In vitro effects of the new oral ß-lactamase inhibitor xeruborbactam in combination with oral ß-lactams against clinical Mycobacterium abscessus isolates.

Non-tuberculosis mycobacteria (NTM), particularly Mycobacterium abscessus subsp. abscessus (M. abscessus), are increasingly being recognized as etiological agents of NTM pulmonary disease. However, treatment options for M. abscessus are limited owing to their natural resistance to most antibiotics, including ß-lactams. M. abscessus produces a class A ß-lactamase, whose activity is inhibited by cyclic boronic acid ß-lactamase inhibitors. We aimed to evaluate the in vitro effects of xeruborbactam, a cyclic boronic acid ß-lactamase inhibitor, against M. abscessus when combined with five ß-lactams (amoxicillin, tebipenem, cefdinir, cefuroxime, and cefoxitin). The drug susceptibilities of 43 M. abscessus clinical isolates obtained from 43 patients between August 2005 and May 2014 were tested. The MIC results for each ß-lactam with or without 4 µg/mL xeruborbactam were examined. Xeruborbactam lowered the MIC90 values of tebipenem, amoxicillin, cefuroxime, and cefdinir by 5, ≥4, 3, and 3 dilutions, respectively. The MIC90 values of cefoxitin without xeruborbactam were 32 µg/mL and did not change upon the addition of xeruborbactam. The lowest MIC90 value was obtained for tebipenem with xeruborbactam. Almost all isolates had an MIC of 4 µg/mL; one isolate had an MIC of 2 µg/mL. With respect to the susceptibility to the same family drug, the number of susceptible isolates increased from 1/43 (2%) to 43/43 (100%) for tebipenem with xeruborbactam. Combining tebipenem and xeruborbactam could be considered an effective all-oral regimen that benefits outpatient treatment of M. abscessus pulmonary disease. IMPORTANCE: Mycobacterium abscessus subsp. abscessus (M. abscessus) disease is treated in two phases; injectable drugs for initial followed by others for continuation. There is a need to develop all-oral treatment methods for M. abscessus infection, especially in the continuation phase. However, treatment options for M. abscessus are limited owing to their natural resistance to most antibiotics. This is the first report to evaluate the in vitro effects of xeruborbactam, a cyclic boronic acid ß-lactamase inhibitor capable of inhibiting the class A ß-lactamase produced by M. abscessus, against 43 M. abscessus clinical isolates when combined with five ß-lactam antibiotics. Xeruborbactam lowered the MIC90 values of tebipenem by five dilutions, and the number of susceptible isolates increased from 1/43 (2%) to 43/43 (100%). We showed that the tebipenem-xeruborbactam combination might be of interest to explore further as a potentially effective oral regimen for outpatient treatment of M. abscessus pulmonary disease.

In Vitro Synergistic Effects of Omadacycline with Other Antimicrobial Agents against Mycobacterium abscessus.

The clinical importance of Mycobacterium abscessus species (MABS) infections has been increasing. However, the standard treatment regimens recommended in the current guidelines often result in unfavorable outcomes. Therefore, we investigated the in vitro activity of omadacycline (OMC), a novel tetracycline, against MABS to explore its potential as a novel therapeutic option. The drug susceptibilities of 40 Mycobacterium abscessus subsp. abscessus (Mab) clinical strains obtained from the sputum of 40 patients from January 2005 to May 2014 were investigated. The MIC results for OMC, amikacin (AMK), clarithromycin (CLR), clofazimine (CLO), imipenem (IPM), rifabutin (RFB), and tedizolid (TZD) alone and their combined effects (with OMC) were examined using the checkerboard method. Additionally, we studied the differences in the effectiveness of the antibiotic combinations based on the colony morphotype of Mab. The MIC50 and MIC90 of OMC alone were 2 and 4 µg/mL, respectively. The combinations of OMC with AMK, CLR, CLO, IPM, RFB, and TZD showed synergy against 17.5%, 75.8%, 25.0%, 21.1%, 76.9%, and 34.4% of the strains, respectively. Additionally, OMC combined with CLO (47.1% versus 9.5%, P = 0.023) or TZD (60.0% versus 12.5%, P = 0.009) showed significantly higher synergy against strains with rough morphotypes than those with smooth morphotypes. In conclusion, the checkerboard analyses revealed that the synergistic effects of OMC were observed most frequently with RFB, followed by CLR, TZD, CLO, IPM, and AMK. Furthermore, OMC tended to be more effective against rough-morphotype Mab strains.

Multiple mutations of Mycobacterium intracellulare subsp. chimaera causing false-negative reaction to the transcription-reverse transcription concerted method for pathogen detection.

OBJECTIVES: To report an isolate of Mycobacterium intracellulare subsp. chimaera with multiple mutations in 16S ribosomal RNA (rRNA) gene, resulting in the false-negative reaction to the transcription-reverse transcription concerted (TRC) method for Mycobacterium avium-intracellulare complex. METHODS: We used TRC, polymerase chain reaction (PCR), and Matrix-assisted laser desorption/ionization Time-of-Flight/Mass Spectrometry (MALDI-TOF/MS) methods to identify a clinical isolate in 2021. Due to the discordant results between TRC and PCR or MALDI-TOF MS methods, 16S rRNA sequencing, whole-genome shotgun (WGS) sequencing, and average nucleotide identity (ANI) analysis were employed to identify the isolate. RESULTS: A mycobacterial isolate from a sputum sample gave negative results for the detection of Mycobacterium tuberculosis complex or M. avium-intracellulare complex by the TRC method. However, the isolate was identified as M. intracellulare by both PCR method and MALDI-TOF MS method. WGS sequencing of 16S rRNA genome revealed eight substitution mutations and one insertion mutation within the region, which could hamper the correct reaction to TRC method. Subsequent ANI analysis between the isolate and various species of nontuberculosis mycobacteria revealed that the isolate could be identified as M. intracellulare subsp. chimaera. CONCLUSION: Rare mutations within the 16S rRNA genome resulted in the false-negative identification of Mycobacterium chimaera by the TRC method. WGS sequencing and ANI analysis was necessary to identify the isolate.

Complete Genome Sequences of 14 Nontuberculous Mycobacteria Type Strains.

Here, we present the complete genome sequences of 14 nontuberculous mycobacteria type strains. The addition of type strain data may provide a concrete basis for further research.

In vitro activity of tedizolid and linezolid against multidrug-resistant Mycobacterium tuberculosis: a comparative study using microdilution broth assay and genomics.

The effects of tedizolid (TZD) against multidrug-resistant Mycobacterium tuberculosis isolates were investigated. This is possibly the first study to evaluate the MIC of TZD against Japanese Mycobacterium tuberculosis isolates. As TZD had a significantly lower MIC than LZD (P < 0.01), it was suggested to be a better, non-toxic alternative to LZD.

Clinical evaluation of the cobas® MTB-RIF/INH reagent and the cobas® 6800 for the detection of isoniazid and rifampicin resistance.

We aimed to validate the performance of a newly developed real-time PCR assay using cobas® MTB-RIF/INH reagent on the cobas® 6800 system for detecting isoniazid (INH) and rifampicin (RIF) resistance, using Japanese Mycobacterium tuberculosis (MTB) isolates. In total, 119 mock sputum specimens spiked with resistant MTB were tested using the cobas® MTB-RIF/INH reagent. The whole genomes of all MTB isolates were sequenced by MiSeq and analysed for mutations/indels causing drug resistance. All isolates were tested for phenotypic drug susceptibility, then MTB negative sputa were collected and pooled to prepare mock sputum specimens for the study. The sensitivity and specificity for INH resistance at a concentration equal to 3 × the limit of detection were 77.8% and 90.0%, respectively; those for RIF resistance were 91.8% and 93.5%, respectively. The sensitivities for INH and RIF were statistically different (P = 0.014), but not the specificities (P = 0.624). Twenty-two false-susceptible and two false-resistant results were obtained in INH; meanwhile, six false-susceptible and three false-resistant results were obtained in RIF. False-resistance for INH and RIF was mainly due to disputed mutations. The cobas® MTB-RIF/INH reagent showed better performance than other rapid molecular tests.

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

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

Efficacy estimation of a combination of triple antimicrobial agents against clinical isolates of Mycobacterium abscessus subsp. abscessus in vitro.

BACKGROUND: Mycobacterium abscessus subsp. abscessus (M. abscessus) is a rapidly growing mycobacterium that is resistant to most antibiotics. The number of patients with pulmonary disease caused by M. abscessus is increasing in several regions, and therapy involves long-term antibiotic combination treatments, although no standard treatment regimen has been established. OBJECTIVES: To examine candidate regimens for maintenance of antimicrobial treatment against M. abscessus by measuring MIC using the three-drug chequerboard method. METHODS: We evaluated the drug susceptibility of 70 clinical isolates of M. abscessus using the three-drug chequerboard method. We tested the antimycobacterial agents bedaquiline, clofazimine, amikacin, and sitafloxacin (which showed a relatively low MIC range when used as single agents) alone and in combinations. RESULTS: The three-drug combinations of bedaquiline/clofazimine/amikacin, and bedaquiline/clofazimine/sitafloxacin were studied. Among isolates for which the fractional inhibitory concentration index (FICI) could be calculated, 29/70 isolates (41%) and 11/70 isolates (16%) showed a synergistic response (FICI ≤0.75) with combined use of bedaquiline/clofazimine/amikacin, or with bedaquiline/clofazimine/sitafloxacin, respectively. CONCLUSIONS: The combination of bedaquiline with clofazimine plus either amikacin or sitafloxacin may be useful as maintenance regimens when treating pulmonary disease caused by M. abscessus.

Pin tract infection caused by Mycobacterium neoaurum in a 14-year-old child: A case report.

Although rapidly growing non-tuberculosis mycobacterium can occasionally cause postoperative infections, Mycobacterium neoaurum is a rare pathogen of surgical site infection. We report a case of pin tract infection caused by M. neoaurum in a 14-year-old girl who was admitted for lengthening of her right fourth metatarsal bone. Pain, redness, and exudate were observed 18 days after external fixator insertion. Repeated exudate cultures revealed M. neoaurum, and she was diagnosed with a mycobacterial pin tract infection. She was initially administered intravenous ciprofloxacin and minocycline, and then was switched to oral trimethoprim-sulfamethoxazole and minocycline for a total of 6 months. Despite the pin tract infection, bone lengthening was completed under antibiotic treatment without removal of the pin; no other complications were noted. There are no prior reports of external fixator pin tract infection by M. neoaurum. While such cases may be rare, this case demonstrates that bone distraction may still be successfully completed using appropriate antibiotic therapy without pin removal.

Mycobacterium europaeum lung disease in an immunocompetent patient without underlying lung disease.

Mycobacterium europaeum (M. europaeum) was recently identified as a nontuberculous mycobacterium belonging to the Mycobacterium simiae complex. There have been only a few reported cases of M. europaeum lung disease, all of which occurred in patients with immunodeficiency or prior lung disease. We herein report a case of M. europaeum lung disease in an otherwise healthy Japanese individual. A 70-year-old woman who had no apparent immunodeficiency or medical history was diagnosed with M. europaeum lung disease by multiple positive sputum cultures. The patient was successfully treated with clarithromycin, rifampin, ethambutol, and amikacin. This report is the first case of M. europaeum lung disease occurring in an individual without predisposing risk factors.

Multidrug Resistant Tuberculosis With Simultaneously Acquired Drug Resistance to Bedaquiline and Delamanid.

This study is the first to report a clinical case of simultaneously acquired resistance to bedaquiline (BDQ) and delamanid (DLM). Whole genome sequencing revealed 2 nucleotide insertions (Rv0678 and fbiC) in the Mycobacterium tuberculosis isolate. The minimum inhibitory concentrations for BDQ and DLM were 0.25 µg/mL and >2.0 µg/mL, respectively.

Ultrasensitive enzyme-linked immunosorbent assay for the detection of MPT64 secretory antigen to evaluate Mycobacterium tuberculosis viability in sputum.

OBJECTIVES: This study examined Mycobacterium tuberculosis (MTB)-secreted MPT64 as a surrogate of bacterial viability for the diagnosis of active pulmonary TB (PTB) and for follow-up treatment. METHODS: In this proof-of-concept prospective study, 50 PTB patients in the Tokyo metropolitan region, between 2017 and 2018, were consecutively included and 30 healthy individuals were also included. Each PTB patient submitted sputum on days 0, 14 and 28 for diagnosis and follow-up, and each healthy individual submitted one sputum sample. The following were performed: smear microscopy, Xpert MTB/RIF, MGIT and solid culture, and MPT64 detection on the sputum samples. Ultrasensitive ELISA (usELISA) was used to detect MPT64. The receiver operating characteristic analyses for diagnosis and follow-up revealed the optimal cut-off value of MPT64 absorbance for detecting culture positivity at multiple intervals. RESULTS: The sensitivity of MPT64 for diagnosing PTB was 88.0% (95% CI 75.7-95.5) and the specificity was 96.7% (95% CI 82.8-99.9). The specificity of MPT64 for predicting negative culture results on day 14 was 89.5% (95% CI 66.9-98.7). The sensitivity of MPT64 for predicting positive culture results on day 28 was 81.0% (95% CI 58.1-94.6). CONCLUSIONS: This study revealed that MPT64 is useful for diagnosing active PTB in patients and predicting treatment efficacy at follow-up.

Antimicrobial susceptibility patterns and MICs among non-photochromogenic rapidly growing Mycobacteroides and Mycolicibacterium species.

Introduction. Non-photochromogenic rapidly growing mycobacteria (NPRGM) that branch distinctly from Mycobacteroides (Myco) and Mycolicibacterium (Mycolici) are increasingly observed clinically and present a complicated treatment challenge; thus, appropriate in vitro susceptibility testing is required.Methodology. We evaluated the MICs of nine antimicrobials used in the treatment of infections of 25 NPRGM type strains. The relation of inducible macrolide resistance with functional erythromycin ribosomal methylase (erm) genes was also investigated.Results. The initial clarithromycin MIC reading at 3 days showed resistance in four of the Mycolici strains. In contrast, the presence of erm genes among Mycolici species differed from previous findings. Both Myco and Mycolici species were highly susceptible to amikacin and linezolid. Myco species were resistant to fluoroquinolones, while Mycolici species were susceptible. Meropenem showed low activity against Myco species, but high activity against Mycolici species.Conclusion. NPRGM clade-specific susceptibility patterns suggest an urgent need to establish distinct breakpoints for Myco and Mycolici species.

Evaluation of Q Gene Mycobacteria: A novel and easy nucleic acid chromatography method for mycobacterial species identification.

OBJECTIVES: A simple, rapid, and new diagnostic test for mycobacteria, named Q Gene Mycobacteria, has been developed. It is based on multiplex PCR using primers harbouring DNA tags combined with a dipstick nucleic acid chromatography method, which does not require the denaturation of PCR products for hybridization and can identify five species of mycobacteria including Mycobacterium tuberculosis complex (MTC), Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium gordonae. This study aimed to evaluate Q Gene Mycobacteria for the accurate identification of these five species. METHODS: A total of 340 mycobacterial strains/isolates were tested, of which 159 were type strains (four MTC and 155 non-tuberculosis mycobacteria (NTM) including four subspecies) and 181 were clinical isolates (18 M. tuberculosis, two Mycobacterium bovis Bacillus Calmette et Guérin (BCG), and 161 NTM comprising 16 species) collected from eight laboratories and hospitals in Japan. Species identification of NTM isolates was performed using the DNA-DNA hybridization method and/or direct sequencing of 16S rRNA, hsp65, and rpoB genes. Q Gene Mycobacteria was compared with above conventional methods for identifying the five species. RESULTS: Q Gene Mycobacteria showed excellent concordance for species identification, specifically 99.4% (158/159) for type strains and 99.4% (180/181) for clinical isolates. The two strains that were misidentified as M. gordonae were Mycobacterium paragordonae. As they are genetically close and there is few case reports of M. paragordonae, it might not be a serious critical issue to distinguish M. paragordonae from M. gordonae. CONCLUSIONS: Q Gene Mycobacteria was able to identify frequently isolated mycobacterial species accurately and easily. Therefore, Q Gene Mycobacteria could be a useful tool for the identification of specific mycobacteria in clinical laboratories.

A case of Mycobacterium tuberculosis laboratory cross-contamination.

SETTING: A laboratory cross-contamination event was suspected because Mycobacterium tuberculosis was unexpectedly detected at a high incidence in the cultures of several clinical specimens at the National Hospital Organization, Tokyo National Hospital, Japan. OBJECTIVE: To describe a case of Mycobacterium tuberculosis laboratory cross-contamination. DESIGN: We reviewed the medical records of 20 patients whose clinical specimens were suspected to have been contaminated by Mycobacterium tuberculosis. Variable number of tandem repeat analysis with 15 loci, the Japan Anti-Tuberculosis Association-12, and three additional hyper-variable loci, was performed to identify the cross-contamination event. RESULTS: The clinical, laboratory, and variable number of tandem repeat data revealed that the cross-contamination had possibly originated from one strongly positive specimen, resulting in false-positive results in 11 other specimens, including a case treated with anti-tuberculosis drugs. CONCLUSION: Clinical and laboratory data must be re-evaluated when cross-contamination is suspected and variable number of tandem repeat analysis should be used to confirm cross-contamination. Furthermore, original isolates should be stored appropriately, without sub-culturing and genotyping should be performed at the earliest possible for better utilization of variable number of tandem repeat for the identification of cross-contamination.

Antimicrobial susceptibility testing of Mycobacteroides (Mycobacterium) abscessus complex, Mycolicibacterium (Mycobacterium) fortuitum, and Mycobacteroides (Mycobacterium) chelonae.

The drug susceptibility of rapidly growing mycobacteria (RGM) varies among isolates. Treatment strategies similarly differ depending on the isolate, and for some, no clear strategy has been identified. This complicates clinical management of RGM. Following Clinical and Laboratory Standards Institute standard M24-A2, we assessed the susceptibility of 140 RGM isolates to 14 different antimicrobial drugs by measuring their minimal inhibitory concentrations (MICs). We also investigated the correlation of clarithromycin (CAM) MICs with the erm(41) and rrl gene mutations in the Mycobacteroides (Mycobacterium) abscessus complex, the rrl mutation in Mycobacteroides (Mycobacterium) chelonae, and the erm(39) mutation in Mycolicibacterium (Mycobacterium) fortuitum to determine the contribution of these mutations to CAM susceptibility. The five species and subspecies examined included 48 M. abscessus subsp. abscessus isolates (34.3%), 35 (25.0%) being M. abscessus subsp. massiliense, and two (1.4%) being M. abscessus subsp. bolletii. The M. abscessus complex accounted for 85 isolates (60.7%) in total, whereas 43 isolates (30.7%) were M. fortuitum, and 12 (8.6%) were M. chelonae. Our results demonstrated species-specific susceptibility to antimicrobials. In most cases, susceptibility to CAM could be predicted based on genetic pattern, but since one isolate did not fit that pattern, MIC values needed to be measured. Some isolates also exhibited rates of resistance to other drugs that differed from those previously reported in other locations, indicating that accurate identification of the bacterial isolate and use of the correct method for determining MIC are both important for the diagnosis of RGM.

A simplified pyrazinamidase test for pyrazinamide drug susceptibility in Mycobacterium tuberculosis.

We modified Wayne's pyrazinamidase test against Mycobacterium tuberculosis to indirectly measure pyrazinamidase activity via pyrazinoic acid in liquid medium. The modified pyrazinamidase test was easy to perform and its results were in complete agreement with those of the conventional Wayne's method, highlighting its potential application in phenotypic pyrazinamide susceptibility testing.

Mycolicibacterium smegmatis, Basonym Mycobacterium smegmatis, Expresses Morphological Phenotypes Much More Similar to Escherichia coli Than Mycobacterium tuberculosis in Quantitative Structome Analysis and CryoTEM Examination.

A series of structome analyses, that is, quantitative and three-dimensional structural analysis of a whole cell at the electron microscopic level, have already been achieved individually in Exophiala dermatitidis, Saccharomyces cerevisiae, Mycobacterium tuberculosis, Myojin spiral bacteria, and Escherichia coli. In these analyses, sample cells were processed through cryo-fixation and rapid freeze-substitution, resulting in the exquisite preservation of ultrastructures on the serial ultrathin sections examined by transmission electron microscopy. In this paper, structome analysis of non pathogenic Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis, was performed. As M. smegmatis has often been used in molecular biological experiments and experimental tuberculosis as a substitute of highly pathogenic M. tuberculosis, it has been a task to compare two species in the same genus, Mycobacterium, by structome analysis. Seven M. smegmatis cells cut into serial ultrathin sections, and, totally, 220 serial ultrathin sections were examined by transmission electron microscopy. Cell profiles were measured, including cell length, diameter of cell and cytoplasm, surface area of outer membrane and plasma membrane, volume of whole cell, periplasm, and cytoplasm, and total ribosome number and density per 0.1 fl cytoplasm. These data are based on direct measurement and enumeration of exquisitely preserved single cell structures in the transmission electron microscopy images, and are not based on the calculation or assumptions from biochemical or molecular biological indirect data. All measurements in M. smegmatis, except cell length, are significantly higher than those of M. tuberculosis. In addition, these data may explain the more rapid growth of M. smegmatis than M. tuberculosis and contribute to the understanding of their structural properties, which are substantially different from M. tuberculosis, relating to the expression of antigenicity, acid-fastness, and the mechanism of drug resistance in relation to the ratio of the targets to the corresponding drugs. In addition, data obtained from cryo-transmission electron microscopy examination were used to support the validity of structome analysis. Finally, our data strongly support the most recent establishment of the novel genus Mycolicibacterium, into which basonym Mycobacterium smegmatis has been classified.

Six species of nontuberculous mycobacteria carry non-identical 16S rRNA gene copies.

Nontuberculous mycobacteria (NTM) can carry two or more 16S rRNA gene copies that are, in some instances, non-identical. In this study, we used a combined cloning and sequencing approach to analyze 16S rRNA gene sequences of six NTM species, Mycobacterium cosmeticum, M. pallens, M. hodleri, M. crocinum, M. flavescens, and M. xenopi. Our approach facilitated the identification of two distinct gene copies in each species. The two M. cosmeticum genes had a single nucleotide difference, whereas two nucleotide polymorphisms were identified in M. hodleri, M. flavescens, and M. xenopi. M. pallens had a difference in four nucleotides and M. crocinum - in 23 nucleotides. Thus, we showed that the six NTM species possess at least two non-identical 16S rRNA gene copies. The full-length sequences of the intraspecies 16S rRNA variants will facilitate NTM identification and sequence analysis of specimens or other samples.

Prevention of aerosol isolation of nontuberculous mycobacterium from the patient's bathroom.

Reinfection of nontuberculous mycobacterium pulmonary disease may be caused by identical and not different genotypes http://ow.ly/62cH30krdpa.