Objectives: Aminoglycoside resistance in bacteria is typically conferred by specific drug-modifying enzymes. Infrequently, such resistance is achieved through 16S ribosomal RNA methyltransferases, such as NpmA and KamB encoded by Escherichia coli and Streptoalloteichus tenebrarius, respectively. These enzymes are not widespread and have not been described in Nocardia species to date. Methods: We report the genomic mining of 18 Nocardia wallacei isolates that were found to be specifically and substantially resistant to amikacin. Results: We identified a gene coding for a protein with very distant homology to NpmA and KamB. However, 3-D modeling revealed that the tertiary structure of these three proteins was highly similar. Cloning and expressing this gene in two susceptible bacteria Nocardia asteroides, and Mycobacterium smegmatis (another Actinobacterium) led to high-level, pan-aminoglycoside resistance in both cases. We named this gene warA (Wallacei Amikacin Resistance A). Conclusions: This is the first description and experimental characterization of a gene of this family in Nocardia, and the first demonstration that such activity could lead to pan-aminoglycoside resistance in Mycobacteria as well. The discovery of this novel gene has important biotechnology and clinical implications.

Mycobacterium abscessus is an emerging pathogen causing severe pulmonary infections. Within chronically infected patients, M. abscessus isolates undergo molecular changes leading to increased virulence and antibiotic resistance. Specifically, mutations in glycopeptidolipid (GPL) synthesis genes, leading to the rough phenotype, are associated with invasive, nonremitting infections and a severe clinical course. It has been unclear whether GPL defects confer antibiotic resistance independently of other molecular changes. We used transposon technology to isolate a rough (GPL-defective; Tn MABS_4099cZeoR) mutant and compare it to a fully isogenic parent strain (ATCC 19977) bearing wild-type zeocin resistance (WTZeoR). Antibiotic susceptibility profiles of Tn_4099cZeoR and WTZeoR were tested and compared using the Sensititre RAPMYCOI antimicrobial susceptibility test plate. MICs were evaluated within clinically relevant values according to the Clinical and Laboratory Standards Institute (CLSI) standards. We found that M. abscessus with rough colony morphotype (Tn_4009c) had comparable antibiotic susceptibility to its smooth isogenic WT counterpart. Small differences (a 1:2 dilution) in MICs were found for imipenem, cefoxitin, and tigecycline, yet those small differences did not change the clinical susceptibility report for these antibiotics, as they fell within the same CLSI cutoffs for resistance. While small alternations in susceptibility to imipenem, cefoxitin, and tigecycline were noted, we conclude that the GPL mutations in M. abscessus did not confer clinically significant antibiotic resistance. Increased antibiotic resistance in the clinical setting may occur in an unrelated and parallel manner to GPL mutations. IMPORTANCE Mycobacterium abscessus chronically infects patients with preexisting lung diseases, leading to progressive deterioration in pulmonary function. The common perception among clinicians is that the rough phenotype is associated with progressive disease and severe clinical course, manifested as a widespread inflammatory response and resistance to antibacterials. However, as clinical isolates accumulate hundreds of mutations over the prolonged course of infection, it is unclear whether the rough phenotype per se is responsible for the antibiotic resistance seen in late-stage infections, or whether the resistance is related to other genetic changes in the bacteria. Previous studies mostly compared rough and smooth clinical isolates. Here, for the first time, we compared WT smooth bacteria to a specific rough, GPL-associated, otherwise-isogenic mutant. We determined that the rough morphotype had essentially identical antibiotic susceptibilities as the parent strain. The mechanistic basis for the antibiotic resistance observed in rough clinical isolates is therefore most probably related to other genetic determinants.

Mycobacterium abscessus causes severe disease in patients with cystic fibrosis. Little is known in M. abscessus about the roles of small regulatory RNAs (sRNA) in gene regulation. We show that the sRNA B11 controls gene expression and virulence-associated phenotypes in this pathogen. B11 deletion from the smooth strain ATCC_19977 produced a rough strain, increased pro-inflammatory signaling and virulence in multiple infection models, and increased resistance to antibiotics. Examination of clinical isolate cohorts identified isolates with B11 mutations or reduced expression. We used RNAseq and proteomics to investigate the effects of B11 on gene expression and test the impact of mutations found in clinical isolates. Over 200 genes were differentially expressed in the deletion mutant. Strains with the clinical B11 mutations showed expression trends similar to the deletion mutant, suggesting partial loss of function. Among genes upregulated in the B11 mutant, there was a strong enrichment for genes with B11-complementary sequences in their predicted ribosome binding sites (RBS), consistent with B11 functioning as a negative regulator that represses translation via base-pairing to RBSs. Comparing the proteomes similarly revealed that upregulated proteins were strongly enriched for B11-complementary sequences. Intriguingly, genes upregulated in the absence of B11 included components of the ESX-4 secretion system, critical for M. abscessus virulence. Many of these genes had B11-complementary sequences at their RBSs, which we show is sufficient to mediate repression by B11 through direct binding. Altogether, our data show that B11 acts as a direct negative regulator and mediates (likely indirect) positive regulation with pleiotropic effects on gene expression and clinically important phenotypes in M. abscessus. The presence of hypomorphic B11 mutations in clinical strains is consistent with the idea that lower B11 activity may be advantageous for M. abscessus in some clinical contexts. This is the first report on an sRNA role in M. abscessus.

The spread of antibiotic resistance is attracting increased attention to combination-based treatments. Although drug combinations have been studied extensively for their effects on bacterial growth1–11, much less is known about their effects on bacterial long-term clearance, especially at cidal, clinically relevant concentrations12–14. Here, using en masse microplating and automated image analysis, we systematically quantify Staphylococcus aureus survival during prolonged exposure to pairwise and higher-order cidal drug combinations. By quantifying growth inhibition, early killing and longer-term population clearance by all pairs of 14 antibiotics, we find that clearance interactions are qualitatively different, often showing reciprocal suppression whereby the efficacy of the drug mixture is weaker than any of the individual drugs alone. Furthermore, in contrast to growth inhibition6–10 and early killing, clearance efficacy decreases rather than increases as more drugs are added. However, specific drugs targeting non-growing persisters15–17 circumvent these suppressive effects. Competition experiments show that reciprocal suppressive drug combinations select against resistance to any of the individual drugs, even counteracting methicillin-resistant Staphylococcus aureus both in vitro and in a Galleria mellonella larva model. As a consequence, adding a β-lactamase inhibitor that is commonly used to potentiate treatment against β-lactam-resistant strains can reduce rather than increase treatment efficacy. Together, these results underscore the importance of systematic mapping the long-term clearance efficacy of drug combinations for designing more-effective, resistance-proof multidrug regimes.

Mycobacterium abscessus is an emerging pathogen causing severe pulmonary infections. While environmental in origin, in the clinical setting M. abscessus often changes to a Rough phenotype associated with severe non-remitting infections. Clinical isolates baring mutations in glycopeptidolipid-synthesis genes, leading to the Rough phenotype, were suggested to have increase bacterial virulence while possibly showing reduced transmissibility on fomites. We set to determine whether an isolated glycopeptidolipid (GPL) defect affects transmissibility. We used transposon technology to create a fully isogenic Rough (GPL-defective) (Tn_4099c) and compare it to the isogenic parent strain (ATCC 19977). Survival on fomites was determined by spotting, drying, and retrieving the isolates at designated time points. This was repeated as a competition experiment using a mixture of differentially fluorescent M. abscessus 19977 (Smooth) and the Tn_4099c mutant (Rough). Survival ability in chlorhexidine solution (Septal Scrub Teva) was performed using a disinfectant killing-assay for mycobacteria. Despite significant bacterial killing in all assays, we found no survival advantage to either GPL-defected Rough or GPL-reserved Smooth morphotype—both on fomites and in chlorhexidine. Our findings suggest that while transmission fitness may be altered due to some within-host evolutionary changes, decreased transmissibility of clinical strains cannot be attributed to the GPL-synthesis defect alone. Further studies are needed to determine the effect of other mutations on the transmission potential of M. abscessus in the clinical setting. IMPORTANCE Mycobacterium abscessus is an emerging pathogen causing severe pulmonary infections. In the clinical setting, M. abscsssus undergoes molecular and genetic changes associated with increased virulence. Specifically, bacterial defects in glycopeptidolipid (GPL) synthesis, creating the “Rough” colony phenotype, have been associated with increased virulence, yet were also presumably observed to have decreased survival on fomites, leading to reduced transmissibility. We set to determine whether GPL-synthesis defects are indeed responsible for reduced transmissibility of clinical isolates. We compared fully isogenic GPL-disrupted versus GPL-preserved strains, and demonstrated no survival advantage for either strain on fomites. Additionally, neither isolate had a survival advantage in chlorhexidine, a widely used disinfectant in health care settings. Our findings suggest that reduced transmissibility of clinical isolates, should it be found, cannot be attributed to GPL-synthesis mutations. While clinical isolates may show changes in transmission potential, more studies are needed to investigate the mechanisms leading to these phenotypic changes.

Mycobacterium abscessus is an emerging pathogen that critically depends on iron for growth and pathogenesis. The acquisition of iron in Mycobacterium tuberculosis is governed by siderophores called mycobactins, synthesized by the mbt gene cluster, but the role of this gene cluster in the adaption of M. abscessus to iron limitation is not characterized. We identified an M. abscessus Tn_mutant with interruption of the mbtE gene (MAB_2248c), a central component of mycobactin biosynthesis. We tested this isolate growth characteristic, dependency on supplements, and transcriptomic response, comparing it to the response of wild-type (WT) bacteria in iron-limiting conditions. We also compare the structure of the mbt gene cluster across several mycobacteria. The Tn_mbtE mutant had a substantial, but not absolute, growth defect, which was more substantial in iron-limited media. Supplementation with mycobactin-J, hemin, blood, and surprisingly, albumin, salvaged the poor growth. Similarly, secreted mature (carboxy)-mycobactins from WT bacteria rescued the Tn_mbtE mutant during iron deprivation. The transcriptomic response of the Tn_mbtE mutant involved the upregulation of genes known to be implicated in iron homeostasis and was comparable to that of WT bacteria grown in iron-limiting conditions. Interestingly, the response was not identical to the response of M. tuberculosis to iron limitation. The mbt gene cluster and mycobactins play important roles in the physiology of M. abscessus. (Carboxy)-mycobactin is secreted from WT bacteria and can serve as “public good.” The role of several iron-homeostasis related genes (like ideR) may differ between M. abscessus and Mtb.

Mycobacterium tuberculosis (Mtb) exposure drives antibody responses, but whether patients with active tuberculosis elicit protective antibodies, and against which antigens, is still unclear. Here we generate monoclonal antibodies from memory B cells of one patient to investigate the B cell responses during active infection. The antibodies, members of four distinct B cell clones, are directed against the Mtb phosphate transporter subunit PstS1. Antibodies p4-36 and p4-163 reduce Mycobacterium bovis-BCG and Mtb levels in an ex vivo human whole blood growth inhibition assay in an FcR-dependent manner; meanwhile, germline versions of p4-36 and p4-163 do not bind Mtb. Crystal structures of p4-36 and p4-170, complexed to PstS1, are determined at 2.1 Å and 2.4 Å resolution, respectively, to reveal two distinctive PstS1 epitopes. Lastly, a prophylactic p4-36 and p4-163 treatment in Mtb-infected Balb/c mice reduces bacterial lung burden by 50%. Our study shows that inhibitory anti-PstS1 B cell responses arise during active tuberculosis.

The antituberculosis drug telacebec is ineffective against Mycobacterium abscessus. A recent study suggested that TB47, a telacebec analogue, potentiated the efficacy of clofazimine against M. abscessus. Here, we report that TB47 not only is ineffective against M. abscessus in vitro but also does not potentiate the activity of clofazimine.

Isoniazid (INH) is a cornerstone of antitubercular therapy. Mycobacterium tuberculosis complex bacteria are the only mycobacteria sensitive to clinically relevant concentrations of INH. All other mycobacteria, including M. marinum and M. avium subsp. paratuberculosis are resistant. INH requires activation by bacterial KatG to inhibit mycobacterial growth. We tested the role of the differences between M. tuberculosis KatG and that of other mycobacteria in INH sensitivity. We cloned the M. bovis katG gene into M. marinum and M. avium subsp. paratuberculosis and measured the MIC of INH. We recombinantly expressed KatG of these mycobacteria and tested in vitro binding to, and activation of, INH. Introduction of katG from M. bovis into M. marinum and M. avium subsp. paratuberculosis rendered them 20 to 30 times more sensitive to INH. Analysis of different katG sequences across the genus found KatG evolution diverged from RNA polymerase-defined mycobacterial evolution. Biophysical and biochemical tests of M. bovis and nontuberculous mycobacteria (NTM) KatG proteins showed lower affinity to INH and substantially lower enzymatic capacity for the conversion of INH into the active form in NTM. The KatG proteins of M. marinum and M. avium subsp. paratuberculosis are substantially less effective in INH activation than that of M. tuberculosis, explaining the relative INH insensitivity of these microbes. These data indicate that the M. tuberculosis complex KatG is divergent from the KatG of NTM, with a reciprocal relationship between resistance to host defenses and INH resistance. Studies of bacteria where KatG is functionally active but does not activate INH may aid in understanding M. tuberculosis INHresistance mechanisms, and suggest paths to overcome them.

Background: Tolerance to antibiotics and persistence are associated with antibiotic treatment failures, chronic-relapsing infections, and emerging antibiotic resistance in various bacteria, including Staphylococcus aureus. Mechanisms of persistence are largely unknown, yet have been linked to physiology under low-ATP conditions and the metabolic-inactive state. EttA is an ATP-binding cassette protein, linked in Eschrechia coli to ribosomal hibernation and fitness in stationary growth phase, yet its role in S. aureus physiology is unknown. Results: Using whole genome sequencing (WGS) of serial clinical isolates, we identified an EttA-negative S. aureus mutant (ettA stop ), and its isogenic wild-type counterpart. We used these two isogenic clones to investigate the role of ettA in S. aureus physiology in starvation and antibiotic stress, and test its role in persistence and antibiotic tolerance. ettA stop and its WT counterpart were similar in their antibiotic resistance profiles to multiple antibiotics. Population dynamics of ettA stop and the WT were similar in low-nutrient setting, with similar recovery from stationary growth phase or starvation. Supra-bacteriocidal concentration of cefazolin had the same killing effect on ettA stop and WT populations, with no difference in persister formation. Conclusions: Lack of ettA does not affect S. aureus antibiotic resistance, beta-lactam tolerance, resilience to starvation or fitness following starvation. We conclude the role of ettA in S. aureus physiology is limited or redundant with another, unidentified gene. WGS of serial clinical isolates may enable investigation of other single genes involved in S. aureus virulence, and specifically persister cell formation.

We present a technically simple, easy-to-perform method for generating the genomic libraries for Himar-1 transposon site sequencing (Tn-seq). In addition to being simpler than present methods in the technical aspect, it also allows more robust and straightforward identification of the insertion site, by generating a longer sequence surrounding the insertion TA in the genome. The method makes Tn-seq more user-friendly and accessible to laboratories with more-limited bioinformatic resources. Finally, we created a saturated transposon-mutant library in Mycobacterium abscessus and demonstrated the usefulness of the method in analysis of genes involved in colony morphology, as well as in analysis of the whole Tn-mutant library, with identification of over 8,000 unique mutants. IMPORTANCE Transposon insertion sequencing is a powerful tool, but many researchers are discouraged by the apparent technical complexity of preparing the genomic library for deep sequencing and by the complicated computational analysis needed for insertion site identification. Our proposed method makes the preparation of the library easy and straightforward, relying on well-known molecular biology techniques. In addition, the results obtained from the deep sequencing are easily analyzed in terms of transposon insertion site identification, placing library preparation and analysis within the reach of more researchers in the microbiology community, including those with less computational and bioinformatic resources and experience. This is demonstrated by analysis of the most saturated Tn-mutant library created to date in the emerging pathogen Mycobacterium abscessus.

M. tuberculosis (Mtb), which causes tuberculosis disease, continues to be a major global health threat. Correct identification of valid drug targets is important for the development of novel therapeutics that would shorten the current 6–9 month treatment regimen and target resistant bacteria. Methionine aminopeptidases (MetAP), which remove the N-terminal methionine from newly synthesized proteins, are essential in all life forms (eukaryotes and prokaryotes). The MetAPs contribute to the cotranslational control of proteins as they determine their half life (N-terminal end rule) and facilitate further modifications such as acetylation and others. Mtb (and M. bovis) possess two MetAP isoforms, MetAP1a and MetAP1c, encoded by the mapA and mapB genes, respectively. Conflicting evidence was reported in the literature on which of the two variants is essential. To resolve this question, we performed a targeted genetic deletion of each of these two genes. We show that a deletion mutant of mapA is viable with only a weak growth defect. In contrast, we provide two lines of genetic evidence that mapB is indispensable. Furthermore, construction of double-deletion mutants as well as the introduction of point mutations into mapB resulting in proteins with partial activity showed partial, but not full, redundancy between mapB and mapA.We propose that it is MetAP1c (mapB) that is essentially required for mycobacteria and discuss potential reasons for its vitality.

Background: Co-translational processes in bacteria are attractive drug targets, but while some processes are essential, others are not. The essentiality of Peptide Deformylase (PDF, def) for vitality of mycobacteria was speculated, but never unequivocally proven. Results: Here we show by targeted deletion experiments that def can only be deleted from M. smegmatis when an additional copy is present; that prior deletion of tRNAfMet-Formyl Transferase (FMT, encoded by fmt) renders def completely dispensable; and that re-introduction of fmt into a Δdef mutant is not possible-constituting a definitive proof for the essentiality of def in mycobacteria. Conclusions: Peptide deformylase is essential in M. smegmatis, but the fact that inactivation of fmt renders the gene completely dispensable, and thus any inhibitor of def useless, casts doubt on the usefulness of PDF as a drug-target in mycobacteria.

Treating M. abscessus infection is challenging due to the potent β-lactamase Bla Mab (Beta-lactamase of M. abscessus ) . Avibactam is a non-β-lactam, β-lactamase inhibitor shown to inhibit Bla Mab . We tested whether avibactem can render piperacillin effective against M. Abscessus. In-vitro, avibactam enhanced the activity of piperacillin by 16-32 fold, with no significant effect on meropenem. In an in-vivo Galleria mellonella model, meropenem and piperacillin/avibactam significantly decreased infection burden compared to untreated controls. Neither piperacillin nor avibactam alone had a significant effect.

Treatment of Mycobacterium abscessus infections is extremely challenging due to its intrinsic resistance to most antibiotics, and research of pathogenesis is limited due to a lack of a practical in vivo model of infection. The objective of this study was to establish a simple in vivo model for M. abscessus infection, virulence, and drug testing in Galleria mellonella larvae. We inoculated larvae with M. abscessus bacteria and assessed histopathology, CFU count, and mortality with and without antibiotic treatment. We also constructed a luminescent, recombinant M. abscessus mutant, mDB158, and imaged infected larvae using the IVIS in vivo imaging system. M. abscessus proliferated and induced granuloma-like responses in infected larvae, leading to larval mortality. The G. mellonella model was further validated successfully by demonstration of the expected favorable antimicrobial effect of treatment with meropenem and the superiority of combination treatment (meropenem and tigecycline) over that with single agents. We then used IVIS imaging of larvae infected with luminescent M. abscessus, allowing live real-time assessment of bacterial load. We used this method to compare the antimicrobial effects of various antibiotics (meropenem, amikacin, linezolid, levofloxacin, etc.) on bacterial proliferation and larval survival. Meropenem and amikacin had the most favorable effects, correlating well with common clinical practice guidelines. These findings suggest G. mellonella to be an excellent in vivo model for research of M. abscessus infection, pathogenesis, and treatment. Luminescent M. abscessus and IVIS imaging further facilitates this model. Results obtained in this model clearly substantiated common clinical practice, thus validating the model as a predictor of treatment efficacy and outcome.

Background. KLRG1 is a marker of terminally differentiated CD8+ T cells in viral infection, but its role in human Mycobacterium tuberculosis infection remains elusive. Methods. A set of cohorts of patients with tuberculosis was designed, and the expression profiles and functions of KLRG1+CD4+ T cells were determined with and without antibody blocking. Results. KLRG1 expression on CD4+ T cells was significantly increased in patients with active tuberculosis, compared with healthy controls and patients without tuberculosis. Upon M. tuberculosis–specific stimulation, the ability to secrete interferon γ, interleukin 2, and tumor necrosis factor α was significantly greater in KLRG1-expressing CD4+ T cells than in their KLRG-negative counterparts and was accompanied by a decreased proportion of regulatory T cells and increased Akt signaling. However, KLRG1-expressing CD4+ T cells had a shorter life-span, which was associated with a higher apoptosis rate but a similar proliferative response. Blockade of KLRG1 signaling significantly enhanced interferon γ and interleukin 2 secretion without affecting either cell apoptosis or multiplication. Addition of a specific Akt inhibitor prevented this increased cytokine response, implicating the Akt signaling pathway. Conclusions. Our study delineated the profile of KLRG1+CD4+ T cells in patients with tuberculosis and suggests that M. tuberculosis infection drives CD4+ T cells to acquire increased effector function in a terminally differentiated state, which is restrained by KLRG1 via KLRG1/Akt signaling pathway.

Tuberculosis remains a major global health problem and efforts to develop a more effective vaccine have been unsuccessful so far. Targeting antigens (Ags) to dendritic cells (DCs) in vivo has emerged as a new promising vaccine strategy. In this approach, Ags are delivered directly to DCs via antibodies that bind to endocytic cell-surface receptors. Here, we explored DC-specific-ICAM3-grabbing-nonintegrin (DC-SIGN) targeting as a potential vaccine against tuberculosis. For this, we made use of the hSIGN mouse model that expresses human DC-SIGN under the control of the murine CD11c promoter. We show that in vitro and in vivo delivery of anti-DC-SIGN antibodies conjugated to Ag85B and peptide 25 of Ag85B in combination with anti-CD40, the fungal cell wall component zymosan, and the cholera toxin-derived fusion protein CTA1-DD induces strong Ag-specific CD4+ T-cell responses. Improved anti-mycobacterial immunity was accompanied by increased frequencies of Ag-specific IFN-γ+ IL-2+ TNF-α+ polyfunctional CD4+ T cells in vaccinated mice compared with controls. Taken together, in this study we provide the proof of concept that the human DC-SIGN receptor can be efficiently exploited for vaccine purposes to promote immunity against mycobacterial infections.

Nitrogen metabolism plays a central role in the physiology of microorganisms, and Glutamine Synthetase (GS) genes are present in virtually all bacteria. In M. Tuberculosis, four GS genes are present, but only glnA1 is essential, whereas glnA2 was shown to be non-essential for in-vitro as well as in-vivo growth and pathogenesis, and is postulated to be involved in D-glutamine and iso-glutamine synthesis. Whilst investigating the activity of an antimicrobial compound in M. Smegmatis, we found a spontaneous temperature-sensitive mutant in glnA2 (I133F), and used it to investigate the role of glnA2 in M. Smegmatis. We deleted the native glnA2 and replaced it with a mutated allele. This re-created the temperature sensitivity-as after 3-4 seemingly normal division cycles, glnA2 became essential for growth. This essentiality could not be salvaged by neither L, D-nor iso-glutamine, suggesting an additional role of glnA2 in M. Smegmatis over its role in M. Tuberculosis. We also found that overexpression of the global nitrogen regulator glnR enabled bypassing the essentiality of glnA2, allowing the creation of a complete deletion mutant. The discrepancy between the importance of glnA2 in Mtb and M. Smegmatis stresses the caution in which results in one are extrapolated to the other.