Annals of Clinical & Laboratory Science 37:330-334 (2007)
© 2007 Association of Clinical Scientists
A Chromogenic Substrate Culture Plate for Early Identification of Vibrio vulnificus and Isolation of Other Marine Vibrios
Yukari Nakashima1,
Megumi Oho1,
Kouji Kusaba1,
Zenzo Nagasawa1,
Osamu Komatsu2,
Isao Manome2,
Kazukuni Araki3,
Hirotaka Oishi4 and
Mikio Nakashima3
1 Department of Clinical Laboratory Medicine, Saga University Hospital, Saga; 2 Biochemical Research Laboratory, Eiken Chemical Co., Ltd., Tokyo; 3 Department of Anesthesiology and Critical Care Medicine, Saga University Faculty of Medicine, Saga; 4 Ariake Sea Research Project, Saga University, Saga, Japan
Address correspondence to Hirotaka Oishi, M.D., Ph.D., Ariake Sea Research Project, Saga University, 5-1-1, Nabeshima, Saga City, Saga, 849-8501, Japan; tel 81 952 34 2373; fax 81 952 34 2056; e-mail ooishih{at}cc.saga-u.ac.jp.
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Abstract
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Vibrio vulnificus infection can result in necrotizing fasciitis and sepsis, which have short latentcy periods and high mortality rates. Thus, an easy and quick detection method is needed to improve the outcome. To distinguish V. vulnificus from other pathogens that cause necrotizing fasciitis, we developed a selective isolation culture agar plate (Chromochecker Vibrio Agar-1; CVA-1) for use in environmental monitoring and in the clinical setting. One hundred four strains of V. vulnificus, already identified biochemically, showed typical colony form and color when grown on CVA-1. Thirty-six of 51 marine bacteria samples suspected to be V. vulnificus on CVA-1 were subsequently identified as V. vulnificus by a biochemical identification system. Of 8 bacteria known to cause necrotizing fasciitis, only V. vulnificus grew on CVA-1. In addition, growth on CVA-1 allowed ready differentiation of Vibrio species. CVA-1 can be used to distinguish pathogenic Vibrios according to colony form and chromatic differences.
Keywords: Vibrio vulnificus, chromogenic substrate culture plate, necrotizing fasciitis, sepsis
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Introduction
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Vibrio vulnificus, categorized in 1979 by Farmer [1], is a Gram-negative rod bacterium associated with septicemia and necrotizing fasciitis in humans. Infection occurs mainly through ingestion of raw seafood. Individuals with underlying liver disease or immunodeficiency are particularly susceptible to this life-threatening infection. Klontz et al [2] reported a mortality rate of 33% in cases of V. vulnificus infection with treatment initiated within 24 hr. The mortality rate is 100% in cases without treatment or treatment initiated after 72 hr. Thus, early identification of the bacterial species is needed for the treatment of this type of infection.
Fatal V. vulnificus infection is relatively rare, but many cases of this infection have been reported since 1980 in the southwestern prefectures of Japan. In a previous study, we found that approximately 40% of patients in Japan were reported from hospitals around the Ariake Sea [3]. In particular, the Saga prefecture (located near the deepest part of the Ariake Sea) was the location of more than 30 patients. A quick and easy isolation method that can be used to detect V. vulnificus bacteria in various environments, such as in marine waters and in seafood, is necessary.
We report the development of a modified agar plate containing a chromogenic substrate for the identification of Vibrios. This culture agar plate (Chromochecker Vibrio Agar-1; CVA-1) can readily distinguish between pathogenic Vibrios, including V. vulnificus, according to colony characteristics and chromatic differences.
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Materials and Methods
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The CVA-1 medium (Eiken Chemical Co., Ltd., Tokyo, Japan) contained the following: 0.12 g chromogenic substrate, 10 g peptone, 3 g yeast extract, 15 g NaCl, 8 g Na3C6H5O7, 40 g sucrose, 6 g bile salt, and 13 g agar in 1 L distilled water. The medium was adjusted to pH 8.8, heated to boiling, and poured into Petri plates.
Standard culture methods were used. Bacterial medium concentration was adjusted to McFarland No. 1. Plated bacteria were cultured for 18 – 24 hr at 35 °C under aerobic conditions. Results were assessed according to colony growth, color, and form. All isolates were identified by biochemical property using the VITEK II automated system (Japan bioMerieux, Tokyo, Japan) or the Phoenix automated system (Nippon Becton Dickinson, Tokyo, Japan).
To validate the performance of CVA-1 for the identification of V. vulnificus, we compared CVA-1 results with those of the biochemical identification system. The determination of the color and form of colonies on CVA-1 was performed by one medical technologist. One hundred four strains of V. vulnificus (22 from clinical sources, 2 from fish, 31 from oyster shells, and 49 from seawater), which had previously ben identified by the biochemical identification system, were inoculated onto CVA-1.
We also investigated bacterial samples from seawater in the Ariake Sea from July to March. Of 207 samples that formed colonies on CVA-1, 51 suspected to be V. vulnificus were identified by the biochemical identification system.
To determine the ability of CVA-1 to differentiate bacteria that cause necrotizing fasciitis, the following 8 strains were examined: V. vulnificus, Haemophilus influenzae, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Aeromonas hydrophila, and Streptococcus pneumoniae. V. vulnificus and the seven strains of non-Vibrio bacteria were obtained from patients with primary septicemia and necrotizing fasciitis at our hospital. We compared colony growth and color in 3 agar culture plates (CVA-1, blood agar, and chocolate agar). All of the plates were purchased from Eiken Chemical Co., Ltd. In addition, in order to examine the usefulness of the CVA-1 plate in the clinical setting, we used this plate in hospitals to identify bacteria in patients with necrotizing fasciitis.
We also examined the growth of Vibrio species on 3 different culture plates for Vibrio detection. We examined colony growth and color on CVA-1, thiosulfate, citrate, bile salts, and sucrose (TCBS) agar (Eiken Chemical Co., Ltd), and CHROM agar Vibrio (Kanto Chemical Co., Tokyo, Japan). The following 8 Vibrio species were examined: V. vulnificus (Eiken catalog no. EKN-6543), V. parahaemolyticus (EKN-234), V. alginolyticus (EKN-Toh2), V. cholerae (EKN-313), V. mimicus (EKN-915), and V. fluvialis (EKN-914).
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Results
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V. vulnificus formed flat colonies on CVA-1 agar culture plates (Fig. 1
). Navy blue plaques with bright halos were observed.
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Fig. 1. Growth of Vibrio vulnificus on CVA-1. Colonies show a flat form and navy blue plaques with bright halo.
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Performance of CVA-1.
One hundred four V. vulnificus isolates, which had previously been identified by the biochemical identification system, were examined on CVA-1 plates. All samples showed typical colony form and color.
Fifty-one isolates of suspected V. vulnificus grown on CVA-1 were subsequently identified with the biochemical identification system. Thirty-six of the colonies (70.6%) were identified as V. vulnificus. The other 15 samples were identified as Aeromonas salmonicida (n = 3), Pseudomonas stutzeri (n = 1), Photobacterium damselae (n = 1), Sphingomonas paucimobilis (n = 9), or V. parahaemolyticus (n = 1).
Identification of bacteria causing necrotizing fasciitis.
Eight bacterial strains that cause necrotizing fasciitis were cultured on blood agar (Fig. 2A), chocolate agar (Fig. 2B), and CVA-1 (Fig. 2C). All of the strains, with the exception of H. influenzae, grew on blood agar, and all of the strains grew on chocolate agar. However, only V. vulnificus grew on CVA-1.
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Fig. 2. Growth of 8 types of bacteria that cause necrotizing fasciitis on blood agar (A), chocolate agar (B), and CVA-1 (C). Only Vibrio vulnificus grew on CVA-1.
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Usefulness in the clinical setting.
A 73-yr-old Japanese man was admitted to the hospital with skin eruptions and severe pain in the lower legs. The patient had a history of alcoholic liver cirrhosis and surgery for esophageal cancer. On the day before he developed leg symptoms, the patient had consumed raw oysters at home. The next day, he began to experience severe pain and edema of the lower legs. Throughout the day, his symptoms worsened, and he visited his family physician. He was admitted to the hospital at 9 pm. V. vulnificus infection was suspected due to his underlying liver disease and recent consumption of shellfish. Samples of the skin lesions were obtained for diagnosis and inoculated onto CVA-1 plates at 10 pm. At 8:30 am on the next day (12 hr after hospital admission), flat navy blue colonies were evident on CVA-1. The isolate was identified as V. vulnificus by the biochemical identification system 48 hr after hospital admission.
Growth of Vibrio species on different culture plates.
Eight Vibrio species were cultured on CVA-1, TCBS agar, and CHROM agar Vibrio plates (Fig. 3). Colony size was similar on the 3 types of plates. On CVA-1, the colors and forms were as follows: V. vulnificus, navy blue and flat; V. parahaemolyticus, light blue and mucoid; V. cholerae, brown and flat; V. alginolyticus, yellow and mucoid; V. mimicus, light blue and flat; and V. fluvialis, light yellow and flat. Each Vibrio species was readily differentiated from the other Vibrio strains on CVA-1.
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Fig. 3. Growth of eight strains of pathogenic Vibrio spp. on CVA-1, TCBS agar, and CHROM agar Vibrio. Each Vibrio spp. is differentiated from the other Vibrio strains on CVA-1.
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Discussion
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We developed CVA-1 as a novel agar culture plate for the detection of V. vulnificus, which causes food-borne infection through exposure of persons to seawater and fishery products. Our results demonstrated that all of the V. vulnificus samples, which had previously been identified by the biochemical identification system, showed typical colony form and color on CVA-1. On the other hand, 70.6% of colonies suspected to be V. vulnificus on CVA-1 were identified as V. vulnificus by the biochemical identification system. Thus, V. vulnificus could be identified on CVA-1 with high sensitivity and specificity.
Necrotizing fasciitis was first described as a life-threatening infection by Meleney in 1924 [4]. Progression, particularly in cases with V. vulnificus infection, is rapid, and the mortality rate is extremely high [5,6]. To decrease morbidity and mortality associated with severe soft-tissue damage in patients with compromised conditions, preventive measures should be taken, for example, education of high-risk individuals. In addition, early identification of the disease, aggressive and appropriate antibiotic therapy, aggressive debridement, and support care are necessary. One surgical option for the treatment of necrotizing fasciitis is amputation. However, amputation in the absence of identification of the pathogen would be inappropriate.
Our results show that CVA-1 can be used to identify V. vulnificus among several bacteria species that cause necrotizing fasciitis. In the present case of necrotizing fasciitis, V. vulnificus was identified on CVA-1 approximately 12 hr after hospital admission. Biochemical identification with systems, such as the VITEK II or Phoenix system after agar plate culture and colony isolation, is the standard method for the identification of bacterial species. However, this method requires 48 hr or longer.
Another advantage of CVA-1 is the detection of pathogenic Vibrio spp. We found that Vibrio spp. grow on CVA-1 as well as other commercial culture plates and can be readily differentiated from each other. V. parahaemolyticus is one of the most important causative organisms of food poisoning in Japan, and V. cholerae is an important pathogen in the tropical world, particularly in Southeast Asia, India, Bangladesh, and Africa. Although definitive identification requires a biochemical identification system or genetic screening, CVA-1 can provide rapid first-pass screening and does not require expensive laboratory equipment or complicated experimental procedures. CVA-1 is useful not only for the detection of V. vulnificus but also for other Vibrio species. We were able to detect the presence of pathogenic Vibrios in seawater with CVA-1. This has resulted in the distribution of CVA-1 to medical institutions in an attempt to prevent future outbreaks.
In conclusion, we have described a rapid and easy -to-use culture plate system for the identification of Vibrio spp. CVA-1 plates may be useful in various scientific applications, such as marine environmental monitoring, food hygiene, early clinical diagnosis in hospitals, and in preventive medicine, particularly in developing countries.
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Acknowledgment
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We thank Ms. Miwako Isoda, St. Mary’s Hospital, for helpful suggestions.
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References
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- Farmer JJ 3rd. Vibrio ("Beneckea") vulnificus, the bacterium associated with sepsis, septicaemia, and the sea. Lancet 1979;2:903.[Medline]
- Klontz KC, Lieb S, Schreiber M, Janowski HT, Baldy LM, Gunn RA. Syndromes of Vibrio vulnificus infections. Clinical and epidemiologic features in Florida cases, 1981–1987. Ann Intern Med 1988;109:318–323.[Abstract/Free Full Text]
- Oishi H, Ura Y, Mitsumizo S, Nakashima M. A collective review of Vibrio vulnificus infection in Japan (in Japanese). Kansenshogaku Zasshi 2006;80:680–689.[Medline]
- Meleney F. Hemolytic Streptococcus gangrene. Arch Surg 1924;9:31.
- Blake PA, Merson MH, Weaver RE, Hollis DG, Heublein PC. Disease caused by a marine Vibrio: clinical characteristics and epidemiology. NEJM 1979;300:1–5.[Medline]
- Hollis DG, Weaver RE, Baker CN, Thornsberry C. Halophilic Vibrio species isolated from blood cultures. J Clin Microbiol 1976;3:425–431.[Abstract/Free Full Text]
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Abstract
Introduction
