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

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.


Members
of the Mycobacterium chelonaeabscessus complex represent Mycobacterium species that cause invasive infections in immunocompetent and immunocompromised hosts. We report the detection of a new pathogen that had been misidentifi ed as M. chelonae with an atypical antimicrobial drug susceptibility profi le. 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 identifi cation of Mycobacterium spp. to detect new pathogens implicated in human disease.  (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). Defi nitive identifi cation 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 identifi cation a great challenge for clinical laboratories.
In 2007, we detected a group of clinical isolates that were misidentifi ed as M. chelonae with an atypical antimicrobial drug susceptibility profi le. All isolates were from Pennsylvania, and as an interim identifi cation, 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. chelonaeabscessus 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. chelonaeabscessus complex that was isolated from 26 patients in the United States. We discuss its potential role in human disease.

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 identifi ed 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 defi ned as isolates that were cefoxitin susceptible or showed intermediate resistance, and identifi ed 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)

Standard for Identifi cation
Final species identifi cations 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 identifi cation to a type strain sequence. Species identifi cation for rpoB gene was based on an identity of 98.0%-100% as outlined by Adekambi et al. (12,15).

DNA-DNA Hybridization
Purifi ed 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 [ 32 P] 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 fi rst 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). 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 identifi ed 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. Confi rming 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.

Identifi cation 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.    Table  1 in the online Technical Appendix (www.cdc.gov/EID/ content/17/9/101667-Techapp.pdf).

Deep Multilocus Sequencing
Full-length genes were successfully assembled for 43 genes from 5 type strains and 1 representative clinical CV isolate. Gene names and corresponding GenBank accession numbers are provided in the supplementary tables (online Technical Appendix Table 2). Pair-wise alignments of the DNA and amino acid sequences were performed for each isolate (online Technical Appendix Tables 3, 4

Clinical Spectrum and Characteristics of CV Isolates
The most common source for the CV isolates was respiratory (n = 20) (online Technical Appendix Table  6). The remaining 6 CV isolates had clinical sources that included skin (n = 2), granulomatous liver lesion (n = 1), central line infections (n = 2), and an unspecifi ed body fl uid. Most isolates (n = 15) were recovered from patients seen in 4 different hospitals or clinics in Pennsylvania. Eight patients with CV infection were seen at the Hospital of the University of Pennsylvania and 7 of 8 patients were adult females (ages 41-74 years) who acquired the mycobacterial infection as outpatients. Six charts were available for review, and partial information was available for 2 additional patients (patients CV007, CV008, CV010, CV012-CV014, CV034, and CV036).
The medical histories of the 6 patients with complete information fell into 2 groups: those with chronic sinusitis (2 patients) and those with lower respiratory symptoms (4 patients). All patients with lower respiratory symptoms and cultures positive for CV had underlying pulmonary disease (cystic fi brosis, primary ciliary dyskinesia, lung cancer, chronic obstructive pulmonary disease, recurrent pneumonia/bronchiectasis). No patients with lower respiratory symptoms received specifi c antibiotic therapy aimed at treating rapidly growing mycobacterial infection, although 1 patient received long term antimicrobial drug therapy for concomitant M. avium infection. Two patients with sinusitis were not treated with antimicrobial drugs, but both had sinus surgery with symptomatic improvement. One patient had 3 positive sputum cultures for a rapidly growing Mycobacterium over a 3-year period, 2 of which were shown by genetic sequencing to be CV organisms (CV014 was selected for further study). No other patient had >1 positive culture for CV, if follow-up cultures were performed. Three patients had sequential or concomitant infections with M. abscessus, M. avium-intracellulare, or both bacteria.
The fi rst isolate identifi ed as a CV was discovered in 2005 from a patient in New York. The next isolate was not identifi ed until 2007 from Pennsylvania. Overall, 23 (88%) of 26 were isolated in patients in the northeastern United States, and the remaining 3 isolates were recovered from patients in Minnesota, Oregon, and Colorado.

Discussion
We describe the discovery of a new human pathogen with clinical features similar to M. abscessus that is implicated as a cause of infection for patients with chronic lung diseases, intravascular catheters, and chronic sinusitis. On the basis of our investigations, we propose CV isolates become a new member of the M. chelonae-abscessus complex and be named Mycobacterium franklinii sp. nov. M. franklinii (frank li′ ni I, N.L. masc. gen. n. franklinii of Franklin, pertaining to Benjamin Franklin, statesman, founder of the University of Pennsylvania, inventor, and scientist who helped create the nation's fi rst public hospital in Philadelphia, Pennsylvania, USA, the origin of the isolates).
The microorganisms are acid-fast, gram-positive bacilli, and colony morphology alone is not suffi cient for differentiation from other rapidly growing Mycobacterium spp. Colonies are nonpigmented appearing on 5% sheep blood agar, Middlebrook 7H10 agar and egg-based Lowenstein-Jensen slants in 2-5 days at temperatures between 24 and 37°C (optimally at 30°C). Even with molecular techniques, underrecognition of this new pathogen is not surprising because it shares 100% full 16S rRNA gene identity with M. chelonae and has sequence variation in the hsp65 gene that results in inconsistent amplifi cation with typical diagnostic primers for sequence or PCR restriction fragment length polymorphism analysis (10,11). However, diagnosis of M. franklinii infection is essential because it is more susceptible to antimicrobial drugs than other members of the M. chelonae-abscessus complex, and its susceptibility pattern with cefoxitin was a distinguishing characteristic leading to its discovery.
Multilocus sequencing on a population of M. chelonaeabscessus complex isolates using 5 DNA regions enabled us to examine a population of closely related isolates to accurately assess species variability. M. franklinii shared complete 16S rRNA gene sequence identity with the type strain of M. chelonae, but was differentiated from M. chelonae and other members of the M. chelonae-abscessus complex by partial sequencing of rpoB, hsp65, sodA, and ITS DNA targets. Concatenated analysis of 43 genes (≈40,000 bp) from deeper sequencing of M. franklinii demonstrated that this novel species shares <90.5% identity with any other M. chelonae-abscessus group member. DNA-DNA hybridization analysis also supports the novel classifi cation with its low relative binding ratios and higher percent divergence from all other M. chelonaeabscessus complex type strains. Cefoxitin susceptibility or intermediate susceptibility is another distinguishing feature. Preliminary testing on 6 M. franklinii isolates revealed inducible resistance (data not shown) in 50% of the isolates upon prolonged clarithromycin incubation (14 days). This fi nding suggests that similar to M. abscessus, isolates of this species may have an inducible erm gene.
The pathophysiology of diseases associated with M. franklinii is largely unknown. Most M. franklinii isolates were from respiratory sources and from patients with underlying lung conditions. Three of these disorders (cystic fi brosis, primary ciliary dyskinesia, and recurrent pneumonia) had associated bronchiectasis, which is a known risk factor for M. abscessus and M. massiliense nodular lung disease but not for M. chelonae (6,25). It is unclear whether this microorganism causes respiratory tract disease, or simply colonizes damaged airways and sinuses. Similar to patients with M. abscessus and M. chelonae infections, we found 2 cases each of sinusitis and catheter-associated infection from M. franklinii (26). The association with chronic sinusitis presumably relates to sinus washes using tap water rinses in previously diseased sinuses. Although the exact reservoir of M. franklinii is unknown, a recent study in the Netherlands by Van  sequence. The observation in the Netherlands suggests an environmental source for this organism, and it is likely that the novel species is regionally specifi c and can survive in municipal water sources. This hypothesis would be supported by the large number of cases in a focused region in Pennsylvania. Our population analyses of clinical isolates of M. chelonae demonstrate a lack of taxonomic clarity. Additionally, our investigations lend further evidence that species distinctions for M. bolletii and M. massiliense may be inappropriate and support the recent proposal to modify their classifi cations (5,28). Taxonomic uncertainty likely arises as our understanding of microbial phylogeny expands with rapid advances in technologies and often results in inconsistent standards being applied for species designations. For example, DNA-DNA hybridization is a relatively standard technique, but upon review of 14 species descriptions in 2009 only 5 were supported by using DNA-DNA hybridization (29)(30)(31)(32)(33)(34)(35)(36).
The discovery of an emerging pathogen should be taken in the context of microbial ecology and evolution, the interaction between host and microbe, and factors of virulence. This investigation underscores the need for accurate identifi cation of Mycobacterium spp. for detection of a new pathogen. The interplay between colonization and disease is not clearly defi ned, but we demonstrate its role in central line infections and for patients with sinopulmonary disease. M. franklinii may have newly emerged as a human pathogen over the past 5 years, or it has been involved in human disease previously and was unrecognized. In order to further our understanding of this pathogen and its role in disease, greater surveillance and awareness is necessary. At this time, clinical laboratories can identify M. franklinii by sequencing based assays that target either the ITS region (between 16S and 23S rRNA genes), hsp65, rpoB, and sodA genes, or by complete 16S rRNA gene sequence analysis in conjunction with cefoxitin and minocycline susceptibility patterns. The type strain, CV002 (ATCC [pending] and DSMZ 45524), was isolated from a skin lesion.