation of an MMA strain that had acquired resistance to CAM. Intravenous IPM/CS, AMK, and oral linezolid were added to the treatment regimen along with oral CAM and STFX. However, he was not fully cured and died 6 months later. Neither the full-length erythromycin ribosome methyltransferase (erm)(41) gene nor the rrl or rpIV gene mutations were found by Sanger sequencing in the pre- and post-treatment strains. Whole-genome sequence analysis of the post-treatment strain revealed mutations in genes with no previous reports of association with macrolide resistance. CONCLUSIONS: Aortic endograft infection caused by MMA strain is extremely rare; nonetheless, MMA should be suspected as the causative microorganism when broad-spectrum antimicrobials are ineffective.

Genome-Scale Characterization of Mycobacterium abscessus Complex Isolates from Portugal.

The Mycobacterium abscessus complex (MABC) is an emerging, difficult to treat, multidrug-resistant nontuberculous mycobacteria responsible for a wide spectrum of infections and associated with an increasing number of cases worldwide. Dominant circulating clones (DCCs) of MABC have been genetically identified as groups of strains associated with higher prevalence, higher levels of antimicrobial resistance, and worse clinical outcomes. To date, little is known about the genomic characteristics of MABC species circulating in Portugal. Here, we examined the genetic diversity and antimicrobial resistance profiles of 30 MABC strains isolated between 2014 and 2022 in Portugal. The genetic diversity of circulating MABC strains was assessed through a gene-by-gene approach (wgMLST), allowing their subspecies differentiation and the classification of isolates into DCCs. Antimicrobial resistance profiles were defined using phenotypic, molecular, and genomic approaches. The majority of isolates were resistant to at least two antimicrobials, although a poor correlation between phenotype and genotype data was observed. Portuguese genomes were highly diverse, and data suggest the existence of MABC lineages with potential international circulation or cross-border transmission. This study highlights the genetic diversity and antimicrobial resistance profile of circulating MABC isolates in Portugal while representing the first step towards the implementation of a genomic-based surveillance system for MABC at the Portuguese NIH.

Clinically relevant mutations in the PhoR sensor kinase of host-adapted Mycobacterium abscessus isolates impact response to acidic pH and virulence.

IMPORTANCE: Difficult-to-treat pulmonary infections caused by nontuberculous mycobacteria of the Mycobacterium abscessus group have been steadily increasing in the USA and globally. Owing to the relatively recent recognition of M. abscessus as a human pathogen, basic and translational research to address critical gaps in diagnosis, treatment, and prevention of diseases caused by this microorganism has been lagging behind that of the better-known mycobacterial pathogen, Mycobacterium tuberculosis. To begin unraveling the molecular mechanisms of pathogenicity of M. abscessus, we here focus on the study of a two-component regulator known as PhoPR which we found to be under strong evolutionary pressure during human lung infection. We show that PhoPR is activated at acidic pH and serves to regulate a defined set of genes involved in host adaptation. Accordingly, clinical isolates from chronically infected human lungs tend to hyperactivate this regulator enabling M. abscessus to escape macrophage killing.

Population heterogeneity in Mycobacterium smegmatis and Mycobacterium abscessus.

Bacteria use population heterogeneity, the presence of more than one phenotypic variant in a clonal population, to endure diverse environmental challenges - a 'bet-hedging' strategy. Phenotypic variants have been described in many bacteria, but the phenomenon is not well-understood in mycobacteria, including the environmental factors that influence heterogeneity. Here, we describe three reproducible morphological variants in M. smegmatis - smooth, rough, and an intermediate morphotype that predominated under typical laboratory conditions. M. abscessus has two recognized morphotypes, smooth and rough. Interestingly, M. tuberculosis exists in only a rough form. The shift from smooth to rough in both M. smegmatis and M. abscessus was observed over time in extended static culture, however the frequency of the rough morphotype was high in pellicle preparations compared to planktonic culture, suggesting a role for an aggregated microenvironment in the shift to the rough form. Differences in growth rate, biofilm formation, cell wall composition, and drug tolerance were noted among M. smegmatis and M. abscessus variants. Deletion of the global regulator lsr2 shifted the M. smegmatis intermediate morphotype to a smooth form but did not fully phenocopy the naturally generated smooth morphotype, indicating Lsr2 is likely downstream of the initiating regulatory cascade that controls these morphotypes. Rough forms typically correlate with higher invasiveness and worse outcomes during infection and our findings indicate the shift to this rough form is promoted by aggregation. Our findings suggest that mycobacterial population heterogeneity, reflected in colony morphotypes, is a reproducible, programmed phenomenon that plays a role in adaptation to unique environments and this heterogeneity may influence infection progression and response to treatment.

Silencing essential gene expression in Mycobacterium abscessus during infection.

IMPORTANCE: Mycobacterium abscessus represents the most common rapidly growing mycobacterial pathogen in cystic fibrosis and is extremely difficult to eradicate. Essential genes are required for growth, often participate in pathogenesis, and encode valid drug targets for further chemotherapeutic developments. However, assessing the function of essential genes in M. abscessus remains challenging due to the limited spectrum of efficient genetic tools. Herein, we generated a Tet-OFF-based system allowing to knock down the expression of mmpL3, encoding the mycolic acid transporter in mycobacteria. Using this conditional mutant, we confirm the essentiality of mmpL3 in planktonic cultures, in biofilms, and during infection in zebrafish embryos. Thus, in this study, we developed a robust and reliable method to silence the expression of any M. abscessus gene during host infection.

A complicated infection by cutaneous Nocardia wallacei and pulmonary Mycobacterium abscessus in a Chinese immunocompetent patient: a case report.

Nocardiosis is an infectious disease caused by Nocardia that primarily affects immunocompromised hosts. Mycobacterium abscessus is a common opportunistic pathogen that causes disease in humans, including pulmonary and extrapulmonary infection. Nocardia spp. infection is uncommon, and infection with Nocardia wallacei and Mycobacterium abscessus is even rarer. A 59-year-old immunocompetent woman with risk factors for environmental exposure developed nocardiosis and presented to the hospital with a cough, shortness of breath, hemoptysis, and a back abscess. An enhanced computed tomography (CT) of the chest revealed partial destruction of the right lung, as well as consolidation of the right upper lobe. Rare pathogens N. wallacei and Mycobacterium abscessus were detected by metagenomic next-generation sequencing (mNGS) from abscess on the back and lung puncture tissue, respectively. She was treated with a combination of antibiotics and was finally discharged with a good prognosis. In this case, we present a patient who was successfully diagnosed with N. wallacei and Mycobacterium abscessus infection using mNGS. This importance of using mNGS in pathogen detection and the effective use of antibiotics in treating patients with long-term rare infections is highlighted in this report.

Clinical characteristics and drug susceptibility profiles of Mycobacterium abscessus complex infection at a medical school in Thailand.

OBJECTIVES: This study investigated the differences in epidemiological and clinical data, and antimicrobial susceptibilities among different subspecies of Mycobacterium abscessus complex (MABSC) clinical isolates at a medical school in Thailand. METHODS: A total of 143 MABSC clinical isolates recovered from 74 patients were genotypically analyzed for erm(41), rrl, and rrs mutations, and antimicrobial susceptibilities were determined using a broth microdilution method. Patient characteristics and clinical outcomes were reviewed from the medical records. RESULTS: Seventy-four patients were infected with 28/74 (37.8%) M. abscessus subspecies abscessus (MAB), 43/74 (58.1%) M. abscessus subsp. massiliense (MMA), and 3/74 (4.1%) M. abscessus subsp. bolletii (MBO). The clinical findings and outcomes were generally indistinguishable between the three subspecies. All three subspecies of MABSC clinical isolates exhibited high resistance rates to ciprofloxacin, doxycycline, moxifloxacin, TMP/SMX, and tobramycin. MAB had the highest resistance rates to clarithromycin (27.8%, 20/72) and amikacin (6.9%, 5/72) compared to MBO and MMA, with p < 0.001 and p = 0.004, respectively. In addition, the rough morphotype was significantly associated with resistance to amikacin (8.9%, 5/56), clarithromycin (26.8%, 15/56), and imipenem (76.8%, 43/56) (p < 0.001), whereas the smooth morphotype was resistant to linezolid (57.1%, 48/84) (p = 0.002). In addition, T28 of erm(41), rrl (A2058C/G and A2059C/G), and rrs (A1408G) mutations were detected in 87.4% (125/143), 16.1% (23/143), and 9.1% (13/143) of MABSC isolates, respectively. CONCLUSIONS: Three MABSC subspecies caused a variety of infections in patients with different underlying comorbidities. The drug susceptibility patterns of the recent circulating MABSC strains in Thailand were different among the three MABSC subspecies and two morphotypes.