Nontuberculous mycobacteria (NTM) include all mycobacteria species that do not belong to Mycobacterium tuberculosis (Mtb). They are a heterogeneous group of bacteria that are ubiquitously distributed in diverse environments such as water, sewage, soil, air, dust, food, animals and humans [1, 2]. To date, more than 150 species of NTM have so far been identified, but most of them are still poorly characterized and their clinical relevance is poorly understood.

In recent years, a number of species of NTM have been found to be associated with a wide range of human infections such as pulmonary, cutaneous and disseminated diseases [3, 4] They also pose a threat to people with various underlying medical conditions such as cystic fibrosis, bronchiectasis, prior tuberculosis and immune suppression [3, 4]. The emergence of the HIV/AIDS epidemic in the past few decades has resulted in the rise of NTM infections in the human population [4]. Together with Mycobacterium tuberculosis, the NTM species are common opportunistic infections in people with HIV/AIDS [5, 6].

Reliable and sensitive laboratory methods for identification of infection by NTM species are therefore normally required. The identification of NTM using traditional methods such as culture and biochemical analysis is not very sensitive and specific. Molecular methods such as polymerase chain reaction and sequencing of specific genes have recently offered the best approach to NTM speciation [7, 8]. The objective of this study was to identify NTM species stored at NMRL in Harare which were previously isolated during a national TB survey in Zimbabwe using 16S ribosomal RNA gene sequencing.

In the Zimbabwe National TB prevalence survey of 2014, a total of 963 NTM samples were isolated from the participants and stored at the National Microbiology Reference Laboratory in Harare. In this study, we retrieved 99 of the 963 isolates for speciation using 16S ribosequencing molecular method.

Polymerase chain reaction of the 16S ribosomal RNA gene was successfully performed on the 99 NTM DNA samples (Fig. 1). An expected DNA band of approximately 460 bp was observed on agarose gel for most samples. Out of the 99 samples, 94 showed successful amplification of the ribosomal RNA gene. Of these 94 amplicons, 90 were sequenced. Bioinformatic analysis was performed on 81 of the NTM samples that showed perfect sequence results.

Fig. (1). A representative gel of 16S ribosomal DNA amplicons from 9 NTM isolates. M is the molecular weight marker; lanes 1 to 8 show positive NTM rRNA gene amplicons, and lane 9 showed no amplification of the NTM rRNA gene. The expected PCR band for the 5¢-end of the 16S rRNA was approximately 460 bp.

Analysis of the DNA sequences of the 81 NTM isolates using bioinformatics revealed interesting findings. From the 81 sequence samples analyzed, 40 (49.4%) belonged to the Mycobacterium avium-intracellulare complex (MAC) species and the other 41 (50.6%) were non-MAC species (Table 1). The 40 samples (coded: B3, B5, B7, B8, B19, B21, B22, B25, B26, B27, B28, B29, B30, B32, B33, B36, B37, B39, B40, B41, B42, B43, B44, B45, B46, B48, B49, B51, B54, B56, B61, B63, B65, B67, B68, B70, B73, B76, B81, B88) had (except one) sequence similarity of 100% with at least one of the MAC species found in Genbank.

The MAC species that had sequence similarities with our samples were mainly M. avium, M. intracellulare, M. colombiense, M. yongonense, M. marseillense, M. vulneris and M. indicus from the Genbank. Further phylogenetic analysis of the 40 MAC samples showed minor sequence variations (Fig. 2). Some of the sequences were 100% similar, while others differed by few bases. Sequences B7, B19, B28, B29, B32, B33, B40, B41, B42, B46, B65 and B75 were identical and they had 100% sequence similarity with M. intracellulare and M. vulneris from the Genbank. Another group of sequences (B3, B8, B25, B21, B27, B39, B43, B54, B56, B88) had 100% similarity with M. avium, M. intracellulare, M. yongonense, M. colombiense, M. bouchedurhonense. Sequences B26, B37, B44, B51, B61, B68 were 100% similar with those of M. avium, M. intracellulare, M. yongonense, M. marseillense. Sequences of B5, B22, B36, B48, B76 were similar with those of M. avium, M. intracellulare, M. yongonense, M. colombiense and M. bouchedurhonense in the Genbank. Sequences of B63, B67, B70, B81 were similar with only those of M. avium and M. intracellulare. Sequences of B30, B45, B49 were closely related, but differed slightly and were similar with a number of MAC species in the Genbank.

The other Mycobacterium species (n = 41) (50.6%) other than MAC that were identified in this study were M. lentiflavum (6.2%), M. terrae complex (4.9%), M. paraense (4.9%), M. kansasii (3.7%), M. moriokaense 3.7%), M. asiaticum (2.5%), M. novocastrense (2.5%), M. brasiliensis (2.5%), M. elephantis (2.5%), M. paraffinicum (1.2%), M. bohemicum (1.2%), M. manitobense (1.2%), M. intermedium (1.2%), M. tuberculosis complex (1.2%), M. parakoreense (1.2%), M. florentinum (1.2%), M. litorale (1.2%), M. fluoranthenivorans (1.2%), M. sherrisii (1.2%), M. fortuitum (1.2%) and M septicum (1.2%) (Table 1). Two isolates (B64 and B69) could not be identified, but were found to be closely related to M. montefiorense and M. phlei respectively (Table 1). Phylogenetic analysis of the 41 sequences showed that some of the isolates were closely related while others were distantly related (Fig. 3).

Fig. (2). The 16S rRNA gene-based phylogenetic tree analysis of the 40 MAC species. The tree was rooted with Mtb sequence (Accession number KF796660).

NTM are emerging pathogens in the human population especially in resource-limited countries [10]. They are now known to cause a number of opportunistic infections in both immunosuppressed and immunocompetent individuals [11]. No previous studies have been done to identify NTM species prevalent in the human population in Zimbabwe. In this study, we set out to identify NTM species circulating in Zimbabwe. We therefore used NTM samples that were stored at the NMRL and were previously collected during the national TB survey in 2014 by the Ministry of Health and Child Care. The main objective of the survey had been to determine the prevalence of Mtb in Zimbabwe. However, during the survey, NTM bacteria were also isolated. The use of ribosomal genes of bacteria such as 16S, 5S and 23S in species identification has gained momentum in the past few decades [12, 13]. These genes have domains which are species-specific and hence can be used in speciation. 16S ribosequencing is the most commonly used method in identification of species of NTM from various sources [14, 15]. Other different methods which are also used in identifying NTM species include sequencing of rpoB, hsp65 and sod genes [7, 16].

This study highlighted the existence of several NTM species in the human population in Zimbabwe. Species belonging to the MAC group were the commonest in this study. The MAC group of NTM is made up of closely related Mycobacterium species, mainly M. avium and M. intracellulare, together with other few less-defined species such as M. colombiense, M. yongonense, M. marseillense, M. vulneris, M. indicus and M. paratuberculosis. These species are ubiquitous in environmental sources such as natural and municipal waters, soil, animals and droppings of infected birds [17]. Humans can get infected from these contaminated sources. It is now known that MAC species are responsible for the majority of infections caused by NTM in both developed and developing countries. Both immunocompetent and immunocompromized human populations are at risk of getting infected opportunistically by MAC species [10, 11, 18, 19]. People who have HIV/AIDS have a higher risk of getting infected [20]. Generally, infections due to Mycobacterium tuberculosis and MAC are the most commonly reported in HIV/AIDS patients [21, 22].

Fig. (3). The 16S rRNA gene-based phylogenetic tree analysis of the 41 non-MAC species. The tree was rooted with Mtb sequence (Accession number KF796660). B3 was used as a representative of the MAC species.

Other than MAC, we also identified other Mycobacterium species that could be of human health importance. They were M. lentiflavum, M. terrae, M. paraense, M. kansasii, M. moriokaense, M. asiaticum, M. novocastrense, M. brasiliensis, M. elephantis, M. paraffinicum, M. bohemicum, M. manitobense, M. intermedium, M. tuberculosis complex, M. parakoreense, M. florentinum, M. litorale, M. fluoranthenivorans, M. sherrisii, M. fortuitum and M septicum (Table 1). In this study, there was no clinical data such as HIV status of the subjects where the NTM were isolated. The clinical relevance of the NTM species identified in Zimbabwe could not therefore be ascertained. However, the species we identified were found to be associated with specific infections in other studies. M. lentiflavum which is considered to be an emerging human pathogen has been found in children with cervical lymphadenitis and in immuno compromised patients [23, 24]. M. lentiflavum can also cause chronic lung disease in immuno competent people [25]. The M. terrae complex (M. triviale, M. nonchromogenicum, and M. terrae) can cause some minor diseases such chronic bronchopulmonary disease, lymphadenitis, skin and soft tissue disease, skeletal (bone, joint, and tendon) disease, and disseminated and catheter-related infections [26]. M. nonchromogenicum has been associated with arthritis, tenosynovitis, meningitis and pulmonary infection [27]. M. paraense was previously isolated from pulmonary samples from Brazil, but its clinical importance is still poorly known [28]. M. kansasii is a photochromogenic bacterium and is known to be the second commonest (after MAC) cause of NTM disease in HIV/AIDS patients [22, 29]. M. moriokaense was found to be associated with chronic pneumonia, cutaneous and wound infections [30, 31]. This rare NTM species was recently isolated from water supplies [32]. M. asiaticum is regarded as a cause of pulmonary disease, childhood lymphadenitis, olecranon bursitis and wound infections [33]. M. moriokaense species was shown to be associated with chronic pneumonia [34]. Clinical importance of Mycobacterium brasiliensis is still scarce. M. elephantis was originally detected from a sick elephant that that died from chronic respiratory disease and is associated with respiratory diseases in humans [35]. M. paraffinicum has been associated with symptomatic pulmonary infections [36]. M. bohemicum has been associated with cervical lymphadenitis in children [37]. M. manitobense is a rapidly growing mycobacterium that is potentially pathogenic to humans [38]. M. intermedium has been found in cutaneous and pulmonary infections [39, 40]. One sample (B62) aligned with sequences of the Mtb complex (M. tuberculosis, M. bovis, M. aficanum, M. canetti and M. microti). The sequencing of 16S ribosomal RNA gene cannot discriminate species of the Mtb complex and hence more molecular methods would be required to identify the species [16, 41]. M. parakoreense has been associated with pulmonary infections [42]. M. florentinum has been detected in people with various pulmonary disorders and cervical lymphadenitis [43, 44]. M. litorale was recently isolated from soil and its medical importance is not yet studied [45]. M. fluoranthenivorans is a fluorenthene and aflotoxin-degrading mycobacterium and has no known clinical relevance [46]. M. neoaurum is known to be associated with bacteraemia and other pulmonary infections [47, 48]. M. cosmeticum is an opportunistic pathogen in humans that is associated with cutaneous granulomatous lesions and gastrointestinal tract problems [49, 50]. M. sherrisii has been associated with pulmonary and disseminated disease in HIV/AIDS patients [51, 52]. M. fortuitum was sometimes associated with catheter infections [53]. M. septicum was previously found in sepsis and bacteraemia [54, 55]. In this study, two isolates could not be identified. Further analysis showed that they were closely related to M. montefiorense and M. phlei respectively. M. montefiorense causes skin lesions in moray eels [56]. Its clinical relevance to humans is still to be established. M. phlei is widely distributed in the environment and can cause opportunistic infections [57].

The sources of human infections by NTM in Zimbabwe were not investigated in this study. Some epidemiologic studies elsewhere have suggested that water (natural and municipal water) was one of the main sources of human exposure to NTM [58]. Water has also been found to be the main source of M. avium [59, 60]. Other sources of human infections include soil, sewage and wastewater, aerosols, plants, food, protozoa, animals, birds and humans [1, 2, 4, 61, 62]. It is therefore the overlap of human and mycobacterial ecology that will continue to make NTM infections in the human population unavoidable [4]. A number of further studies will need to be done to establish the scope of the NTM problem in Zimbabwe.

The study highlighted the importance of identifying species of NTM in Zimbabwe. The MAC species were found to be the most common NTM in humans during the national TB survey in Zimbabwe. Further studies are necessary to ascertain the true prevalence and clinical relevance of NTM species in the Zimbabwe especially in people with HIV/AIDS. Further studies are also necessary to establish the sources of human infections by NTM.