Metagenomic Detection of Bacterial Zoonotic Pathogens among Febrile Patients, Tanzania, 2007-20091

Abstract

Bacterial zoonoses are established causes of severe febrile illness in East Africa. Within a fever etiology study, we applied a high-throughput 16S rRNA metagenomic assay validated for detecting bacterial zoonotic pathogens. We enrolled febrile patients admitted to 2 referral hospitals in Moshi, Tanzania, during September 2007-April 2009. Among 788 participants, median age was 20 (interquartile range 2-38) years. We performed PCR amplification of V1-V2 variable region 16S rRNA on cell pellet DNA, then metagenomic deep-sequencing and pathogenic taxonomic identification. We detected bacterial zoonotic pathogens in 10 (1.3%) samples: 3 with Rickettsia typhi, 1 R. conorii, 2 Bartonella quintana, 2 pathogenic Leptospira spp., and 1 Coxiella burnetii. One other sample had reads matching a Neoerhlichia spp. previously identified in a patient from South Africa. Our findings indicate that targeted 16S metagenomics can identify bacterial zoonotic pathogens causing severe febrile illness in humans, including potential novel agents.

Keywords: Bacteria; Bartonella; Coxiella; East Africa; Ehrlichia; Leptospira; Rickettsia; Tanzania; bacterial zoonoses; metagenomics; vector-borne diseases; zoonoses.

Figure 1

Phylogenetic tree for Candidatus Neoehrlichia spp. identified during metagenomic detection of bacterial zoonotic pathogens among febrile patients, Tanzania, 2007–2009. Bold text indicates the sequence from this study. Numbers in parentheses indicate GenBank accession numbers. A 1,467-bp 16S sequence amplified from a bone marrow aspirate from a patient from South Africa (GenBank accession no. OP208838) matched 100% over the 296-bp variable regions 1 and 2 target sequence amplified in this study (18). Scale bar indicates nucleotide substitutions per site.

Figure 2

Phylogenetic tree of Rickettsia spp. sequences detected in metagenomic analysis of bacterial zoonotic pathogens among febrile patients, Tanzania, 2007–2009. The tree compares sequences from the 16S variable regions 1 and 2 (V1–V2) of the Rickettsia cohort from this study (bold text) to sequences from closely related Rickettsia species. Numbers in parentheses indicate GenBank accession numbers. The sequence from the study sample with R. conorii aligned 100% R. conorii strain Malish (accession no. NC003103.1) and was distinct from R. africae (accession no. NC012633.1). All 3 R. typhi strains from this study aligned 100% with R. typhi reference strain (accession no. NC017066.1) and were distinct from R. prowazekii (accession no. NC017049.1). The V1–V2 16S target is not sufficient to differentiate between R. conorii subsp. heilogjiangensis and R. japonica, or between R. rickettsia, R. peacockii, R. philipii, and R. slovaca. However, the V1–V2 16S target is sufficient to differentiate between R. conorii conorii and R. africae because 2 single-neucleotide differences would be expected between R. conorii conorii and R. africae and a TTT insertion in R. africae. Scale bar indicates nucleotide subsitutions per site.

Figure 3

Phylogenetic tree of Leptospira sequences detected in metagenomic analysis of bacterial zoonotic pathogens among febrile patients, Tanzania, 2007–2009. The tree compares sequences from the 16S V1–V2 of the L. kerchnerii and L. borgpetersenii cohort from this study (bold text) to sequences from closely related Leptospira species. Numbers in parentheses indicate GenBank accession numbers. Scale bar indicates nucleotide subsitutions per site.