W. Ray Butler

PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions

ABSTRACT The classical Mycobacterium tuberculosis complex (MtbC) subspecies include Mycobacterium tuberculosis, Mycobacterium africanum (subtypes I and II), Mycobacterium bovis (along with the attenuated M. bovis bacillus Calmette-Guérin [BCG]), and Mycobacterium microti; increasingly recognized MtbC groupings include Mycobacterium bovis subsp. caprae and “Mycobacterium tuberculosis subsp. canettii.” Previous investigations have documented each MtbC subspecies as a source of animal and/or human tuberculosis. However, study of these organisms is hindered by the lack of a single protocol that quickly and easily differentiates all of the MtbC groupings. Towards this end we have developed a rapid, simple, and reliable PCR-based MtbC typing method that makes use of MtbC chromosomal region-of-difference deletion loci. Here, seven primer pairs (which amplify within the loci 16S rRNA, Rv0577, IS1561′, Rv1510, Rv1970, Rv3877/8, and Rv3120) were run in separate but simultaneous reactions. Each primer pair either specifically amplified a DNA fragment of a unique size or failed, depending upon the source mycobacterial DNA. The pattern of amplification products from all of the reactions, visualized by agarose gel electrophoresis, allowed immediate identification either as MtbC composed of M. tuberculosis (or M. africanum subtype II), M. africanum subtype I, M. bovis, M. bovis BCG, M. caprae, M. microti, or “M. canettii” or as a Mycobacterium other than MtbC (MOTT). This MtbC PCR typing panel provides an advanced approach to determine the subspecies of MtbC isolates and to differentiate them from clinically important MOTT species. It has proven beneficial in the management of Mycobacterium collections and may be applied for practical clinical and epidemiological use.

Mycolic Acid Analysis by High-Performance Liquid Chromatography for Identification of Mycobacterium Species

SUMMARY Mycobacterium tuberculosis is the etiologic agent of tuberculosis and can be accurately detected by laboratories using commercial genetic tests. Nontuberculosis mycobacteria (NTM) causing other mycobacterioses can be difficult to identify. The identification processes are confounded by an increasing diversity of newly characterized NTM species. The ubiquitous nature of NTM, combined with their potential to be opportunistic pathogens in immunocompromised as well as nonimmunodeficient patients, further complicates the problem of their identification. Since clinical case management varies depending on the etiologic agent, laboratories must identify the species in a timely manner. However, only a few identification methods can detect the species diversity within the Mycobacterium genus. Over the last decade, high-performance liquid chromatography analysis of the mycolic acids has become an accepted method for identification of mycobacteria. In this review, we assess its development and usefulness as an identification technique for Mycobacterium species.

Discovery of a Novel Mycobacterium tuberculosis Lineage That Is a Major Cause of Tuberculosis in Rio de Janeiro, Brazil

ABSTRACT The current study evaluated Mycobacterium tuberculosis isolates from Rio de Janeiro, Brazil, for genomic deletions. One locus in our panel of PCR targets failed to amplify in ∼30% of strains. A single novel long sequence polymorphism (>26.3 kb) was characterized and designated RDRio. Homologous recombination between two similar protein-coding genes is proposed as the mechanism for deleting or modifying 10 genes, including two potentially immunogenic PPE proteins. The flanking regions of the RDRio locus were identical in all strains bearing the deletion. Genetic testing by principal genetic group, spoligotyping, variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR), and IS6110-based restriction fragment length polymorphism analysis cumulatively support the idea that RDRio strains are derived from a common ancestor belonging solely to the Latin American-Mediterranean spoligotype family. The RDRio lineage is therefore the predominant clade causing tuberculosis (TB) in Rio de Janeiro and, as indicated by genotypic clustering in MIRU-VNTR analysis, the most significant source of recent transmission. Limited retrospective reviews of bacteriological and patient records showed a lack of association with multidrug resistance or specific risk factors for TB. However, trends in the data did suggest that RDRio strains may cause a form of TB with a distinct clinical presentation. Overall, the high prevalence of this genotype may be related to enhanced virulence, transmissibility, and/or specific adaptation to a Euro-Latin American host population. The identification of RDRio strains outside of Brazil points to the ongoing intercontinental dissemination of this important genotype. Further studies are needed to determine the differential strain-specific features, pathobiology, and worldwide prevalence of RDRioM. tuberculosis.

Comparison of Methods for Identification of Mycobacterium abscessus and M. chelonae Isolates

ABSTRACT Mycobacterium abscessus and Mycobacterium chelonae are two closely related species that are often not distinguished by clinical laboratories despite the fact they cause diseases requiring different treatment regimens. Multilocus enzyme electrophoresis, PCR-restriction fragment length polymorphism analysis of the 65-kDa heat shock protein gene, biochemical tests, and high-performance liquid chromatography of mycolic acids were used to identify 75 isolates as either M. abscessus or M. chelonae that were originally submitted for drug susceptibility testing. Only 36 of these isolates were submitted with an identification at the species level. Using the above methods, 46 of the isolates were found to be M. abscessus and 29 were identified as M. chelonae. Eight isolates originally submitted as M. chelonae were identified as M. abscessus, and one isolate submitted as M. abscessuswas found to be M. chelonae. The four identification methods were in agreement in identifying 74 of the 75 isolates. In drug susceptibility testing, all isolates of M. abscessusexhibited resistance to tobramycin (MIC of 8 to ≥16 μg/ml), while all isolates of M. chelonae were susceptible to this drug (MIC of ≤4 μg/ml). The results suggest that once an identification method is selected, clinical laboratories should be able to easily identify isolates of M. abscessus and M. chelonae.

Multiphasic Approach Reveals Genetic Diversity of Environmental and Patient Isolates of Mycobacterium mucogenicum and Mycobacterium phocaicum Associated with an Outbreak of Bacteremias at a Texas Hospital

ABSTRACT Between March and May 2006, a Texas hospital identified five Mycobacterium mucogenicum bloodstream infections among hospitalized oncology patients using fluorescence high-performance liquid chromatography analysis of mycolic acids. Isolates from blood cultures were compared to 16 isolates from environmental sites or water associated with this ward. These isolates were further characterized by hsp65, 16S rRNA, and rpoB gene sequencing, hsp65 PCR restriction analysis, and molecular typing methods, including repetitive element PCR, random amplified polymorphic DNA PCR, and pulsed-field gel electrophoresis (PFGE) of large restriction fragments. Three of five patient isolates were confirmed as M. mucogenicum and were in a single cluster as determined by all identification and typing methods. The remaining two patient isolates were identified as different strains of Mycobacterium phocaicum by rpoB sequence analysis. One of these matched an environmental isolate from a swab of a hand shower in the patients room, while none of the clinical isolates of M. mucogenicum matched environmental strains. Among the other 15 environmental isolates, 11 were identified as M. mucogenicum and 4 as M. phocaicum strains, all of which were unrelated by typing methods. Although the 16S rRNA gene sequences matched for all 14 M. mucogenicum isolates, there were two each of the hsp65 and rpoB sequevars, seven PCR typing patterns, and 12 PFGE patterns. Among the seven M. phocaicum isolates were three 16S rRNA sequevars, two hsp65 sequevars, two rpoB sequevars, six PCR typing patterns, and six PFGE patterns. This outbreak represents the first case of catheter-associated bacteremia caused by M. phocaicum and the first report of clinical isolates from a U.S. hospital. The investigation highlights important differences in the available typing methods for mycobacteria and demonstrates the genetic diversity of these organisms even within narrow confines of time and space.

Clinical and Microbiological Assessment of Mycobacterium simiae Isolates from a Single Laboratory in Southern Arizona

Mycobacterium simiae was the third most common mycobacterium identified over a 2-year period from a single clinical laboratory in southern Arizona. Thirty-three isolates from 25 patients were identified over 1 year. The isolation of M. simiae was considered clinically significant for only two of 23 evaluable patients. None of five patients with human immunodeficiency virus infection had clinical disease associated with M. simiae. Twenty isolates were available for detailed study. All but one of the 20 isolates were niacin-negative, and 11 were nonphotochromogenic. All 20 isolates had a triple-cluster pattern consistent with M. simiae by high-performance liquid chromatography, and restriction fragment patterns were identical for 16 isolates. Analysis of 16S rDNA confirmed the identity of all the tested isolates as M. simiae. In this study, M. simiae was a frequent clinical isolate but was rarely associated with disease. The organisms isolated were confirmed to be M. simiae but appeared to be phenotypically distinct strains of low virulence.

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.

First Isolations of Segniliparus rugosus from Patients with Cystic Fibrosis

ABSTRACT We report three cases of the new genus Segniliparus isolated from patients with cystic fibrosis. All isolates were unambiguously identified by 16S rRNA gene sequencing as Segniliparus rugosus (GenBank accession no. AY 60892). Drug susceptibility results that may enhance treatment for cystic fibrosis patients with this opportunistic pathogen are presented.

Differentiation of a rapidly growing, scotochromogenic, polycyclic-aromatic-hydrocarbon-metabolizing strain of Mycobacterium sp. from other known Mycobacterium species

A rapidly-growing, acid-alcohol fast, scotochromogenic, polycyclic-aromatic-hydrocarbon-degrading Mycobacterium sp. isolate, Pyr-1, which was different from known Mycobacterium species based on biochemical tests, was further analyzed to compare its mycolic acids, cellular proteins, and nucleic acids with those of known species. Mass spectral analysis of the mycolic acids of Mycobacterium sp. Pyr-1 indicated that its mycolic acids were C60H120O3 and C62H124O3. The mycolic acid pattern from this bacterium was compared to those of 29 rapidly-growing, scotochromogenic species and 31 other species of Mycobacterium by reversed-phase high-performance liquid chromatography (HPLC). The mycolic acid pattern was unique, most closely resembling M. austroafricanum but also resembling M. parafortuitum and M. gilvum. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of soluble cellular proteins also readily differentiated this isolate from other species. The polypeptide pattern of Mycobacterium sp. Pyr-1 most closely resembled that of M. austroafricanum. Restriction enzyme analysis and Southern blot hybridization, however, revealed differences between the chromosomal DNA of our isolate and that of M. austroafricanum. The unique biochemical characteristics, mycolic acid pattern, polypeptide fingerprints, DNA restriction digest patterns, and DNA homology indicate that this strain is different from previously known species of mycobacteria. Since this bacterium is efficient in the metabolism of polycyclic aromatic hydrocarbons, its characteristics and relationships to other Mycobacterium species are reported here.

Characterization of a novel rapidly growing Mycobacterium species associated with sepsis.

ABSTRACT A rapidly growing mycobacterium was isolated five times from blood cultures from a 6-year-old female patient with relapsed pre-B-cell acute lymphocytic leukemia. All five isolates had identical nucleotide sequences for the first 500 bp of the 16S rRNA gene, indicative of a single species. High-performance liquid chromatography analysis of mycolic acids indicated that the species was similar to Mycobacterium smegmatis. Sequence analysis of the 16S rRNA gene (1,455 bp) for one isolate demonstrated that the species was closely related to Mycobacterium diernhoferi. Based on the phenotypic features and phylogenetic analysis, it was concluded that the isolates represented a novel rapidly growing Mycobacterium species. The name “Mycobacterium hackensackense” is proposed for this unique strain, 147-0552T, which was deposited in the American Type Culture Collection as ATCC BAA-823T.