Mycobacterium kumamotonense Sp. Nov. recovered from clinical specimen and the first isolation report of Mycobacterium arupense in Japan: Novel slowly growing, nonchromogenic clinical isolates related to Mycobacterium terrae complex

Three mycobacterium strains isolated from clinical specimens in Japan were provisionally assigned to the genus Mycobacterium based on their phenotypical characteristics. These isolates were further investigated to determine their specific taxonomic statuses. Mycolic acid analysis and 16S rRNA gene, rpoB, and hsp65 sequence data for the isolates showed that they are most similar to M. terrae complex. DNA-DNA hybridization studies indicated that the three strains were of two species and were distinguishable from M. terrae, M. nonchromogenicum, and M. hiberniae. Therefore, these strains represent two novel species within the genus Mycobacterium. However, one potential new species should have been considered as M. arupense with the 16S rRNA gene and hsp65 sequences similarities of 99.8% and 100% respectively; it was isolated from human specimens in the United States and was proposed in June 2006 as a new species. This report describes the first isolation of M. arupense in Japan, suggesting that the organism is clinically relevant. In addition, we propose the novel species designation Mycobacterium kumamotonense sp. nov. The type strain is CST 7247(T) (=GTC 2729(T), =JCM 13453(T), =CCUG 51961(T)).     

Comparative study of two typing methods, hsp65 PRA and ITS sequencing, revealed a possible evolutionary link between Mycobacterium kansasii type I and II isolates

One hundred and ninety-eight clinical isolates of Mycobacterium kansasii collected between 2003 and 2004 in Japan were genotyped by PCR and restriction enzyme analysis (PRA) and 16S-23S internal transcribed spacer (ITS) sequencing. The results demonstrated that clinical isolates of M. kansasii in Japan are almost exclusively of the type I PRA genotype, as is the case in other countries. Although the results of subtyping using the 16S-23S ITS sequence were generally consistent with subtyping using hsp65 PRA, four strains showed a discrepancy between the two methods. Sequence analysis of the hsp65, gyrB and 16S rRNA genes and the ITS sequence of the four strains suggests that they branched from type II and could be considered an ancestral strain of the type I strain. The newly recognized strains were designated as intermediate type I.     

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

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 patient's 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.     

Isolation and identification of mycobacteria from soils at an illegal dumping site and landfills in Japan

In order to study the diversity and community of genus Mycobacterium in polluted soils, we tried to isolate mycobacteria from 11 soil samples collected from an illegal dumping site and 3 landfills in Japan. Using culture methods with or without Acanthamoeba culbertsoni, a total of 19 isolates of mycobacteria were obtained from 5 soil samples and 3 of them were isolated only by the co-culture method with the amoeba. Conventional biochemical tests and sequencing of the hsp65, rpoB, and 16S rRNA genes were performed for species identification of 17 of the 19 isolates. Among the 17 isolates, there was one isolate each of Mycobacterium vanbaalenii, Mycobacterium mageritense, Mycobacterium frederiksbergense, M. vanbaalenii or Mycobacterium austroafricanum, and Mycobacterium chubuense or Mycobacterium chlorophenolicum. The remaining 12 isolates could not be precisely identified at the species level. A phylogenic tree based on the hsp65 sequences indicated that 2 of the 12 isolates were novel species. In addition, 4 isolates were phylogenically close to species that degrade polycyclic aromatic hydrocarbons, which induce some cancers in humans. These results demonstrated that there were many hitherto-unreported mycobacteria in the polluted soils, and suggested that some mycobacteria might play roles in the natural attenuation and engineered bioremediation of contaminated sites with other microorganisms.     

Mycobacterium paraterrae sp. nov. recovered from a clinical specimen: novel chromogenic slow growing mycobacteria related to Mycobacterium terrae complex

A previously unidentified, slowly growing scotochromogenic Mycobacterium was isolated from a Korean patient with symptomatic pulmonary infection. Phenotypically, this strain was generally similar to Mycobacterium terrae complex strains, however it uniquely produced orange pigmentation. Unique mycolic acid profiles and phylogenetic analyses based on three alternative chronometer molecules, 16S rRNA gene, hsp65 and rpoB , confirmed the taxonomic status of this strain as a novel species. These results support that this strain represents a novel Mycobacterium species. The name Mycobacterium paraterrae sp. nov. is proposed. The type strain is 05-2522 (= DSM 45127 = KCTC 19556).     

Strain variation in Mycobacterium marinum fish isolates

A molecular characterization of two Mycobacterium marinum genes, 16S rRNA and hsp65, was carried out with a total of 21 isolates from various species of fish from both marine and freshwater environments of Israel, Europe, and the Far East. The nucleotide sequences of both genes revealed that all M. marinum isolates from fish in Israel belonged to two different strains, one infecting marine (cultured and wild) fish and the other infecting freshwater (cultured) fish. A restriction enzyme map based on the nucleotide sequences of both genes confirmed the divergence of the Israeli marine isolates from the freshwater isolates and differentiated the Israeli isolates from the foreign isolates, with the exception of one of three Greek isolates from marine fish which was identical to the Israeli marine isolates. The second isolate from Greece exhibited a single base alteration in the 16S rRNA sequence, whereas the third isolate was most likely a new Mycobacterium species. Isolates from Denmark and Thailand shared high sequence homology to complete identity with reference strain ATCC 927. Combined analysis of the two gene sequences increased the detection of intraspecific variations and was thus of importance in studying the taxonomy and epidemiology of this aquatic pathogen. Whether the Israeli M. marinum strain infecting marine fish is endemic to the Red Sea and found extremely susceptible hosts in the exotic species imported for aquaculture or rather was accidentally introduced with occasional imports of fingerlings from the Mediterranean Sea could not be determined.     

Mycobacterium species identification--a new approach via dnaJ gene sequencing

The availability of the dnaJ1 gene for identifying Mycobacterium species was examined by analyzing the complete dnaJ1 sequences (approximately 1200 bp) of 56 species (54 of them were type strains) and comparing sequence homologies with those of the 16S rRNA gene and other housekeeping genes (rpoB, hsp65). Among the 56 Mycobacterium species, the mean sequence similarity of the dnaJ1 gene (80.4%) was significantly less than that of the 16S rRNA, rpoB and hsp65 genes (96.6%, 91.3% and 91.1%, respectively), indicating a high discriminatory power of the dnaJ1 gene. Seventy-one clinical isolates were correctly clustered to the corresponding type strains, showing isolates belonging to the same species. In order to propose a method for strain identification, we identified an area with a high degree of polymorphism, bordered by conserved sequences, that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (approximately 350 bp) allows accurate species identification and may be used as a new tool for the identification of Mycobacterium species.     

Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium

16S rRNA sequences from Mycobacterium tuberculosis, M. avium, M. gastri, M. kansasii, M. marinum, M. chelonae, M. smegmatis, M. terrae, M. gordonae, M. scrofulaceum, M. szulgai, M. intracellulare, M. nonchromogenicum, M. xenopi, M. malmoense, M. simiae, M. flavescens, M. fortuitum, and M. paratuberculosis were determined and compared. The sequence data were used to infer a phylogenetic tree, which provided the basis for a systematic phylogenetic analysis of the genus Mycobacterium. The groups of slow- and fast-growing mycobacteria could be differentiated as distinct entities. We found that M. simiae occupies phylogenetically an intermediate position between these two groups. The phylogenetic relatedness within the slow-growing species did not reflect the Runyon classification of photochromogenic, scotchromogenic, and nonchromogenic mycobacteria. In general, the phylogenetic units identified by using rRNA sequences confirmed the validity of phenotypically defined species; an exception was M. gastri, which was indistinguishable from M. kansasii when this kind of analysis was used.     

A Study of the Taxonomy of the Mycobacterium nonchromogenicum Complex and Report of Six Cases of Lung Infection Due to Mycobacterium nonchromogenicum

In numerical classification, four species of the Mycobacterium nonchromogenicum complex, Mycobacterium nonchromogenicum, M. terrae, M. novum, and M. triviale, formed one cluster. These four species appeared to be reduced to one species, Mycobacterium nonchromogenicum. Furthermore, relationships between the species were numerically analyzed by using the hypothetical median organism pattern. The results showed that the M. nonchromogenicum complex can be divided into two subgroups: M. nonchromogenicum and the other three. These two subgroups were differentiated from each other by scores based on two or more positive reactions in the following three characteristics: resistance to bleomycin (5 μg/ml); heat-stable acid phosphatase activity; nicotinamidase or pyrazinamidase activity or both activities. M. nonchromogenicum gave two or three positive reactions among these three, and M. terrae, M. novum, and M. triviale gave two or three negative reactions. Three cases of lung infection due to M. nonchromogenicum, as well as three other cases of probable lung infection due to M. nonchromogenicum, were observed in this study. Only one organism isolated from one doubtful case was M. terrae. Up to now, M. nonchromogenicum was considered a nonpathogen. It was shown, however, that this organism causes lung infection in humans.     

Characterization of photochromogenic Mycobacterium spp. from Chesapeake Bay striped bass Morone saxatilis

A large diversity of Mycobacterium spp. has been isolated from striped bass Morone saxatilis in Chesapeake Bay, USA. The new species M. shottsii and M. pseudoshottsii are the dominant isolates, while the classical fish pathogen M. marinum is found much less frequently. M. fortuitum and M. chelonae, other Mycobacterium spp. known to commonly infect fishes, have not yet been aseptically isolated from striped bass within Chesapeake Bay. While M. pseudoshottsii and M. shottsii have been phenotypically and genotypically characterized, other less common mycobacterial isolates have not. In the present study, we describe 17 photochromogenic isolates from Chesapeake Bay striped bass using phenotypic characterization and multilocus sequencing of 16S rRNA, hsp65 and rpoB genes. Genetic characterization reveals that these isolates are related to widely divergent portions of the mycobacterial phylogeny; however, some interesting trends are observed, such as a majority of isolates (10/17) belonging to the M. simiae-related grouping. Five additional isolates were assigned to the slow-growing mycobacteria (including 2 identified as M. marinum), while 2 are clearly shown to belong genetically to the fast-growing mycobacteria.     

Comparison of proteins from Mycobacterium fortuitum, Mycobacterium nonchromogenicum and Mycobacterium terrae using flat bed electrophoresis.

Polyacrylamide gel electrophoresis of bacterial lysates in a flat bed gives a linear relationship between 1n mol. wt of the proteins and the square root of their migration distances, thereby allowing standardization of different electrophoresis runs and precise comparison between homologous bands. The results obtained with Mycobacterium fortuitum, M. terrae and M. nonchromogenicum strains were used in numerical analysis. Mycobacterium fortuitum and M. nonchromogenicum showed a greater internal similarity than M. terrae, while two strains of the latter clustered with M. nonchromogenicum. The method described allows the comparison of mycobacteria with different generation times and provides a large number of good characters for numerical taxonomy.     

Large-restriction-fragment polymorphism analysis of Mycobacterium chelonae and Mycobacterium terrae isolates

Mycobacterium chelonae and Mycobacterium terrae were reported to be frequently present in the environment of the Mycobacterium bovis BCG trial area in south India. Six isolates of M. chelonae and four isolates of M. terrae obtained from different sources in this area were analyzed by pulsed-field gel electrophoresis (PFGE) to examine large-restriction-fragment (LRF) polymorphism using the chromosomal DNA digested with DraI and XbaI restriction enzymes. With the exception of one isolate of M. terrae, DNA from all other isolates could be digested with DraI and XbaI and resulted in separable fragments. Visual comparison of the LRFs showed a unique pattern for each of the isolates tested. A computer-assisted dendrogram of the percent similarity demonstrated a high degree of genetic diversity in this group of isolates. This study demonstrates that species of nontuberculous mycobacteria, particularly M. chelonae and M. terrae, can be successfully typed by their LRF pattern using PFGE, which does not require species-specific DNA probes.     

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.     

Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean

The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.     

Diversity of Mycobacterium species from marine sponges and their sensitivity to antagonism by sponge-derived rifamycin-synthesizing actinobacterium in the genus Salinispora

Eleven isolates of Mycobacterium species as well as an antimycobacterial Salinispora arenicola strain were cultured from the sponge Amphimedon queenslandica. The 16S rRNA, rpoB, and hsp65 genes from these Mycobacterium isolates were sequenced, and phylogenetic analysis of a concatenated alignment showed the formation of a large clade with Mycobacterium poriferae isolated previously from another sponge species. The separation of these Mycobacterium isolates into three species-level groups was evident from sequence similarity and phylogenetic analyses. In addition, an isolate that is phylogenetically related to Mycobacterium tuberculosis was recovered from the sponge Fascaplysinopsis sp. Several different mycobacteria thus appear to co-occur in the same sponge. An actinobacterium closely related to S. arenicola, a known producer of the antimycobacterial rifamycins, was coisolated from the same A. queenslandica specimen from which mycobacteria had been isolated. This Salinispora isolate was confirmed to synthesize rifamycin and displayed inhibitory effects against representatives from two of three Mycobacterium phylotype groups. Evidence for antagonism of sponge-derived Salinispora against sponge-derived Mycobacterium strains from the same sponge specimen and the production of antimycobacterial antibiotics by this Salinispora strain suggest that the synthesis of such antibiotics may have functions in competition between sponge microbial community members.     

Development of multiplex PCR assays based on the 16S-23S rRNA internal transcribed spacer for the detection of clinically relevant nontuberculous mycobacteria

Aims: To accelerate the identification and differentiation of clinically relevant nontuberculous mycobacteria (NTM) with two sets of multiplex PCR (mPCR) targeting the 16S–23S rRNA internal transcribed spacer (ITS) region for timely patient management. Methods and Results: Two mPCR assays were developed: Slow‐Growers (SG) mPCR was used for the detection of slow‐growing mycobacteria, which included Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium gordonae and Mycobacterium xenopi whilst the other mPCR assay labelled as Fast‐Growers (FG) mPCR was used for the detection of Mycobacterium fortuitum complex, Mycobacterium abscessus and Mycobacterium chelonae. In these assays, a common forward primer based on a conserved section of the 16S rRNA region was used in conjunction with species‐specific reverse primers. The mPCRs were tested against 247 clinical mycobacterial isolates and demonstrated 100% specificity and sensitivity. Identification of the mycobacterial species was also validated by DNA sequencing of the 16S–23S ITS region and when further confirmation was needed, hsp65 sequencing was performed. Conclusions: The mPCR assays could be a potentially useful diagnostic tool for the rapid and accurate identification of clinically relevant NTM. Significance and Impact of the Study: In this study, we looked at the frequency of hospital isolated NTM over the last 5 years (2005–2010), and an mPCR targeting the ITS region was developed for NTM species that appeared to be more prevalent in the context of Singapore.     

Combined rpoB duplex PCR and hsp65 PCR restriction fragment length polymorphism with capillary electrophoresis as an effective algorithm for identification of Mycobacterial species from clinical isolates

ABSTRACT: BACKGROUND: Mycobacteria can be quickly and simply identified by PCR restriction-enzyme analysis (PRA), but misidentification can occur because of similarities in band sizes that are critical for discriminating among species. Capillary electrophoresis can provide computer-aided band discrimination. The aim of this research was to develop an algorithm for identifying mycobacteria by combined rpoB duplex PRA (DPRA) and hsp65 PRA with capillary electrophoresis. RESULTS: Three hundred and seventy-six acid-fast bacillus smear-positive BACTEC cultures, including 200 Mycobacterium tuberculosis complexes (MTC) and 176 non-tuberculous mycobacteria (NTM) were analyzed. With combined hsp65 and rpoB DPRA, the accuracy rate was 100 % (200 isolates) for the MTC and 91.4 % (161 isolates) for the NTM. Among the discordant results (8.6 %) for the NTM, one isolate of Mycobacterial species and the an isolate of M. flavescens were found as new sub-types in hsp65 PRA. CONCLUSIONS: This effective and novel identification algorithm using combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can rapidly identify mycobacteria and find new sub-types in hsp65 PRA. In addition, it is complementary to 16S rDNA sequencing.     

Atypical Helicobacter canadensis strains associated with swine

Forty-two Helicobacter isolates were isolated from swine feces in The Netherlands and Denmark. All 12 isolates sequenced (16S rRNA gene) formed a robust clade with Helicobacter canadensis ( approximately 99% similarity). Species-specific PCR indicated that all of the isolates were H. canadensis isolates. Although the appearance of the porcine isolates was similar to the appearance of H. canadensis, only one of these isolates was able to hydrolyze indoxyl acetate, a cardinal characteristic of this taxon. Examination of the 23S rRNA and hsp60 genes revealed high levels of similarity between the porcine isolates and H. canadensis. However, amplified fragment length polymorphism genomic typing showed that isolates recovered from swine feces were genetically distinct from H. canadensis strains obtained from humans and geese.     

Expanding the mycobacterial diversity of metalworking fluids (MWFs): evidence showing MWF colonization by Mycobacterium abscessus

Nontuberculous mycobacteria (NTM) have been associated with hypersensitivity pneumonitis in machinists. Only two species of NTM, namely Mycobacterium immunogenum and Mycobacterium chelonae, have been reported thus far to have the ability to colonize contaminated metalworking fluids (MWFs). Here, we report, for the first time, the presence and characterization (phenotypic and genotypic) of a third species, Mycobacterium abscessus, colonizing these harsh alkaline machining fluids. Two Mycobacterium morphotypes, smooth (S) and rough (R), were isolated (two isolates each) from an in-use industrial MWFs. Biocide susceptibility analysis using triclosan as a model yielded the same minimal inhibitory concentration for the two morphotypes. PCR-restriction analysis-based speciation of the morphotypes confirmed their identity as M. abscessus. Genotyping based on partial DNA sequences corresponding to the variable regions of the hsp65 gene and 16S-23S rRNA operon internal transcribed spacer region and randomly amplified polymorphic DNA-PCR analysis showed that both morphotypes belong to a single genotype. In addition, we isolated and confirmed two novel mycobacterial genotypes, one each of M. immunogenum and M. chelonae from additional in-use MWF screening. Taken together, this study expands the known mycobacterial species- and strain-diversity colonizing MWF. Furthermore, the study emphasizes the need for including M. abscessus species in the existing mycobacterial screening of contaminated MWF.     

Genetic diversity in clinical isolates of Mycobacterium avium complex from Guinea-Bissau, West Africa

Isolates of Mycobacterium avium complex (MAC) were cultured from sputum samples obtained from patients in Guinea-Bissau, West Africa. Twenty-eight isolates hybridising with MAC probe (AccuProbe) were further characterised by different molecular techniques: hybridisation with species-specific probes (AccuProbe) for M. avium and M. intracellulare, partial sequencing of 16S rRNA gene and PCR detection of the DT1-DT6 sequences and the macrophage-induced gene (mig). Only one of the 28 isolates reacted with the M. avium probe and four with the M. intracellulare probe. Two isolates expressed the DT1 sequence, and three the DT6. The mig was detected in 18 (64%) of the isolates. Sequencing of 16S rRNA had the greatest discriminative power of the typing methods applied, without strong correlation with any other technique. Clinical MAC isolates from Guinea-Bissau demonstrated a wide genetic diversity among the members of M. avium complex that might reflect on biotope variation.