Members Of Mycobacterium Avium Complex Research Articles

Mycobacteriosis in slaughter pigs from South Africa from 1991 to 2002: Mycobacterium spp. diversity and Mycobacterium avium complex genotypes.

Mycobacterium avium complex (MAC) bacteria are the most prominent etiological agents of lymphadenitis in pigs. M. avium subspecies hominissuis (MAH) is a member of MAC and has been reported in many parts of the world to be the most prevalent non-tuberculous mycobacteria (NTM) to cause mycobacteriosis in humans, mainly in children. Thus, the economic and zoonotic impact of MAC species are increasingly being recognized. In South Africa, little is known about the distribution of NTM and the molecular epidemiology of M. avium in pigs. In this study, lymph nodes including mandibular, mesenteric, submandibular, and retropharyngeal, with tuberculosis-like lesions were collected during routine meat inspection of slaughter pigs with no disease symptoms (n = 132), between 1991 and 2002. These pigs were slaughtered at 44 abattoirs distributed across seven of the nine South African provinces. Mycobacterial culture, polymerase chain reaction (PCR), and sequencing of the Mycobacterium specific 577 bp 16S rRNA gene fragment were performed for species and subspecies identification. The majority of the isolates (each per sample); 114 (86.4%) were identified as MAH, 8 (6%) as MAA/M. avium subsp. silvaticum, 4 (3%) were Mycobacterium tuberculosis, 2 (1.5%) as Mycobacterium intracellulare, and 1 (0.75%) as Mycobacterium bovis. The other isolates were identified as Mycobacterium lentiflavum (0.75%), Mycobacterium novocastrense (0.75%), and a Micrococcus spp. (0.75%). Using an eight-marker MLVA typing tool, we deciphered at least nine MIRU VNTR INMV types of MAH and MAA. Identification of known zoonotic mycobacteria, including MAH, MAA, M. intracellulare, M. bovis, and M. tuberculosis, from slaughter pigs has a potential public health impact and also strengthens recognition of the potential economic impact of MAC. This study has also for the first time in South Africa, revealed MAC MIRU VNTR INMV genotypes which will aid in the future epidemiological investigation of MAC in South Africa.

Investigating in vivo Mycobacterium avium subsp. paratuberculosis microevolution and mixed strain infections.

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's Disease (JD) in ruminants, which is responsible for significant economic loss to the global dairy industry. Mixed strain infection (MSI) refers to the concurrent infection of a susceptible host with genetically distinct strains of a pathogen, whereas within-host changes in an infecting strain leading to genetically distinguishable progeny is called microevolution. The two processes can influence host-pathogen dynamics, disease progression and outcomes, but not much is known about their prevalence and impact on JD. Therefore, we obtained up to 10 MAP isolates each from 14 high-shedding animals and subjected them to whole-genome sequencing. Twelve of the 14 animals examined showed evidence for the presence of MSIs and microevolution, while the genotypes of MAP isolates from the remaining two animals could be attributed solely to microevolution. All MAP isolates that were otherwise isogenic had differences in short sequence repeats (SSRs), of which SSR1 and SSR2 were the most diverse and homoplastic. Variations in SSR1 and SSR2, which are located in ORF1 and ORF2, respectively, affect the genetic reading frame, leading to protein products with altered sequences and computed structures. The ORF1 gene product is predicted to be a MAP surface protein with possible roles in host immune modulation, but nothing could be inferred regarding the function of ORF2. Both genes are conserved in Mycobacterium avium complex members, but SSR1-based modulation of ORF1 reading frames seems to only occur in MAP, which could have potential implications on the infectivity of this pathogen. IMPORTANCE Johne's disease (JD) is a major problem in dairy animals, and concerns have been raised regarding the association of Mycobacterium avium subsp. paratuberculosis (MAP) with Crohn's disease in humans. MAP is an extremely slow-growing bacterium with low genome evolutionary rates. Certain short sequence repeats (SSR1 and SSR2) in the MAP chromosome are highly variable and evolve at a faster rate than the rest of the chromosome. In the current study, multiple MAP isolates with genetic variations such as single-nucleotide polymorphisms, and more noticeably, diverse SSRs, could simultaneously infect animals. Variations in SSR1 and SSR2 affect the products of the respective genes containing them. Since multiple MAP isolates can infect the same animal and the possibility that the pathogen undergoes further changes within the host due to unstable SSRs, this could provide a compensative mechanism for an otherwise slow-evolving pathogen to increase phenotypic diversity for overcoming host responses.

Detection of the Mycobacterium avium complex in dogs with lymphadenitis.

Mycobacterium avium complex (MAC) is connected to human immunosuppressive diseases, including HIV-AIDS, and may pose a zoonotic threat. MAC causes lymphadenopathy in children, respiratory infection in adults, and generalized infection in immunocompromised individuals. Infection with nontuberculous Mycobacteria (NTM) in humans is now primarily brought on by MAC. Recently, MAC members have emerged as pathogenic organisms for animals and humans. While dogs are generally resistant to mycobacterial infections, there have been some cases of infection that result in systemic or disseminated diseases. The organisms can be transmitted to dogs through oral contact, and their faeces can be a possible source of infection for dog owners. It is important to note that this ailment is zoonotic, especially if infected pet dogs are in prolonged contact with their humans. The study was planned to demonstrate the occurrence of MAC organisms and other Mycobacteria in dogs associated with lymphadenopathy cases with special emphasis on lymphadenitis. A total of 123 samples (100 lymph node aspirates, 15 lymph node tissues, and 8 blood samples) from 83 dogs suspected of lymphadenitis accompanied by gastroenteritis, chronic skin infections, immunosuppression, chronic pulmonary diseases, and other chronic undiagnosed diseases were studied. The samples were processed for cytological and microscopic examination by Ziehl-Neelsen staining. Following the decontamination procedure, the aspiration and lymph node tissue samples were inoculated into Middlebrook 7H11 media for up to 8 weeks. The aspirated material was also directly used for molecular detection by triplex-nested polymerase chain reaction (nPCR) assay. A cytological study revealed pyogranulomatous inflammation of the lymph node tissue. Impression smears from lymph node tissues displayed the presence of acid-fast organisms. Out of 83 cases of dogs, 8 were found to be positive for Mycobacterium spp. Among those 8 positive cases, 3 were confirmed to belong to MAC, and 5 belonged to the Mycobacterium tuberculosis complex (MTB complex). MAC and MTB are the underestimated bacteria that could be the causative agents of lymphadenitis in animals.

Comparative Genomics of Mycobacterium avium Complex Reveals Signatures of Environment-Specific Adaptation and Community Acquisition.

ABSTRACTNontuberculous mycobacteria, including those in the Mycobacterium avium complex (MAC), constitute an increasingly urgent threat to global public health. Ubiquitous in soil and water worldwide, MAC members cause a diverse array of infections in humans and animals that are often multidrug resistant, intractable, and deadly. MAC lung disease is of particular concern and is now more prevalent than tuberculosis in many countries, including the United States. Although the clinical importance of these microorganisms continues to expand, our understanding of their genomic diversity is limited, hampering basic and translational studies alike. Here, we leveraged a unique collection of genomes to characterize MAC population structure, gene content, and within-host strain dynamics in unprecedented detail. We found that different MAC species encode distinct suites of biomedically relevant genes, including antibiotic resistance genes and virulence factors, which may influence their distinct clinical manifestations. We observed that M. avium isolates from different sources—human pulmonary infections, human disseminated infections, animals, and natural environments—are readily distinguished by their core and accessory genomes, by their patterns of horizontal gene transfer, and by numerous specific genes, including virulence factors. We identified highly similar MAC strains from distinct patients within and across two geographically distinct clinical cohorts, providing important insights into the reservoirs which seed community acquisition. We also discovered a novel MAC genomospecies in one of these cohorts. Collectively, our results provide key genomic context for these emerging pathogens and will facilitate future exploration of MAC ecology, evolution, and pathogenesis.IMPORTANCE Members of the Mycobacterium avium complex (MAC), a group of mycobacteria encompassing M. avium and its closest relatives, are omnipresent in natural environments and emerging pathogens of humans and animals. MAC infections are difficult to treat, sometimes fatal, and increasingly common. Here, we used comparative genomics to illuminate key aspects of MAC biology. We found that different MAC species and M. avium isolates from different sources encode distinct suites of clinically relevant genes, including those for virulence and antibiotic resistance. We identified highly similar MAC strains in patients from different states and decades, suggesting community acquisition from dispersed and stable reservoirs, and we discovered a novel MAC species. Our work provides valuable insight into the genomic features underlying these versatile pathogens.

Molecular and Serological Footprints of Mycobacterium avium Subspecies Infections in Zoo Animals.

Background: Mycobacteria of the Mycobacterium avium complex (MAC) pose a significant risk to zoological collections. Mycobacterium avium subspecies paratuberculosis (MAP) is a member of MAC and the causative agent of Johne’s disease. Despite many reports in animals kept in zoological gardens, systemic surveillance has rarely been reported. Methods: In this study, archived serum samples collected from animal species at the Wilhelma Zoological and Botanical Gardens in Stuttgart, Germany, were screened for the presence of antibodies against MAC and MAP. In addition, molecular investigations were performed on necropsy, fecal, and environmental samples. Results: In total, 30/381 serum samples of various mammalian species were positive for MAC antibodies in ELISA, while one sample of a reticulated giraffe (Giraffa camelopardalis reticulata) was positive in MAP-specific ELISA. Samples from many species were positive in pan-Mycobacterium real-time PCR (40/43 fecal samples, 27/43 environmental samples, and 31/90 necropsy samples). Surprisingly, no sample was positive in the MAP-specific molecular assays. However, two environmental samples from primate enclosures were positive in Mycobacterium avium subspecies hominissuis (MAH)-specific real-time PCR. Conclusions: The results reveal serological indications of MAC infections in the zoological collection. However, the presence of a MAP-contaminated environment by a high-shedding individual animal or MAP-infected population is unlikely.

Mycobacterium avium Complex: Addressing Gaps in Diagnosis and Management.

Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and an important cause of disease. The most common species causing pulmonary disease are members of Mycobacterium avium complex (MAC). MAC pulmonary disease (MAC-PD) can be chronic, debilitating, costly, and associated with a high mortality. However, MAC diagnoses are often delayed due to the nonspecific presentation of MAC-PD and radiological findings that overlap with other pulmonary diseases. Patients with risk factors and who meet the diagnostic criteria—which include clinical, radiological, and microbiologic criteria—should be considered for treatment. Diagnosis requires 2 or more positive sputum cultures or 1 bronchoscopic specimen culture. The recommendation for those who are treated is a 3-drug regimen including macrolide, rifamycin, and ethambutol that is continued for 12 months beyond sputum culture conversion to negative. MAC-PD is difficult to treat, with frequent drug-related side effects and suboptimal treatment outcomes. Refractory and recurrent disease is common, leading to lifelong follow-up of patients. There are limited treatment options for patients with macrolide-resistant or refractory disease. Amikacin liposome inhalation suspension is recommended for treatment-refractory patients whose cultures remain positive after 6 months of guideline-based therapy. Among the research priorities to improve patient outcomes and quality of life are developing new, more rapid diagnostic tests, investigating biomarkers associated with disease progression, and identifying new drugs and routes of administration as well as new, shorter, and better-tolerated regimens.

MAC-INMV-SSR: a web application dedicated to genotyping members of Mycobacterium avium complex (MAC) including Mycobacterium avium subsp. paratuberculosis strains

Genotyping of Mycobacterium avium subsp. paratuberculosis (Map) is an indispensable tool for surveillance of this significant veterinary pathogen. For Map, multi-locus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRUs) and other variable number variable-number tandem repeats (VNTRs) was established using 8 markers. In the recent past this standard, portable, reproducible and discriminatory typing method has been frequently applied alone or in combinations with multi-locus short-sequence-repeat (MLSSR) sequencing. With the widespread use of these genotyping methods, standardization between laboratories needs to be managed, and knowledge of existing profiles and newly defined genotypes should be indexed and shared. To meet this need, a web application called “MAC-INMV-SSR database” was developed. This freely accessible service allows users to compare MLVA and MLSSR subtype data of their strains with those of existing reference strains analyzed with the same genotyping methods.

DISTRIBUTION OF MYCOBACTERIUM AVIUM COMPLEX IN BULGARIA

The members of Mycobacterium avium complex (MAC) are opportunistic microorganisms, common in the environment (soil and water) and cause infections in birds, mammals and humans. The objective of the present study was to determine the prevalence of MAC representatives among suspected for tuberculosis patients in Bulgaria over an eight-year period – from 2010 to 2017. The exact epidemiology of pulmonary non-tuberculous mycobacterial (NTM) disease was difficult to determine because unlike cases of infection with M. tuberculosis complex, notification of NTM and MAC in particular, is not mandatory in terms of Ordinance 21 of the Ministry of Health (1) and there is no any accurate information about their prevalence in Bulgaria. The survey was conducted in the National Reference Laboratory of Tuberculosis at the National Centre of Infectious and Parasitic Diseases. MAC was identified using phenotypic methods, biochemical features and molecular genetic assay (LPA).
 A total of 596 NTM strains were identified during the study period and 16.8% (n=100) of them were MAC. In EU countries, MAC representatives areprevalent among NTM isolates, but in Bulgaria they are significantly less in number.

Distribution and clinical significance of Mycobacterium avium complex species isolated from respiratory specimens

Mycobacterium avium complex (MAC) was originally composed of 2 species, M. avium and M. intracellulare. However, several new species closely related to M. intracellulare have recently been identified. In addition, M. avium has been further subdivided into 4 subspecies. The aim of this study was to determine the proportion of different MAC species recovered from respiratory specimens and to elucidate the clinical relevance of these species. Clinical isolates, from 219 patients, that had been initially identified as M. avium or M. intracellulare by non-sequencing methods were reidentified using multilocus sequence typing, and the clinical significance of the identified species was then investigated. Of 91 isolates originally identified as M. intracellulare, 75 (82%) were confirmed to be M. intracellulare, 8 (9%) isolates were identified as M. chimaera, and 4 (4%) isolates each were identified as “M. indicus pranii” and M. yongonense. The 128 isolates originally designated as M. avium were determined to be M. avium subsp. hominissuis. Of the 219 patients, 146 (67%) met the diagnostic criteria for MAC lung disease, and for each MAC species, the proportion of patients meeting these criteria was as follows: M. intracellulare (54/75, 72%), M. chimaera (3/8, 38%), “M. indicus pranii” (3/4, 75%), M. yongonense (2/4, 50%), and M. avium subsp. hominissuis (84/128, 66%). In summary, multilocus sequence typing of respiratory isolates initially identified as MAC revealed that, although most isolates were M. avium subsp. hominissuis or M. intracellulare, approximately 7% were newer MAC members, with clinical evidence supporting their potential pathogenicity for humans.

A fatal case of pulmonary infection by Mycobacterium colombiense in Para State, Amazon Region, Brazil

Mycobacterium avium complex (MAC) is a heterogeneous group of species found in several environmental sources and that exhibit variable degrees of pathogenicity. Among the MAC members, Mycobacterium colombiense has been related to pulmonary disease and disseminated infection in HIV-infected patients in Colombia. Lymphadenopathy cases have also been reported. We have described a fatal case of M. colombiense pulmonary disease in a Brazilian patient without evidence of HIV infection or other known causes of immunosuppression.

Identification of potential biomarkers to distinguish Mycobacterium colombiense from other mycobacterial species

Mycobacterium avium complex (MAC) consists of 9 species of slow-growing mycobacteria with differing degrees of pathogenicity, host preference and environmental distribution. Mycobacterium colombiense is a novel member of MAC that is responsible for disseminated infections in HIV-infected patients in Colombia and lymphadenopathy cases in Europe. At present, methods to easily differentiate novel members of MAC are lacking. In this study, we identified possible biomarkers that are potentially useful for the detection of M. colombiense by PCR or chromatography. The Randomly Amplified Polymorphic DNA (RAPD) technique was used to amplify genomic fragments of M. colombiense CECT 3035 that were subsequently used in the development of a direct colony-specific PCR assay using specific primers. The designed primers amplified a 634-bp fragment of DNA from M. colombiense, which included a 450-bp genomic region that encodes a hypothetical protein of 149 amino acids that is exclusive to M. colombiense. Bioinformatic analyses revealed that this hypothetical protein had no signal peptide, active sites or functional domains to aid its identification or classification. In addition, using thin-layer chromatography, we identified a different profile of mycolates for M. colombiense strains. The test developed in this study has potential applications in the routine identification of M. colombiense and in molecular assays designed for the surveillance of MAC strains.

Complete Genome Sequence of Mycobacterium intracellulare Strain ATCC 13950T

Here we report the first complete genome sequence of Mycobacterium intracellulare ATCC 13950(T), a Mycobacterium avium complex (MAC) strain. This genome sequence will serve as a valuable reference for understanding the epidemiologic, biological, and pathogenic aspects of the disparity between MAC members.

Inter- and Intra-subtype genotypic differences that differentiate Mycobacterium avium subspecies paratuberculosis strains

BackgroundMycobacterium avium subspecies paratuberculosis (Map) is the aetiological agent of Johne’s disease or paratuberculosis and is included within the Mycobacterium avium complex (MAC). Map strains are of two major types often referred to as ‘Sheep’ or ‘S-type’ and ‘Cattle’ or ‘C-type’. With the advent of more discriminatory typing techniques it has been possible to further classify the S-type strains into two groups referred to as Type I and Type III. This study was undertaken to genotype a large panel of S-type small ruminant isolates from different hosts and geographical origins and to compare them with a large panel of well documented C-type isolates to assess the genetic diversity of these strain types. Methods used included Mycobacterial Interspersed Repetitive Units - Variable-Number Tandem Repeat analysis (MIRU-VNTR), analysis of Large Sequence Polymorphisms by PCR (LSP analysis), Single Nucleotide Polymorphism (SNP) analysis of gyr genes, Pulsed-Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism analysis coupled with hybridization to IS900 (IS900-RFLP) analysis.ResultsThe presence of LSPA4 and absence of LSPA20 was confirmed in all 24 Map S-type strains analysed. SNPs within the gyr genes divided the S-type strains into types I and III. Twenty four PFGE multiplex profiles and eleven different IS900-RFLP profiles were identified among the S-type isolates, some of them not previously published. Both PFGE and IS900-RFLP segregated the S-type strains into types I and III and the results concurred with those of the gyr SNP analysis. Nine MIRU-VNTR genotypes were identified in these isolates. MIRU-VNTR analysis differentiated Map strains from other members of Mycobacterium avium Complex, and Map S-type from C-type but not type I from III. Pigmented Map isolates were found of type I or III.ConclusionThis is the largest panel of S-type strains investigated to date. The S-type strains could be further divided into two subtypes, I and III by some of the typing techniques (IS900-RFLP, PFGE and SNP analysis of the gyr genes). MIRU-VNTR did not divide the strains into the subtypes I and III but did detect genetic differences between isolates within each of the subtypes. Pigmentation is not exclusively associated with type I strains.

Polymerase chain reaction–restriction fragment length polymorphism of the rpoB gene for identification of Mycobacterium avium subsp. paratuberculosis and differentiation of Mycobacterium avium subspecies

Mycobacterial speciation by polymerase chain reaction (PCR)–restriction fragment length polymorphism analysis (PRA) of the rpoB gene was evaluated for identification of Mycobacterium avium subsp. paratuberculosis (MAP) and other Mycobacterium avium complex (MAC) members to the species or subspecies level by comparison with conventional methods including hsp65 sequencing, high-performance liquid chromatography, and PCR for accepted species- or subspecies-specific genomic targets. A total of 185 type and clinical mycobacterial strains from humans, animals, and environments were tested. A 360-bp PCR product was subsequently digested with MspI, HaeIII, and SmaI restriction enzymes. The PRA using SmaI restriction showed a unique digestion pattern for MAP distinguishing it from other MAC members and other Mycobacterium spp. Moreover, HaeIII and MspI restriction of the rpoB gene enabled MAC-species and -subspecies discrimination. The rpoB-PRA using SmaI or MspI and HaeIII restriction of the rpoB gene is a simple, convenient, and reliable confirmatory assay for simultaneous identification of MAP and other MAC members.

Mycobacteria in the environment of pig farms in the Czech Republic between 2003 and 2007

In the Czech Republic, most mycobacterial infections in pigs are caused by the <I>Mycobacterium avium</I> complex (<I>MAC</I>) and potentially pathogenic mycobacteria (PPM) derived from the environment. This study was undertaken after the isolation of PPM from various components of the environment of pig herds between 1996 and 2002 (Matlova et al., Veterinarni Medicina, 48, 2003, 343–357). Between 2003 and 2007, a total of 1114 environmentally-derived samples from 24 farms were examined. After staining according to Ziehl-Neelsen, acid-fast rods were found in 42 (3.8%) samples by direct microscopy, and PPM were isolated from 223 (20.0%) samples by culture. PPM occurred primarily in soil from the paddocks (53.8%), peat (53.2%), bedding (28.4%) and biofilm from the pipeline (21.0%). From <I>MAC,</I> <I>M. avium</I> subsp. <I>hominissuis</I> (<I>MAH</I>) genotype IS<I>901</I>– and IS<I>1245</I>+ was most frequent; <I>M. avium </I>subsp<I>. avium</I> (<I>MAA</I>) genotype IS<I>901</I>+ and IS<I>1245</I>+ and <I>M. intracellulare</I> genotype IS<I>901</I>– and IS<I>1245</I>– were isolated from one (0.4%) and three (1.3%) samples, respectively. The remaining isolates were identified as 19 other mycobacterial species: <I>M. gordonae</I> (<I>n</I> = 8), <I>M. triviale</I> (<I>n</I> = 6), <I>M. flavescens</I> (<I>n</I> = 3), <I>M. nonchromogenicum</I> (<I>n</I> = 3), <I>M. terrae</I> (<I>n</I> = 3), <I>M. xenopi</I> (<I>n</I> = 3), <I>M. fortuitum</I> (<I>n</I> = 2), <I>M. chelonae</I> (<I>n</I> = 2), <I>M. chitae</I> (<I>n</I> = 2), <I>M. abscessus</I> (<I>n</I> = 1), <I>M. gastri</I> (<I>n</I> = 1), <I>M. kumamotonense</I> (<I>n</I> = 1), <I>M. marinum</I> (<I>n</I> = 1), <I>M. parafortuitum</I> (<I>n</I> = 1), <I>M. peregrinum</I> (<I>n</I> = 1), <I>M. porcinum</I> (<I>n</I> = 1), <I>M. scrofulaceum</I> (<I>n</I> = 1), <I>M. smegmatis</I> (<I>n</I> = 1) and <I>M. simiae</I> (<I>n</I> = 1). The remaining 41 isolates of unidentified mycobacterial species did not contain the sequences IS<I>901</I> and/or IS<I>1245,</I> specific for medically important members of <I>MAC</I> (<I>MAA</I> and <I>MAH</I>); a further 44 isolates were not tested due to their contamination or loss of ability to grow <I>in vitro</I>. A farm where <I>MAH</I> was often detected in the lymph nodes of pigs and in the environment between 1996 and 2002 (Period I), was selected for further investigation between 2003 and 2007 (Period II). A comparison of the findings of mycobacteria on the investigated farm in Period I and in the following Period II showed a significant increase (<I>P</I> < 0.01) in the occurrence of mycobacteria other than <I>MAH</I>, especially in peat samples<I>.</I>

Surviving within the amoebal exocyst: the Mycobacterium avium complex paradigm

BackgroundMost of environmental mycobacteria have been previously demonstrated to resist free-living amoeba with subsequent increased virulence and resistance to antibiotics and biocides. The Mycobacterium avium complex (MAC) comprises of environmental organisms that inhabit a wide variety of ecological niches and exhibit a significant degree of genetic variability. We herein studied the intra-ameobal location of all members of the MAC as model organisms for environmental mycobacteria.ResultsType strains for M. avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium arosiense, Mycobacterium marseillense, Mycobacterium timonense and Mycobacterium bouchedurhonense were co-cultivated with the free-living amoeba Acanthamoeba polyphaga strain Linc-AP1. Microscopic analyses demonstrated the engulfment and replication of mycobacteria into vacuoles of A. polyphaga trophozoites. Mycobacteria were further entrapped within amoebal cysts, and survived encystment as demonstrated by subculturing. Electron microscopy observations show that, three days after entrapment into A. polyphaga cysts, all MAC members typically resided within the exocyst.ConclusionsCombined with published data, these observations indicate that mycobacteria are unique among amoeba-resistant bacteria, in residing within the exocyst.

EmergingMycobacteriaspp. in Cooling Towers

To the Editor: The importance of nontuberculous mycobacteria (NTM) in various clinical situations recently has increased. Members of the Mycobacterium avium complex (MAC) cause a high percentage of infections in persons with acquired immunodeficiency syndrome. Some species are considered emerging pathogens, particularly those of the M. chelonae–M. abscessus group. They occur not only in immunocompromised persons but also in persons without predisposing conditions. Although sources of infection are considered to originate from the human environment, until now cooling towers were not clearly demonstrated to be one of these contamination sources, despite being suspected (1). Natural streams, groundwater, brook waters, and swamps already were reported to contain different species of NTM. Constructed environments, such as hospital hot water systems, aerosols from showers, ice machines, swimming pools, dental unit water, endoscopes, and bronchoscopes are other sources (2). Other studies have shown members of MAC in drinking-water distribution systems (3). A 1999 study in South Africa reported the presence of mycobacteria in cooling towers but reported no details about species (4). In 2003, cooling towers were reported to be a potential source of slow-growing mycobacterial species (1). Some mycobacterial species recently were shown to survive within amebae (5). We aimed to study the population of ameba-associated mycobacterial species in 3 cooling towers using a co-coculture method. These cooling towers (E, H, and O), located in downtown Paris, France, were sampled in May 2006. Water was taken in a sample point of the cooling circuit located just before the entrance of the tower basins. Because these cooling towers are regularly treated by oxidizing biocide BCDMH (1-bromo-3-chloro-5,5-dimethylhydantoin) to prevent development of Legionella spp., no Legionella spp. or Legionella-like amebal pathogens were isolated. Two liters of water samples were filtrated through 0.22-µm pore-sized filters that were injected onto amebal microplates as previously described (6) and incubated at 32°C. We screened amebal microplates by examination under inverted microscope and by Ziehl-Nielsen, Gram, and Gimenez staining. Positive wells were subcultured on axenic medium and incubated at 32°C for 10 days. Acid-fast isolates were identified by using partial rpoB gene amplification and sequencing with Myco-F and Myco-R primers (7). These primers did not allow amplification of partial rpoB gene for the 6 M. phocaicum strains. We thus used Myco-F/Myco-Rbon (5′-AGCGGCTGCTGGGTGATCAT-3′) primer pair for M. phocaicum (8). We compared these sequences with the rpoB gene sequence of Mycobacteria type strains available in the GeneBank database by using BLASTn on the NCBI website (www.ncbi.nlm.nih.gov). We observed bacteria growing in amebae. Subculture on axenic media led to polymicrobial cultures and allowed isolation of 33 mycobacterial strains (Table). All these strains were submitted to molecular identification. The 33 isolates corresponded to 5 mycobacterial species: M. fortuitum, M. conceptionense, M. chelonae, M. chimaera, and M. phocaicum (Table). Some of these mycobacteria already had been shown to survive in free-living amebae and to be implicated in human diseases, such as M. chelonae (5). The same author demonstrated recently that 26 environmental mycobacteria survived in the trophozoites and cysts of Acanthamoeba polyphaga (5). The recently described species M. phocaicum was isolated only in samples from humans and was associated with chronic pneumonia (9). However, the natural source of this species is still unknown. M. fortuitum was described as an opportunistic Mycobacterium associated with disseminated infections in a leukemia patient or in furunculosis after footbath in nail salons. M. conceptionense belongs to the M. fortuitum group and was isolated in a posttraumatic osteitis inflammation (10). M. chimaera belongs to MAC and has been isolated only in patients with pulmonary disorders but not from immunocompromised patients. The authors reported that the isolate showed unusually high virulence. We isolated this species for the first time in an environmental sample. It could belong to the transitory flora of the H cooling tower because it was isolated only once in our samples. Cooling towers already had been investigated for slow-growing mycobacteria (1), and our study showed they also can be an environmental source of rapidly growing NTM pathogens. In our procedure for amebal co-cultures, incubation cannot last >10 days because amebae do not survive longer in Page’s amebal saline buffer. Moreover, agar plates were incubated for 10 days only, which explains why we could recover only rapidly growing mycobacteria. The cooling towers, already known as a source of dissemination of Legionella spp., may disseminate mycobacteria in aerosols and be a previously nonrecognized source of infection. Table Identification of isolated nontuberculous mycobacteria according to their rpoB gene sequence similarity and GenBank accession numbers

Further analysis of VNTR and MIRU in the genome of complex, and application to molecular epidemiology of isolates from South America

All members of Mycobacterium avium complex are serious pathogens for humans and animals. The aim of this study was to look for and analyze VNTR-MIRU loci in the genome of M. avium complex and their preliminary application to test these isolates. In the present study, we identified 22 novel VNTR-MIRU by using Tandem Repeat software: five with a structure similar to MIRU and 17 without MIRU structure; these latter were designated as VNTR. Most VNTR were located within predicted coding regions. Most MIRU were intercistronic with their extremities overlapping the termination and initiation codons of their flanking genes. Some of these VNTR-MIRU exhibited polymorphism among M. avium complex isolates due to insertion or deletion of whole repeats and/or of nucleotide sequence degeneration. We determined the variability of six VNTR-MIRU loci in 21 M. avium subsp. hominissuis and 26 M. avium subsp. paratuberculosis. The analysis identified 15 different alleles with the combination of six VNTR-MIRU in the 21 M. avium subsp. hominissuis with 16 different IS1245 RFLP and four different profiles with PCR-restriction analysis of hsp65 (PRA). However, neither the six VNTR-MIRU loci nor the PRA were able to distinguish M. avium subsp. paratuberculosis isolates with five different IS900 RFLP profiles. In conclusion, some of the VNTR-MIRU loci identified were useful to differentiate M. avium subsp. hominissuis but not M. avium subsp. paratuberculosis isolates here included. However, we observed polymorphism in VNTR-MIRU loci between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis genomes, which could be important in the understanding of the obvious differences in the pathogenic effects of these mycobacteria.

Mycobacterial infections in cattle and pigs caused by Mycobacterium aviumcomplex members and atypical mycobacteria in theCzech Republicduring 2000-2004

Bovine tuberculosis was last detected in cattle and pigs in the CzechRepublicin 1995. Since March, 31, 2004(Commission Decision No. 2004/320/EC) the Czech Republichas been included amongst states free from bovine tuberculosis within the European Union. The purpose of the present study was to evaluate results of intravital and post-mortem diagnosis of mycobacterial infections in slaughtered cattle and pigs from 2000 to 2004. When bovine tuberculosis in cattle was investigated, a tuberculin skin test with bovine tuberculin was performed every year and a skin test with avian tuberculin was simultaneously conducted in the animals with a positive response. A total of 2 419 889 animals were examined with a positive response being found in 123 (0.005%) of them. After slaughter, bovine tuberculosis was not detected in any of these animals by gross and/or laboratory examinations. With avian tuberculin, 40 349 animals were tested and positive responses were detected in 43 (0.1%) of them; the incidence was similar in all the years monitored. Tuberculous lesions were detected in 209 (0.01%) of 1 967 211 slaughtered cattle. Mycobacteria were present in 40 (21.3%) of 188 animals examined by laboratory methods: 26 isolates of Mycobacterium avium subsp. avium (18 isolates of serotype 2 and 8 isolates not typeable), 11 isolates  of M. a. hominissuis (1 isolate of serotype 8 and 10 isolates not typeable), and 3 isolates of atypical mycobacteria. Tuberculous lesions were detected in 49 312 (0.22%) of 22 312 580 slaughtered pigs by veterinary-meat inspection. During the 5-year-period monitored, the incidence of tuberculous lesions decreased from 0.37% in 2000 to 0.10% in 2004. The following mycobacteria were isolated from 757 (33.5%) of 2 261 animals whose organs were examined by culture: 203 isolates of M. a. avium (180 isolates of serotype 2, 3 isolates of serotype 2/8, and 20 isolates not typeable), 442 isolates of M. a. hominissuis (1 isolate of serotype 1, 262 isolates of serotype 8, 35 isolates of serotype 9, 1 isolates of mixed serotypes 8/9, and 143 isolates not typeable), and atypical mycobacteria (n = 112). In both animal species, M. avium complex members prevailed: M. a. avium was the most prevalent subspecies in cattle, M. a. hominissuis in pigs. The main sources of M. a. avium and M. a. hominissuis are free living birds and contaminated external and stable environments (i.e. drinking water, feeds, and feed supplements), respectively. During the entire period monitored, miliary or generalized tuberculosis was not detected in any of the animals. The decreased incidence of tuberculous lesions in pigs was particularly a result of preventive measures adopted to control the occurrence of atypical mycobacteria.

Mycobacteria isolated from the environment of pig farms in the Czech Republic during the years 1996 to 2002

Sources of mycobacterial infections in 50 pig herds in the Czech Republicwere investigated during the years 1996 to 2002. A total of 2 412 samples from the external environment (feeds, bedding materials, drinking water, biofilms on drinkers, scrapings from the walls, floors and pen barriers, dust, spider webs, peat, kaolin, faeces, organs of rodents, and birds, etc.) were examined. After staining by the Ziehl-Neelsen technique, acid-fast rods were detected in 95 (3.9%) samples by direct microscopic examination and mycobacteria were cultured from 575 (23.8%) samples. From Mycobacterium avium complex (MAC), M. avium subsp. hominissuis (genotype IS901-, IS1245+) of serotypes 4, 6, 8, and 9 (272; 47.0% isolates), M. a. avium (genotype IS901+, IS1245+) of serotype 2 (13; 2.2% isolates) and M. intracellulare (genotype IS901-, IS1245-) of different serotypes (2; 0.3% isolates) were detected most frequently. Other isolates from among 14 other mycobacterial species ranked as follows: 64 M. gordonae, 47 M. fortuitum, 17 M. chelonae, 14 M. flavescens, 11 M. terrae, seven M. phlei, seven M. scrofulaceum, three M. diernhoferi, three M triviale, three M. smegmatis, two M. xenopi, one M. szulgai, one M. gastri, and one M. ulcerans. The remaining 111 isolates of unidentified species did not contain specific sequences IS901 and IS1245 characteristic for the pathogenic members of MAC (M. a. avium and M. a. hominis­suis). Peat, drinking water, biofilms on drinkers, bedding materials, feeds, free living birds, kaolin and charcoal were identified as potential sources of mycobacterial infections for pigs. Peat given to piglets as a feed supplement was the most important source of mycobacteria (65.1% positive of 327 examined samples); 81.2% of them were positive for M. a. hominissuis of serotypes 4, 6, 8, and 9. By contrast, mycobacteria of other species (M. gordonae, M. fortuitum, M. chelonae, M. flavescens, etc.) were the main isolates obtained from drinking water and biofilms on drinkers for pigs. By culture examination, the detection rate was higher in the biofilm samples (36.4%) than in the samples of drinking water (29.6%). The third group of sites with detected high levels (26.4%) of mycobacterial contamination were various types of beddings of woody material. M. a. hominissuis of serotypes 6, 8, and 9 were the most frequent isolates from sawdust; M. a. avium serotype 2 was sporadically detected. Mycobacterial findings in other samples from the external environment (wall and floor scrapings, dust, soil from the runs, and invertebrates) gave an indication of the pressure of infection in the herds. High contamination levels in faecal samples (15.6%) and in scrapings (18.4%) from respective parts of pens and stables indicated exposure of pigs to mycobacteria. In those materials, isolation of M. a. hominissuis of serotypes 4, 6, 8, and 9 prevailed. Mycobacteria were also detected in 7.9% of 430 samples of various invertebrate species. Various mycobacterial species were identified in the larvae and puparia of Eristalis tenax and Musca spp. and in imagoes of Drosophila spp., Musca spp., family Scatophagidae, Stomoxys calcitrans, E. tenax, and in earthworms. All of the constituents of the external environment that are potential sources of mycobacterial infections should be considered during implementation of preventative measures and the control of mycobacterial infections in pig herds.