Mycobacterium chelonae-abscessus Complex Associated with Sinopulmonary Disease, Northeastern USA

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Learning Objectives

Upon completion of this activity, participants will be able to:

Analyze the M. chelonae-abscessus complex
Distinguish the molecular identity of "M. franklinii"
Identify the most common clinical source of "M. franklinii"
Evaluate the antimicrobial susceptibility of "M. franklinii"

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Charles P. Vega, MD, Associate Professor; Residency Director, Department of Family Medicine, University of California, Irvine. Disclosure: Charles P. Vega, MD, has disclosed no relevant financial relationships.

Authors

Disclosures: Barbara A. Brown-Elliott, MS; Perry G. Ridge; Jacob D. Durtschi, BS; Linda Bridge Mann, BS; E. Susan Slechta; Arnold G. Steigerwalt, BS; Benjamin D. Moser; Anne M. Whitney, PhD; June M. Brown, BS; Karl V. Voelkerding, MD; Karin L. McGowan, PhD; Anne F. Reilly, MD, MPH; W. Ray Butler, MS; Paul H. Edelstein, MD; and Richard J. Wallace Jr., MD, have disclosed no relevant financial relationships. Keith E. Simmon, BS, has disclosed the following relevant financial relationships: employed by: Isentio US/Software company (non-FDA). Thomas J. Kirn, MD, has disclosed the following relevant financial relationships: served as an advisor or consultant for Merck Sharpe and Dohme. Cathy A. Petti, MD, has disclosed the following relevant financial relationships: was an employee for Novartis Diagnostics from 2009–2010.

Volume 17, Number 9–September 2011

Research

Mycobacterium chelonae-abscessus Complex Associated with Sinopulmonary Disease, Northeastern USA

Keith E. Simmon,¹ Barbara A. Brown-Elliott,¹ Perry G. Ridge, Jacob D. Durtschi, Linda Bridge Mann, E. Susan Slechta, Arnold G. Steigerwalt, Benjamin D. Moser, Anne M. Whitney, June M. Brown, Karl V. Voelkerding, Karin L. McGowan, Anne F. Reilly, Thomas J. Kirn, W. Ray Butler, Paul H. Edelstein, Richard J. Wallace, Jr., and Cathy A. Petti
Author affiliations: Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA (K.E. Simmon, P.G. Ridge, J.D. Durtschi, E.S. Slechta, K.V. Voelkerding, C.A. Petti); University of Texas Health Science Center, Tyler, Texas, USA (B.A. Brown-Elliott, L.B. Mann, R.J. Wallace, Jr.); Centers for Disease Control and Prevention, Atlanta, Georgia, USA (A.G. Steigerwalt, B.D. Moser, A.M. Whitney, J.M. Brown, W.R. Butler); University of Utah School of Medicine, Salt Lake City, Utah, USA (K.V. Voelkerding, C.A. Petti); University of Pennsylvania School of Medicine at Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA (K.F. McGowan, A.F. Reilly); Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA (T.J. Kirn); and University of Pennsylvania Medical Center, Philadelphia (P.H. Edelstein)
Suggested citation for this article

Abstract
Members of the Mycobacterium chelonae-abscessus complex represent Mycobacterium species that cause invasive infections in immunocompetent and immunocompromised hosts. We report the detection of a new pathogen that had been misidentified as M. chelonae with an atypical antimicrobial drug susceptibility profile. The discovery prompted a multicenter investigation of 26 patients. Almost all patients were from the northeastern United States, and most had underlying sinus or pulmonary disease. Infected patients had clinical features similar to those with M. abscessus infections. Taxonomically, the new pathogen shared molecular identity with members of the M. chelonae-abscessus complex. Multilocus DNA target sequencing, DNA-DNA hybridization, and deep multilocus sequencing (43 full-length genes) support a new taxon for these microorganisms. Because most isolates originated in Pennsylvania, we propose the name M. franklinii sp. nov. This investigation underscores the need for accurate identification of Mycobacterium spp. to detect new pathogens implicated in human disease.

Infections caused by members of the Mycobacterium chelonae-abscessus complex remain a serious public health problem, and their role has expanded, with growing numbers of therapeutic interventions that disrupt the competency of the human immune system. Before 2001, only 2 species, M. chelonae and M. abscessus, were recognized as members of the complex. Since that time, 4 new species have been added to the complex: M. immunogenum, M. massiliense, M. bolletii, and M. salmoniphilum. With the exception of M. salmoniphilum, all members of the complex have been implicated in human disease (1–4). Recently investigators have noted a lack of separation of M. bolletii and M. massiliense from M. abscessus and have proposed they be classified as a subspecies of M. abscessus (5).
Members of the M. chelonae-abscessus complex represent Mycobacterium species that cause invasive skin and soft tissue infections, pneumonia, bloodstream infections, and abscesses in immunocompetent and immunocompromised hosts (6,7). Definitive identification by the clinical laboratory is needed for outbreak detection and for performance of susceptibility testing for patient management. Currently, the taxonomic relationships among members of the M. chelonae-abscessus complex lack clarity. The species are biochemically inert and their genetic signatures by partial 16S rRNA gene sequencing are often similar, which makes identification a great challenge for clinical laboratories.
In 2007, we detected a group of clinical isolates that were misidentified as M. chelonae with an atypical antimicrobial drug susceptibility profile. All isolates were from Pennsylvania, and as an interim identification, we labeled these isolates as CV for M. chelonae variant. Our discovery prompted a large multistate investigation that involved obtaining clinical correlation, retrospective and prospective collections of isolates with similar CV characteristics, and examination of a large set of known clinical isolates and type strains from the M. chelonae-abscessus complex. Given the taxonomic complexities and current ambiguities within the M. chelonae-abscessus group, we performed a comprehensive analysis of the potentially new species, including DNA-DNA hybridization, multilocus sequencing, and deep multilocus sequencing. We describe a new pathogen, M. franklinii sp. nov., a proposed new member of the M. chelonae-abscessus complex that was isolated from 26 patients in the United States. We discuss its potential role in human disease.

Methods

Isolates

All available type strains of M. chelonae-abscessus complex were obtained from American Type Culture Collection (ATCC), Collection of Institut Pasteur, or Culture Collection, University of Göteborg, Sweden. Previously identified clinical isolates of M. abscessus, and M. chelonae by partial 16S rRNA gene sequencing and internal transcribed spacer (ITS) PCR were retrieved for comparative analysis. A subset of these isolates was described in a prior study (8). CVs were defined as isolates that were cefoxitin susceptible or showed intermediate resistance, and identified as M. chelonae by partial 16S rRNA gene sequencing and ITS PCR (9) by Associated Regional and University Pathologists Laboratories and the Hospital of the University of Pennsylvania or by failure to amplify hsp65 by PCR restriction fragment length polymorphism analysis (10,11) by the University of Texas Health Science Center. Microbiologic and medical records were reviewed for clinical information for select isolates. Clinical case reviews were conducted under institutional review board approved protocols at Associated Regional and University Pathologists Laboratories, Children's Hospital of Philadelphia, University of Pennsylvania, and the University of Texas Health Science Center. Study isolates CV002 (ATCC [pending] and DSMZ 45524) and CV005 (ATCC [pending]) have been deposited into culture collections.

Multilocus Sequencing

DNA extractions, PCR, and sequencing reactions were performed as described (8). Amplifications and sequencing reactions were performed by using primers specific for ≈1,400 bp of the 16S rRNA 5F (5′-TTGGAGAGTTTGATCCTGGCTC-3′) and 1492R (5′-ACGGITACCTTGTTACGACTT-3′), ≈700 bp of rpoB, ≈400 bp of sodA (12), ≈240 bp of the ITS (13), and ≈400 bp of hsp65 (11) genes or region. Sequence alignments and phylogenetic trees were constructed by using neighbor-joining method with Kimura 2-parameter distance correction model and 1,000 bootstrap replications in MEGA4 (14). Only unique sequences (sequevars) were included in the trees.

Standard for Identification

Final species identifications were based on comparisons of sequences for the full 16S rRNA and the partial rpoB genes to GenBank references of type strains. Full-length 16S rRNA sequences were used in this study, and we used 99.5% shared identity for identification to a type strain sequence. Species identification for rpoB gene was based on an identity of 98.0%–100% as outlined by Adekambi et al. (12,15).

DNA-DNA Hybridization

Purified DNA of the type strains and the patient isolates CV002, CV004, CV005, CV006, and CV005 was prepared as described (16). CV002 and CV005 strains were labeled with [³²P] dCTP using the Nick Translation Kit (Invitrogen, Carlsbad, CA, USA). Labeled DNA from the CV002 was hybridized with unlabeled DNA from isolates CV002, CV004, CV005, CV006, and CV015 and with unlabeled DNA from the type strains. Labeled DNA from patient isolate CV005 was then hybridized with unlabeled DNA from isolates CV002 and CV005. The reciprocal experiment was performed because of the nearness of CV005 to the 70% cut-off designated by Wayne (17) and the 0% divergence obtained in the first experiment. Hybridization was performed as previously described (18). All reactions were performed in duplicate at 70°C. The relative binding ratio (RBR) was calculated by using the method of Brenner et al. (19). The percentage divergence (calculated to the nearest 0.5%) was determined by assuming that each degree of heteroduplex instability, when compared with the melting temperature of the homologous duplex, was caused by 1% unpaired bases (19).

Deep Multilocus Sequencing

Single-end DNA libraries of M. bolletii CIP 108541^T, M. chelonae ATCC 35752^T, M. immunogenum CIP 106684^T, M. massiliense CCUG 48898^T, and CV002 were prepared using Illumina DNA Sample Kit (Illumina, Inc., San Diego, CA, USA) according to manufacturer's recommendations. Sequencing was performed in individual flow cell lanes on the Illumina Genome Analyzer (Illumina, Inc.) at the University of Utah Huntsman Cancer Institute Core Sequencing Facility.
De novo assembly of raw Illumina sequence data was achieved by using Velvet software (20). Velvet was run in 2 parts, velveth and velvetg. For velveth, the hash length was set at 23 (value was selected by calculations in the software manual); default settings were used for all other parameters. In velvetg, the –cov cutoff value was set to auto (setting allows software to automate appropriate coverage cutoff), and –min_contig_lgth was set to 100; all other settings were default.
The genome of M. abscessus CIP 104536^T (21) was used as the source reference set of 123 genes that were identified as likely core genome components for the phylum Actinobacter by Ventura et al. (22). The set of reference genes was randomly divided into 5 similarly sized sets to facilitate analysis. SeqMan (DNASTAR Inc., Madison, WI, USA) was used to align the assembled contigs from the sequenced species against each of the sets of reference genes. DNA and inferred amino acid sequences were aligned using MEGA. Only near full-length genes were used in future comparisons. Confirming the translation of the gene was in the correct reading frame relative to the different isolates substantiated quality of each assembled gene. The DNA and amino acid sequences were concatenated for each isolate and sequences alignments and phylogenetic trees were constructed in MEGA using the neighbor-joining method. Kimura 2-parameter distance correction was used for DNA and Poisson correction model was used for amino acid trees; 1,000 bootstrap replications were used for each tree constructed.

Susceptibility Testing

We determined antimicrobial susceptibility by broth microdilution using the recommended Clinical and Laboratory Standards Institute guidelines for rapidly growing Mycobacterium spp. (23). Some isolates were not tested for all antimicrobial agents and the concentrations of antimicrobial agents that were tested varied in the panels. MICs of clarithromycin were assessed at 3 days.

Results

Identification of Isolates by Multilocus Sequencing

We obtained 6 type strains representing all members of the M. chelonae-abscessus complex. All type strains and all 127 archived isolates underwent multilocus sequencing. For the 127 archived clinical isolates, we identified 64 M. abscessus, 58 M. chelonae, and 5 M. massiliense isolates. We designated an additional 26 isolates as CV on the basis of our case definition. All 26 CV isolates underwent rpoB gene sequencing, and a subset (n = 11) underwent multilocus sequencing. Unique sequevars are designated among the species M. abscessus, M. massiliense, M. bolletii, M. chelonae, and CV isolates.
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