Mycolic acid-producing bacteria isolated from the respiratory tract of human and non-human mammals were recently assigned as a distinct genus, Segniliparus, because they diverge from rhodococci and mycobacteria in genetic and chemical features. Using high accuracy mass spectrometry, we determined the chemical composition of 65 homologous mycolic acids in two Segniliparus species and separately analyzed the three subclasses to measure relative chain length, number and stereochemistry of unsaturations and cyclopropyl groups within each class. Whereas mycobacterial mycolate subclasses are distinguished from one another by R groups on the meromycolate chain, Segniliparus species synthesize solely non-oxygenated alpha-mycolates with high levels of cis unsaturation. Unexpectedly Segniliparus alpha-mycolates diverge into three subclasses based on large differences in carbon chain length with one bacterial culture producing mycolates that range from C58 to C100. Both the overall chain length (C100) and the chain length diversity (C42) are larger than previously seen for mycolic acid-producing organisms and provide direct chemical evidence for assignment of Segniliparus as a distinct genus. Yet, electron microscopy shows that the long and diverse mycolates pack into a typical appearing membrane. Therefore, these new and unexpected extremes of mycolic acid chemical structure raise questions about the modes of mycolic acid packing and folding into a membrane.

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.

BACKGROUND: Healthcare-associated outbreaks and pseudo-outbreaks of rapidly growing mycobacteria (RGM) are frequently associated with contaminated tap water. A pseudo-outbreak of Mycobacterium chelonae-M. abscessus in patients undergoing bronchoscopy was identified by 2 acute care hospitals. RGM was identified in bronchoscopy specimens of 28 patients, 25 of whom resided in the same skilled nursing facility (SNF). An investigation ruled out bronchoscopy procedures, specimen collection, and scope reprocessing at the hospitals as sources of transmission. OBJECTIVE: To identify the reservoir for RGM within the SNF and evaluate 2 water system treatments, hyperchlorination and point-of-use (POU) membrane filters, to reduce RGM. DESIGN: A comparative in situ study of 2 water system treatments to prevent RGM transmission. SETTING: An SNF specializing in care of patients requiring ventilator support. METHODS: RGM and heterotrophic plate count (HPC) bacteria were examined in facility water before and after hyperchlorination and in a subsequent 24-week assessment of filtered water by colony enumeration on Middlebrook and R2A media. RESULTS: Mycobacterium chelonae was consistently isolated from the SNF water supply. Hyperchlorination reduced RGM by 1.5 log(10) initially, but the population returned to original levels within 90 days. Concentration of HPC bacteria also decreased temporarily. RGM were reduced below detection level in filtered water, a 3-log(10) reduction. HPC bacteria were not recovered from newly installed filters, although low quantities were found in water from 2-week-old filters. CONCLUSION: POU membrane filters may be a feasible prevention measure for healthcare facilities to limit exposure of sensitive individuals to RGM in potable water systems.

High-performance liquid chromatography analysis of mycolic acids and partial gene sequencing for the first 500-bp 5' end of the 16S rRNA gene were used singularly and in combination to evaluate the final identification of species. Examination of 200 cultures revealed 100 strains of slowly growing mycobacteria (SGM), 91 strains of rapidly growing mycobacteria (RGM), and 9 strains of other genera. SGM were discriminated in complexes with both methods for 56 strains, composed primarily of the Mycobacterium spp.: Mycobacterium avium, Mycobacterium terrae, and Mycobacterium simiae-Mycobacterium lentiflavum. For RGM, 73 strains were associated with complexes designated as Mycobacterium abscessus-Mycobacterium chelonae, Mycobacterium fortuitum-Mycobacterium peregrinum, and Mycobacterium mucogenicum-Mycobacterium phocaicum. Consistent identification of all the isolates differentiated to single species within the Mycobacterium genus was not possible with either test method. Sequencing results often distinguished complexes containing fewer species, and combining the results from each method increased the confidence of identifying the correct species.

The omission of the name 'Mycobacterium paraffinicum' from the 1980 Approved List of Bacterial Names was due to phenotypic confusion surrounding a close relationship with Mycobacterium scrofulaceum. Correspondingly 'M. paraffinicum' strains grew slowly in >7 days, stained acid-alcohol fast, produced yellow-pigmented smooth waxy colonies in the dark at an optimal temperature of 35 degrees C. However 'M. paraffinicum' strains demonstrated no activity for urease, nicotinamidase or pyrazinamidase, and lacked growth at 42 degrees C as compared to M. scrofulaceum. The mycolic acid pattern as determined by high performance liquid chromatography (HPLC) clustered 'M. paraffinicum' with M. scrofulaceum, Mycobacterium avium, and Mycobacterium parascrofulaceum. Strains were fully susceptible to linezolid, rifabutin, clarithromycin and amikacin. Examination of the historical reference strain of 'M. paraffinicum' ATCC 12670 and five additional isolates using comparative gene studies with 16S rRNA, hsp65, rpoB and concatenated sequences demonstrated separate, monophyletic tree branching that was distinct from similar nontuberculous mycobacteria and formed a tight taxonomical group with the classical reference strain of 'M. paraffinicum'. Multilocus enzyme electrophoresis (MEE) analysis confirmed a close association of the five additional isolates with the reference strain of 'M. paraffinicum' with a genetic distance of 0.12 and was distinct from other closely related species. These genetic results provided unambiguous evidence of the uniqueness of the slow-growing, scotochromogenic species and supported the revival of the name as Mycobacterium paraffinicum (ex Davis, Chase and Raymond 1956) sp. nov., nom. rev. We propose the previously deposited reference strain located in the worldwide collections as the type strain ATCC 12670T =DSM 44181T =NCIMB10420T.

OBJECTIVE: To investigate a pseudo-outbreak of "Mycobacterium paraffinicum" (unofficial taxon) infection and/or colonization, using isolates recovered from clinical and environmental specimens. DESIGN: Outbreak investigation. SETTING: University-affiliated, tertiary-care hospital. METHODS: M. paraffinicum, a slow-growing, nontuberculous species of mycobacteria, was recovered from 21 patients and an ice machine on a single patient care unit over a 2.5-year period. The clinical, epidemiological, and environmental investigation of this pseudo-outbreak is described. RESULTS: Twenty-one patients with pulmonary symptoms and possible risk factors for tuberculosis were admitted to inpatient rooms that provided airborne isolation conditions in 2 adjacent hospital buildings. In addition, 1 outpatient had induced sputum cultured for mycobacteria in the pulmonary function laboratory. Of the samples obtained from these 21 patients, 26 isolates from respiratory samples and 1 isolate from a stool sample were identified as M. paraffinicum. Environmental isolates obtained from an ice machine in the patient care unit where the majority of the patients were admitted were also identified as M. paraffinicum. CONCLUSIONS: An epidemiological investigation that used molecular tools confirmed the suspicion of a pseudo-outbreak of M. paraffinicum infection and/or colonization. The hospital water system was identified as the source of contamination.