Development of 2nd generation aminomethyl spectinomycins that overcome native efflux in Mycobacterium abscessus.

Mycobacterium abscessus (Mab), a nontuberculous mycobacterial (NTM) species, is an emerging pathogen with high intrinsic drug resistance. Current standard-of-care therapy results in poor outcomes, demonstrating the urgent need to develop effective antimycobacterial regimens. Through synthetic modification of spectinomycin (SPC), we have identified a distinct structural subclass of N-ethylene linked aminomethyl SPCs (eAmSPCs) that are up to 64-fold more potent against Mab over the parent SPC. Mechanism of action and crystallography studies demonstrate that the eAmSPCs display a mode of ribosomal inhibition consistent with SPC. However, they exert their increased antimicrobial activity through enhanced accumulation, largely by circumventing efflux mechanisms. The N-ethylene linkage within this series plays a critical role in avoiding TetV-mediated efflux, as lead eAmSPC 2593 displays a mere fourfold susceptibility improvement against Mab ΔtetV, in contrast to the 64-fold increase for SPC. Even a minor shortening of the linkage by a single carbon, akin to 1st generation AmSPC 1950, results in a substantial increase in MICs and a 16-fold rise in susceptibility against Mab ΔtetV. These shifts suggest that longer linkages might modify the kinetics of drug expulsion by TetV, ultimately shifting the equilibrium towards heightened intracellular concentrations and enhanced antimicrobial efficacy. Furthermore, lead eAmSPCs were also shown to synergize with various classes of anti-Mab antibiotics and retain activity against clinical isolates and other mycobacterial strains. Encouraging pharmacokinetic profiles coupled with robust efficacy in Mab murine infection models suggest that eAmSPCs hold the potential to be developed into treatments for Mab and other NTM infections.

Drug susceptibility distributions of Mycobacterium chimaera and other non-tuberculous mycobacteria.

Recent outbreaks of cardiac surgery-associated Mycobacterium chimaera infections have highlighted the importance of species differentiation within the Mycobacterium avium complex and pointed to a lack of antibiotic susceptibility data for M. chimaera Using the MGIT 960/EpiCenter TB eXiST platform, we have determined antibiotic susceptibility patterns of 48 clinical M. chimaera isolates and 139 other non-tuberculous mycobacteria including 119 members of the M. avium complex and 20 Mycobacterium kansasii towards clofazimine and other drugs used to treat infections with slowly growing nontuberculous mycobacteria (NTM). MIC50, MIC90 and tentative epidemiological cutoff (ECOFF) values for clofazimine were 0.5 mg/L, 1 mg/L and 2 mg/L for M. chimaera. Comparable values were observed for other M. avium complex members, lower MIC50 (≤0.25 mg/L), MIC90 (0.5 mg/L) and ECOFF (1 mg/L) values were found for M. kansasii Susceptibility to clarithromycin, ethambutol, rifampin, rifabutin, amikacin, moxifloxacin and linezolid was in general similar for M. chimaera and other members of the M. avium complex but increased for M. kansasii The herein determined MIC distributions, MIC90 and ECOFF values of clofazimine for M. chimaera and other NTM provide the basis for the definition of clinical breakpoints. Further studies are needed to establish correlation of in vitro susceptibility and clinical outcome.

Phenolic Substitution in Fidaxomicin: A Semisynthetic Approach to Antibiotic Activity Across Species.

Fidaxomicin (Fdx) is a natural product antibiotic with potent activity against Clostridioides difficile and other Gram-positive bacteria such as Mycobacterium tuberculosis. Only a few Fdx derivatives have been synthesized and examined for their biological activity in the 50 years since its discovery. Fdx has a well-studied mechanism of action, namely inhibition of the bacterial RNA polymerase. Yet, the targeted organisms harbor different target protein sequences, which poses a challenge for the rational development of new semisynthetic Fdx derivatives. We introduced substituents on the two phenolic hydroxy groups of Fdx and evaluated the resulting trends in antibiotic activity against M. tuberculosis, C. difficile, and the Gram-negative model organism Caulobacter crescentus. As suggested by the target protein structures, we identified the preferable derivatisation site for each organism. The derivative ortho-methyl Fdx also exhibited activity against the Gram-negative C. crescentus wild type, a first for fidaxomicin antibiotics. These insights will guide the synthesis of next-generation fidaxomicin antibiotics.

In Vitro Bedaquiline and Clofazimine Susceptibility Testing in Mycobacterium abscessus.

Bedaquiline and clofazimine are increasingly used to treat infections with Mycobacterium abscessus. We determined distributions of MICs by broth microdilution for bedaquiline and clofazimine for 61 M. abscessus clinical isolates using different media and incubation times. We show that incubation time and growth media critically influence the MIC. Our data will aid in defining future clinical breakpoints for in vitro susceptibility testing for bedaquiline and clofazimine in M. abscessus.

Apramycin Overcomes the Inherent Lack of Antimicrobial Bactericidal Activity in Mycobacterium abscessus.

Antibiotic therapy of infections caused by the emerging pathogen Mycobacterium abscessus is challenging due to the organism's inherent resistance to clinically available antimicrobials. The low bactericidal potency of currently available treatment regimens is of concern and testifies to the poor therapeutic outcomes for pulmonary M. abscessus infections. Mechanistically, we demonstrate here that the acetyltransferase Eis2 is responsible for the lack of bactericidal activity of amikacin, the standard aminoglycoside used in combination treatment. In contrast, the aminoglycoside apramycin, with a distinct structure, is not modified by any of the pathogen's innate aminoglycoside resistance mechanisms and is not affected by the multidrug resistance regulator WhiB7. As a consequence, apramycin uniquely shows potent bactericidal activity against M. abscessus. This favorable feature of apramycin is reflected in a mouse model of pulmonary M. abscessus infection, which demonstrates superior activity, compared with amikacin. These findings encourage the development of apramycin for the treatment of M. abscessus infections and suggest that M. abscessus eradication in pulmonary disease may be within therapeutic reach.

Aquimarins, Peptide Antibiotics with Amino-Modified C-Termini from a Sponge-Derived Aquimarina sp. Bacterium.

Genome mining and bioactivity studies suggested the sponge-derived bacterium Aquimarina sp. Aq135 as a producer of new antibiotics. Activity-guided isolation identified antibacterial peptides, named aquimarins, featuring a new scaffold with an unusual C-terminal amino group and chlorine moieties. Responsible for the halogenation is the FeII /α-ketoglutarate-dependent chlorinase AqmA that halogenates up to two isoleucine residues in a carrier protein-dependent fashion. Total syntheses of two natural aquimarins and eight non-natural variants were developed. Structure-activity relationship (SAR) studies with these compounds showed that the synthetically more laborious chlorinations are not required for antibacterial activity but enhance cytotoxicity. In contrast, variants lacking the C-terminal amine were virtually inactive, suggesting diamines similar to the terminal aquimarin residue as candidate building blocks for new peptidomimetic antibiotics.

Rifabutin Is Inactivated by Mycobacterium abscessus Arr.

Mycobacterium abscessus exhibits Arr (ADP-ribosyltransferase)-dependent rifampin resistance. In apparent contrast, rifabutin (RBT) has demonstrated promising activity in M. abscessus infection models, implying that RBT might not be inactivated by Arr. RBT susceptibility testing of M. abscessusΔarr revealed a strongly decreased MIC. Our findings suggest that the efficacy of RBT might be enhanced by rendering RBT resilient to Arr-dependent modification or by blocking M. abscessus Arr activity.

BATF3-dependent dendritic cells drive both effector and regulatory T-cell responses in bacterially infected tissues.

The gastric lamina propria of mice that have been experimentally infected with the pathobiont Helicobacter pylori hosts a dense network of myeloid cells that includes BATF3-dependent CD103+ dendritic cells (DCs). We show here that CD103+ DCs are strictly required for gastric Th1 responses to H. pylori and for H. pylori infection control. A similar dependence of type 1 immunity on CD103+ DCs is observed in a Mycobacterium bovis BCG infection model, and in a syngeneic colon cancer model. Strikingly, we find that not only the expansion and/or recruitment of Th1 cells, but also of peripherally induced, neuropilin-negative regulatory T-cells to sites of infection requires BATF3-dependent DCs. A shared feature of the examined models is the strongly reduced production of the chemokines and CXCR3 ligands CXCL9, 10 and 11 in BATF3-deficient mice. The results implicate BATF3-dependent DCs in the recruitment of CXCR3+ effector and regulatory T-cells to target tissues and in their local expansion.

Increased drug permeability of a stiffened mycobacterial outer membrane in cells lacking MFS transporter Rv1410 and lipoprotein LprG.

The major facilitator superfamily transporter Rv1410 and the lipoprotein LprG (Rv1411) are encoded by a conserved two-gene operon and contribute to virulence in Mycobacterium tuberculosis. Rv1410 was originally postulated to function as a drug efflux pump, but recent studies suggested that Rv1410 and LprG work in concert to insert triacylglycerides and lipoarabinomannans into the outer membrane. Here, we conducted microscopic analyses of Mycobacterium smegmatis lacking the operon and observed a cell separation defect, while surface rigidity measured by atomic force microscopy was found to be increased. Whereas Rv1410 expressed in Lactococcus lactis did not confer drug resistance, deletion of the operon in Mycobacterium abscessus and M. smegmatis resulted in increased susceptibility toward vancomycin, novobiocin and rifampicin. A homology model of Rv1410 revealed a periplasmic loop as well as a highly conserved aspartate, which were found to be essential for the operon's function. Interestingly, influx of the fluorescent dyes BCECF-AM and calcein-AM in de-energized M. smegmatis cells was faster in the deletion mutant. Our results unambiguously show that elevated drug susceptibility in the deletion mutant is caused by increased drug influx through a defective mycobacterial cell envelope and not by drug efflux mediated by Rv1410.

Natural Polymorphisms in Mycobacterium tuberculosis Conferring Resistance to Delamanid in Drug-Naive Patients.

Mutations in the genes of the F420 signaling pathway of Mycobacterium tuberculosis complex, including dnn, fgd1, fbiA, fbiB, fbiC, and fbiD, can lead to delamanid resistance. We searched for such mutations among 129 M. tuberculosis strains from Asia, South America, and Africa using whole-genome sequencing; 70 (54%) strains had at least one mutation in one of the genes. For 10 strains with mutations, we determined the MIC of delamanid. We found one strain from a delamanid-naive patient carrying the natural polymorphism Tyr29del (ddn) that was associated with a critical delamanid MIC.

Whole-Genome Sequencing for Drug Resistance Profile Prediction in Mycobacterium tuberculosis.

Whole-genome sequencing allows rapid detection of drug-resistant Mycobacterium tuberculosis isolates. However, the availability of high-quality data linking quantitative phenotypic drug susceptibility testing (DST) and genomic data have thus far been limited. We determined drug resistance profiles of 176 genetically diverse clinical M. tuberculosis isolates from the Democratic Republic of the Congo, Ivory Coast, Peru, Thailand, and Switzerland by quantitative phenotypic DST for 11 antituberculous drugs using the BD Bactec MGIT 960 system and 7H10 agar dilution to generate a cross-validated phenotypic DST readout. We compared DST results with predicted drug resistance profiles inferred by whole-genome sequencing. Classification of strains by the two phenotypic DST methods into resistotype/wild-type populations was concordant in 73 to 99% of cases, depending on the drug. Our data suggest that the established critical concentration (5 mg/liter) for ethambutol resistance (MGIT 960 system) is too high and misclassifies strains as susceptible, unlike 7H10 agar dilution. Increased minimal inhibitory concentrations were explained by mutations identified by whole-genome sequencing. Using whole-genome sequences, we were able to predict quantitative drug resistance levels for the majority of drug resistance mutations. Predicting quantitative levels of drug resistance by whole-genome sequencing was partially limited due to incompletely understood drug resistance mechanisms. The overall sensitivity and specificity of whole-genome-based DST were 86.8% and 94.5%, respectively. Despite some limitations, whole-genome sequencing has the potential to infer resistance profiles without the need for time-consuming phenotypic methods.

Molecular Mechanisms of Intrinsic Streptomycin Resistance in Mycobacterium abscessus.

Streptomycin, the first drug used for the treatment of tuberculosis, shows limited activity against the highly resistant pathogen Mycobacterium abscessus We recently identified two aminoglycoside-acetylating genes [aac(2') and eis2] which, however, do not affect susceptibility to streptomycin. This suggests the existence of a discrete mechanism of streptomycin resistance. M. abscessus BLASTP analysis identified MAB_2385 as a close homologue of the 3″-O-phosphotransferase [APH(3″)] from the opportunistic pathogen Mycobacterium fortuitum as a putative streptomycin resistance determinant. Heterologous expression of MAB_2385 in Mycobacterium smegmatis increased the streptomycin MIC, while the gene deletion mutant M. abscessus ΔMAB_2385 showed increased streptomycin susceptibility. The MICs of other aminoglycosides were not altered in M. abscessus ΔMAB_2385. This demonstrates that MAB_2385 encodes a specific and prime innate streptomycin resistance determinant in M. abscessus We further explored the feasibility of applying rpsL-based streptomycin counterselection to generate gene deletion mutants in M. abscessus Spontaneous streptomycin-resistant mutants of M. abscessus ΔMAB_2385 were selected, and we demonstrated that the wild-type rpsL is dominant over the mutated rpsLK43R in merodiploid strains. In a proof of concept study, we exploited this phenotype for construction of a targeted deletion mutant, thereby establishing an rpsL-based counterselection method in M. abscessus.

Intrinsic rifamycin resistance of Mycobacterium abscessus is mediated by ADP-ribosyltransferase MAB_0591.

OBJECTIVES: Rifampicin, a potent first-line TB drug of the rifamycin group, shows only little activity against the emerging pathogen Mycobacterium abscessus. Reportedly, bacterial resistance to rifampicin is associated with polymorphisms in the target gene rpoB or the presence of enzymes that modify and thereby inactivate rifampicin. The aim of this study was to investigate the role of the MAB_0591 (arrMab)-encoded rifampicin ADP-ribosyltransferase (Arr_Mab) in innate high-level rifampicin resistance in M. abscessus. METHODS: Recombinant Escherichia coli and Mycobacterium tuberculosis strains expressing MAB_0591 were generated, as was an M. abscessus deletion mutant deficient for MAB_0591. MIC assays were used to study susceptibility to rifampicin and C25 carbamate-modified rifamycin derivatives. RESULTS: Heterologous expression of MAB_0591 conferred rifampicin resistance to E. coli and M. tuberculosis Rifamycin MIC values were consistently lower for the M. abscessus ΔarrMab mutant as compared with the M. abscessus ATCC 19977 parental type strain. The rifamycin WT phenotype was restored after complementation of the M. abscessus ΔarrMab mutant with arrMab Further MIC data demonstrated that a C25 modification increases rifamycin activity in WT M. abscessus However, MIC studies in the M. abscessus ΔarrMab mutant suggest that C25 modified rifamycins are still subject to modification by Arr_Mab CONCLUSIONS: Our findings identify Arr_Mab as the major innate rifamycin resistance determinant of M. abscessus. Our data also indicate that Arr_Mab-mediated rifamycin resistance in M. abscessus can only in part be overcome by C25 carbamate modification.

Effect of ß-lactamase production and ß-lactam instability on MIC testing results for Mycobacterium abscessus.

OBJECTIVES: Limited treatment options available for Mycobacterium abscessus infections include the parenteral ß-lactam antibiotics cefoxitin and imipenem, which show moderate in vitro activity. Other ß-lactam antibiotics (except meropenem) have no considerable in vitro activity, due to their rapid hydrolysis by a broad-spectrum ß-lactamase (Bla_Mab). We here addressed the impact of ß-lactamase production and ß-lactam in vitro stability on M. abscessus MIC results and determined the epidemiological cut-off (ECOFF) values of cefoxitin, imipenem and meropenem. METHODS: By LC high-resolution MS (LC-HRMS), we assessed the in vitro stability of cefoxitin, imipenem and meropenem. M. abscessus ATCC 19977 strain and its isogenic blaMab deletion mutant were used for MIC testing. Based on MIC distributions for M. abscessus clinical strains, we determined ECOFFs of cefoxitin, imipenem and meropenem. RESULTS: A functional Bla_Mab increased MICs of penicillins, ceftriaxone and meropenem. LC-HRMS data showed significant degradation of cefoxitin, imipenem and meropenem during standard antibiotic susceptibility testing procedures. MIC, MIC50 and ECOFF values of cefoxitin, imipenem and meropenem are influenced by incubation time. CONCLUSIONS: The results of our study support administration of imipenem, meropenem and cefoxitin, for treatment of patients infected with M. abscessus. Our findings on in vitro instability of imipenem, meropenem and cefoxitin explain the problematic correlation between in vitro susceptibility and in vivo activity of these antibiotics and question the clinical utility of susceptibility testing of these chemotherapeutic agents.

Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes.

Objectives: Mycobacterium abscessus is innately resistant to a variety of drugs thereby limiting therapeutic options. Bacterial resistance to aminoglycosides (AGs) is conferred mainly by AG-modifying enzymes, which often have overlapping activities. Several putative AG-modifying enzymes are encoded in the genome of M. abscessus . The aim of this study was to investigate the molecular basis underlying AG resistance in M. abscessus . Methods: M. abscessus deletion mutants deficient in one of three genes potentially involved in AG resistance, aac(2 ' ) , eis1 and eis2 , were generated by targeted gene inactivation, as were combinatorial double and triple deletion mutants. MICs were determined to study susceptibility to a variety of AG drugs and to capreomycin. Results: Deletion of aac(2 ' ) increased susceptibility of M. abscessus to kanamycin B, tobramycin, dibekacin and gentamicin C. Deletion of eis2 increased susceptibility to capreomycin, hygromycin B, amikacin and kanamycin B. Deletion of eis1 did not affect drug susceptibility. Equally low MICs of apramycin, arbekacin, isepamicin and kanamycin A for WT and mutant strains indicate that these drugs are not inactivated by either AAC(2 ' ) or Eis enzymes. Conclusions: M. abscessus expresses two distinct AG resistance determinants, AAC(2 ' ) and Eis2, which confer clinically relevant drug resistance.

Adverse Events Following International Normalized Ratio Reversal in Intracerebral Hemorrhage.

BACKGROUND: Prothrombin complex concentrates (PCCs) are frequently used to reverse the effect of vitamin K antagonists (VKAs) in patients with non-traumatic intracerebral hemorrhage (ICH). However, information on the rate of thromboembolic events (TEs) and allergic events after PCC therapy in VKA-ICH patients is limited. METHODS: Consecutive VKA-ICH patients treated with PCC at our institution between December 2004 and June 2014 were included into this retrospective observational study. We recorded international normalized ratio (INR) values before and after PCC treatment, baseline clinical characteristics including the premorbid modified Rankin Scale (pmRS) score, TE and allergic event that occurred during the hospital stay. All events were classified by 3 reviewers as being 'related', 'probably related', 'possibly related', 'unlikely related' or 'not related' to treatment with PCC. To identify factors associated with TEs, log-rank analyses were applied. RESULTS: Two hundred and five patients were included. Median INR was 2.8 (interquartile range (IQR) 2.2-3.8) before and 1.3 (IQR 1.2-1.4) after PCC treatment and a median of 1,500 IU PCC (IQR 1,000-2,500) was administered. Nineteen TEs were observed (9.3%); none were classified 'related' but 9 were classified as 'possibly' or 'probably related' to PCC infusion (4.4%). One allergic reaction (0.5%), 'unlikely related' to PCC, was observed. In the whole cohort, PCC doses >2,000-3,000 IU, ICH volumes >40 ml, National Institute of Health Stroke Scale values >10 and a pmRS >2 were associated with the development of TEs (p = 0.031, p = 0.034, p = 0.050 and p = 0.036, respectively). CONCLUSIONS: Overall, INR reversal with PCC appears safe. Though no clear relationship between higher PCC dosing and TEs was observed, PCC doses between >2,000 and 3,000 IU and higher morbidity at ICH onset were associated with TEs. Hence, individual titration of PCC to avoid exposure to unnecessarily high doses using point-of-care devices should be prospectively explored.

Treatment With Prothrombin Complex Concentrate to Enable Emergency Lumbar Puncture in Patients Receiving Vitamin K Antagonists.

STUDY OBJECTIVE: Lumbar punctures are frequently necessary in neurologic emergencies, but effective oral anticoagulation with vitamin K antagonists represents a contraindication. We report the effectiveness of prothrombin complex concentrates to reverse vitamin K antagonist to enable emergency lumbar punctures, as well as evaluate lumbar puncture- and prothrombin complex concentrates-related complications. METHODS: Consecutive patients treated with prothrombin complex concentrates between December 2004 and June 2014 to enable emergency lumbar puncture were included. International normalized ratio (INR) before and after prothrombin complex concentrates treatment and the time between start of reversal treatment and lumbar puncture were recorded. A target INR of less than or equal to 1.5 was defined as effective prothrombin complex concentrates treatment. Bleeding events, thromboembolic events, and allergic reactions after prothrombin complex concentrates treatment were identified and classified as "related," "probably," "possibly," "unlikely related," or "not related" to the lumbar puncture and prothrombin complex concentrates infusion. RESULTS: Thirty-seven patients were included (64.9% men; median age 76.0 years; interquartile range [IQR] 71.0 to 84.0 years). The intervention with prothrombin complex concentrates was effective in 33 of 37 patients (89.2%; 95% confidence interval [CI], 78.4% to 97.3%). The median INR was 2.2 (IQR 1.8 to 2.9; 95% CI, 1.9 to 2.5) before and 1.3 (IQR 1.2 to 1.4; 95% CI, 1.2 to 1.3) after prothrombin complex concentrates treatment. The median time between start of prothrombin complex concentrates treatment and lumbar puncture was 135 minutes (IQR 76 to 266 minutes; 95% CI, 84 to 198 minutes). One clinically irrelevant intracranial subdural hematoma "related" to the lumbar puncture developed. No allergic reaction was observed, but 2 of 37 patients (5.4%; 95% CI, 0% to 13.5%) experienced a thromboembolic event (1 ischemic stroke, classified "unlikely related," and 1 myocardial infarction, "possibly related" to prothrombin complex concentrates treatment). CONCLUSION: Reversing the effect of vitamin K antagonist with prothrombin complex concentrates to enable emergency lumbar puncture appears effective and safe, particularly in regard to bleeding events.

Discovery of the first potent and selective Mycobacterium tuberculosis Zmp1 inhibitor.

The Mycobacterium tuberculosis extracellular zinc metalloprotease 1 (Zmp1) has been proposed to play a key role in phagosome maturation and to enhance the survival of Mycobacterium tuberculosis in the host. Consequently, small molecule inhibitors of Zmp1 are of pivotal importance as a tool to better understand the pathogenicity of Zmp1 and as lead candidates for pharmacological intervention. Here we combined in silico structure-based inhibitor design with biochemical studies to discover and characterize the first potent competitive Zmp1 inhibitor showing a Ki of 94 nM and a high selectivity for Zmp1 with respect to human Neprilysin.