Quantitative evaluation of environmental Vibrio cholerae population dynamics over temporal and spatial scales

  • Author / Creator
    Nasreen, Tania
  • Cholera, a severe life-threatening waterborne diarrheal disease, has been endemic to the Ganges delta for centuries. Vibrio cholerae, the causative agent of this disease, is a natural inhabitant of brackish water. Amongst the 200 serogroups identified so far, only O1 and O139 were found responsible for most of the cholera endemics. Transmission of cholera mainly occurs through fecal-oral route from compromised water management systems, poor sanitation and personal hygiene. Effective control of cholera outbreaks relies on timely detection of the pathogen from clinical samples and environmental sources. Although cholera epidemiology is well studied, information on the abundance of V. cholerae and other pathogenic Vibrio species in environmental sources is minimal and rapid detection is still a challenge. Moreover, Vibrio metoecus, closely related to V. cholerae, often coexists with the latter within aquatic environments and has recently been described to cause infections in humans. Therefore, the relative abundance of V. metoecus and V. cholerae, along with their population dynamics in aquatic reservoirs is critical to understand the virulence of these bacteria. Consistent environmental monitoring of pathogenic Vibrio species and the pandemic generating (PG) O1 serogroup of V. cholerae could facilitate the identification of their actual distributions in aquatic reservoirs, thus would help predict an outbreak before it strikes. The difficulties in substantial temporal and spatial environmental sampling and lack of specific quantitative methods made this goal difficult until now.

    I developed a multiplex qPCR assay with a limit of detection (LOD) of three copies per reaction to simultaneously quantify total V. metoecus and V. cholerae populations, as well as the toxigenic and O1 serogroup subpopulations of V. cholerae in environmental samples by targeting four different genes as specific markers. Analysis of water samples from four different geographic locations, including cholera-endemic (Dhaka, Kuakata and Mathbaria, Bangladesh) and non-endemic (Oyster Pond in Falmouth, Massachusetts, USA), showed that V. metoecus was present seasonally in the USA site only. The non-toxigenic O1 serogroup comprised up to 18% of the total V. cholerae population in the USA coastal sites. V. cholerae toxigenic O1 serogroup was consistently present as a high proportion of the total V. cholerae populations in inland waters but rarely present in coastal waters of the cholera-endemic region studied (Bangladesh). Large numbers (>90% of the total) of both the Vibrio species were found attached to host/particle (>63 μm fraction size samples). This is the first study that used a culture-independent method to quantify V. cholerae or V. metoecus directly in environmental samples from cholera endemic and non-endemic areas. This culture-independent multiplex qPCR-based detection and quantification method was validated to allow direct quantification of pathogenic V. cholerae in environmental water samples on-site, in low resource settings and obtained a limit of detection (LOD) of 6×10^3 cells/L of water. Analysis of the environmental water samples collected from a site endemic for cholera (Gabtoli area, Dhaka, Bangladesh) showed O1 serogroup comprises 15% of total V. cholerae. Portability of the equipment, the stability of the reagents at 4 °C, user-friendly online software and easy set-up make this assay extremely useful for field research and thus fast quantitative analysis of the abundance of these organisms in the environment could be possible.
    Finally, spatial and temporal dynamics of the PG lineage and other lineages of the V. cholerae species in Dhaka’s waters were evaluated by sequences analysis coupled with real-time qPCR of a species-specific, highly diverse protein-coding gene (vibriobactin utilization protein, viuB) amplified from aquatic biomass DNA. This method provided subspecies level resolution of the abundance and lineage composition of V. cholerae populations. The total abundance of V. cholerae was found to be very stable, ranging from 2 to 5× 10^5 cells/L, being highest in the most densely populated site of the seven locations sampled in Dhaka for six consecutive months. The PG lineage of the O1 serogroup comprised 24 to 92% of the total V. cholerae population, which was consistently present and showed occasional but sudden reductions in abundance. In these rare instances in which PG O1 lost its dominance, another lineage underwent a rapid expansion while the total V. cholerae population size remained unchanged. This suggests there is intra-species competition among the lineages of V. cholerae within a niche. Environmental parameters like salinity and total dissolved solids (TDS) were found to correlate with V. cholerae lineage richness which positively indicates more diversity at the subspecies level. However, the abundance of PG O1 showed a negative correlation with salinity. Overall this result suggests that population composition can be influenced by intra-lineage interactions as well as by environmental factors.

  • Subjects / Keywords
  • Graduation date
    Fall 2019
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
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