Genetic diversity of Vibrio cholerae in Chesapeake Bay determined by amplified fragment length polymorphism fingerprinting

Abstract

Vibrio cholerae is indigenous to the aquatic environment, and serotype non-O1 strains are readily isolated from coastal waters. However, in comparison with intensive studies of the O1 group, relatively little effort has been made to analyze the population structure and molecular evolution of non-O1 V. cholerae. In this study, high-resolution genomic DNA fingerprinting, amplified fragment length polymorphism (AFLP), was used to characterize the temporal and spatial genetic diversity of 67 V. cholerae strains isolated from Chesapeake Bay during April through July 1998, at four different sampling sites. Isolation of V. cholerae during the winter months (January through March) was unsuccessful, as observed in earlier studies (J. H. L. Kaper, R. R. Colwell, and S. W. Joseph, Appl. Environ. Microbiol. 37:91-103, 1979). AFLP fingerprints subjected to similarity analysis yielded a grouping of isolates into three large clusters, reflecting time of the year when the strains were isolated. April and May isolates were closely related, while July isolates were genetically diverse and did not cluster with the isolates obtained earlier in the year. The results suggest that the population structure of V. cholerae undergoes a shift in genotype that is linked to changes in environmental conditions. From January to July, the water temperature increased from 3 degrees C to 27.5 degrees C, bacterial direct counts increased nearly an order of magnitude, and the chlorophyll a concentration tripled (or even quadrupled at some sites). No correlation was observed between genetic similarity among isolates and geographical source of isolation, since isolates found at a single sampling site were genetically diverse and genetically identical isolates were found at several of the sampling sites. Thus, V. cholerae populations may be transported by surface currents throughout the entire Bay, or, more likely, similar environmental conditions may be selected for a specific genotype. The dynamic nature of the population structure of this bacterial species in Chesapeake Bay provides new insight into the ecology and molecular evolution of V. cholerae in the natural environment.

FIG. 1

Chesapeake Bay sampling stations included in this study. F, Susquehanna River flats; S, SERC; K, Kent Island; H, Horn Point laboratory.

FIG. 2

Environmental parameters measured at the Susquehanna River flats, Horn Point, Kent Island, and SERC stations between January and July 1998. (A) Salinity. (B) Bacterial abundance and temperature. Bacterial abundance (bar graph) was determined by epifluorescence microscopy and is presented as the number of bacteria per milliliter of seawater. Temperature was determined in situ. (C) Chlorophyll a (measured fluorometrically in micrograms per milliliter of seawater).

FIG. 3

Frequency of V. cholerae isolation among total bacterial isolates on TCBS at the Susquehanna River flats, Horn Point, Kent Island, and SERC stations in Chesapeake Bay between January and July 1998. V. cholerae was identified by biochemical testing and confirmed by PCR amplification with V. cholerae-specific primers.

FIG. 4

AFLP fingerprints of V. cholerae isolated from Chesapeake Bay by using adapters and primer sequences described in Table 1. Lanes: M, reference marker; 1, H19; 2, H8; 3, H12; 4, H23; 5, H30; 6, H21; 7, H11; 8, H27; 9, S20, 10, H10.

FIG. 5

Similarity analysis of AFLP fingerprints for 67 V. cholerae isolates from the Susquehanna River flats, Horn Point, Kent Island, and SERC stations in Chesapeake Bay collected between April and July 1998. The dendrogram was created by computing the similarity values according to the position of the bands with Molecular Analyst/Fingerprinting software (Bio-Rad Laboratories). The isolates were either free-living bacteria from the water column or associated with smaller phytoplankton (>20-μm- to <64-μm-mesh fraction) or larger plankton (>64-μm-mesh fraction).