Genotypic characterization of virulent Vibrio species and their endemic bacteriophages
Abstract
Vibrio are gram negative curved bacteria commonly found in aquatic environments. Some species are pathogenic to humans as well as to aquatic animals and have been associated with serious public health complications in their hosts. Water quality is presently a great issue of public health concern globally as well as in South Africa. Water-borne diseases still pose significant threats to public health and a number of outbreaks have been associated with pathogenic Vibrio species. The aim of this study was to determine the virulence profiles of pathogenic Vibrio species and assess the bio-control potential of Vibrio species-specific endemic bacteriophages against environmental isolates. A total of 136 water samples were randomly collected from areas around North West Province, South Africa and 57 presumptive Vibrio isolates were obtained using the Thiosulfate bile salt sucrose agar. Typical yellow and green colonies were selected, purified and subjected to both preliminary and confirmatory tests which are specific to Vibrio species. All the isolates were Gram negative rods while only 38% of the isolates were oxidase positive. All the presumptive isolates were further subjected to PCR in order to amplify bacterial 16S rRNA gene fragments and all isolates were positive for this gene. Of the 57 presumptive isolates a relatively small proportion of 22 (38.60%) isolates were confirmed as Vibrio species based on the presence of vhh (45.45%), flaE (22.72%), sodB (13.64%), sodB (1) (9.09%) and hsp (9.09%) gene fragments. Furthermore, isolates were subjected to a panel of 11 antimicrobial agents in order to determine their antibiotic resistant profiles using the CLSI guidelines. Results revealed that isolates displayed high levels of resistance to Cephalothin (95.45%), Ampicillin (77.27%) and Streptomycin (40.91%). On the contrary, small proportions (4.55% to 27.27%) of the isolates were resistant to Chloramphenicol, Ciprofloxacin, Tetracyline, Gentamycin. Kanamycin, Nalidixic acid, Trimethoprim-Sulfamethoxazde and Trimethoprim. All the 22 confirmed Vibrio isolates were screened for the presence of virulence genes. Out of 6 virulence genes targeted, only 3 were positively amplified. Vibrio parahaemolyticus possessed tdh (18.18%) and trh (18.18%) virulence genes while Vibrio cholerae and Vibrio mimicus harboured (13.64%) and (4.55%) of the zot gene fragment respectively. The presence of these genes might indicate the public health implication these isolates may pose on humans. The biofilm formation potentials of 22 Vibrio species was determined using microtiter plate assay at different temperatures. The Vibrio species displayed potentials to form biofilm at different temperatures. Although the isolates were not able to form strong biofilms at 37 ?C, small proportions (4.55% and 27.27%) of the isolates produced strong biofilms at 25 °Cand 35 °C respectively. These findings indicate that the isolates of this study may be capable of colonizing a wide range of ecological niches and persist when associated with human infections. A further objective of the study was to assess the virulence potentials of Vibrio species-specific bacteriophages as biocontrol agents against antibiotic resistant and virulent environmental Vibrio isolates obtained in this study. Phage virulence assays showed that the phages isolated in this study presented lytic potentials. Two of the employed bacteriophages Phage A and Phage B were able to inhibit bacterial growth of more than one host strain. These findings indicate that phages isolated in this study have presented potential characteristics of being used as effective agents for biological control of environmental Vibrio bacterial host strains that also possess multidrug resistant determinants. These findings are of great epidemiological significance implications associated with Vibrio infections.