African Journal of Microbiology Res

African Journal of Microbiology Research Vol. 6(20), pp. 4256-4260, 30 May, 2012 Available online at http://www.academicjournals.org/AJMR
DOI: 10.5897/AJMR11.651
ISSN 1996-0808 ©2012 Academic Journals





Full Length Research Paper
Effect of Ultraviolet-C (UV-C) irradiation on the virulence genes expression in Vibrio parahaemolyticus and Vibrio alginolyticus
Rihab Lagha1#, Fethi Ben Abdallah1, 2*#, Ali Ellafi1, Héla Kallel2 and Amina Bakhrouf1
1Laboratoire d’Analyse, Traitement et Valorisation des Polluants de l’Environnement et des Produits, Faculté de Pharmacie Rue Avicenne. Monastir 5000, Tunisie.
2Unité de Fermentation et de Développement de Vaccins Virologiques, Institut Pasteur de Tunis.13 place Pasteur, 1002, Tunisie.

Accepted 27 December, 2011

In this study, Vibrio parahaemolyticus and Vibrio alginolyticus, marine foodborne pathogens, were treated with Ultraviolet-C (UV-C) irradiation (240 J.m-2) to evaluate alterations in their virulence genes expression levels. Firstly, we searched for the presence of eight Vibrio cholerae virulence genes, toxR,
toxS, toxRS, ctxA, zot, ace, toxT, and virulence pathogenicity island (VPI), in the genome of investigated strains. The expression of toxR and toxS genes in UVC-irradiated bacteria, studied by reverse transcriptase polymerase chain reaction, was found to be altered. These variations were manifested by an increase or a decrease in the expression level of tested virulence genes. Further, the mRNA quantities of VPI and ace genes remained stable after treatment.

Key words: Vibrio, Ultraviolet-C (UV-C), alteration, virulence genes expression, RT-PCR.


INTRODUCTION


Ultraviolet-C (UV-C) radiation has been suggested as one of the successful disinfection practices for water treatment. Therefore, UV-sterilization has become a practical solution for safe disinfection of water (Said et al., 2010). The effectiveness of UV light in biological inacti- vation arises primarily from the fact that Deoxyribonucleic acid (DNA) molecules absorb UV photons between 200 and 300 nm, with peak absorption at 254 nm (Jeffrey et al., 1990). This absorption creates damage in the DNA by altering the nucleotide base pairing, thereby creating new linkages between adjacent nucleotides on the same DNA strand. This damage occurs particularly between pyrimidine bases. Two types of mutagenic lesions in DNA were determined: cyclobutane pyrimidine dimers (CPD) formed between the C-4 and C-5 positions of adjacent




*Corresponding author. E-mail: [e-mail]. Tel: + 21 6 73 466 244. Fax: + 216 73 461 830.

# These authors have equally contributed to this work

thymidine or cytosine residues, and pyrimidine (6 to 4) pyrimidone (6 to 4) photoproducts formed between the C6 and C4 position of adjacent pyrimidine residues, most often between T-C and C-C residues (Zimmer and Slawson, 2002). UV radiation in the range of 250 to 260 nm is lethal to most micro-organisms, including bacteria, viruses, protozoa, mycelial fungi, yeasts and algae. Among the bacteria, V. alginolyticus and V. parahaemolyticus, marine foodborne pathogen, frequently involved in epizootic outbreaks in cultured gilt- head sea bream and sea bass, causing fish mortality in Tunisian aquaculture farms (Abdallah et al., 2009a). In marine environment, V. alginolyticus and V. parahaemolyticus were considered as an important reservoir of Vibrio cholerae virulence genes (Abdallah et al., 2009b). According to Boyd et al. (2000) these genes may be horizontally transferred to V. alginolyticus in an aquatic environment. Indeed, the mobility of virulence genes may cause the transformation of non pathogenic strain to pathogenic strain. Xie et al. (2005) reported that
V. alginolyticus often possess homologues of the V. parahaemolyticus and V. cholerae virulence genes such





Abdallah et al. 4257




as toxR, tlh and VPI, suggesting that V. alginolyticus may be an important reservoir of many known virulence genes of other Vibrio species in the aquatic environment. It is probably that the aquatic environment harbours different virulence-associated genes scattered among environmental Vibrios.
In this work we searched, by polymerase chain reaction (PCR), for the presence of eight V. cholerae virulence genes, toxR, toxS, toxRS, ctxA, zot, ace, toxT, and virulence pathogenicity island (VPI), in the genome of V. alginolyticus and V. parahaemolyticus strains. The expression level of transferred virulence genes under UVC irradiation was achieved by reverse transcriptase polymerase chain reaction (RT-PCR).


MATERIALS AND METHODS

Bacterial strains

Six Vibrio strains were used in this study including three reference strains: V. alginolyticus ATCC 33787 (S1), V. alginolyticus ATCC 17749 (S2), and V. parahaemolyticus ATCC 17802 (S5). In addition, V. parahaemolyticus strain (S6), isolated from the Calich estuary (Alghero, Italy), and two V. alginolyticus strains (S3 and S4) isolated, respectively from the internal organs of aquacultured diseased gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax), in Tunisian aquaculture farm (Abdallah et al., 2009a), were included in this work.


UVC treatment

V. alginolyticus and V. parahaemolyticus were cultivated at 30°C in Tryptic soy broth 1% NaCl (TSB 1%) with shaking (150 rpm). The cultures of Vibrio strains grown to late log phase (OD600 = 0.6) were diluted and spread on Tryptic soy 1% NaCl (TSA 1%, Pronadisa, Spain) agar plates in glass Petri dishes. After 18 h of incubation at 30°C, bacterial colonies that appeared on the plates were exposed, in triplicate, to UV light according to the method described previously (Wang et al., 2004). The plates with even bacterial growth were covered with a piece of glass for non-UV treatment. The plates (covered and non-covered) were exposed to a 4-W UV lamp with a wavelength of 254 nm. The applied dose was 240
Joules m-2. After exposure, 250-ml Erlenmeyer flasks containing 100 ml of TSB 1% were inoculated with a loopful of colonies from
control and UV treated bacteria. All flasks were kept in at 30°C for 18 h with a shaking.



PCR detection of Vibrio cholerae virulence genes in V. alginolyticus and V. parahaemolyticus strains

Bacteria were cultured on TSA 1% for 24 h at 30°C. One colony was cultured in TSB 1% for 24 h at 30°C, and 1.5 mL was cen- trifuged. DNA was extracted using a Wizard genomic purification kit (Promega, Madison, WI) according to the manufacturer’s instructions. The primers of V. cholerae virulence genes used in this study (Sechi et al., 2000) are listed in Table 1. Polymerase chain reaction (PCR) were performed in 25 µL containing 50 ng of extracted DNA, 5 µL Green Go Taq buffer (5×), 0.25 µL of each deoxynucleoside triphosphate (10 mM), 0.5 µL MgCl2 (50 mM), 1
µL of each primer (25 pM), and 1U of Go Taq DNA polymerase
(Promega). Reaction mixtures were incubated for 5 min at 94°C; followed by 35 cycles at 94°C for 45 s; annealing at 52°C for 45 s

for toxS, toxR, and virulence pathogenicity island (VPI); 72°C for 1 min; and a final extension at 72°C for 10 min. The annealing temperature for the detection of the toxRS and toxT genes was 58°C, whereas for ctxA, ace, and zot the temperature was 60°C. PCR products (5 µL) were analyzed on 1% agarose gels stained with ethidium bromide (0.5 mg/mL) at 90 V for 1 h and viewed under ultraviolet transillumination. All PCR-positive strains, indicating the presence of the virulence genes, were confirmed by repeating the PCR three times.


RT-PCR for virulence gene expression

To study the level of expression of V. alginolyticus and V. parahaemolyticus virulence genes before and after UV irradiation, semi-quantitative RT-PCR method was used. RNA from control and irradiated cells was extracted by SV total RNA isolation system (Promega) according to the manufacturer’s instructions. RNA was quantified by Ultraspec spectrophotometer (Ultraspec 2100 pro; Amersham Biosciences Europe GmbH, France). RT-PCR was
performed in triplicate using SuperScriptTM One-Step RT-PCR with
Platinum® Taq kit according to the manufacturer’s recom- mendations (Invitrogen, Carlsbad, CA). For cDNA synthesis, 100 ng of RNA served as template. RT-PCR (25 µL reaction volume) was
performed as follows: 50°C for 30 min; 94°C for 2 min; 35 cycles at 94°C for 45 s; annealing at 52°C for 45 s for toxS, toxR, and VPI; 72°C for 1 min; and a final extension at 72°C for 10 min. The annealing temperature for ace gene was 60°C. RT-PCR products (5
µL) were analyzed on 1% agarose gel stained with ethidium bromide (0.5 mg/mL) at 90 V for 1 h and viewed under ultraviolet transillumination. The amplification products were photographed and their sizes determined with 100 bp molecular size marker (Promega). Quantitative analysis of DNA bands was performed using imaging software (Gene Tools, Sygene, UK).



RESULTS

Virulence genes expression

PCR amplification of the eight V. cholerae virulence genes in investigated Vibrio strains showed that only V. alginolyticus (S1 and S3) and V. parahaemolyticus (S5 and S6) were positive for toxR and toxS genes. In addition, V. alginolyticus S3 and S4 were positive for VPI and ace genes, respectively.
Expression levels of detected genes before and after treatments with UVC irradiation were analyzed by semi- quantitative RT-PCR (Figure 1). After treatment, we observed a decrease in the expression level of toxS gene in V. parahaemolyticus (S6) isolated from the Calich estuary. In addition, the expression level of toxR gene was decreased in V. parahaemolyticus S6 but it was increased in V. alginolyticus S3. Further, the mRNA quantities of VPI and ace genes remained stable in V. alginolyticus strains S3 and S4, respectively.


DISCUSSION

The results developed in this work indicate wide dissemination among V. alginolyticus and V. parahaemolyticus of different V. cholerae virulence genes





4258 Afr. J. Microbiol. Res.



Table 1. PCR primers sele

Kết quả (Việt) 1:

African Journal of Microbiology Research Vol. 6(20), pp. 4256-4260, 30 May, 2012 Available online at http://www.academicjournals.org/AJMRDOI: 10.5897/AJMR11.651ISSN 1996-0808 ©2012 Academic JournalsFull Length Research PaperEffect of Ultraviolet-C (UV-C) irradiation on the virulence genes expression in Vibrio parahaemolyticus and Vibrio alginolyticusRihab Lagha1#, Fethi Ben Abdallah1, 2*#, Ali Ellafi1, Héla Kallel2 and Amina Bakhrouf11Laboratoire d’Analyse, Traitement et Valorisation des Polluants de l’Environnement et des Produits, Faculté de Pharmacie Rue Avicenne. Monastir 5000, Tunisie.2Unité de Fermentation et de Développement de Vaccins Virologiques, Institut Pasteur de Tunis.13 place Pasteur, 1002, Tunisie.Accepted 27 December, 2011In this study, Vibrio parahaemolyticus and Vibrio alginolyticus, marine foodborne pathogens, were treated with Ultraviolet-C (UV-C) irradiation (240 J.m-2) to evaluate alterations in their virulence genes expression levels. Firstly, we searched for the presence of eight Vibrio cholerae virulence genes, toxR,toxS, toxRS, ctxA, zot, ace, toxT, and virulence pathogenicity island (VPI), in the genome of investigated strains. The expression of toxR and toxS genes in UVC-irradiated bacteria, studied by reverse transcriptase polymerase chain reaction, was found to be altered. These variations were manifested by an increase or a decrease in the expression level of tested virulence genes. Further, the mRNA quantities of VPI and ace genes remained stable after treatment.Từ khóa: Vibrio, tia cực tím-C (UV-C), thay đổi, biểu hiện gen virulence, RT-Đảng Cộng sản Romania.GIỚI THIỆU Bức xạ tia cực tím-C (UV-C) đã được đề xuất là một trong những cách thức thành công khử trùng để xử lý nước. Vì vậy, UV-khử trùng đã trở thành một giải pháp thực tế cho an toàn khử trùng nước (Said và ctv., 2010). Hiệu quả của UV ánh sáng trong sinh học inacti-vation phát sinh chủ yếu từ thực tế rằng Deoxyribonucleic acid (DNA) phân tử hấp thụ tia UV photon giữa 200 và 300 nm, với đỉnh hấp thụ tại 254 nm (Jeffrey và ctv., 1990). Hấp thu này tạo ra thiệt hại trong DNA bằng cách thay đổi các nucleotide cơ sở ghép nối, do đó việc tạo ra mối liên kết mới giữa liền kề nucleotide trên chuỗi ADN cùng. Thiệt hại này xảy ra đặc biệt là giữa pyrimidine căn cứ. Hai loại mutagenic tổn thương trong DNA đã được xác định: cyclobutan pyrimidine dimer (CPD) hình thành giữa các vị trí C-4 và C-5 của liền kề * Tác giả tương ứng. E-mail: [e-mail]. Điện thoại: + 21 6 73 466 244. Số Fax: + 216 73 461 830.# Các tác giả đã đều góp phần vào công việc này thymidine or cytosine residues, and pyrimidine (6 to 4) pyrimidone (6 to 4) photoproducts formed between the C6 and C4 position of adjacent pyrimidine residues, most often between T-C and C-C residues (Zimmer and Slawson, 2002). UV radiation in the range of 250 to 260 nm is lethal to most micro-organisms, including bacteria, viruses, protozoa, mycelial fungi, yeasts and algae. Among the bacteria, V. alginolyticus and V. parahaemolyticus, marine foodborne pathogen, frequently involved in epizootic outbreaks in cultured gilt- head sea bream and sea bass, causing fish mortality in Tunisian aquaculture farms (Abdallah et al., 2009a). In marine environment, V. alginolyticus and V. parahaemolyticus were considered as an important reservoir of Vibrio cholerae virulence genes (Abdallah et al., 2009b). According to Boyd et al. (2000) these genes may be horizontally transferred to V. alginolyticus in an aquatic environment. Indeed, the mobility of virulence genes may cause the transformation of non pathogenic strain to pathogenic strain. Xie et al. (2005) reported thatV. alginolyticus often possess homologues of the V. parahaemolyticus and V. cholerae virulence genes such Abdallah et al. 4257 as toxR, tlh and VPI, suggesting that V. alginolyticus may be an important reservoir of many known virulence genes of other Vibrio species in the aquatic environment. It is probably that the aquatic environment harbours different virulence-associated genes scattered among environmental Vibrios.In this work we searched, by polymerase chain reaction (PCR), for the presence of eight V. cholerae virulence genes, toxR, toxS, toxRS, ctxA, zot, ace, toxT, and virulence pathogenicity island (VPI), in the genome of V. alginolyticus and V. parahaemolyticus strains. The expression level of transferred virulence genes under UVC irradiation was achieved by reverse transcriptase polymerase chain reaction (RT-PCR).


MATERIALS AND METHODS

Bacterial strains

Six Vibrio strains were used in this study including three reference strains: V. alginolyticus ATCC 33787 (S1), V. alginolyticus ATCC 17749 (S2), and V. parahaemolyticus ATCC 17802 (S5). In addition, V. parahaemolyticus strain (S6), isolated from the Calich estuary (Alghero, Italy), and two V. alginolyticus strains (S3 and S4) isolated, respectively from the internal organs of aquacultured diseased gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax), in Tunisian aquaculture farm (Abdallah et al., 2009a), were included in this work.


UVC treatment

V. alginolyticus and V. parahaemolyticus were cultivated at 30°C in Tryptic soy broth 1% NaCl (TSB 1%) with shaking (150 rpm). The cultures of Vibrio strains grown to late log phase (OD600 = 0.6) were diluted and spread on Tryptic soy 1% NaCl (TSA 1%, Pronadisa, Spain) agar plates in glass Petri dishes. After 18 h of incubation at 30°C, bacterial colonies that appeared on the plates were exposed, in triplicate, to UV light according to the method described previously (Wang et al., 2004). The plates with even bacterial growth were covered with a piece of glass for non-UV treatment. The plates (covered and non-covered) were exposed to a 4-W UV lamp with a wavelength of 254 nm. The applied dose was 240
Joules m-2. After exposure, 250-ml Erlenmeyer flasks containing 100 ml of TSB 1% were inoculated with a loopful of colonies from
control and UV treated bacteria. All flasks were kept in at 30°C for 18 h with a shaking.



PCR detection of Vibrio cholerae virulence genes in V. alginolyticus and V. parahaemolyticus strains

Bacteria were cultured on TSA 1% for 24 h at 30°C. One colony was cultured in TSB 1% for 24 h at 30°C, and 1.5 mL was cen- trifuged. DNA was extracted using a Wizard genomic purification kit (Promega, Madison, WI) according to the manufacturer’s instructions. The primers of V. cholerae virulence genes used in this study (Sechi et al., 2000) are listed in Table 1. Polymerase chain reaction (PCR) were performed in 25 µL containing 50 ng of extracted DNA, 5 µL Green Go Taq buffer (5×), 0.25 µL of each deoxynucleoside triphosphate (10 mM), 0.5 µL MgCl2 (50 mM), 1
µL of each primer (25 pM), and 1U of Go Taq DNA polymerase
(Promega). Reaction mixtures were incubated for 5 min at 94°C; followed by 35 cycles at 94°C for 45 s; annealing at 52°C for 45 s

for toxS, toxR, and virulence pathogenicity island (VPI); 72°C for 1 min; and a final extension at 72°C for 10 min. The annealing temperature for the detection of the toxRS and toxT genes was 58°C, whereas for ctxA, ace, and zot the temperature was 60°C. PCR products (5 µL) were analyzed on 1% agarose gels stained with ethidium bromide (0.5 mg/mL) at 90 V for 1 h and viewed under ultraviolet transillumination. All PCR-positive strains, indicating the presence of the virulence genes, were confirmed by repeating the PCR three times.


RT-PCR for virulence gene expression

To study the level of expression of V. alginolyticus and V. parahaemolyticus virulence genes before and after UV irradiation, semi-quantitative RT-PCR method was used. RNA from control and irradiated cells was extracted by SV total RNA isolation system (Promega) according to the manufacturer’s instructions. RNA was quantified by Ultraspec spectrophotometer (Ultraspec 2100 pro; Amersham Biosciences Europe GmbH, France). RT-PCR was
performed in triplicate using SuperScriptTM One-Step RT-PCR with
Platinum® Taq kit according to the manufacturer’s recom- mendations (Invitrogen, Carlsbad, CA). For cDNA synthesis, 100 ng of RNA served as template. RT-PCR (25 µL reaction volume) was
performed as follows: 50°C for 30 min; 94°C for 2 min; 35 cycles at 94°C for 45 s; annealing at 52°C for 45 s for toxS, toxR, and VPI; 72°C for 1 min; and a final extension at 72°C for 10 min. The annealing temperature for ace gene was 60°C. RT-PCR products (5
µL) were analyzed on 1% agarose gel stained with ethidium bromide (0.5 mg/mL) at 90 V for 1 h and viewed under ultraviolet transillumination. The amplification products were photographed and their sizes determined with 100 bp molecular size marker (Promega). Quantitative analysis of DNA bands was performed using imaging software (Gene Tools, Sygene, UK).



RESULTS

Virulence genes expression

PCR amplification of the eight V. cholerae virulence genes in investigated Vibrio strains showed that only V. alginolyticus (S1 and S3) and V. parahaemolyticus (S5 and S6) were positive for toxR and toxS genes. In addition, V. alginolyticus S3 and S4 were positive for VPI and ace genes, respectively.
Expression levels of detected genes before and after treatments with UVC irradiation were analyzed by semi- quantitative RT-PCR (Figure 1). After treatment, we observed a decrease in the expression level of toxS gene in V. parahaemolyticus (S6) isolated from the Calich estuary. In addition, the expression level of toxR gene was decreased in V. parahaemolyticus S6 but it was increased in V. alginolyticus S3. Further, the mRNA quantities of VPI and ace genes remained stable in V. alginolyticus strains S3 and S4, respectively.


DISCUSSION

The results developed in this work indicate wide dissemination among V. alginolyticus and V. parahaemolyticus of different V. cholerae virulence genes





4258 Afr. J. Microbiol. Res.



Table 1. PCR primers sele