Vibrio cholerae endemic to the lower Rio Grande Delta segregate into urban and rural phylotypes

Abstract

The lifestyle of Vibrio cholerae is primarily environmental, yet a chance encounter with a human host can lead to cholera, a potentially lethal form of diarrhea. Strains belonging to O1 and O139 serogroups have pandemic potential, but the contribution of non-O1/non-O139 serovars towards the genesis of cholera remains unclear. Endemic V. cholerae lineages were investigated given several historical accounts describing cholera epidemics and sporadic, contemporary cholera-like outbreaks along the lower Rio Grande Delta (LRGD). Seven isolates were recovered from an urban segment of the Rio Grande and six from a rural segment where the river empties into the Gulf of Mexico. Urban isolates all encode ß-lactamase, and with one exception are phylogenetically closely related, rough (do not express O-antigen), harbor identical plasmids, exhibit a disabled Type VI Secretion System (T6SS), and decreased protease activity. In contrast, rural strains belong to distinct serogroups, are sensitive to ß-lactams, express proteases, and kill Escherichia coli in T6SS competition assays. Genome-scale phylogenetics and multilocus sequence typing indicate that urban and rural isolates belong to distinct and novel phylogroups. These results suggest that an urban niche heavily impacted by anthropogenic pressures and a downstream protected rural niche are inhabited by distinct V. cholerae phylotypes.

Keywords: Vibrio cholerae; Antibiotic resistance; Cholera environmental reservoir; Lower Rio Grande Delta; Novel phylogroups; Phylotypes.

Fig. 1

V. cholerae was isolated at two locations along the U.S.-Mexico border in the LRGD. (A) The inset highlights the lower Rio Grande Delta where the collection sites are located relative to the broader Texas/Mexico border and the Gulf of Mexico. Site 21 (white area) is a heavily populated and transited urban segment of the Rio Grande that joins Matamoros, Tamaulipas, Mexico; with Brownsville, Texas, U.S.A that collectively hosts 1.1 million inhabitants. Site 42 is a rural, protected coastal region of the Gulf of Mexico where the Rio Grande empties into the ocean 87.5 km of river path downstream of Site 21. (B) GPS coordinates and environmental variables monitored at the sampling location at the time of collection. *Salinity is reported in practical salinity units (PSUs).

Fig. 2

Agarose electrophoresis of alkaline lysis isolated DNA reveals the presence of episomes in rough, urban and in one smooth rural V. cholerae isolate from the LRGD. (A) All rough isolates (DL2111–DL2113 and DL2115–DL2117) collected from the urban setting harbored extra-chromosomal elements. The episomes we named p21L displayed an electrophoretic migration rate of ~ 2.5 Kb, p21S of ~ 2.0 Kb and the small episome p42 migrated at ~ 1.3 Kb. Pandemic O1 classical O395 and El Tor N1691 served as controls (cont). (B) Isolation and Illumina sequencing revealed that the episomes are plasmids coding annotated ORFs.

Fig. 3

Profile of antibiotic resistance detected in the 13 V. cholerae endemic to the LRGD and pandemic control strains. (A) Zones of Growth inhibition were determined by Kirby-Bauer method on LB agar plates (p < 0.01). = ◯ Ampicillin, ∇ = Carbenicillin, = ☐ Erythromycin, ◊ = Trimpethroprim. Red and blue symbols represent urban and rural isolates respectively; green symbols are control V. cholerae strains. (B) Detection of genes from draft genomes matching the CARD RGI perfect and strict criteria conducted in silico.

Fig. 4

SDS-PAGE and Western blots display OmpU polymorphisms among smooth V. cholerae from the lower Rio Grande Delta. (A) Total protein SDS-PAGE patterns of endemic strains were similar to control strain N16961 except for considerable polymorphisms displayed by prominent bands suggestive of OmpU migrating at ~ 37 kDa marker (bracket). (B) Western blots probed with OmpU antisera confirmed that the prominent bands diverging from the control (c), in most cases are OmpU and that polymorphisms were predominant in smooth, rural isolates. Surprisingly, DL4213 belonging to serogroup O109 and DL4215 belonging to serogroup O113 did not express a protein recognized by OmpU antiserum. Cultures were normalized by OD₆₀₀ prior to sample preparation. A representative experiment from three independent replicates is shown.

Fig. 5

Zymographs of V. cholerae from the Rio Grande Delta exhibit variable protease activities. (A) Protease activities of cell pellets of the smooth urban DL2114, and of the rural isolates are enhanced compared to rough urban and control V. cholerae. A dominant protease activity at ~ 48 kDa visible in all strains is highlighted by the * symbol. (B) Similar to cell pellets, protease activities are higher in cell-free supernatants of urban DL2114 and the rural DL4211-4216 smooth isolates. In addition to the dominant protease activity from cell pellets (*), supernatants also exhibit prominent substrate degradation at ~ 100 kDa migration rate (+) and additional bands migrating at intermediate rates. All bacterial cultures were normalized by OD₆₀₀ prior to sample preparation. Representative gels from three independent experimental replicates are shown.

Fig. 6

V. cholerae isolated from the rural lower Rio Grande Delta site display enhanced T6SS activity compared to urban isolates. (A) All isolates from the rural site express Hcp, and with the exception of DL4214 and DL4216 secrete the protein conspicuously in culture supernatants suggestive of functional T6SS activity. In contrast none of the rough isolates from the urban location express Hcp except smooth DL2114 belonging to the O74 serogroup. (B) All smooth V. cholerae isolated from the rural location (blue) and DL2114 from the urban location (red symbols) engage their T6SS to kill E. coli MG1655 prey bacteria comparably to control strain V52 (green, second lane). The ΔvasK V52 strain (green; first lane) is a negative control that cannot engage T6SS. Rough strains from the urban location except for DL2114 harbor a frame-shift mutation in vasH that debilitates their T6SS. (C) Only two rural strains DL4211 and DL4215 prevent plaque formation by D. discoideum amoeba grazing comparably to control strain V52 (green; second lane). Other rural (blue) and all urban (red) V. cholerae are preyed upon with efficiency comparable to K. aerogenes control (green; first lane). Shown are results from two independent experiments performed in duplicates, horizontal bars represent the median.

Fig. 7

Genome-scale maximum-likelihood tree depicts relatedness of the 13 V. cholerae strains endemic in the LRGD and 29 globally diverse strains. Node labels show the bootstrap support and branch lengths represent the average number of substitutions per site. The tree was rooted to closely related V. mimicus ATCC 33,655 and V. paracholerae SIO was included as a deep branching nearest neighbor. LRGD endemic urban isolates are highlighted in red and rural V. cholerae in blue.