Mycobacterium avium is the most commonly encountered mycobacterium species among non-Mycobacterium tuberculosis complex (nontuberculous mycobacteria) isolates worldwide and frequently causes lymphadenitis in children. During a multi-centre study in The Netherlands that was performed to determine the optimal treatment for mycobacterial lymphadenitis, concern was expressed in the media about the possible role of birds as sources of these M. avium infections, referred to as ‘bird tuberculosis.’ To examine the involvement of birds in mycobacterial lymphadenitis, 34 M. avium isolates from lymphadenitis cases were subjected to IS1245 restriction fragment length polymorphism (RFLP) typing. This genotyping method enables the distinction of the subspecies M. avium subsp. hominissuis and the ‘bird-type’ M. avium spp. avium. Highly variable RFLP patterns were found among the lymphadenitis M. avium isolates, and all belonged to the M. avium hominissuis subspecies. A relation to pet birds in the etiology of mycobacterial lymphadenitis could not be established, and the source of the infections may be environmental.
Members of the Mycobacterium avium complex (MAC) are ubiquitous bacteria that can be found in water, food, and other environmental samples and are considered opportunistic pathogens for numerous animal species, mainly birds and pigs, as well as for humans. We have recently demonstrated the usefulness of a PCR-based mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing for the molecular characterization of M. avium subsp. paratuberculosis and M. avium strains exclusively isolated from AIDS patients. In the present study we extended our analysis, based on eight MIRU-VNTR markers, to a strain collection comprehensively comprising the other M. avium subspecies, including M. avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. silvaticum, isolated from numerous animal species, HIV-positive and HIV-negative humans, and environmental sources. All strains were fully typeable, with the discriminatory index being 0.885, which is almost equal to that obtained by IS1311 restriction fragment length polymorphism (RFLP) typing as a reference. In contrast to IS1311 RFLP typing, MIRU-VNTR typing was able to further discriminate M. avium subsp. avium strains. MIRU-VNTR alleles strongly associated with or specific for M. avium subspecies were detected in several markers. Moreover, the MIRU-VNTR typing-based results were consistent with a scenario of the independent evolution of M. avium subsp. avium/M. avium subsp. silvaticum and M. avium subsp. paratuberculosis from M. avium subsp. hominissuis, previously proposed on the basis of multilocus sequence analysis. MIRU-VNTR typing therefore appears to be a convenient typing method capable of distinguishing the three main subspecies and strains of the complex and providing new epidemiological knowledge on MAC.
Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply.
Mycobacterium avium includes the subspecies avium, silvaticum, paratuberculosis and hominissuis, and M. avium subspecies has been isolated from various environments all over the world including from biofilms in water distribution systems. The aim of this study was to examine isolates of M. avium subsp. avium and M. avium subsp. hominissuis of different origin for biofilm formation and to look for correlations between biofilm formation and RFLP-types, and to standardise the method to test for biofilm formation. In order to determine the best screening method, a panel of 14 isolates of M. avium subsp. avium and M. avium subsp. hominissuis, were tested for their ability to form biofilm in microtiter plates under different conditions. Subsequently, 83 additional isolates from humans, swine and birds were tested for biofilm formation. The isolates were tested for the presence of selected genes involved in the synthesis of glycopeptidolipids (GPLs) in the cell wall of M. avium, which is believed to be important for biofilm formation. Colony morphology and hsp65 sequvar were also determined.
Nine isolates from swine produced biofilm. There was a significant higher frequency of porcine isolates forming biofilm compared to human isolates. All isolates were previously characterised by IS1311- and IS1245-RFLP typing. The ability to form biofilm did not correlate with the RFLP-type, hsp65 sequevar, colony morphology or the presence of gene sequences related to GPL synthesis.
The observed differences in biofilm forming abilities between porcine and human isolates raises questions regarding the importance of biofilm formation for infectious potential. The optimised method worked well for screening of multiple isolates.
“Mycobacterium avium subsp. hominissuis” often causes cervical lymphadenitis in children; its prompt and accurate identification enables adequate therapy, tracing, and prevention. The aims of this study were to determine the causative agent of lymphadenitis using culture, PCR, and triplex quantitative real-time PCR (qPCR) methods with DNA directly isolated from tissue, as well as to identify possible sources of infection from the environment. We confirmed the diagnoses by detecting M. avium subsp. hominissuis using qPCR with DNA directly isolated from lymph node biopsy specimens of two patients. In order to trace the source of infection from the environment, a method of DNA isolation from soil and other environmental samples, such as dust, cobwebs, and compost, was developed. The triplex qPCR examination revealed the presence of M. avium subsp. hominissuis in a high proportion of the environmental samples (42.8% in the first patient's house and 47.6% in the second patient's house). Both patients were also exposed to M. avium subsp. avium, which was present due to the breeding of infected domestic hens. The high infectious dose of M. avium subsp. hominissuis or the increased susceptibility of humans to M. avium subsp. hominissuis compared to M. avium subsp. avium could be the reason why the children were infected with M. avium subsp. hominissuis.
Nontuberculous mycobacteria (NTM) are ubiquitous and have been isolated from a variety of environmental sources, including water. Various NTM were isolated from biofilms in drinking water distribution systems in two urban and two semiurban areas in South Africa. Most of the isolates belonged to opportunistic pathogenic species of the NTM group, but none belonged to the Mycobacterium avium complex.
Background: Non-tuberculous mycobacteria (NTM) are ubiquitous environmental organisms. Patients with pre-existing lung damage are susceptible to NTM, but their prevalence in bronchiectasis is unknown. Distinguishing between lung colonisation and disease can be difficult.
Methods: A prospective study of 100 patients with bronchiectasis was undertaken to evaluate the prevalence of NTM in sputum, and a retrospective analysis of clinical, microbiological, lung function and radiology data of our clinic patients with NTM sputum isolates over 11 years was performed.
Results: The prevalence of NTM in this population of patients with bronchiectasis was 2%. Patients in the retrospective study were divided into three groups: bronchiectasis + multiple NTM isolates (n = 25), bronchiectasis + single isolates (n = 23), and non-bronchiectasis + multiple isolates (n = 22). Mycobacterium avium complex (MAC) species predominated in patients with bronchiectasis compared with non-bronchiectasis lung disease (72% v 9%, p<0.0001). Single isolates were also frequently MAC (45.5%). Multiple isolates in bronchiectasis were more often smear positive on first sample than single isolates (p<0.0001). NTM were identified on routine screening samples or because of suggestive radiology. No particular bronchiectasis aetiology was associated with an NTM. Pseudomonas aeruginosa and Staphylococcus aureus were frequently co-cultured. Six (25%) of multiple NTM patients had cavities of which five were due to MAC. Half the patients with multiple isolates were treated, mostly due to progressive radiology.
Conclusions: NTM are uncommon in non-cystic fibrosis bronchiectasis. Routine screening identifies otherwise unsuspected patients. MAC is the most frequent NTM isolated.
Mycobacterium avium subsp. avium (Maa) and Mycobacterium avium subsp. hominissuis (Mah) are opportunistic pathogens that may infect several species, including humans and pigs. Mah is however more frequently isolated from pigs than Maa, and it is unclear if this is due to difference in virulence or in exposure to the two organisms. Clinical isolates of each subspecies were administered perorally to ten domestic pigs, respectively. The animals were sacrificed at six and 12 weeks after inoculation. At necropsy, macroscopic lesions were recorded, and tissue samples were collected for mycobacterial culture, IS1245 real time PCR and histopathological examination. Culturing was also performed on faecal samples collected at necropsy.
Macroscopic and histopathological lesions were detected in pigs infected with each subspecies, and bacterial growth and histopathological changes were demonstrated, also in samples from organs without gross pathological lesions. Six weeks after inoculation, live Mah was detected in faeces, as opposed to Maa. The presence of live mycobacteria was also more pronounced in Mah infected tonsils. In comparison, the Maa isolate appeared to have a higher ability of intracellular replication in porcine macrophages compared to the Mah isolate.
The study shows that both subspecies were able to infect pigs. Additionally, the more extensive shedding of Mah might cause pig-to-pig transmission and contribute to the higher incidence of infection caused by this subspecies.
Mycobacterium avium; Experimentally infected pigs; Transmission; Source of infection
Mycobacterium avium subsp hominissuis, hereafter referred to as M. avium, forms biofilm, a property that, in mice, is associated with lung infection via aerosol. As M. avium might co-inhabit the respiratory tract with other pathogens, treatment of the co-pathogen-associated infections, such as in bronchiectasis, would expose M. avium to therapeutic compounds which may have their origin in other organisms sharing the natural environments. Incubation of M. avium with two compounds produced by environmental organisms, streptomycin and tetracycline in vitro at sub-inhibitory concentrations increased biofilm formation in a number of M. avium strains, although exposure to ampicillin, moxifloxacin, rifampin, and TMP/SMX had no effect on biofilm. No selection of genotypically resistant clones was observed. While bacteria incubation in presence of streptomycin upregulates the expression of biofilm-associated genes, the response to the antibiotics had no association with a regulation of a regulator (LysR) linked to the formation of biofilm in M. avium.
Biofilms are made of planktonic and sessile bacteria. While planktonic M. avium is susceptible to clarithromycin and ethambutol (clinically used antimicrobials), sessile bacteria are at least 3- to 4-fold more resistant to antibiotics. The sessile phenotype, though, is reversible, and no selection of resistant clones was observed. Mice infected through the airway with both phenotypes were infected with a similar number of bacteria, demonstrating no phenotype advantage.
M. avium biofilm formation is enhanced by commonly used compounds and, in the sessile bacterial phenotype, is resistant to clarithromycin and ethambutol, in a reversible manner.
M. avium; biofilm; gene expression; antibiotics
We used HPLC and AdvanSure real-time PCR (LG Life Sciences, Korea) to retrospectively analyze non-tuberculous mycobacteria (NTM) in 133 clinical specimens. The specimens were culture-positive for NTM and the HPLC method identified 130 strains of mycobacteria from the cultures (97.7%) at the species level. Among the isolates, 48 Mycobacterium. kansasii (36.1%), 39 M. intracellulare (29.3%), 17 M. avium (12.8%), 16 M. abscessus (12.0%), 6 M. fortuitum (4.5%), 2 M. szulgai (1.5%), 2 M. gordonae (1.5%), and 3 unclassified NTM strains (2.3%) were identified. The real-time PCR assay identified 60 NTM-positive specimens (45.1%), 65 negative specimens (48.9%), and 8 M. tuberculosis (TB)-positive specimens (6.0%). The real-time PCR assay is advantageous because of its rapid identification of NTM. However, in our study, the real-time PCR assay showed relatively low sensitivity (45.1%) when using direct specimens including sputum and bronchoalveolar lavage (BAL) fluid. HPLC is useful as it discriminates NTM at the species level, although it is time-consuming and requires specific equipment and technical expertise. A combination of both methods will be helpful for the rapid and accurate identification of mycobacteria in clinical laboratories.
Non-tuberculous mycobacteria; Real-time PCR; HPLC
Mycobacterium avium is the principal etiologic agent of non-tuberculous lymphadenitis in children. It is also a known pathogen for birds and other animals. Genetic typing of M. avium isolates has led to a proposal to expand the set of subspecies to include M. avium subsp. hominissuis. Isolates associated with disease in humans belong to this subspecies.
Peripheral blood mononuclear cells from six healthy blood donors were stimulated in vitro with ten isolates of M. avium avium and 11 isolates of M. avium hominissuis followed by multiplex bead array quantification of cytokines in supernatants. M. avium hominissuis isolates induced significantly more IL-10 and significantly less IL-12p70, TNF, IFN-γ and IL-17 when compared to M. avium avium isolates. All strains induced high levels of IL-17, but had very low levels of IL-12p70.
The strong association between M. avium subsp. hominissuis and disease in humans and the clear differences in the human immune response to M. avium subsp. hominissuis compared to M. avium subsp. avium isolates, as demonstrated in this study, suggest that genetic differences between M. avium isolates play an important role in the pathogenicity in humans.
The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species.
The Mycobacterium avium complex (MAC) comprises genomically similar but phenotypically divergent bacteria that inhabit diverse environments and that cause disease in different hosts. In this study, a whole-genome approach was used to examine the polymorphic PE (Pro-Glu) and PPE (Pro-Pro-Glu) gene families, implicated in immunostimulation and virulence. The four major groups of MAC organisms were examined, including the newly sequenced type strains of M. intracellulare and M. avium subsp. avium, plus M. avium subsp. paratuberculosis and M. avium subsp. hominissuis, for the purpose of finding genetic differences that could be exploited to design diagnostic tests specific to these groups and that could help explain their divergence in pathogenesis and host specificity. Unique and missing PPE genes were found in all MAC members except M. avium subsp. avium. Only M. intracellulare had a unique PE gene. Apart from this, most PE and PPE sequences were conserved, with average nucleotide sequence identities of 99.1 and 98.1%, respectively, among the M. avium subspecies, but only 82.9 and 79.7% identities with the PE and PPE sequences of M. intracellulare, respectively. A detailed analysis of the amino acid sequences was performed between M. avium subsp. paratuberculosis and M. avium subsp. hominissuis. Most differences were detected in the PPE proteins, with amino acid substitutions and frame shifts leading to unique amino acid sequences. In conclusion, several unique PPE proteins were identified in MAC organisms next to numerous polymorphisms in both the PE and PPE gene families. These substantial differences could help explain the divergence in phenotypes within the MAC and could lead to diagnostic tests with better discriminatory abilities.
The importance of non-tuberculous mycobacteria (NTM) infections in humans and animals in sub-Saharan Africa at the human-environment-livestock-wildlife interface has recently received increased attention. NTM are environmental opportunistic pathogens of humans and animals. Recent studies in pastoral ecosystems of Uganda detected NTM in humans with cervical lymphadenitis and cattle with lesions compatible with bovine tuberculosis. However, little is known about the source of these mycobacteria in Uganda. The aim of this study was to isolate and identify NTM in the environment of pastoral communities in Uganda, as well as assess the potential risk factors and the public health significance of NTM in these ecosystems.
A total of 310 samples (soil, water and faecal from cattle and pigs) were examined for mycobacteria. Isolates were identified by the INNO-Lipa test and by 16S rDNA sequencing. Additionally, a questionnaire survey involving 231 pastoralists was conducted during sample collection. Data were analysed using descriptive statistics followed by a multivariable logistic regression analysis.
Forty-eight isolates of NTM were detected; 25.3% of soil samples, 11.8% of water and 9.1% from animal faecal samples contained mycobacteria. Soils around water sources were the most contaminated with NTM (29.8%). Of these samples, M. fortuitum-peregrinum complex, M. avium complex, M. gordonae, and M. nonchromogenicum were the most frequently detected mycobacteria. Drinking untreated compared to treated water (OR = 33), use of valley dam versus stream water for drinking and other domestic use (OR = 20), sharing of water sources with wild primates compared to antelopes (OR = 4.6), sharing of water sources with domestic animals (OR = 5.3), and close contact with cattle or other domestic animals (OR = 13.8) were the most plausible risk factors for humans to come in contact with NTM in the environment.
The study detected a wide range of potentially pathogenic NTM from the environment around the pastoral communities in Uganda. Drinking untreated water and living in close contact with cattle or other domestic animals may be risk factors associated with the possibility of humans and animals acquiring NTM infections from these ecosystems.
Tuberculosis (TB) remains a major international health problem. Rapid differentiation of Mycobacterium tuberculosis complex (MTB) from non-tuberculous mycobacteria (NTM) is critical for decisions regarding patient management and choice of therapeutic regimen. Recently we developed a 20-compound model to distinguish between MTB and NTM. It is based on thermally assisted hydrolysis and methylation gas chromatography-mass spectrometry and partial least square discriminant analysis. Here we report the validation of this model with two independent sample sets, one consisting of 39 MTB and 17 NTM isolates from the Netherlands, the other comprising 103 isolates (91 MTB and 12 NTM) from Stellenbosch, Cape Town, South Africa. All the MTB strains in the 56 Dutch samples were correctly identified and the model had a sensitivity of 100% and a specificity of 94%. For the South African samples the model had a sensitivity of 88% and specificity of 100%. Based on our model, we have developed a new decision-tree that allows the differentiation of MTB from NTM with 100% accuracy. Encouraged by these findings we will proceed with the development of a simple, rapid, affordable, high-throughput test to identify MTB directly in sputum.
To characterize the distribution of nontuberculous mycobacteria (NTM) species isolated from pulmonary samples from persons in Asia and their association with pulmonary infections, we reviewed the literature. Mycobacterium avium complex bacteria were most frequently isolated (13%–81%) and were the most common cause of pulmonary NTM disease (43%–81%). Also pathogenic were rapidly growing mycobacteria (M. chelonae, M. fortuitum, M. abscessus). Among all NTM isolated from pulmonary samples, 31% (582/1,744) were considered clinically relevant according to American Thoracic Society diagnostic criteria. Most patients were male (79%) and had a history of tuberculosis (37%). In Asia, high prevalence of rapidly growing mycobacteria and a history of tuberculosis are distinct characteristics of pulmonary NTM disease. This geographic variation is not well reflected in the American Thoracic Society criteria for NTM infections and could be incorporated in future guidelines.
Atypical mycobacteria; Mycobacterium; infections; Asia; respiratory tract infections; review; tuberculosis and other mycobacteria; bacteria; synopsis
Mycobacterium avium subsp hominissuis (previously Mycobacterium avium subsp avium) is an environmental organism associated with opportunistic infections in humans. Mycobacterium hominissuis infects and replicates within mononuclear phagocytes. Previous study characterized an attenuated mutant in which the PPE gene (MAV_2928) homologous to Rv1787 was inactivated. This mutant, in contrast to the wild-type bacterium, was shown both to have impaired the ability to replicate within macrophages and to have prevented phagosome/lysosome fusion.
MAV_2928 gene is primarily upregulated upon phagocytosis. The transcriptional profile of macrophages infected with the wild-type bacterium and the mutant were examined using DNA microarray, which showed that the two bacteria interact uniquely with mononuclear phagocytes. Based on the results, it was hypothesized that the phagosome environment and vacuole membrane of the wild-type bacterium might differ from the mutant. Wild-type bacterium phagosomes expressed a number of proteins different from those infected with the mutant. Proteins on the phagosomes were confirmed by fluorescence microscopy and Western blot. The environment in the phagosome of macrophages infected with the mutant differed from the environment of vacuoles with M. hominissuis wild-type in the concentration of zinc, manganese, calcium and potassium.
The results suggest that the MAV_2928 gene/operon might participate in the establishment of bacterial intracellular environment in macrophages.
Non-tuberculous mycobacteria (NTM) are increasingly important as opportunistic infections after major and minor surgical procedures, likely because they are ubiquitous and not effectively killed by many commonly used disinfectants. Outbreaks of soft tissue infections with NTM appeared related to the use of commercial disinfectants based on quaternary ammonium compounds (QACs).
We studied the survival of clinical and environmental isolates of Mycobacterium abscessus, Mycobacterium massiliense, Mycobacterium chelonae and Mycobacterium fortuitum after 20 min, 60 min or 24 h exposures to different QACs, and the surviving bacteria were then re-exposed to QACs to see if the percentage of surviving bacteria had increased. The bacteria were labelled with a dnaA–gfp fusion and their level of QAC resistance monitored as increasing fluorescence. The QAC-resistant bacteria were then serially restreaked onto non-selective medium and retested for QAC survival.
The frequency of survivors was <1 in 105 bacteria with Mycobacterium smegmatis, but >1 in 100 with the other mycobacteria studied. Different environmental and clinical isolates had similar QAC MICs, but QAC survivors of each strain were resistant. The QAC-surviving strains reverted to the original, non-resistant phenotype after several passages on non-selective medium.
QACs should not be used in settings where even minimally invasive procedures are performed, as they select for a non-genetically determined reversible resistant phenotype that appears at high frequency with several rapidly growing mycobacterial species associated with healthcare-related infections. M. smegmatis behaves differently and is not an adequate model for testing the activity of disinfectants against NTM.
quaternary ammonium compounds; disinfectants; NTM; resistance; tolerance; persistence
The multiplication of opportunistic pathogens in drinking water supplies might pose a threat to public health. In this study, distributed unchlorinated drinking water from eight treatment plants in the Netherlands was sampled and analyzed for fungi, nontuberculous mycobacteria (NTM), and several opportunistic pathogens by using selective quantitative PCR methods. Fungi and NTM were detected in all drinking water samples, whereas Legionella pneumophila, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Aspergillus fumigatus were sporadically observed. Mycobacterium avium complex and Acanthamoeba spp. were not detected. Season had no influence on the occurrence of these organisms, except for NTM and S. maltophilia, which were present in higher numbers in the summer. Opportunistic pathogens were more often observed in premise plumbing water samples than in samples from the distribution system. The lowest number of these organisms was observed in the finished water at the plant. Thus, fungi, NTM, and some of the studied opportunistic pathogens can multiply in the distribution and premise plumbing systems. Assimilable organic carbon (AOC) and/or total organic carbon (TOC) had no clear effects on fungal and NTM numbers or on P. aeruginosa- and S. maltophilia-positive samples. However, L. pneumophila was detected more often in water with AOC concentrations above 10 μg C liter−1 than in water with AOC levels below 5 μg C liter−1. Finally, samples that contained L. pneumophila, P. aeruginosa, or S. maltophilia were more frequently positive for a second opportunistic pathogen, which shows that certain drinking water types and/or sampling locations promote the growth of multiple opportunistic pathogens.
Mycobacterial opportunistic infections are a major cause of morbidity and mortality among patients living with HIV (PLHIV) worldwide. Nontuberculous mycobacterial (NTM) infection is one of the leading causes of opportunistic infection in patients with advanced acquired immunodeficiency syndrome i.e., with CD4 count less than 50/cu.mm. Mycobacterium avium complex (MAC) is among the most common opportunistic bacterial infections in those patients with advanced immunodeficiency apart from cryptococcal meningitis, progressive multifocal leukoencephalopathy, etc. Common presentations of mycobacterium avium complex are fever, lymphadenitis and respiratory disease. Immune reconstitution disease is also known to manifest with MAC infections in PLHIV on highly active antiretroviral therapy. Very few cases of central nervous system involvement due to NTM infection have been described. We are reporting a case of advanced acquired immunodeficiency who presented with brain abscess due to Mycobacterium avium intracellulare.
Brain abscess; mycobacterium avium intracellulare; non tuberculous mycobacterium
Mycobacterium avium comprises genetically related yet phenotypically distinct subspecies. Consistent with their common origin, whole-genome sequence comparisons have revealed extensive synteny among M. avium organisms. However, the sequenced strains also display numerous regions of heterogeneity that likely contribute to the diversity of the individual subspecies. Starting from a phylogenetic framework derived by multilocus sequence analysis, we examined the distribution of 25 large sequence polymorphisms across a panel of genetically defined M. avium strains. This distribution was most variable among M. avium subsp. hominissuis isolates. In contrast, M. avium subsp. paratuberculosis strains exhibited a characteristic profile, with all isolates containing a set of genomic insertions absent from other M. avium strains. The emergence of the pathogen from its putative M. avium subsp. hominissuis ancestor entailed the acquisition of approximately 125 kb of novel genetic material, followed by a second phase, characterized by reductive genomics. One genomic deletion is common to all isolates while additional deletions distinguish two major lineages of M. avium subsp. paratuberculosis. For the average strain, these losses total at least 38 kb (sheep lineage) to 90 kb (cattle lineage). This biphasic pattern of evolution, characterized by chromosomal gene acquisition with subsequent gene loss, describes the emergence of M. avium subsp. paratuberculosis and may serve as a general model for the origin of pathogenic mycobacteria.
To evaluate the frequency and clinical significance of non-tuberculous mycobacteria (NTM) isolates among children investigated for pulmonary tuberculosis in a rural South African community.
Children were investigated for pulmonary tuberculosis as part of a tuberculosis vaccine surveillance program (2001–2005). The clinical features of children in whom NTM were isolated, from induced sputum or gastric lavage, were compared to those with culture-proven M. tuberculosis.
Mycobacterial culture demonstrated 114 NTM isolates from 109 of the 1,732 children investigated, a crude yield of 6% (95% CI 5–7). The comparative yield of positive NTM cultures from gastric lavage was 40% (95% CI 31–50), compared to 67% (95% CI 58–76) from induced sputum. 95% of children with NTM isolates were symptomatic. Two children were HIV-infected. By contrast, M. tuberculosis was isolated in 187 children, a crude yield of 11% (95% CI 9–12). Compared to those with culture-proven M. tuberculosis, children with NTM isolates were less likely to demonstrate acid-fast bacilli on direct smear microscopy (OR 0.19; 95% 0.0–0.76). Children with NTM were older (p<0.0001), and more likely to demonstrate constitutional symptoms (p = 0.001), including fever (p = 0.003) and loss of weight or failure to gain weight (p = 0.04), but less likely to demonstrate a strongly positive tuberculin skin test (p<0.0001) or radiological features consistent with pulmonary tuberculosis (p = 0.04).
NTM were isolated in 6% of all children investigated for pulmonary tuberculosis and in more than one third of those with a positive mycobacterial culture. NTM may complicate the diagnosis of PTB in regions that lack capacity for mycobacterial species identification. The association of NTM isolates with constitutional symptoms suggestive of host recognition requires further investigation.
Mycobacterium avium comprises organisms that share the same species designation despite considerable genomic and phenotypic variability. To determine the degree and nature of variability between subspecies and strains of M. avium, we used multilocus sequencing analysis, studying 56 genetically diverse strains of M. avium that included all described subspecies. In total, 8,064 bp of sequence from 10 gene loci were studied, with 205 (2.5%) representing variable positions. The majority (149/205) of these variations were found among M. avium subsp. hominissuis organisms. Recombination was also evident in this subspecies. In contrast, there was comparatively little variability and no evidence of recombination within the pathogenic subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. silvaticum. Phylogenetic analysis showed that M. avium subsp. avium and M. avium subsp. silvaticum strains clustered together on one branch, while a distinct branch defined M. avium subsp. paratuberculosis organisms. Despite the independent origin of these pathogenic subspecies, an analysis of their rates of nonsynonymous (dN) to synonymous (dS) substitutions showed increased dN/dS ratios for both: 0.67 for M. avium subsp. paratuberculosis and 0.50 for M. avium subsp. avium/M. avium subsp. silvaticum, while the value was 0.08 for M. avium subsp. hominissuis organisms. In conclusion, M. avium subsp. hominissuis represents a diverse group of organisms from which two pathogenic clones (M. avium subsp. paratuberculosis and M. avium subsp. avium/M. avium subsp. silvaticum) have evolved independently.
Chronic diarrhea is one of the most frequent gastro-intestinal manifestations in acquired immunodeficiency syndrome (AIDS). Protozoa and nontuberculous mycobacteria (NTM) are opportunistic pathogens that can easily infect these patients. Among the NTM, Mycobacterium avium complex (MAC) is the most frequently observed pathogen in HIV-infected patients. However, NTMs other than MAC have not been reported as a gastrointestinal pathogen as yet. We present a case of chronic diarrhea in an AIDS patient in whom Mycobacterium ulcerans and cryptosporidium co-infection is evidenced from colonic tissue.
Mycobacterium ulcerans; Cryptosporidium; HIV; Chronic diarrhea
Smear microscopy, a mainstay of tuberculosis (TB) diagnosis in developing countries, cannot differentiate M. tuberculosis complex from NTM infection, while pulmonary TB shares clinical signs with NTM disease, causing clinical and diagnostic dilemmas. This study used molecular assays to identify species and assess genotypic diversity of non-tuberculous mycobacteria (NTM) isolates from children investigated for pulmonary tuberculosis at a demographic surveillance site in rural eastern Uganda.
Children were investigated for pulmonary tuberculosis as part of a TB vaccine surveillance program (2009–2011). Two cohorts of 2500 BCG vaccinated infants and 7000 adolescents (12–18 years) were recruited and followed up for one to two years to determine incidence of tuberculosis. Induced sputum and gastric aspirates were processed by the standard N-acetyl L-cysteine (NALC)-NaOH method. Sediments were cultured in the automated MGIT (Becton Dickson) liquid culture system and incubated at 37°C for at least six weeks. Capilia TB assay was used to classify mycobacteria into MTC and NTM. The GenoType CM/AS assays were performed to identify species while Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR genotyping was used to assess genetic diversity of the strains within each species.
Among 2859 infants and 2988 adolescents screened, the numbers of TB suspects were 710 and 1490 infants and adolescents respectively. The prevalence of NTM in infant suspects was 3.7% (26/710) (95% CI 2.5–5.2) while that in adolescent suspects was 4.6% (69/1490) (95% CI 3.6–5.8). On culture, 127 isolates were obtained, 103 of which were confirmed as mycobacteria comprising of 95 NTM and eight M. tuberculosis complex. The Genotype CM/AS assay identified 63 of the 95 NTM isolates while 32 remained un-identified. The identified NTM species were M. fortuitum (40 isolates, 63.5%), M. szulgai (9 isolates, 14.3%), M. gordonae (6 isolates, 9.5%), M. intracellulare (3 isolates, 4.7%), M. scrofulaceum (2 isolates, 3.2%), M. lentiflavum (2 isolates, 3.2%), and M. peregrinum (1 isolate, 1.6%). Genotyping did not reveal any clustering in M. intracellulare, M. gordonae and M. szulgai species. M. fortuitum, on the other hand, had two clusters, one with three isolates of M. fortuitum 1 and the other with two isolates of M. fortuitum 2 subspecies. The remaining 35 of the 40 isolates of M. fortuitum had unique fingerprint patterns.
M. fortuitum is the most common cause of infection by NTM among Infants and adolescents in rural Uganda. There is a varied number of species and genotypes, with minimal clustering within species, suggesting ubiquitous sources of infection to individuals in this community.