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J. Trop. Agric. and Fd. Sc. 30(1)(2002): 31–37 Characterization of Vibrio vulnificus isolated from retail cockle and
shrimp by plasmid profiling and antibiotic susceptibility test
(Pencirian Vibrio vulnificus yang dipencilkan daripada kerang dan udang dengan
profil plasmid dan ujian kerentanan antibiotik)
S. Radu*, T.A.F.T. Ahmad* and A.M. Sahilah** Key words: Vibrio vulnificus, shrimp, cockle, antibiotic resistance, plasmid Abstrak
Daripada 148 sampel kerang (Anadara granosa) dan 433 sampel udang (Paneus
indicus) yang dikaji, 27% dan 6.9% masing-masing didapati positif mengandungi
Vibrio vulnificus. Sebanyak 29 pencilan daripada kerang dan 21 pencilan daripada
udang dikaji untuk kerintangan terhadap antibiotik. Semua pencilan menunjukkan
kerintangan kepada satu atau lebih antibiotik yang diuji. Dalam ujian
transkonjugasi, tiada kaitan didapati antara plasmid dengan berat molikul yang
tinggi (35.8 Mda) yang dikesan di dalam beberapa pencilan, dengan fenotip
kerintangan. Ini menunjukkan kerintangan antibiotik adalah berasaskan
kromosom. Profil plasmid dan corak kerintangan antibiotik digunakan sebagai
pendekatan awal untuk mencirikan, pada tahap pencilan, kesemua pencilan
daripada kerang dan udang yang dikaji. Analisis corak kerintangan antibiotik
menunjukkan polimorfisma fenotipik yang tinggi. Walau bagaimanapun oleh
sebab banyak pencilan yang tidak ada plasmid, teknik ini kurang berguna dalam
pencirian. Keputusan ini menunjukkan bahawa pencilan V. vulnificus yang
mempunyai kerintangan pelbagai dan mempamerkan variasi genotip dan fenotip,
amat mudah diperoleh daripada kerang dan udang. Keadaan ini berpotensi
membahayakan kesihatan awam.
Abstract
Of the 148 cockle (Anadara granosa) and 433 shrimp (Paneus indicus) samples
examined, 27% and 6.9% were positive for Vibrio vulnificus, respectively.
Twenty-nine and 21 isolates from cockles and shrimps were examined for their
antibiotic resistance. All isolates showed resistance to one or more of the
antibiotics tested. In transconjugation tests, no relationship was found between
the high molecular weight plasmid (35.8 MDa) detected in several isolates and
their resistance phenotypes, indicating that their antibiotic resistance is
chromosomal. Plasmid profiles and antibiotic resistance patterns were used as a
preliminary approach to type, at strain level, the isolates from cockles and
shrimps. Analysis by antibiotic resistance patterns showed a high phenotypic
polymorphism. However, the high number of isolates devoid of plasmid rendered
this technique less useful. These results indicate that multiresistant V. vulnificus
*Department of Biotechnology, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia,43400 Serdang, Selangor, Malaysia**Strategic Resources Research Centre, MARDI Headquarters, Serdang, P.O. Box 12301, 50774 Kuala Lumpur,MalaysiaAuthors' full names: Son Radu, Tg Ahbrizal Farizal Tg Ahmad and Sahilah Abdul MutalibE-mail: [email protected] Agricultural Research and Development Institute 2002 Vibrio vulnificus in cockles and shrimps isolates exhibiting genotypic or phenotypic variations are easily recovered fromcockles and shrimps in the study area, posing a potential public health risk.
Introduction
Materials and methods
Vibrio vulnificus is a naturally occurring, Sample collection, isolation and
free-living inhabitant of brackish water and identification
salt water. It prefers tropical to subtropical climates and proliferates in areas or during months where the water temperature exceeds distilled water and the cockle shells were 1993; Hoi et al. 1998). Though the number of people infected with V. vulnificus is low illnesses, this bacterium has the ability to intravulvar fluid were collected in individual sterile stomacher bags. The samples (50 g) (Morris and Black 1985; Hlady 1997).
were enriched in 450 mL of alkaline peptone or undercooked seafood, especially shellfish, (Stomacher Lab-Blender 400). After 18 h of serially diluted with alkaline peptone water Klontz 1996). Most cases of V. vulnificus and 0.1 mL of 10–3, 10– 4, 10–5 and 10– 6 infections are sporadic and characterized by dilutions were plated onto thiosulfate citrate bile salts agar (TCBS) plates (Oxoid Ltd, include gastroenteritis, wound infection and incubated for 18–24 h at 37 °C. Five non- fermenting sucrose colonies (green coloured virulence factors of V. vulnificus has yet to colonies) per samples were transferred onto duplicate plates of TCBS agar to obtain pure molecular diversity of V. vulnificus strains should offer insight into their ecology and standard biochemical tests as described by epidemiology of V. vulnificus as a result ofavailability of various molecular techniques Antibiotic susceptibility tests
Disc diffusion tests were performed three Ryang et al. 1999), relatively little effort has Malaysia. Thus, in the study reported here, National Committee for Clinical Laboratory patterns and plasmid profiling were used to Standards (1997). All strains were tested characterize the V. vulnificus isolated from against 15 antibiotics: bacitracin (10 µg), cockles (Anadara granosa) and shrimps carbenicillin (100 µg), cefoperazone (75 µg), ceftazidime (30 µg), ceftriaxone (30 µg),cephalothin (30 µg), chloramphenicol(30 µg), erythromycin (15 µg), gentamicin(10 µg), nalidixic acid (30 µg), norfloxacin been found to harbour 1–7 plasmids with sizes ranging from 1.5 to 35.8 megadalton(MDa) (Table 1). Plasmid analysis grouped Plasmid DNA isolation
Plasmid DNA of the V. vulnificus strains was groups, with three isolates showing the same extracted three times from each isolate by the mini-preparation method of Sambrook et unique plasmid profiles. In genetic transfer electrophoresed in 0.7% agarose gel. The gel photographed under UV transillumination.
phenotypes or plasmid. Antibiotic resistance Plasmids of Escherichia coli V517 were patterns allowed division of the 50 isolates used to determine molecular weight of each plasmid (Macrina et al. 1978).
Conjugation tests
Discussion
Multiresistant V. vulnificus isolates Although occurrence of foodborne V. vulnificus infection has not been recognized in Malaysia, it has been widely recognized studies. The selected resistant isolates, as donors, were incubated overnight at 37 °C consumption of various V. vulnificus- with a nalidixic acid-resistant E. coli K12 as contaminated seafoods (Hlady 1997; Morena the recipient on nutrient agar plate. The positive for V. vulnificus is not surprising as Transconjugants were selected by plating the diluted mating mixtures (10–1 to 10– 4) on molluscs such as oysters, clams, mussels and nutrient agar plates containing 50 µg/mL of nalidixic acid and inhibiting concentration of agent to which the donor isolates had been numbers of positive shrimp positive (30/433)were small, shrimp as a primary source of V. vulnificus contamination cannot be ruled out. Therefore, more research is needed to positive for V. vulnificus, yielding 80 contamination with V. vulnificus.
only 30 were positive for V. vulnificus, Vibrio vulnificus includes two biotypes differences in biochemical property (Tison V. vulnificus were randomly selected from et al. 1982). In this study, V. vulnificus sources. Only biotype 1 has been associated showed the presence of both V. vulnificus biotype 1 and 2 from both sources (Table 1).
previously identified as only pathogenic for Isolates were resistant to one or more of the 15 antibiotics tested; exhibiting a wide range opportunistic infections in humans (Amaro of multiresistance distribution and resistance to as many as 12 antibiotics (Table 1).
Vibrio vulnificus in cockles and shrimps source of the contamination, additional tests V. vulnificus biotype 3, a newly identified virulent clone of V. vulnificus causing because of the possibility for sample-to- outbreak of wound infection and bacteremia sample transmission. However, the sources associated with exposure to pond-cultured of V. vulnificus and the routes of contamination of the product examined are system for V. vulnificus in Malaysia. The Vibrio vulnificus has been reported to ingrained tradition of consuming foods and chloramphenicol, aminoglycoside, and third- increase the risk of infection, especially generation cephalosporins (Tacket et al.
1984; Morris and Black 1985). These results situation is of special interest risk, since ran contrary to our finding of V. vulnificus there are certain practices that encourage (Table 1). Such resistance might be due to the indiscriminate use of these antibiotics.
survival of Vibrio spp. during heat-treatment Despite the fact that the usage patterns of of cockles and in street foods indicate their antibiotics in animals or humans in the study potential as sources of infection (Rusul et al.
area is still not clear, it cannot be disputed 1997; Liew et al. 1998). Regardless of the Table 1. Vibrio vulnificus isolates examined in this study aNumber in parenthesis indicate biotypes of the isolates. VC and VS denoteisolates from cockle and shrimp, respectivelybTested for bacitracin (B), carbenicillin (Cb), cefoperazone (Cfp),ceftazidime (Caz), ceftriaxone (Cro), cephalothin (Cf), chloramphenicol (Cm),erythromycin (Er), gentamicin (Gm), kanamycin (Km), nalidixic acid (Na),norfloxacin (Nor), penicillin (P), streptomycin (Sm) and tetracycline (Te) multiresistant V. vulnificus strains in cockles phenotypes in bacteria and usually the high prompts the need to evaluate their potential antimicrobial therapy would be required.
independent experiments the five isolates Multiresistance to antibiotics is therefore a transconjugation, an observation indicating epidemiological interest, but of overriding that resistance phenotypes of these isolates concern is the efficacy of these antibiotics in the treatment of multiresistant V. vulnificus infections. Every source of resistance must more than one genotype or phenotype could safeguard the efficiency of antibiotics in the resistance patterns. It is also worth noting examined shared some related genotypes or phenotypes. Jackson et al. (1997) reported weight plasmid of 35.8 MDa were tested for on the V. vulnificus infections caused by their abilities in transconjugation. In general, Vibrio vulnificus in cockles and shrimps populations in shellfish. Therefore, multiple Acknowledgement
isolates from a single sample type should be This study was supported by the Malaysian typed in epidemiological and contamination Government through the Intensification of Research in Priority Areas (IRPA) grant. The authors gratefully acknowledge Dora Bulan V. vulnificus strains tested, the present results in the preparation of the manuscript.
indicate that there were only a limitednumber of isolates recovered from cockles References
and shrimps sharing the same genotypes or Amaro, C., Biosca, E.G., Esteve, C., Fonz, B. and phenotypes (Table 1) and they were Toranzo, A.E. (1992). Comparative study of phenotypic and virulence properties of V.
vulnificus biotypes 1 and 2 obtained from a European eel farm experiencing mortalities.
could not be excluded. Looking through the Dis. Aqua. Organ. 13: 29–35
antibiotic resistance patterns and plasmid Arias, C.R., Verdonck, L., Swings, J., Aznar, R. and profiles, it was observed that there were Garay, E. (1997). A polyphasic approach to study the intraspecific diversity amongst Vibrio vulnificus isolates. Syst. Appl.
Microbiol. 20: 622–33
Austin, B. and Austin, D.A. (1999). Bacterial fish similar plasmid profiles, but had different pathogens: disease of farmed and wild fish.
antibiotic resistance patterns. These results suggested that antibiotic resistance pattern is Bisharat, N., Agmon, V., Finkelstein, R., Raz, R., not influenced by the presence of plasmids, Colodner, R., Cameron, D.N., Wykstra, D.L.,Swerdlow, D.L., Farmer III, J.J. and for the Israeli Vibrio Study Group. (1999). Clinical, resistant phenotypes of the isolates tested epidemiological and microbiological features of Vibrio vulnificus biogroup 3 causing carefully the typing technique used as they bacteraemia in Israel. The Lancet 354:
could provide a different insight into the Hlady, W.G. (1997). Vibrio infections associated with raw oyster consumption in Florida,
1981–1994. J. Food Prot. 60: 353–7
Conclusion
epidemiology of vibrio infections in Florida, characterization of V. vulnificus strain using 1981–1993. J. Infect. Dis. 173: 1176– 83
phenotypic (antibiotic resistance patterns) Hoi, L., Larsen, J.L., Dalsgaard, I. and Dalsgaard, A. (1998). Occurrence of Vibrio vulnificus biotypes in Danish marine environments.
Appl. Environ. Microbiol. 64: 7–13
methods to differentiate V. vulnificus of the Jackson, J.K., Murphree, R.L. and Tamplin, M.L.
same or different biotypes. The strains from (1997). Evidence that mortality from Vibrio vulnificus infection results from single strains which allowed for a simple determination of among heterogeneous population in shellfish.
J. Clin. Microbiol. 35: 2098 –101
inter- and intra-biotype genetic differences.
Kasper, C.W. and Tamplin, M.L. (1993). Effects of The possibility of using antibiotic resistance temperature and salinity on the survival of patterns, and to a less extent the plasmid Vibrio vulnificus in seawater and shellfish.
profiles as an adjunct, for differentiation of Appl. Environ. Microbiol. 59: 2425 –9
V. vulnificus strains can provide knowledge Liew, W.S., Leisner, J.J., Rusul, G., Son, R. and of their diversity in epidemiological studies.
Rasip, A. (1998). Survival of Vibrio spp.
including inoculated V. cholerae O139 during
heat-treatment of cockles (Anadara granosa).
Internat. J. Food Microbiol. 42: 167–73
Macrina, F.L., Kopecko, D.J., Jones, K.R., Ayers, Sambrook, J., Fritsch, E.F. and Maniatis, M. (1989).
D.J. and McCowen, S.M. (1978). A multiple- Molecular cloning: a laboratory manual. 2nd plasmid-containing Escherichia coli strain: convenient source of size reference plasmid molecules. Plasmid 1: 417–20
Strom, M.S. and Paranjpye, R.N. (2000).
Morena, M.L. and Landgraf, M. (1998). Virulence Epidemiology and pathogenesis of Vibrio factors and pathogenicity of Vibrio vulnificus vulnificus. Microb. Infect. 2: 177– 88
strains isolated from seafood. J. Appl. Tacket, C.O., Breener, F. and Black, P.A. (1984).
Microbiol. 84: 747–51
Clinical features and an epidemiological study Morris, Jr. J.G. and Black, R.E. (1985). Cholera and of Vibrio vulnificus. J. Infect. Dis. 149:
other vibrioses in the United States. New Engl. J. Med. 312: 343–50
Tison, D.L., Nishibuchi, M., Greenwood, J.D. and National Committee for Clinical Laboratory Seidler, R.J. (1982). Vibrio vulnificus Standards. (1997). Approved standards biogroup 2: a new biogroup pathogenic for M2–A6. Performance standards for eels. Appl. Environ. Microbiol. 44: 640 – 6
antimicrobial disc susceptibility tests. 6th ed.
Veenstra, P.J., Rietra, J.P.G.M., Stoutenbeck, C.P., Coster, J.M., De Hier, H.H.W. and Dirks-Go, Oliver, D. (1989). Vibrio vulnificus. In: Foodborne S. (1992). Infection by an indole-negative bacterial pathogens. p. 569– 99. (Doyle, M.P., variants of Vibrio vulnificus transmitted by eels. J. Infect. Dis. 16: 209 –10
Rusul, G., Chun, C.K. and Son, R. (1997). Survival Vickery, M.C.L., Smith, A.L., DePaola, A., Jones, and growth of Vibrio cholerae O139 in D.D., Steffan, R.J. and Bej, A.K. (1998).
selected Malaysian street foods. J. Food Prot. 60: 644 – 8
Ryang, D.W., Koo, S.B., Shin, M.G., Shin, J.H. and intra-species differentiation of Vibrio Suh, S.P. (1999). Molecular typing of Vibrio vulnificus. J. Microbiol. Meth. 33: 181– 9
vulnificus isolated from clinical specimens by
pulsed-field gel electrophoresis and random
amplified polymorphic DNA analysis.
Japanese J. Infect. Dis. 52: 38 – 41
Accepted for publication on 20 February 2002

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