Clinical and Environmental Vibrio cholerae Non-O1, Non-O139 Strains from Australia Have Similar Virulence and Antimicrobial Resistance Gene Profiles

Abstract

Cholera caused by pathogenic Vibrio cholerae is still considered one of the major health problems in developing countries including those in Asia and Africa. Australia is known to have unique V. cholerae strains in Queensland waterways, resulting in sporadic cholera-like disease being reported in Queensland each year. We conducted virulence and antimicrobial genetic characterization of O1 and non-O1, non-O139 V. cholerae (NOVC) strains (1983 to 2020) from Queensland with clinical significance and compared these to environmental strains that were collected as part of a V. cholerae monitoring project in 2012 of Queensland waterways. In this study, 87 V. cholerae strains were analyzed where O1 (n = 5) and NOVC (n = 54) strains from Queensland and international travel-associated NOVC (n = 2) (61 in total) strains were sequenced, characterized, and compared with seven previously sequenced O1 strains and 18 other publicly available NOVC strains from Australia and overseas to visualize the genetic context among them. Of the 61 strains, three clinical and environmental NOVC serogroup strains had cholera toxin-producing genes, namely, the CTX phage (identified in previous outbreaks) and the complete Vibrio pathogenicity island 1. Phylogenetic analysis based on core genome analysis showed more than 10 distinct clusters and interrelatedness between clinical and environmental V. cholerae strains from Australia. Moreover, 30 (55%) NOVC strains had the cholix toxin gene (chxA) while only 11 (20%) strains had the mshA gene. In addition, 18 (34%) NOVC strains from Australia had the type three secretion system and discrete expression of type six secretion system genes. Interestingly, four NOVC strains from Australia and one NOVC strain from Indonesia had intSXT, a mobile genetic element. Several strains were found to have beta-lactamase (bla_CARB-9) and chloramphenicol acetyltransferase (catB9) genes. Our study suggests that Queensland waterways can harbor highly divergent V. cholerae strains and serve as a reservoir for various V. cholerae-associated virulence genes which could be shared among O1 and NOVC V. cholerae strains via mobile genetic elements or horizontal gene transfer. IMPORTANCE Australia has its own V. cholerae strains, both toxigenic and nontoxigenic, that are associated with cholera disease. This study aimed to characterize a collection of clinical and environmental NOVC strains from Australia to understand their virulence and antimicrobial resistance profile and to place strains from Australia in the genetic context of international strains. The findings from this study suggest the toxigenic V. cholerae strains in the Queensland River water system are of public health concern. Therefore, ongoing monitoring and genomic characterization of V. cholerae strains from the Queensland environment are important and would assist public health departments to track the source of cholera infection early and implement prevention strategies for future outbreaks. Understanding the genomics of V. cholerae could also inform the natural ecology and evolution of this bacterium in natural environments.

Keywords: Vibrio cholerae; antimicrobial activity; clinical strains; environmental strains; gene sequencing; virulence factors.

FIG 1

Maximum likelihood tree showing phylogenetic relationships between 87 clinical (human and animal infections) and environmental V. cholerae strains: O1 (n = 13), NOVC (n = 56), and publicly available (n = 18) V. cholerae strains with origin of isolates, source, year of collection (YI), serogroup and virulence genes, Vibrio pathogenicity islands 1 and 2 (VPI-1 and -2), and Vibrio seventh-pandemic islands I and II (VSP-I and -II). Gene profiles were generated using iTOL (https://itol.embl.de/) and represented as a colored box for presence and a white box for absence. Strains sequenced in this study were noted with an asterisk (*). Description of origin, source, and year of isolation in red represents clinical samples, and that in green represents environmental samples.

FIG 2

Multiple-sequence alignment of CtxB amino acid sequences from three NOVC strains (243712, M12590, and M138351) compared to two previously sequenced O1 serogroup strains from Australia (101_1 and 4287_St). Black-edge rectangles represent the position of ctxB genotype 2-distinguishing amino acid sequences.

FIG 3

Presence or absence display annotation on iTOL for the clinical and environmental O1 and NOVC strains from Australia and other countries, their antimicrobial resistance genes, and mobile genetic element profile. The source of clinical V. cholerae strains is highlighted in red and that of environmental strains in green. Blue stars represent the strains sequenced in this study.

FIG 4

Sequence comparison of CTX phage regions integrated into the chromosome of O1 and NOVC strains. M138351 and M12590 CTX phage sequences are from NOVC strains sequenced in this study and compared to the CTX phage sequence of O1 El Tor strain Si_F from Australia and strain N16961 from Bangladesh. The intensity of color represents the level of identity as shown in parentheses.