Proposal of Mycobacterium peregrinum sp. nov., nom. rev., and elevation of Mycobacterium chelonae subsp. abscessus (Kubica et al.) to species status: Mycobacterium abscessus comb. nov.

We studied the taxonomic positions of the rapidly growing organism Mycobacterium fortuitum and phenotypically related organisms. We confirmed that "Mycobacterium peregrinum" ATCC 14467T (T = type strain) is genetically independent of M. fortuitum ATCC 6841T by using various DNA hybridization conditions. Strains that were genetically identified as "M. peregrinum" were phenotypically differentiated from M. fortuitum ATCC 6841T. Thus, we propose that "M. peregrinum" should be revived as an independent species, Mycobacterium peregrinum sp. nov., nom. rev. The type strain is strain ATCC 14467. M. fortuitum subsp. acetamidolyticum ATCC 35931T exhibited a high level of DNA relatedness to M. fortuitum ATCC 6841T. The hybridized DNAs maintained stable heteroduplexity at high stringency; thus, we confirmed that M. fortuitum subsp. acetamidolyticum is identical to M. fortuitum ATCC 6841T. We found that M. chelonae subsp. abscessus ATCC 19977T is genetically different from M. chelonae subsp. chelonae NCTC 946T on the basis of the results of quantitative hybridization even under optimal conditions. There was no reason to maintain this organism as a subspecies of M. chelonae. Thus, we propose that M. chelonae subsp. abscessus should be elevated to species status as Mycobacterium abscessus (Kubica et al.) comb. nov. The type strain is strain ATCC 19977.     

Activities of Linezolid against nontuberculous mycobacteria

The activity of linezolid (Pfizer, USA) was tested by broth microdilution against 53 clinical isolates of non-tuberculous mycobacteria (NTM), including the common disease producing species Mycobacterium avium, M. intracellulare, M. fortuitum, M. chelonae and M. abscessus, obtained from western Turkey. The isolates of M. abscessus and M. intracellulare were the least susceptible, M. mucogenicum, M. gordonae and M. avium were the most susceptible to linezolid of the common species of NTM. Linezolid showed a variable sensitivity in all strains; therefore, each species and strain must be individually evaluated, and it is always advisable to perform in vitro sensitivity tests before using the drug for human therapy.     

Numerical classification of rapidly growing nonphotochromogenic mycobacteria

Numerical analysis of 211 strains of rapidly growing, nonphotochromogenic mycobacteria was carried out by using 116 characters. New species reported after 1981 and not yet compared with known species by numerical classification were included. At the level of 90% of the matching coefficient, the following species could be differentiated from each other and were shown as distinct clusters: Mycobacterium pulveris, M. moriokaense, M. chitae, M. diernhoferi, M. porcinum, M. fortuitum, M. chelonae subspecies chelonae, M. chelonae subspecies abscessus, M. smegmatis, M. agri, M. fallax, M. parafortuitum, M. fortuitum subspecies acetamidolyticum. Four taxa, M. fortuitum, M. porcinum, M. chelonae subsp. chelonae, and M. chelonae subsp. abscessus, were regarded as distinct. However, these were combined into one large cluster at a level of 90% of the matching coefficient and, therefore, were regarded as forming one series or complex. M. fortuitum subsp. acetamidolyticum was reported as a subspecies of M. fortuitum, but this was differentiated clearly from the other species including M. fortuitum.     

PCR with subsequent sequencing of the 16S gene rRNA in species identification of mycobacteria of the non-tuberculosis complex

One hundred of mycobacterium cultures were assayed by the method of PCR with subsequent sequencing of the 16S rRNA region. The below mycobacterium species were identified: M. tuberculosis complex (n = 55), M. avium (n = 17), M. intracellulare (n = 4), M. scrofaleceum (n = 2), M. kansasii - M. gastri (n = 3), M. gordonae (n = 3), M. ulcerans - M. marinum (n = 1), M. smegmatis (m = 2), M. fortuitum (n = 11), M. peregrinum (n = 1) and M. chelonae - M. abscessus (n = 1). The method enabled the differentiation of species M. avium from M. intracellulare and M. peregrinum from M. fortuitum, which could not be differentiated by using the classic biochemical and bacteriological methods. Genetic heterogeneity of the mycobacterium strains of M. avium, M. fortuitum and M. gordonae was also established by PCR plus sequencing of the 16S rRNA region.     

Pathogenesis of Mycobacterium spp. in zebrafish (Danio rerio) from research facilities

One of the most common diseases that we have diagnosed in zebrafish is mycobacteriosis, caused by several Mycobacterium spp. The severity of the disease ranged from severe outbreaks to incidental infections. We conducted an in vivo study to evaluate the pathogenesis of six isolates of Mycobacterium from zebrafish with mycobacteriosis from four research facilities and one wholesale supplier of zebrafish in the United States: Mycobacterium abscessus, Mycobacterium peregrinum, Mycobacterium chelonae (2 isolates), and Mycobacterium marinum. We also included two isolates of M. marinum from other fishes. Fish were exposed by intraperitoneal injection at a target does of 5 x 10(4) bacteria/fish, and were held in static aquaria at 28 degrees C for 8 weeks. Fish were examined by histology and culture, and mortalities were recorded. The M. marinum isolates caused 100% infection and mortality between 30% and 100%. None of the other Mycobacterium species caused significant mortalities, but several of these fish had granulomatous lesions in visceral organs. Mycobacteria were consistently recovered in culture from fish exposed to M. marinum, and from only 9% of fish exposed to the other species. This study suggests that, of the isolates tested, only M. marinum is highly pathogenic and virulent to healthy zebrafish.     

Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory

Aims:  To facilitate efficient identification of commonly encountered mycobacteria species ( Mycobacterium tuberculosis , Mycobacterium avium , Mycobacterium intracellulare , Mycobacterium fortuitum complex , Mycobacterium chelonae/abscessus , Mycobacterium kansasii , Mycobacterium gordonae ) in high throughput laboratories, a 16s rDNA sequence based real-time PCR assay was developed and evaluated.
Methods and Results:  Oligonucleotide primers and hybridization probes were designed based on sequence differences of the mycobacterial 16S rDNA gene. This assay was evaluated with 1649 suspected non-tuberculosis mycobacterial isolates. Apart from 3 out of 40  M. avium isolates that showed false signal with M. intracellulare specific probe, 100% specificity was obtained for all tested probes. Assay sensitivity varied from 88·9 to 100% depending on species. Average cost for obtaining a definite identification was only USD 1·1 with an average turn around time of less than 3 days.
Conclusions:  A rapid, simple and inexpensive real-time PCR assay was developed for the identification of common encountered mycobacteria in a high throughput laboratory setting.
Significance and Impact of the Study:  With this assay, more than 80% of the clinically isolated nontuberculous mycobacteria could be identified in a highly cost effective manner. This helped to save resources for other laboratory activities especially in high throughput mycobacterial laboratories.     

Mycobacterium marinum infections in humans and tracing of its possible environmental sources

The low frequency of nontuberculous mycobacterial infections, nonspecific symptoms for individual mycobacteria, and the lack of specific identification methods could alter correct diagnosis. This study presents a combined microbiology and molecular-based approach for Mycobacterium?marinum detection in four aquarists with cutaneous mycobacterial infection. Simultaneously, ecology screening for M.?marinum presence in the aquarists' fish tanks was performed. A total of 38 mycobacterial isolates originated from four human patients (n?=?20), aquarium animals (n?=?8), and an aquarium environment (n?= 10). Isolate identification was carried out using 16S?rRNA sequence analysis. A microbiology-based approach, followed by 16S rRNA sequence analysis, was successfully used for detection of M.?marinum in all four patients. Animal and environmental samples were simultaneously examined, and a total of seven mycobacterial species were isolated: Mycobacterium chelonae , Mycobacterium fortuitum , Mycobacterium gordonae , Mycobacterium?kansasii , Mycobacterium mantenii , Mycobacterium marinum , and Mycobacterium?peregrinum . The presence of M.?marinum was proven in the aquarium environments of two patients. Although M.?marinum is described as being present in water, it was detected only in fish.     

Mycobacteriosis in zebrafish (Danio rerio) research facilities

The Zebrafish International Resource Center was established to support the zebrafish research community, and includes a diagnostic service. One of the most common diseases that we have diagnosed is mycobacteriosis, which represented 18% of the diagnostic cases submitted from November 1999 to June 2003. We describe here the severity of the disease and associated pathological changes of 24 diagnostic cases from 14 laboratories. Identifications of the bacteria are provided for seven of these cases. For two cases in which culture of the organism was not successful, these identifications were based on ribosomal DNA (rDNA) sequence analysis obtained directly from infected tissues. Biochemical characteristics and rDNA sequence analysis from cultures are reported for the other isolates. Two severe outbreaks from different facilities on different continents were associated with an organism identified as Mycobacterium haemophilum based on rDNA sequence from tissues. Another severe outbreak was associated with an organism most closely related to Mycobacterium peregrinum. These species are recognized pathogens of humans, but this is the first report of them from fish. Bacteria identified as Mycobacterium chelonae or M. abscessus were recovered from fish in cases categorized as moderate disease or as an incidental finding. These findings indicate that species of Mycobacterium previously undescribed from fish (i.e., M. haemophilum and M. peregrinum) may pose significant health problems in zebrafish research facilities, whereas species and strains that are already recognized as common in fish usually cause limited disease on a population basis in zebrafish.     

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.     

2018—2020年平顶山地区非结核分枝杆菌菌种鉴定及耐药性分析

本文旨在观察2018—2020年河南省平顶山地区非结核分枝杆菌(nontuberculous mycobacteria,NTM)的菌种分布及耐药情况。收集2018年1月—2020年12月平顶山市传染病医院分离到的326株NTM,采用DNA微阵列芯片鉴定菌种,改良罗氏培养基比例法进行药敏试验。结果显示,从61～80岁患者中分离的NTM菌株最多,其次是41～60岁患者。共鉴定出8个NTM菌种,分别为胞内分枝杆菌(35.28%)、龟/脓肿分枝杆菌(24.85%)、鸟分枝杆菌(18.40%)、偶然分枝杆菌(5.21%)、戈登分枝杆菌(1.23%)、堪萨斯分枝杆菌(12.58%)、浅黄分枝杆菌(1.53%)、瘰疬分枝杆菌(0.92%)。NTM对异烟肼的耐药率最高,为97.85%。除戈登分枝杆菌外,其他NTM菌种对异烟肼的耐药率均＞94%;胞内分枝杆菌对丙硫异烟胺的耐药率(8.70%)相对较低,鸟分枝杆菌对丙硫异烟胺的耐药率为10.00%;龟/脓肿分枝杆菌对异烟肼、利福平、链霉素、乙胺丁醇、阿米卡星的耐药率均＞95%;偶然分枝杆菌对左氧氟沙星的耐药率为35.29%,堪萨斯分枝杆菌对左氧氟沙星的耐药率最低(7.32%);戈登分枝杆菌对异烟肼、乙胺丁醇、链霉素、对氨基水杨酸的耐药率均≥50%;浅黄分枝杆菌对乙胺丁醇、左氧氟沙星、阿米卡星、卡那霉素的耐药率均＜50%;瘰疬分枝杆菌对阿米卡星和丙硫异烟胺的耐药率为0。结果提示,2018—2020年河南省平顶山地区鉴定出的8个NTM菌种中,胞内分枝杆菌占比最高,不同菌种对不同抗结核药物的耐药性差异较大,因此菌种鉴定对临床治疗有重要意义。     

Detection of mycobacteria in aquarium fish in Slovenia by culture and molecular methods

Thirty-five aquarium fish were investigated for the presence of mycobacteria by culture and molecular methods. The following species were examined: goldfish Carassius auratus auratus, guppy Poecilia reticulata, 4 three-spot gourami Trichogaster trichopterus, dwarf gourami Colisa lalia, Siamese fighting fish Betta splendens, freshwater angelfish Pterophyllum scalare, African cichlid fish Cichlidae spp., cichlid fish Microgeophagus altispinosus, cichlid fish Pseudotropheus lombardoi, blue streak hap Labidochromis caeruleus, sterlet Acipenser ruthenus, southern platyfish Xiphophorus maculatus, and catfish Corydoras spp. Isolates of mycobacteria were obtained in 29 cases (82.9%). Two specimens were positive using Ziehl-Neelsen (ZN) staining, but the cultivation failed. Four specimens were both ZN- and culture-negative. On the basis of GenoType Mycobacterium assay (Hain Life-science) and restriction enzyme analysis of the amplified products (PCR-RFLP), 23 isolates (79.3%) were identified: 7 as Mycobacterium fortuitum, 6 as M. gordonae, 6 as M. marinum, 3 as M. chelonae, and 1 as M. peregrinum. Five isolates remained unidentified (Mycobacterium spp.). One case probably represented a mixed infection (M. marinum/M. fortuitum). Since M. marinum infections are also detected in humans, the significance of mycobacteria in aquarium fish should not be overlooked.     

Comparison of the Pathogenicity for Mice of Mycobacterium fortuitum and Mycobacterium abscessus

The pathogenicity for mice of 12 strains of Mycobacterium abscessus was compared with that for 8 strains of M. fortuitum. Both species caused lesions in kidneys and produced "spinning disease" resulting from inner ear infections. No major differences in pathogenicity of these two species were demonstrated. Strain to strain variation was marked, especially with M. abscessus. For example, 1.6 x 10(6) organisms of strain 11188 of M. abscessus produced death in four of five animals within 42 days, whereas strain 380 of M. abscessus failed to produce any deaths within 42 days. In the case of M. fortuitum, the greatest mortality observed was one of five animals, yet the incidence of spinning disease and kidney disease occurred earlier postinfection than in mice infected with M. abscessus. Histologically, abscess formation by a strain of M. abscessus was greater than by a strain of M. fortuitum, but this difference cannot be interpreted as a species difference.     

Mycobactins as chemotaxonomic characters for some rapidly growing mycobacteria

Thirty-nine strains of rapidly growing mycobacteria were examined for the production of mycobactins (lipid-soluble, iron-binding compounds) when grown under conditions of iron-limitation on solidified medium. Different growth conditions had little effect on the structure of individual mycobactins, indicating them to be strongly conserved molecules showing intra-species consistency and thus suitable for use as chemotaxonomic characters of high discriminatory power. Strains of Mycobacterium aurum, M. chitae, M. chelonae subsp. abscessus, 'M. diernhoferi', M. duvalii, M. flavescens, M. fortuitum, M. gadium, 'M. gallinarum', M. neoaurum, M. parafortuitum, 'M. peregrinum', M. phlei, M. smegmatis, M. thermoresistible and M. vaccae formed mycobactins which were readily isolated and characterized by a combination of thin-layer and high-performance liquid chromatography. All strains of M. komossense and 'M. kanazawa' failed to produce a mycobactin; some strains of M. aurum, M. chelonae, M. parafortuitum, M. thermoresistible and M. vaccae were similarly negative. Mycobacteria of the M. fortuitum complex (M. fortuitum, M. chelonae and 'M. peregrinum') formed distinctive mycobactins, as did those in the M. parafortuitum complex (M. aurum, M. neoaurum, 'M. diernhoferi', M. vaccae and M. parafortuitum). The mycobactin from 'M. gallinarum' was different from those of the related species M. flavescens, for which four distinct mycobactin patterns were recorded. For routine examination of mycobactins in a diagnostic laboratory with limited resources, thin-layer chromatography used alone offers a simple but adequate means of characterization and final identification of the producing mycobacterium. High-performance liquid chromatography is only needed in those few instances where a high degree of discrimination is required.     

Draft genome sequence of Mycobacterium abscessus subsp. bolletii BD(T)

Mycobacterium abscessus subsp. bolletii is an increasing cause of human pulmonary disease and infections of the skin and soft tissues. Consistent reports of human infections indicate that M. bolletii is a highly pathogenic, emerging species of rapidly growing mycobacteria (RGM). Here we report the first whole-genome sequence of M. abscessus subsp. bolletii BD(T).     

鲟分枝杆菌病及其病原研究

20092010年间,我国人工养殖的中华鲟（Acipenser sinensis）、史氏鲟（Acipenser schrencki）和杂交鲟（hybrid sturgeon:A. baeri-A. gueldenstaedtii）暴发了细菌性疾病。患病鲟通过组织切片观察,病原菌的分离、鉴定以及组织样品中病原菌的检测,结果显示从19条患病鲟中分离到49株分枝杆菌。病原菌经过多个保守基因的测序分析和部分生理生化特征的鉴定,共发现有7种分枝杆菌,分别为龟分枝杆菌（Mycobacterium chelonae）、海分枝杆菌（Mycobacterium marinum）、戈登氏分枝杆菌（Mycobacterium gordonae）、偶发分枝杆菌（Mycobacterium fortuitum）、苏尔加分枝杆菌（Mycobacterium szulgai）、猪分枝杆菌 （Mycobacterium porcinum）和Mycobacterium arpuense。在诊断过程中发现两种或三种分枝杆菌同时存在于同一样品中,分子生物学的诊断结果表明分枝杆菌复合感染十分常见,而海分枝杆菌是分枝杆菌复合感染中最为常见的分枝杆菌。分离的病原菌对斑马鱼的攻毒试验结果表明在以上7种分枝杆菌中海分枝杆菌的毒力最强。以上结果表明海分枝杆菌是鲟分枝杆菌病的主要致病菌,分枝杆菌复合感染是鲟分枝杆菌病的主要感染形式。研究中史氏鲟和中华鲟的分枝杆菌病,以及在病鱼体内分离的猪分枝杆菌和M. arupense在国内外均尚未见报道。       

Mycobacterium fortuitum and Mycobacterium chelonae biofilm formation under high and low nutrient conditions

The rapidly growing mycobacteria (RGM) are broadly disbursed in the environment. They have been recovered from freshwater, seawater, wastewater and even potable water samples and are increasingly associated with non-tuberculous mycobacterial disease. There is scant evidence that non-tuberculous mycobacteria (NTM) and RGM form biofilms. Therefore, an experimental system was designed to assess the ability of RGM to form biofilms under controlled laboratory conditions. A flat plate reactor flow cell was attached to either a high or low nutrient reservoir and monitored by image analysis over time. Two surfaces were chosen for assessment of biofilm growth: silastic which is commonly used in medical settings and high density polyethylene (HDPE) which is prevalent in water distribution systems. The results show that Mycobacterium fortuitum and M. chelonae formed biofilms under both high and low nutrient conditions on both surfaces studied. These results suggest that RGM may form biofilms under a variety of conditions in industrial and medical environments.     

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.     

Various stages in the life cycle of syrphid flies (Eristalis tenax; diptera: Syrphidae) as potential mechanical vectors of pathogens causing mycobacterial infections in pig herds

We defined the role of the syrphid fly Eristalis tenax in the survival and transmission of mycobacteria in pigs. The conditionally pathogenic mycobacterial (CPM) species Mycobacterium chelonae was isolated from 10 % of liquid dung samples, and both M. chelonae and another CPM species M. fortuitum were isolated from 7 (78 %) of the examined E. tenax larvae collected from the same location. Mycobacteriosis of the lymph nodes of pigs from 3 infected farms was caused by M. avium subsp. avium, M. avium subsp. hominissuis, and M. fortuitum. M. avium subsp. avium and M. avium subsp. hominissuis of identical genotype and serotypes and M. fortuitum were isolated from 7 (1.9 %) larvae, 2 (7.4 %) puparia, and one (1.6 %) imago. The count of colony forming units isolated from larval skin covering (pouch) was higher (p < or = 0.01) than that isolated from the internal organs of larvae. These results showed the potential for E. tenax larvae to spread mycobacteria throughout pig herds and the surrounding environment.     

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.