Distribution of environmental mycobacteria in Karonga District, northern Malawi

Abstract

The genus Mycobacterium includes many species that are commonly found in the environment (in soil and water or associated with plants and animals), as well as species that are responsible for two major human diseases, tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). The distribution of environmental mycobacteria was investigated in the context of a long-term study of leprosy, tuberculosis, Mycobacterium bovis BCG vaccination, and the responses of individuals to various mycobacterial antigens in Karonga District, northern Malawi, where epidemiological studies had indicated previously that people may be exposed to different mycobacterial species in the northern and southern parts of the district. A total of 148 soil samples and 24 water samples were collected from various locations and examined to determine the presence of mycobacteria. The detection method involved semiselective culturing and acid-fast staining, following decontamination of samples to enrich mycobacteria and reduce the numbers of other microorganisms, or PCR with primers specific for the mycobacterial 16S rRNA gene, using DNA extracted directly from soil and water samples. Mycobacteria were detected in the majority of the samples, and subsequent sequence analysis of PCR products amplified directly from soil DNA indicated that most of the products were related to known environmental mycobacteria. For both methods the rates of recovery were consistently higher for dry season samples than for wet season samples. All isolates cultured from soil appeared to be strains of Mycobacterium fortuitum. This study revealed a complex pattern for the environmental mycobacterial flora but identified no clear differences between the northern and southern parts of Karonga District.

FIG. 1.

Phylogenetic tree showing relationships between environmental sequences and isolates from Karonga District and known mycobacterial species. The tree is based on a comparison of a 473-bp sequence from the mycobacterial 16S rRNA gene, using maximum likelihood. Bootstrap values greater than 70% are indicated at the nodes. Bar = 0.1 substitution per site. The superscript numbers are branch numbers. Branch 1 contained the outgroup Nocardia asteroides strain DSM 43255 (accession no. AF430026). Branch 2 contained a sequence amplified from soil sample S119 from Chilumba in southern Karonga (AJ785782). Branch 3 contained M. heidelbergense ITG 7971 (AJ422048) and Mycobacterium sp. strain IWGMT 90174 (X88908). Branch 4 contained a sequence amplified from soil sample S132 from site 3A in Gweleweta in northern Karonga (AJ420144). Branch 5 contained sequences amplified from soil sample 8MJ from Kasyata (AJ420149) and soil sample S160 from Gweleweta (AJ785779) in northern Karonga. Branch 6 contained a sequence amplified from soil sample S128 from Gweleweta in northern Karonga (AJ420152). Branch 7 contained a cultured isolate from soil sample S132 from Gweleweta in northern Karonga (AJ785766), isolate 2525 from sputum obtained in Chilumba in southern Karonga (AJ420160), sequences amplified from soil samples S128, S132, and S133 from Gweleweta in northern Karonga (AJ420161, AJ785777, AJ420147, and AJ420162), M. chlorophenolicum PCP-1 (X79094), M. chubuense ATCC 27278 (AF480597), M. duvalii ATCC 43910 (U94745), M. lacticola ATCC 9626 (AF480582), M. mageritense DSM 44476 (AJ699399) and 1336 (AJ011335), M. moriokaense DSM 44221^T (AJ429044), M. psychrotolerans (AJ534886), M. pulveris DSM 44222^T (AJ429046), and Mycobacterium spp. strains SM7.6.1 (AF247497), WF2 (U90877), DSM 3803 (AY147261), HE5 (AJ012738), and PX3 (AY337605). Branch 8 contained M. chlorophenolicum DSM 43826 (X79292). Branch 9 contained a sequence amplified from soil sample S126 from Gweleweta in northern Karonga (AJ420150). Branch 10 contained a sequence amplified from soil sample S68 from Mzang'unya in southern Karonga (AJ420163). Branch 11 contained M. rhodesiae JS60 (AF498650). Branch 12 contained M. petroleophilum ATCC 21497 (AF480587), M. rhodesiae DSM44223^T (AJ429047), and Mycobacterium spp. strains PAH135 (X84978), GP1 (AJ012626), RJG11 (U30661), and TA27 (AB028482). Branch 13 contained a sequence amplified from soil sample S134 from Gweleweta in northern Karonga (AJ785778). Branch 14 contained M. interjectum MB739 (AJ272088), M. asiaticum ATCC 25276 (AF480595), and Mycobacterium spp. strains IWGMT 90100 (X88915), IWGMT90093 (X88915), and IWGMT 90203 (X88910). Branch 15 contained M. brisbanense ATCC 49938 (AY012577). Branch 16 contained M. wolinskyi ATCC 700009 (Y12871) and M. peregrinum ATCC 14467 (AF058712). Branch 17 contained a sequence amplified from soil sample S134 from Gweleweta in northern Karonga (AJ420164). Branch 18 contained a sequence amplified from soil sample S64 from Mzang'unya in southern Karonga (AJ785780). Branch 19 contained M. mucogenicum strains ATCC 49650 (AF480586) and ATCC 49649 (AF480585), M. ratisbonense (AF055331), SD4 (AJ271863), and Mycobacterium spp. soil isolates EM3 (AY436799) and BPC5 (AY162040). Branch 20 contained a cultured isolate from soil sample S78 from Mgyoyera in southern Karonga (AJ785776), sequences amplified from soil samples GKPS and S115 from Chilumba (AJ420153 and AJ420155), from samples S58 and S93 from Mwamtawale (AJ420156, AJ420154), and from sample S66 from Mzang'unya (AJ420157) in southern Karonga, M. fortuitum strain ATCC 49403 (AF480580), M. smegmatis strain DSM 43756 (AJ536041), M. farcinogenes strain NCTC 10955^T (AY457084), M. goodii strain M069 (Y12872), M. senegalense CIP 104941^T (AY457081), and United Kingdom soil isolate Mycobacterium sp. CF1 (AJ509013). Branch 21 contained M. parafortuitum DSM 43528 (X93183) and M. septicum MB804 (AJ416909). Branch 22 contained M. phlei ATCC 11758 (AF480603). Branch 23 contained M. frederiksbergense DSM 44346 (AJ276274). Branch 24 contained cultured isolates from soil samples GKPS, S115, and S119 from Chilumba in southern Karonga (AJ785767, AJ785768, and AJ785769) and from samples S133 and S160 from Gweleweta (AJ785773 and AJ785772) and samples S175 and S188 from Kasyata (AJ785771 and AJ785770) in northern Karonga, and M. fortuitum CIP 104534 (AY457066). Branch 25 contained a cultured isolate from soil sample S72 from Mzang'unya in southern Karonga (AJ785775). Branch 26 contained sequences amplified from soil sample S134 from Gweleweta (AJ420148) and sample 8MJ from Kasyata (AJ420151) in northern Karonga, M. poriferae ATCC 35087 (AF480589), and soil isolate Mycobacterium sp. strain JS621 (AF498659). Branch 27 contained a sequence amplified from soil sample S119 from Chilumba in southern Karonga (AJ785783). Branch 28 contained M. celatum DSM 44243 (AJ536040), ATCC 51130 (L08170), and ATCC 51131 (L08169). Branch 29 contained a sequence amplified from soil sample S160 from Gweleweta in northern Karonga (AJ420159). Branch 30 contained M. neoaurum ATCC 25795 (AF480593). Branch 31 contained a sequence amplified from soil sample S78 from Mgoyera in southern Karonga (AJ785781). Branch 32 contained a sequence amplified from soil sample S132 from Gweleweta in northern Karonga (AJ420143), M. canariense 502329 (AY255478), and M. cosmeticum ATCC BAA-878 (AY449728). Branch 33 contained M. fortuitum ATCC 49404 (AF480581), M. boenickei ATCC 49935 (AY012573), M. farcinogenes ATCC 35753 (AF055333), M. houstonense ATCC 49403 (AY012579), M. neworleansense ATCC 49404 (AY012575), M. porcinum ATCC 33776 (AF480588), and M. senegalense ATCC 35796 (AF480596). Branch 34 contained a cultured isolate from soil sample S209 from Kaporo in northern Karonga (AJ785774), M. fortuitum MB44 (AJ416910), M. concordense ATCC BAA-329 (AY012576), M. peregrinum ATCC 14467 (AJ42246), M. septicum HXN1900 (AJ457055), HXN500 (AJ457055), and W4964 (AF111809), Mycobacterium sp. strain NB01 (AY188086), and Mycobacterium sp. strain DhA-55 (AJ011510). Branch 35 contained M. aichiense JS618 (AF498656). Branch 36 contained M. austroafricanum ATCC 33464 (X93182) and M. vanbaalenii DSM 7251 (AY636003). Branch 37 contained a sequence amplified from soil sample S164 from Kasyata in northern Karonga (AJ420145), M. tokaiense ATCC 27282 (AF480590), and Mycobacterium spp. strains MA-112/96 (Y08857) and PP1 (AB009578). Branch 38 contained M. leprae TN (AL583920). Branch 39 contained M. tuberculosis CDC 1551 (AE007009), M. avium NIPHL070703A (AY360329), M. avium subsp. paratuberculosis k10 (AE017236), M. africanum ATCC 25420 (AF480605), M. bovis NIPHL 110703 (AY360331), M. bohemicum E743 (AJ277284), M. gastri ATCC 15754 (AF480602), M. intracellulare MB971 (AJ422047), M. intermedium (X67847), M. kansasii ATCC 12478 (AF480601), M. liflandii (AY500838), M. marinum ATCC 927 (AF456240), M. microti ATCC 19422 (AF480584), M. pinnipedi (AF502574), M. scrofulaceum ATCC 19981 (AF480604), and M. ulcerans Borstel 10564/70 (X58954).