Molecular characterization of Burkholderia pseudomallei and its seroprevalence in Bangladesh

Abstract

The Gram negative bacillus, Burkholderia pseudomallei causes melioidosis, a rare but serious infection that can be fatal if untreated or misdiagnosed. The organism is widely distributed in the soil and water of tropical and subtropical countries. Melioidosis is naturally acquired through environmental contact with the bacterium. Although melioidosis is largely restricted to the Southeast Asia and Northern Australia, the disease has been increasingly reported from Bangladesh. However, its actual prevalence in Bangladesh is largely unknown due to the lack of systematic study and awareness of the medical community about the disease and the organism. So far, no systematic study has been done to find out the extent of exposure of B. pseudomallei infection among healthy individuals and to detect the source of this organism in environmental samples of Bangladesh. In order to address the issue, the first phase of the present study was designed to determine the magnitude of exposure by detecting antibodies to B. pseudomallei among the healthy population of selected regions of Bangladesh. In the second phase of the study, attempt was made to detect the organism in the soil by culture and molecular methods. The clinical and environmental isolates were further characterized by molecular techniques to determine the relatedness of the organisms. In the first phase of the study, blood samples were collected from healthy population residing in rural area of four districts (Mymensingh, Sylhet, Narayangange and Kishoregange) to determine the seroprevalence of B. pseudomallei infection. We have used both sonicated whole cell antigen (SWCA) and recombinant truncated flagellin antigen (RTFA) of B. pseudomallei in an in-house indirect enzyme linked immunosorbent assay (ELISA). The cut off optical density (OD) value for SWCA and RTFA was 0.8 and 0.4 respectively. The cut off OD value was determined by mean OD of negative control + 3x SD. In the second phase of the study, we have determined the source of the organism in the soil samples from four northeastern districts of Bangladesh by culture and polymerase chain reaction (PCR). Multiple soil samples from 5–7 sampling points of 3–5 sites were collected from rural areas of four northeastern districts (Mymensingh, Sylhet, Gazipur, and Narayangange) of Bangladesh from where culture confirmed melioidosis cases were detected earlier. Approximately 200 gm soil was taken from each point from a depth of about 20-30 cm using a shell augur disinfected with 70% alcohol in between soil collection. Collected soil was placed into a sterile plastic bag and sealed with rubber band to prevent moisture loss and was transported to the laboratory as soon as possible. In the second phase of the study, total 179 soil samples were collected and cultured in Ashdown selective media and sub-cultured in MacConkey`s agar medium. The suspected organisms which grew on MacConkey`s agar medium at 420C were identified as B. pseudomallei by typical colony morphology, Gram staining (bipolar staining), motility, biochemical tests (including API 20NE), arabinose assimilation and resistance to colistin and aminoglycoside. Monoclonal antibody based latex agglutination test (Melioidosis Research Center, Khon Kaen, Thailand) was performed for the final identification and confirmation of the suspected colonies of B. pseudomallei. Phenotypically suspected colonies of B. pseudomallei were further confirmed by PCR using 16s rRNA specific primers and orf2 gene of type three secretion system1 (TTS1). In the present study, molecular analysis was performed to characterize the strains isolated from clinical specimens and environmental sources. All these isolates were analyzed by real-time PCR assay targeting TTS1, Yersinia-like fimbrial (YLF) gene cluster and B. thailandensis-like flagellum and chemotaxis (BTFC) gene cluster. Real-time PCR assay targeting TTS1 was performed to confirm phenotypically identified organisms. YLF and BTFC gene clusters were examined to demonstrate the diversity of B. pseudomallei from various geographical regions and sources. Multilocus sequence typing (MLST) with seven house-keeping genes was performed with all the isolates to clarify the genetic relationships between B. pseudomallei isolated from clinical specimens and environmental sources and also between strains isolated from Bangladesh and other countries. The primers used in the PCR amplification and sequencing of the seven housekeeping gene fragments were ace-up and ace-dn, -gltB-up and gltB-dn, gmhD-up and gmhD-dn, lepA-up and lepA –dn, lipA-up and lipA –dn, narK-up and narK-dn, ndh-up and ndh-dn. Out of 940 blood samples, anti- B. pseudomallei IgG antibody against SWCA was detected in 21.5% individuals. However, ELISA using RTFA protein showed the seropositivity rate as 13.7%, which was lower than the seropositivity (21.5%) detected by ELISA using SWCA. Seropositivity rate by using SWCA was 22.6%-30.8% in three districts from where melioidosis cases were detected earlier, compared to 9.8% in a district (Kishoregange) where no melioidosis case was either detected or reported (p<0.01) previously. Seropositivity increased with the advancement of age from 5.3% to 30.4% among individuals aged 1–10 years and > 50 years respectively. The seropositivity rates were 26.0% and 20.6% in male and female respectively, while it was 20–27% among different occupational groups. No significant association was observed with gender (χ2 = 3.441, p = 0.064) or any occupational group (χ2 = 3.835, p = 0.280). Out of 179 soil samples, B. pseudomallei was isolated from two (2) samples from paddy field of Gazipur district, which is located 54 km north of capital Dhaka city. Both the isolates were phenotypically identical and arabinose negative and were positive for 16s rRNA and orf2 (TTS1) gene by conventional and real-time PCR assay respectively. Gene cluster analysis targeting YLF and BTFC gene demonstrated that all the isolates from Bangladesh contained YLF gene cluster. None of the isolates was positive for BTFC gene cluster. YLF gene cluster is predominantly found among B. pseudomallei derived from Southeast Asia. Phylogenetic analysis of 24 B. pseudomallei isolates by MLST revealed thirteen different sequence types (STs) of which 4 STs (ST- 1352, 1124, 761 and 756) were of novel types and identified for the first time. All these isolates were from Bangladeshi patients. Strains having the above STs were isolated from patients with abscess in different organs (liver, soft tissue, lungs). It is to be noted that in the present study, ST 56 (5 cases), ST 1007 (4 cases) and ST 1005 were the most frequently isolated types. ST 56 which was present in 5 clinical isolates out of 22, was the most common variant present in Bangladesh, followed by ST 1007 revealed from 4 cases and ST 1005, found in 2 clinical and 2 environmental isolates. Presence of ST 1005 in soil of Gazipur district as well as its presence in melioidosis patients from the same district indicated soil as the source and reservoir. All the strains containing ST 56 was isolated from patients with septicemia. ST 56 was also detected earlier in 1999 from a Bangladeshi patient living abroad. Apart from the four novel STs described above, all other STs that have been detected in the present study are also present in Thailand, Cambodia, China and Vietnam and other neighboring countries. The present study has demonstrated that a large proportion of people residing in the rural area of four districts are exposed to the organism as determined by serology and have a potential for developing overt diseases during their lifetime. The study has also identified for the first time the presence of B. pseudomallei in the soil samples of Bangladesh and determined soil as the source of B. pseudomallei infection in this region. All B. pseudomallei isolated in Bangladesh posses YLF gene, which confirms their Asian origin. As YLF strains are more virulent than BTFC strains, so people in this region are at higher risk of severe form of infection. MLST study has revealed that a number of novel STs of B. pseudomallei exist in Bangladesh. Presence of unique STs of B. pseudomallei in our environment demands further study to understand their importance to the biology of these organisms. Presence of same ST from the soil and clinical isolates indicates soil as the source and reservoir of this organism. It is likely that human and/or animal movements between these areas played pivotal role in the dissemination of B. pseudomallei, however analysis of broader range of isolates from these region are required for confirmation. Further large scale study is necessary to find out the magnitude of the infection in different areas of Bangladesh and its different reservoirs in the environment along with phylogenetic distribution.