Sruthi Vasireddy

Absence of Mycobacterium intracellulare and Presence of Mycobacterium chimaera in Household Water and Biofilm Samples of Patients in the United States with Mycobacterium avium Complex Respiratory Disease

ABSTRACT Recent studies have shown that respiratory isolates from pulmonary disease patients and household water/biofilm isolates of Mycobacterium avium could be matched by DNA fingerprinting. To determine if this is true for Mycobacterium intracellulare, household water sources for 36 patients with Mycobacterium avium complex (MAC) lung disease were evaluated. MAC household water isolates from three published studies that included 37 additional MAC respiratory disease patients were also evaluated. Species identification was done initially using nonsequencing methods with confirmation by internal transcribed spacer (ITS) and/or partial 16S rRNA gene sequencing. M. intracellulare was identified by nonsequencing methods in 54 respiratory cultures and 41 household water/biofilm samples. By ITS sequencing, 49 (90.7%) respiratory isolates were M. intracellulare and 4 (7.4%) were Mycobacterium chimaera. In contrast, 30 (73%) household water samples were M. chimaera, 8 (20%) were other MAC X species (i.e., isolates positive with a MAC probe but negative with species-specific M. avium and M. intracellulare probes), and 3 (7%) were M. avium; none were M. intracellulare. In comparison, M. avium was recovered from 141 water/biofilm samples. These results indicate that M. intracellulare lung disease in the United States is acquired from environmental sources other than household water. Nonsequencing methods for identification of nontuberculous mycobacteria (including those of the MAC) might fail to distinguish closely related species (such as M. intracellulare and M. chimaera). This is the first report of M. chimaera recovery from household water. The study underscores the importance of taxonomy and distinguishing the many species and subspecies of the MAC.

Emended description of mycobacterium abscessus mycobacterium abscessus subsp. Abscessus and mycobacterium abscessus subsp. bolletii and designation of mycobacterium abscessus subsp. massiliense comb. nov.

The taxonomic position of members of the Mycobacterium abscessus complex has been the subject of intensive investigation and, in some aspects confusion, in recent years as a result of varying approaches to genetic data interpretation. Currently, the former species Mycobacterium massiliense and Mycobacterium bolletii are grouped together as Mycobacterium abscessus subsp. bolletii. They differ greatly, however, as the former M. bolletii has a functional erm(41) gene that confers inducible resistance to macrolides, the primary therapeutic antimicrobials for M. abscessus, while in the former M. massiliense the erm(41) gene is non-functional. Furthermore, previous whole genome studies of the M. abscessus group support the separation of M. bolletii and M. massiliense. To shed further light on the population structure of Mycobacterium abscessus, 43 strains and three genomes retrieved from GenBank were subjected to pairwise comparisons using three computational approaches: verage ucleotide dentity, enome to enome istance and single nucleotide polymorphism analysis. The three methods produced overlapping results, each demonstrating three clusters of strains corresponding to the same number of taxonomic entities. The distances were insufficient to warrant distinction at the species level, but met the criteria for differentiation at the subspecies level. Based on prior erm(41)-related phenotypic data and current genomic data, we conclude that the species M. abscessus encompasses, in adjunct to the presently recognized subspecies M. abscessus subsp. abscessus and M. abscessus subsp. bolletii, a third subspecies for which we suggest the name M. abscessus subsp. massiliense comb. nov. (type strain CCUG 48898T=CIP 108297T=DSM 45103T=KCTC 19086T).

Utility of Sequencing the erm(41) Gene in Isolates of Mycobacterium abscessus subsp. abscessus with Low and Intermediate Clarithromycin MICs

ABSTRACT The erm(41) gene confers inducible macrolide resistance in Mycobacterium abscessus subsp. abscessus, calling into question the usefulness of macrolides for treating M. abscessus subsp. abscessus infections. With an extended incubation (14 days), isolates with MICs of ≥8 μg/ml are considered macrolide resistant by current CLSI guidelines. Our goals were to determine the incidence of macrolide susceptibility in U.S. isolates, the validity of currently accepted MIC breakpoints, and the erm(41) sequences associated with susceptibility. Of 349 isolates (excluding those with 23S rRNA gene mutations), 85 (24%) had clarithromycin MICs of ≤8 μg/ml. Sequencing of the erm(41) genes from these isolates, as well as from isolates with MICs of ≥16 μg/ml, including ATCC 19977T, revealed 10 sequevars. The sequence in ATCC 19977T was designated sequevar (type) 1; most macrolide-resistant isolates were of this type. Seven sequevars contained isolates with MICs of >16 μg/ml. The T28C substitution in erm(41), previously associated with macrolide susceptibility, was identified in 62 isolates (18%) comprising three sequevars, with MICs of ≤2 (80%), 4 (10%), and 8 (10%) μg/ml. No other nucleotide substitution was associated with macrolide susceptibility. We recommend that clarithromycin susceptibility breakpoints for M. abscessus subsp. abscessus be changed from ≤2 to ≤4 μg/ml and that isolates with an MIC of 8 μg/ml have repeat MIC testing or erm sequencing performed. Our studies suggest that macrolides are useful for treating approximately 20% of U.S. isolates of M. abscessus subsp. abscessus. Sequencing of the erm gene of M. abscessus subsp. abscessus will predict inducible macrolide susceptibility.

Mycobacterium arupense, Mycobacterium heraklionense, and a Newly Proposed Species, “Mycobacterium virginiense” sp. nov., but Not Mycobacterium nonchromogenicum, as Species of the Mycobacterium terrae Complex Causing Tenosynovitis and Osteomyelitis

ABSTRACT Mycobacterium terrae complex has been recognized as a cause of tenosynovitis, with M. terrae and Mycobacterium nonchromogenicum reported as the primary etiologic pathogens. The molecular taxonomy of the M. terrae complex causing tenosynovitis has not been established despite approximately 50 previously reported cases. We evaluated 26 isolates of the M. terrae complex associated with tenosynovitis or osteomyelitis recovered between 1984 and 2014 from 13 states, including 5 isolates reported in 1991 as M. nonchromogenicum by nonmolecular methods. The isolates belonged to three validated species, one new proposed species, and two novel related strains. The majority of isolates (20/26, or 77%) belonged to two recently described species: Mycobacterium arupense (10 isolates, or 38%) and Mycobacterium heraklionense (10 isolates, or 38%). Three isolates (12%) had 100% sequence identity to each other by 16S rRNA and 99.3 to 100% identity by rpoB gene region V sequencing and represent a previously undescribed species within the M. terrae complex. There were no isolates of M. terrae or M. nonchromogenicum, including among the five isolates reported in 1991. The 26 isolates were susceptible to clarithromycin (100%), rifabutin (100%), ethambutol (92%), and sulfamethoxazole or trimethoprim-sulfamethoxazole (70%). The current study suggests that M. arupense, M. heraklionense, and a newly proposed species (“M. virginiense” sp. nov.; proposed type strain MO-233 [DSM 100883, CIP 110918]) within the M. terrae complex are the major causes of tenosynovitis and osteomyelitis in the United States, with little change over 20 years. Species identification within this complex requires sequencing methods.

Emergence of mmpT5 variants during bedaquiline treatment of mycobacterium intracellulare lung disease

ABSTRACT Bedaquiline (BDQ), a diarylquinoline antibiotic that targets ATP synthase, is effective for the treatment of Mycobacterium tuberculosis infections that no longer respond to conventional drugs. While investigating the off-label use of BDQ as salvage therapy, seven of 13 patients with Mycobacterium intracellulare lung disease had an initial microbiological response and then relapsed. Whole-genome comparison of pretreatment and relapse isolates of M. intracellulare uncovered mutations in a previously uncharacterized locus, mmpT5. Preliminary analysis suggested similarities between mmpT5 and the mmpR5 locus, which is associated with low-level BDQ resistance in M. tuberculosis. Both genes encode transcriptional regulators and are adjacent to orthologs of the mmpS5-mmpL5 drug efflux operon. However, MmpT5 belongs to the TetR superfamily, whereas MmpR5 is a MarR family protein. Targeted sequencing uncovered nonsynonymous mmpT5 mutations in isolates from all seven relapse cases, including two pretreatment isolates. In contrast, only two relapse patient isolates had nonsynonymous changes in ATP synthase subunit c (atpE), the primary target of BDQ. Susceptibility testing indicated that mmpT5 mutations are associated with modest 2- to 8-fold increases in MICs for BDQ and clofazimine, whereas one atpE mutant exhibited a 50-fold increase in MIC for BDQ. Bedaquiline shows potential for the treatment of M. intracellulare lung disease, but optimization of treatment regimens is required to prevent the emergence of mmpT5 variants and microbiological relapse.

Absence of a Functional erm Gene in Isolates of Mycobacterium immunogenum and the Mycobacterium mucogenicum Group, Based on In Vitro Clarithromycin Susceptibility

ABSTRACT Macrolide resistance has been linked to the presence of a functional erythromycin ribosomal methylase (erm) gene in most species of pathogenic rapidly growing mycobacteria (RGM). For these Mycobacterium isolates, extended incubation in clarithromycin is necessary to determine macrolide susceptibility. In contrast, the absence of a detectable erm gene in isolates of M. chelonae, M. senegalense, and M. peregrinum and a nonfunctional erm gene in M. abscessus subsp. massiliense and 15% to 20% of M. abscessus subsp. abscessus isolates renders these species intrinsically macrolide susceptible. Not all RGM species have been screened for the presence of an erm gene, including the Mycobacterium mucogenicum group (M. mucogenicum, M. phocaicum, and M. aubagnense) and Mycobacterium immunogenum. A total of 356 isolates of these two pathogenic RGM taxa from two reference laboratories (A.R.U.P. Reference Laboratories and the Mycobacteria/Nocardia Laboratory at the University of Texas Health Science Center at Tyler) underwent clarithromycin susceptibility testing with readings at 3 to 5 days and 14 days. Only 13 of the 356 isolates had resistant clarithromycin MICs at initial extended MIC readings, and repeat values on all available isolates were ≤2 μg/ml. These studies suggest that these two additional RGM groups do not harbor functional erm genes and, like M. chelonae, do not require extended clarithromycin susceptibility testing. We propose to the Clinical Laboratory and Standards Institute that isolates belonging to these above-mentioned six rapidly growing mycobacterial groups based on molecular identification with no known functional erm genes undergo only 3 to 5 days of susceptibility testing (to exclude mutational resistance).

Treatment of mycobacterium abscessus subsp. Massiliense tricuspid valve endocarditis

To the Editor: Mycobacterium abscessus is a ubiquitous, rapidly growing mycobacteria (RGM) found in water supplies, soil, and dust. M. abscessus is considered the most pathogenic and difficult to treat of the RGM and is most often associated with pulmonary, skin, and soft tissue infections; it has also been reported to cause ocular infections, otitis, lymphadenitis, arthritis, osteomyelitis, disseminated disease, and prosthetic valve endocarditis (1,2). Most prosthetic valve endocarditis cases have been fatal. M. abscessus subsp. massiliense is 1 of 3 subspecies of M. abscessus. M. abscessus subsp. massiliense has an identical 16S rRNA gene sequence to the other 2 subspecies, Mycobacterium abscessus subsp. bolletii and Mycobacterium abscessus subsp. abscessus, but can be differentiated by rpoβ and erm41 gene sequencing (3,4). M. abscessus subsp. massiliense grows readily in blood culture media and on sheep’s blood agar within 2–4 days. Care should be taken in interpreting Gram staining of isolates because RGM is not identifiable by this method and could be mistaken for corynebacteria or diphtheroids (5,6). Such isolates could be further tested by acid-fast staining and, if positive, sent to a reference laboratory for definitive identification and susceptibility testing. Five cases of M. abscessus native valve endocarditis have been reported; 4 were fatal and 1 was lost to follow-up (1,5–9). One of the 4 fatal cases also involved the tricuspid valve and was associated with intravenous heroin abuse (9). We report a case of M. abscessus subsp. massiliense native tricuspid valve endocarditis successfully treated with antimicrobial therapy and surgical debridement. A 52-year-old man who used intravenous drugs was admitted to our hospital describing a 25-pound weight loss, fever, and night sweats. He reported injecting crushed opioid tablets mixed with tap water. He had tachycardia and pitting edema of the legs and feet. Laboratory data revealed elevated minotransferase levels, thrombocytopenia, and opiates in the urine. Computerized chest tomographic scan showed cavitary right upper lobe and lingular nodules. Routine blood cultures (BacT/ALERT3D; bioMerieux, Marcy l’Etoile, France) of samples drawn at admission and on hospital day 3, before the initiation of antimicrobial drug therapy, grew acid-fast bacilli (AFB) in broth medium on days 3 and 4 of incubation. A transthoracic echocardiogram on hospital day 5 revealed a 1-cm vegetation on the tricuspid valve. An empiric regimen for RGM consisting of intravenous cefoxitin and amikacin and oral clarithromycin and moxifloxacin were administered. Based on preliminary (3-day) susceptibility test results showing susceptibility to amikacin, resistance to the quinolones, and intermediate susceptibility to cefoxitin, linezolid, and imipenem, the regimen was changed to tigecycline, linezolid, clarithromycin, and amikacin (10). Routine blood cultures on hospital days 11 and 17 were negative. On hospital day 19, linezolid was stopped, and imipenem was added. A transthoracic echocardiogram on hospital day 31 showed the vegetation had enlarged to 1.5 × 0.5 cm. We concluded that antibiotics alone were unlikely to be curative; cardiac catheterization was performed on hospital day 38. On the basis of hemodynamic findings, the cardiologist inferred that valve replacement would be of no value and recommended valvectomy alone. Surgery on hospital day 41 revealed a 2-cm nodule on each anterior and posterior leaflet and a 2–3 mm nodule on the septal leaflet of the tricuspid valve. The anterior and posterior leaflets were removed, and the septal leaflet was segmentally resected. Routine cultures of valve tissues, in which M. abscessus would have grown, were negative. Pathologic examination confirmed suppurative vegetations with numerous bacterial colonies consistent with AFB; AFB staining disclosed numerous mycobacteria. Identification and final susceptibilities of the RGM from the original blood culture isolate revealed M. abscessus subsp. massiliense by hsp65 PCR and erm gene sequencing (4) and 14-day susceptibility to clarithromycin (10). M. abscessus subsp. massiliense has a nonfunctional (truncated) macrolide-inactivating gene (erm41), and untreated isolates are susceptible to the macrolides (4). Repeat chest tomographic scan on hospital day 69 showed nearly complete resolution of the RUL cavitary and lingular nodules/infiltrates. Tigecycline, amikacin, imipenem, and clarithromycin were continued until hospital day 77, when amikacin was stopped because of moderate hearing loss. The patient was discharged without antibiotics after 2 months of postoperative antibiotic therapy. At follow-up visits 2 and 8 weeks later he was doing well except for peripheral edema. AFB and routine blood cultures drawn at both visits were negative. He is periodically seen in the cardiology clinic; his edema has resolved with diuretic therapy. Cure of M. abscessus native valve endocarditis has not been previously reported. A case of M. chelonae native tricuspid valve endocarditis associated with a pacemaker lead was successfully treated with wire removal, valve debridement, and antimicrobial therapy (11). The patient in the current study likely acquired his infection from the tap water diluent he injected. Clinicians should consider the possibility of mycobacterial endocarditis when evaluating a septic patient with intravenous drug use history or cardiac prosthetic devices. We successfully treated mycobacterial native tricuspid valve endocarditis with combination antimicrobial therapy and surgical debridement. The location of the infection in the tricuspid valve and favorable hemodynamics enabled debridement without implantation and the subsequent possibility of intraoperative infection of a prosthetic valve.

Mycobacterium abscessus, a taxonomic puzzle

Adekambi et al. questioned our data produced, performing the wet lab DDH test with five replicates [11]. Instead they proposed results (Table 2 of [10]) inferred from rpoB similarity using the above mentioned formula. In our view, it is not valid to replace a complex assay that takes into account the whole genome (which is still considered the gold standard), with a formula based on just a single gene. Bioinformatic algorithms are available and validated in multiple studies, which can be used to infer the DDH from genomic data. The best known, in addition to Average Nucleotide Identity (ANI) [12], are the Genome to Genome Distance (GGD) [13, 14] and the Genomic Signature-Delta Difference (GS-DD) [15].

Evaluation of the Vitek MS v3.0 Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Mycobacterium and Nocardia Species

ABSTRACT This multicenter study was designed to assess the accuracy and reproducibility of the Vitek MS v3.0 matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry system for identification of Mycobacterium and Nocardia species compared to DNA sequencing. A total of 963 clinical isolates representing 51 taxa were evaluated. In all, 663 isolates were correctly identified to the species level (69%), with another 231 (24%) correctly identified to the complex or group level. Fifty-five isolates (6%) could not be identified despite repeat testing. All of the tuberculous mycobacteria (45/45; 100%) and most of the nontuberculous mycobacteria (569/606; 94%) were correctly identified at least to the group or complex level. However, not all species or subspecies within the M. tuberculosis, M. abscessus, and M. avium complexes and within the M. fortuitum and M. mucogenicum groups could be differentiated. Among the 312 Nocardia isolates tested, 236 (76%) were correctly identified to the species level, with an additional 44 (14%) correctly identified to the complex level. Species within the N. nova and N. transvalensis complexes could not always be differentiated. Eleven percent of the isolates (103/963) underwent repeat testing in order to get a final result. Identification of a representative set of Mycobacterium and Nocardia species was highly reproducible, with 297 of 300 (99%) replicates correctly identified using multiple kit lots, instruments, analysts, and sites. These findings demonstrate that the system is robust and has utility for the routine identification of mycobacteria and Nocardia in clinical practice.

Correction for Vasireddy et al., Mycobacterium arupense, Mycobacterium heraklionense, and a Newly Proposed Species, “Mycobacterium virginiense” sp. nov., but Not Mycobacterium nonchromogenicum, as Species of the Mycobacterium terrae Complex Causing Tenosynovitis and Osteomyelitis

Volume 54, no. 5, p. [1340–1351][1], 2016, . Page 1349, column 1: The last three paragraphs preceding the Acknowledgments section should be replaced with the following. Mycobacterium virginiense (vir.gi.ni.en′se. N.L. neut. adj. virginiense, of or