DETECTION OF VIRULENCE GENES IN ENVIRONMENTAL STRAINS
                    OF <i>Vibrio cholerae</i> FROM ESTUARIES IN NORTHEASTERN
                    BRAZIL

Menezes, Francisca Gleire Rodrigues de; Neves, Soraya da Silva; Sousa, Oscarina Viana de; Vila-Nova, Candida Machado Vieira Maia; Maggioni, Rodrigo; Theophilo, Grace Nazareth Diogo; Hofer, Ernesto; Vieira, Regine Helena Silva dos Fernandes

doi:10.1590/S0036-46652014000500010

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Microbiology • Rev. Inst. Med. Trop. São Paulo 56 (5) • Sep-Oct 2014 • https://doi.org/10.1590/S0036-46652014000500010 linkcopy

DETECTION OF VIRULENCE GENES IN ENVIRONMENTAL STRAINS OF Vibrio cholerae FROM ESTUARIES IN NORTHEASTERN BRAZIL

Detecção de genes de virulência em estirpes de Vibrio cholerae isolados de estuários no Nordeste do Brasil

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstracts

The objectives of this study were to detect the presence of Vibrio cholerae in tropical estuaries (Northeastern Brazil) and to search for virulence factors in the environmental isolates. Water and sediment samples were inoculated onto a vibrio-selective medium (TCBS), and colonies with morphological resemblance to V. cholerae were isolated. The cultures were identified phenotypically using a dichotomous key based on biochemical characteristics. The total DNA extracted was amplified by PCR to detect ompW and by multiplex PCR to detect the virulence genes ctx, tcp, zot and rfbO1. The results of the phenotypic and genotypic identification were compared. Nine strains of V. cholerae were identified phenotypically, five of which were confirmed by detection of the species-specific gene ompW. The dichotomous key was efficient at differentiating environmental strains of V. cholerae. Strains of V. cholerae were found in all four estuaries, but none possessed virulence genes.

Cholera; Estuaries; Pathogenicity; Genes

Technique	Genes	Primer sequence (5′- 3′)	Thermocycling conditions	Amplicons (bp)^f	Source
PCR	ompW ^a	F: 5′ –cac caa gaa ggt gac ttt att gtg- 3′		304
PCR	ompW ^a	R:5′ – ggt ttg tcg aat tag ctt cac c - 3′.		304
Multiplex PCR	ctxAB ^b	F:5′ –gcc ggg ttg tgg gaa tgc tcc aag - 3′	94°C/10 min. 30 cycles (94°C/1 min., 59°C/1min., 72°C/ 2 min.) 72°C/10 min.	536	GOEL et al., (2007)
	ctxAB ^b	R:5′ – gcc ata cta att gcg gca atc gca tg - 3′		536
	tcp ^c	F:5′ – cgt tgg cgg tca gtc ttg- 3′		805
	tcp ^c	R:5′ – cgg gct ttc ttc ttg ttc g - 3′		805
	rfbO1 ^d	F:5′ – tct atg tgc tgc gat tgg tg - 3′		638
	rfbO1 ^d	R:5′ – ccc cga aaa cct aat gtg ag - 3′;		638
	zot ^e	F:5′- tcg ctt aac gat ggc gcg ttt t - 3′		947
	zot ^e	R:5′– aac ccc gtt tca ctt cta ccc a - 3′		947

Reagents of the reaction*	PCR		Multiplex PCR
	Species-specific gene		Virulence genes
	ompW	ctxAB	tcp	rfbO1	zot
Buffer 10X	20 mM Tris pH 8.4, 50 mM KCl		20 mM Tris pH 8.4, 50 mM KCl
dNTP's (2.5 mM)	0.25 µM		0.25 µM
Primer F (10 µM)	0.4 µM	0.4 µM	0.4 µM	0.4 µM	0.4 µM
Primer R (10 µM)	0.4 µM	0.4 µM	0.4 µM	0.4 µM	0.4 µM
MgCl2 (50 mM)	1.5 mM		1.5 mM
Taq polymerase (500 U)	4 U		4 U
Sample	20-35 ng**		20-35 ng**
Final reaction volume	25 µL		25 µL

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Instituto de Medicina Tropical de São Paulo Av. Dr. Enéas de Carvalho Aguiar, 470, 05403-000 - São Paulo - SP - Brazil, Tel. +55 11 3061-7005 - São Paulo - SP - Brazil
E-mail: revimtsp@usp.br

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[TFN01t01] ompW^a = V. cholerae-specific gene; ctxAB^b = cholera toxin; tcp^c = toxin coregulated pilus; rfbO1^d = serogroup O1 identification gene; zot^e = zonula occluden s toxin; (bp)^f = base pair.