- Email: [email protected]
Quantification of pathogenic marine vibrio using membrane filter technique
JOURNAlOF ELSEVIER
Journal of Microbiological Methods 21 (1995) 143-149
Quantification of pathogenic marine vibrio using membrane filter technique Jorge
Hernhndez-Lopez, Francisco
Ma. Antonia Guzman-Murillo, Vargas-Albores*
Center for Biological Research, Department of Marine Pathology, P.O. Box 128, La Paz, BCS 23000, M&co
Received 5 May 1994; accepted 30 May 1994
Abstract
A rapid and simple methodology based on a membrane filter technique was designed to detect and quantify marine vibrios involved in fish and shellfish diseases, as well as human gastrointestinal disorders. By placing the membrane in TCBS medium and incubating it at 40-4’232, it was possible to differentiate between pathogenic and non-pathogenic vibrios. A presumptive identification was also done by growing cultures at different salinities and by using a few biochemical tests. Keywords:
1.
Bacteria quantification;
Pathogenic
vibrio; Marine vibrio
Introduction
Diseases caused by Vibrio spp., and to a lesser extent other Gram-negative bacilli, are important to marine fish and shellfish farming worldwide. Vibrio epidemics are a frequent problem in the culture of shrimp [l-5] were epizootic disease has caused over 90% mortality [6]. During 1984 in Japan, production loss caused by vibriosis, was valued at 231.4 million yen [7]. The principal bacteria identified in epidemic diseases, of marine fishes and shellfishes are Vibrio anguillarum, V. alginolyticus, V. Jluvialis, V. furnissii, V. harveyi, V. parahaemolyticus, V. vulnificus, Vibrio sp., Aeromonas sp. and Pseudomonas sp. These bacteria are also important to public health. Besides V. cholerae 01, *Corresponding author. [email protected].
Tel.:
+52 (112) 536-33 Ext 759. Fax:
0167-7012/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDZ 0167-7012(94)00042-6
+52
(112)
547-10.
e-mail:
144
.I. Hernrindez-Ldpez
et ul. i Journal
of Microhiologlcal
Methods 21 (iW5)
143-149
several vibrio species are capable of producing gastrointestinal disorders in humans who have ingested contaminated fish and shellfish [S-12]. The most frequently isolated vibrios from patients with gastroenteric disorders are: V. parahaemolyticus, V. furnissii, V. alginolyticus. V. fluvialis, V. cholerae, non-01, V. mimicus and V. vulni’cus. Thus, the list of pathogenic vibrios for fishes, shellfishes and human is the same and contains from 6 to 8 species. Interestingly, bacterial species recovered from diseased organisms have also been isolated from the body of healthy organisms [13-151 as well as from the water and sediment in hatcheries and ponds [1,16]. These reports agree with the wide distribution of Vibrio in marine environments, and give evidence to their opportunism. Except for V. cholerae in humans, infections by pathogenic vibrios are dependent on the animal’s physiological condition, and are highly influenced by the environment. To obtain a rapid and simple methodology for the detection and quantification of marine vibrio involved in diseased fish and shellfish and human gastrointestinal disorders, we implemented a procedure based on a membrane filter technique. Two criteria were used to differentiate between pathogenic and non-pathogenic vibrios: the capability for growth at 40-42°C and the tolerance to high NaCl concentrations.
2. Materials 2.1.
and methods
Culture media
TCBS agar (thiosulfate citrate bile sucrose, Difco) was prepared according to the manufacturer’s instructions. Petri dishes (10 x 60 mm) for membrane filtration were filled with 5 ml of TCBS medium; for the plate count, 12 ml were placed on 10 x 100 mm plates. For other culture media, 96-well plates (Nunc) with 200 ~1 per well were used. To determine salinity tolerance, 1% tryptone with different NaCl concentrations (0, 3, 6, 8 and 10% w/v) were prepared according to [17]. Moller base medium for ornithine and lysine decarboxylation, base medium for carbohydrate fermentation and MR/VP medium were modified by adding 3% NaCl. To establish the feasibility of using membrane filtration with TCBS as culture medium, different dilutions of V. parahaemolyticus (ATCC 17802) were analyzed and compared with the plate count technique. The pre-inoculum was prepared in marine broth by incubating for 24 h at 30°C and 150 rpm. One ml (ASsOn,,,= 1.0) of the inoculum was placed in 25 ml of marine broth and incubated under the same conditions for 18 h. Different dilutions were prepared, and 0.1 ml aliquots were used for quantification in both the plate count and membrane filter techniques. The plates were incubated at 35°C for 7 and 18 h periods for membrane filter and plate count methods, respectively.
J. Hermindez-Ldpez
2.2.
et ai. I Journal of Microbiological
Methoak 21 (1995) 143-149
145
Samples
Marine water was collected from a shrimp culture tile pond in sterile 250 ml flasks, and bacteriological analyses were done 30 min thereafter. To aliquots of 0.1 or 1 ml, sterile artificial seawater was added to obtain a final volume of 10 ml. In other cases, 10 ml aliquots were used. The samples were filtered under negative pressure using 47 mm diameter membranes with 0.45 pm pores (Nalgene, Cat. no. 2054045). The membranes were placed in 60 X 15 mm Petri dishes containing TCBS agar. These plates were incubated at 35.5”C or 41.5”C for 7 h, before determining the number of colony forming units (CFU). Only plates with 20-200 CFU were considered. 2.3. Identification Colonies from each TCBS plates were placed in 200 ~1 marine broth and incubated for 24 h at 35.5”C. From these cultures, 10 ~1 aliquots were used for the following biochemical tests: ornithine and lysine decarboxylation, gas production from glucose, Voges-Proskauer, NaCl request or tolerance, oxidase production, and Gram stain. The oxidase production was determined by putting a drop of culture on a paper filter (Whatman No. 1) and adding one drop of Kovacs reagent (1% w/v p-aminedimethylalanine oxalate, in water). All cultures for biochemical testing were incubated at 35.5”C. Cultures in tryptone, Moller medium for ornithine and lysine decarboxylation, and base medium for glucose fermentation were recorded at 7 and 18 h, while those for the Voges-Proskauer were read at 24 h.
3. Results and discussion Using different dilutions of V. parahaemolyticus (ATCC 17802), the plate count and membrane filter techniques were compared. The data in Table 1 shows that values obtained with the membrane filter were higher (30-50%) than those obtained by the plate count method, however a correlation between both techniques could be established. Based on these results, we believe the membrane Table 1 The number of bacteria in different dilutions of V. parahaemolyticus culture was determined by plate count and membrane filter methods; in both cases the plates were incubated at 35S”C during 24 h. Mean of triulicates and SD are given Sample
Plate count
Membrane
A B C
405 2 12 140 r 4 59 2 10
580 2 16 2OO”lo 120 t 3
filter
146
J. Hernrindez-Ldpez
et ul. I Journul of Microhiologicul
Methods 31 (1995)
14-1-W
filter using TCBS agar as a culture medium is a precise and reliable technique for vibrio quantification. Bacterial recovery from sea water was also successful using the membrane filter and selective medium, TCBS. Considering the abundance of Vibrio in sea water, it was necessary to determine the adequate sample size in order to obtain from 20 to 200 CFU per filter. The vibrio counts varied depending on the incubation temperatures used, thereby permitting us select the sample size (Table 2). In our experiments, 0.1 ml was enough for a total vibrio count (which can be determined vibrios (incubated at 40-42”C), by incubating at 3.5-36”C), while for pathogen volumes from 1 to 10 ml were necessary. Obviously, different dilutions could be tested depending on the sample. During this study, several water samples from a tile pond were analyzed. The number of vibrio varied from 92 to 1680 bacteria/ml and from 2 to 2.5 bacteria/ml for total and pathogenic vibrio, respectively. Correlation between the number of total and pathogenic vibrio was not found, indicating that both kinds of bacteria are distributed independently and that an increase in total vibrios does not necessarily increase the pathogenic vibrio count. On the other hand, the count of V. parahaemolyticus. which is considered pathogenic for human and aquatic organisms, was not affected by the incubation temperature. When different dilutions were filtered and incubated at 35.5 and 41.5”C in triplicate, similar counts were found (34 t 2, 30 + 8; 38? 16, 28 + 13; 50 2 21, 30 + 12), and no significant difference was established by Student’s t-test. This data partially supports the idea that incubation temperature can be used to discriminate non-pathogenic vibrios. As shown in Table 3, pathogenic vibrios are more tolerant to temperature and salinities. They can grow at 40-42°C in TCBS, and in salinities higher than 3% [18,19]. If the membrane after filtration is incubated at 35 or 41”C, it is possible to observe a significant difference in the number of CFUs (Table 2). Thus, pathogenic vibrios can be selected by their tolerance for incubation at 415°C. The characteristic vibrio colonies are visible at 5 h, at both 35 and 41”C, but the colonies are clearer and more defined at 7 h. For the identification of vibrios growing in TCBS bacteria, each colony was placed in marine broth and incubated at 35°C for 12-16 h. From these cultures,
Table 1 Different counts of Vibrro found m marine water depending sample size can be determined from these data Sample
size
Incubation
temperature
(ml) 35.5”C
IN, innumerable
41 .SY
on incubation
temperature.
The adequate
J. Hermindez-L6pez
et al. I Journal of Microbiological
Methods 21 (1995) 143-149
147
Table 3 Vibrios most referred as provoking diseases in culture fishes and shellfishes: the pathogen vibrios are more tolerant to temoerature and salinities Growth at 40-42°C V. V. V. V. V. V. V. V. V.
Growth at NaCl5-10%
alginolyticus cholerae 01 fluvialis furnissii harveyi parahaemolyticus vulnificus anguillarun fisheri
we performed the following tests: oxidase production, NaCl tolerance, amino acids decarboxylation and glucose utilization. Adequate growths were obtained using 200 ~1 of culture medium and 10 ~1 inoculum on 96-well plates. All vibrios isolated during this experiment were oxidase positive and required NaCl for growth. Although V. cholerae and some strains of V. anguillarum, V. fluvialis, V. furnissii and V. metschnikovii are able to grow in the absence of NaCl [18], they were apparently not present in the pond water. The isolates from the membrane incubated at 41°C and requiring NaCl for growth represent the most important group of non-cholerae pathogenic vibrios: V. alginolyticus, V. fluvialis, V. furnissii, V. harvegy, V. parahaemolyticus and v. vulnificus. From this list, only two vibrios (V. fluvia1i.s and V. furnissii) are ODC-negative, although they show many variations of salt tolerance. By this, we felt that ODC was the best test to discriminate both species. On the other hand, V. furnissii produces gas from glucose utilization, while V. fluvialis does not [19]. Thus, based on ODC-negative reaction and gas production, it was possible to make a presumptive identification of these two bacteria. For the rest, three species showed a definite salt tolerance, thus this criterion could be used. V. vulni.cus grow in only 3% and 6% of NaCl, V. parahaemolyticus can grow up to NaCl 8%, while V. alginolyticus is able to grow in all salinities tests (3-10%). The salt tolerance of V. harveyi is less defined (some strains do not grow at either 8 or 10%; others grow at 8, but not at 10; and others grow up to lo%), but could be differentiated by additional tests. For example, V. alginolyticus and some strains of V. harveyi grow at 10% NaCl, but the latter could be differentiated because it is Voges-Proskauer-negative. Using the basic scheme presented in Fig. 1, it was possible to quantify pathogenic vibrios isolated from marine waters after 7 h and tentatively identify them 24 h later. During the analysis of a water sample from the shrimp pond, 5 pathogenic vibrios/ml and 560 total vibrios/ml were obtained. In this sample, v. alginolyficus (ca. 60%) and V. @vialis (ca. 40%) were the most abundant pathogenic vibrios isolated from the 41°C membrane. V. campellii (25%) and V. anguillarum (27%) were also recovered from the membrane incubated at 35°C.
For total Vibrios
For pathogenic Vibrios
4 Filtrate 0.1 ml
i Filtrate 10 ml.
k Incubate 7h at 35.5”C
& Incubate 7h at 41.5%
-+
COUflt
,
i
colonies
1 Transfer each colony to 200 /A of Marine Broth 1 Oxidase test
Incubate 18h at 35.5%
t----.
.&
Gram stain
I Transfer 1 O’pl aliquotes to each biochemical test i 1 I jOj3(6[8
I
I ]lOlO]
0.3.6,8,10 L 0 G V
Fig. I. Scheme of complete presumptive identification.
method
for both
total
I L = = = = =
2
G
NaCl concentration (%). Lyslne decarboxylation Ornithine decarboxylation Gas production of glucose Voges-Proscauer
and
pathogenic
vibrio
quantification
and
their
For aquaculture, this rapid methodology could avoid dissemination and permit rapid treatment with bactericides, or by modifying the physicochemical conditions (temperature, salinity, etc.) of the pond. Of course, for a more definitive identification, other tests must be performed, and the methodology proposed here has not been proven for clinical samples. Nevertheless, for samples of Vibrio affecting humans or for microbiological research proposes, this methodology could be used as a rapid technique for Vibrio recovery and presumptive identification.
Acknowledgements The authors are grateful to Dr. J.L. Ochoa for criticism of the manuscript and to Roy Bowers for clarifying the English. This research was partially supported by the (Secretaria de Pesca. Mexican government) project in tile pond technology.
J. Hernandez-Lopez
et al. I Journal of Microbiological
Methods 21 (1995)
143-149
149
References [ll Lavilla-Pitogo, C.R., Baticados, M.C.L., Cruz-Lacierda. E.R., de la Pena, L.D. and Sunaz, N.A. (1990) Occurrence of luminous bacterial diseases of Penaeus monodon larvae in the Philippines. Aquaculture 91, 1-13. 121 Brock, J.A. and Lightner, D.V. (1990) Microbial pathogens and diseases of marine crustaceans. In: Diseases of Marine Animals (Kinne O., ed.),Vol. III, pp. 245-349, John Wiley & Sons, New York. 131 Takahashi, Y., Shimoyama, Y. and Momoyama, K. (1985) Pathogenicity and characteristics of Vibrio sp. isolated from cultured kuruma prawn Penaeus japonicus Bate. Bull. Jap. Sot. Sci. Fish./nissuishi. 51, 721-730. [41 Anderson, I.G., Shausung, M.N., Shariff, M. and Nash, G. (1988) Bacterial septicemia in juvenile tiger shrimp, Penaeus monodon, in Malaysia brackiswate ponds. Asian Fish. Sci. 2, 93-108. [51 Lightner, D.V. (1988) Diseases of cultured penaeid shrimps and prawns. In: Disease Diagnosis and Control in North American Marine Aquaculture (Sindermann, C.J. and Lightner, D.V., eds.), 2nd edn., pp. 8-127, Elsevier, New York. @I Zheng, G., Shen, Y. and Li, H. (1990) Vibrio anguiliarum as a cause of diseases in Penaeus orientalis Kishinouye. J. Fish China Shuichan Xuebao 14, l-7. [71 Sano, T. and Fukuda, H. (1987) Principal microbial diseases of mariculture in Japan. Aquaculture 67, 59-69. PI Nolan, C.M., Ballard, J., Kaysner, CA., Lilja, J.L., Williams, L.P.J. and Tenover, F.C. (1984) Vibrio parahaemolyticus gastroenteritis. An outbreak associated with raw oysters in the Pacific Northwest. Diagn. Microbial. Infect. Dis. 2, 119-128. [91 Buck, J.D. (1990) Potentially pathogenic marine Vibrio species in sea water and marine animals in the Sarasota, Florida, area. J. Coast. Rev. 6, 943-948. I101 Shirai, H., Ito, H., Hirayama, T., Nakamoto, Y., Nakabayashi, N., Kumagai, K., Takeda, Y. and Nishibuchi, M. (1990) Molecular epidemiologic evidence for association of thermostable direct hemolysin (THD) and TDH-related hemolysin of Vibrio parahaemolyticus with gastroentiritis. Infect. Immun. 58, 3568-3573. [Ill Magalhaes, M., DaSilva, G.P., Magalhaes, V., Antas, M.G., Andrade, M.A. and Tateno, S. (1990) Vibrio fhwialis and Vibrio furnissii associated with infantile diarrhea. Rev. Microbial. 21, 295-298. [121 Magalhaes, V., Magalhaes, M., Lima, R.A., Tateno, S. and Magalhaes, E. (1992) Non-cholera vibrios in enterobacteriologic routine. Rev. Inst. Med. Trop. Sao Paulo 34, 1313-1315. [131 Vanderzant, C., Mroz, E. and Nickelson, R. (1970) Microbial flora on the Gulf of Mexico and pond shrimp. J. Food Milk Technol. 33, 346-350. [I41 Yasuda, K. and Kitao, T. (1980) Bacterial flora in the digestive tract of prawns, Penaeus japonicus Bate. Aquaculture 19, 229-234. 1151 Dempsey, A.C. and Kitting, C.L. (1987) Characteristics of bacteria isolated from penaeid shrimp. Crustacea 52, 90-94. [161 Baticados, M.C.L., Lavilla-Pitogo, C.R., Cruz-Lacierda, E.R., Pena, L.D. and Sunaz, N.A. (1990) Studies on the chemical control of luminous bacteria Vibrio harveyi and V. splendidus isolated from diseased Penaeus monodon larvae and rearing water. Dis. Aquat. Org. 9, 133-139. [171 Lee, J.V. (1979) Identification of Aeromonas, Vibrios and related organism. In: Identification methods for microbiologists (Skinner, F.A. and Lovelock, D.W., eds.), pp. 151-175, Academic Press, London. 1181 Bauman, P., Furniss, A.L. and Lee, J.V. (1984) Genus I. Vibrio. In: Bergey’s Manual of Systematic Bacteriology, Vol. 1 (Krieg, N.R. and Holt, J.G.. eds.), pp. 518-538, Williams & Wilkins, Baltimore. [I91 Brenner, D.J., Hickman-Brenner, F.W., Lee, J.V., Steigerwalt, A.G., Fanning, R.G.. Hollis, D.G., Farmer III, J.J., Weaver, R.E., Joseph S.W. and Seidler, R.J. (1983) Vibrio furnissii (formerly aerogenic biogroup of Vibrio @vialis), a new species isolated from human fete and the environment. J. Clin. Microbial. 18, 816-824.
Journal of Microbiological Methods 21 (1995) 143-149
Quantification of pathogenic marine vibrio using membrane filter technique Jorge
Hernhndez-Lopez, Francisco
Ma. Antonia Guzman-Murillo, Vargas-Albores*
Center for Biological Research, Department of Marine Pathology, P.O. Box 128, La Paz, BCS 23000, M&co
Received 5 May 1994; accepted 30 May 1994
Abstract
A rapid and simple methodology based on a membrane filter technique was designed to detect and quantify marine vibrios involved in fish and shellfish diseases, as well as human gastrointestinal disorders. By placing the membrane in TCBS medium and incubating it at 40-4’232, it was possible to differentiate between pathogenic and non-pathogenic vibrios. A presumptive identification was also done by growing cultures at different salinities and by using a few biochemical tests. Keywords:
1.
Bacteria quantification;
Pathogenic
vibrio; Marine vibrio
Introduction
Diseases caused by Vibrio spp., and to a lesser extent other Gram-negative bacilli, are important to marine fish and shellfish farming worldwide. Vibrio epidemics are a frequent problem in the culture of shrimp [l-5] were epizootic disease has caused over 90% mortality [6]. During 1984 in Japan, production loss caused by vibriosis, was valued at 231.4 million yen [7]. The principal bacteria identified in epidemic diseases, of marine fishes and shellfishes are Vibrio anguillarum, V. alginolyticus, V. Jluvialis, V. furnissii, V. harveyi, V. parahaemolyticus, V. vulnificus, Vibrio sp., Aeromonas sp. and Pseudomonas sp. These bacteria are also important to public health. Besides V. cholerae 01, *Corresponding author. [email protected].
Tel.:
+52 (112) 536-33 Ext 759. Fax:
0167-7012/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDZ 0167-7012(94)00042-6
+52
(112)
547-10.
e-mail:
144
.I. Hernrindez-Ldpez
et ul. i Journal
of Microhiologlcal
Methods 21 (iW5)
143-149
several vibrio species are capable of producing gastrointestinal disorders in humans who have ingested contaminated fish and shellfish [S-12]. The most frequently isolated vibrios from patients with gastroenteric disorders are: V. parahaemolyticus, V. furnissii, V. alginolyticus. V. fluvialis, V. cholerae, non-01, V. mimicus and V. vulni’cus. Thus, the list of pathogenic vibrios for fishes, shellfishes and human is the same and contains from 6 to 8 species. Interestingly, bacterial species recovered from diseased organisms have also been isolated from the body of healthy organisms [13-151 as well as from the water and sediment in hatcheries and ponds [1,16]. These reports agree with the wide distribution of Vibrio in marine environments, and give evidence to their opportunism. Except for V. cholerae in humans, infections by pathogenic vibrios are dependent on the animal’s physiological condition, and are highly influenced by the environment. To obtain a rapid and simple methodology for the detection and quantification of marine vibrio involved in diseased fish and shellfish and human gastrointestinal disorders, we implemented a procedure based on a membrane filter technique. Two criteria were used to differentiate between pathogenic and non-pathogenic vibrios: the capability for growth at 40-42°C and the tolerance to high NaCl concentrations.
2. Materials 2.1.
and methods
Culture media
TCBS agar (thiosulfate citrate bile sucrose, Difco) was prepared according to the manufacturer’s instructions. Petri dishes (10 x 60 mm) for membrane filtration were filled with 5 ml of TCBS medium; for the plate count, 12 ml were placed on 10 x 100 mm plates. For other culture media, 96-well plates (Nunc) with 200 ~1 per well were used. To determine salinity tolerance, 1% tryptone with different NaCl concentrations (0, 3, 6, 8 and 10% w/v) were prepared according to [17]. Moller base medium for ornithine and lysine decarboxylation, base medium for carbohydrate fermentation and MR/VP medium were modified by adding 3% NaCl. To establish the feasibility of using membrane filtration with TCBS as culture medium, different dilutions of V. parahaemolyticus (ATCC 17802) were analyzed and compared with the plate count technique. The pre-inoculum was prepared in marine broth by incubating for 24 h at 30°C and 150 rpm. One ml (ASsOn,,,= 1.0) of the inoculum was placed in 25 ml of marine broth and incubated under the same conditions for 18 h. Different dilutions were prepared, and 0.1 ml aliquots were used for quantification in both the plate count and membrane filter techniques. The plates were incubated at 35°C for 7 and 18 h periods for membrane filter and plate count methods, respectively.
J. Hermindez-Ldpez
2.2.
et ai. I Journal of Microbiological
Methoak 21 (1995) 143-149
145
Samples
Marine water was collected from a shrimp culture tile pond in sterile 250 ml flasks, and bacteriological analyses were done 30 min thereafter. To aliquots of 0.1 or 1 ml, sterile artificial seawater was added to obtain a final volume of 10 ml. In other cases, 10 ml aliquots were used. The samples were filtered under negative pressure using 47 mm diameter membranes with 0.45 pm pores (Nalgene, Cat. no. 2054045). The membranes were placed in 60 X 15 mm Petri dishes containing TCBS agar. These plates were incubated at 35.5”C or 41.5”C for 7 h, before determining the number of colony forming units (CFU). Only plates with 20-200 CFU were considered. 2.3. Identification Colonies from each TCBS plates were placed in 200 ~1 marine broth and incubated for 24 h at 35.5”C. From these cultures, 10 ~1 aliquots were used for the following biochemical tests: ornithine and lysine decarboxylation, gas production from glucose, Voges-Proskauer, NaCl request or tolerance, oxidase production, and Gram stain. The oxidase production was determined by putting a drop of culture on a paper filter (Whatman No. 1) and adding one drop of Kovacs reagent (1% w/v p-aminedimethylalanine oxalate, in water). All cultures for biochemical testing were incubated at 35.5”C. Cultures in tryptone, Moller medium for ornithine and lysine decarboxylation, and base medium for glucose fermentation were recorded at 7 and 18 h, while those for the Voges-Proskauer were read at 24 h.
3. Results and discussion Using different dilutions of V. parahaemolyticus (ATCC 17802), the plate count and membrane filter techniques were compared. The data in Table 1 shows that values obtained with the membrane filter were higher (30-50%) than those obtained by the plate count method, however a correlation between both techniques could be established. Based on these results, we believe the membrane Table 1 The number of bacteria in different dilutions of V. parahaemolyticus culture was determined by plate count and membrane filter methods; in both cases the plates were incubated at 35S”C during 24 h. Mean of triulicates and SD are given Sample
Plate count
Membrane
A B C
405 2 12 140 r 4 59 2 10
580 2 16 2OO”lo 120 t 3
filter
146
J. Hernrindez-Ldpez
et ul. I Journul of Microhiologicul
Methods 31 (1995)
14-1-W
filter using TCBS agar as a culture medium is a precise and reliable technique for vibrio quantification. Bacterial recovery from sea water was also successful using the membrane filter and selective medium, TCBS. Considering the abundance of Vibrio in sea water, it was necessary to determine the adequate sample size in order to obtain from 20 to 200 CFU per filter. The vibrio counts varied depending on the incubation temperatures used, thereby permitting us select the sample size (Table 2). In our experiments, 0.1 ml was enough for a total vibrio count (which can be determined vibrios (incubated at 40-42”C), by incubating at 3.5-36”C), while for pathogen volumes from 1 to 10 ml were necessary. Obviously, different dilutions could be tested depending on the sample. During this study, several water samples from a tile pond were analyzed. The number of vibrio varied from 92 to 1680 bacteria/ml and from 2 to 2.5 bacteria/ml for total and pathogenic vibrio, respectively. Correlation between the number of total and pathogenic vibrio was not found, indicating that both kinds of bacteria are distributed independently and that an increase in total vibrios does not necessarily increase the pathogenic vibrio count. On the other hand, the count of V. parahaemolyticus. which is considered pathogenic for human and aquatic organisms, was not affected by the incubation temperature. When different dilutions were filtered and incubated at 35.5 and 41.5”C in triplicate, similar counts were found (34 t 2, 30 + 8; 38? 16, 28 + 13; 50 2 21, 30 + 12), and no significant difference was established by Student’s t-test. This data partially supports the idea that incubation temperature can be used to discriminate non-pathogenic vibrios. As shown in Table 3, pathogenic vibrios are more tolerant to temperature and salinities. They can grow at 40-42°C in TCBS, and in salinities higher than 3% [18,19]. If the membrane after filtration is incubated at 35 or 41”C, it is possible to observe a significant difference in the number of CFUs (Table 2). Thus, pathogenic vibrios can be selected by their tolerance for incubation at 415°C. The characteristic vibrio colonies are visible at 5 h, at both 35 and 41”C, but the colonies are clearer and more defined at 7 h. For the identification of vibrios growing in TCBS bacteria, each colony was placed in marine broth and incubated at 35°C for 12-16 h. From these cultures,
Table 1 Different counts of Vibrro found m marine water depending sample size can be determined from these data Sample
size
Incubation
temperature
(ml) 35.5”C
IN, innumerable
41 .SY
on incubation
temperature.
The adequate
J. Hermindez-L6pez
et al. I Journal of Microbiological
Methods 21 (1995) 143-149
147
Table 3 Vibrios most referred as provoking diseases in culture fishes and shellfishes: the pathogen vibrios are more tolerant to temoerature and salinities Growth at 40-42°C V. V. V. V. V. V. V. V. V.
Growth at NaCl5-10%
alginolyticus cholerae 01 fluvialis furnissii harveyi parahaemolyticus vulnificus anguillarun fisheri
we performed the following tests: oxidase production, NaCl tolerance, amino acids decarboxylation and glucose utilization. Adequate growths were obtained using 200 ~1 of culture medium and 10 ~1 inoculum on 96-well plates. All vibrios isolated during this experiment were oxidase positive and required NaCl for growth. Although V. cholerae and some strains of V. anguillarum, V. fluvialis, V. furnissii and V. metschnikovii are able to grow in the absence of NaCl [18], they were apparently not present in the pond water. The isolates from the membrane incubated at 41°C and requiring NaCl for growth represent the most important group of non-cholerae pathogenic vibrios: V. alginolyticus, V. fluvialis, V. furnissii, V. harvegy, V. parahaemolyticus and v. vulnificus. From this list, only two vibrios (V. fluvia1i.s and V. furnissii) are ODC-negative, although they show many variations of salt tolerance. By this, we felt that ODC was the best test to discriminate both species. On the other hand, V. furnissii produces gas from glucose utilization, while V. fluvialis does not [19]. Thus, based on ODC-negative reaction and gas production, it was possible to make a presumptive identification of these two bacteria. For the rest, three species showed a definite salt tolerance, thus this criterion could be used. V. vulni.cus grow in only 3% and 6% of NaCl, V. parahaemolyticus can grow up to NaCl 8%, while V. alginolyticus is able to grow in all salinities tests (3-10%). The salt tolerance of V. harveyi is less defined (some strains do not grow at either 8 or 10%; others grow at 8, but not at 10; and others grow up to lo%), but could be differentiated by additional tests. For example, V. alginolyticus and some strains of V. harveyi grow at 10% NaCl, but the latter could be differentiated because it is Voges-Proskauer-negative. Using the basic scheme presented in Fig. 1, it was possible to quantify pathogenic vibrios isolated from marine waters after 7 h and tentatively identify them 24 h later. During the analysis of a water sample from the shrimp pond, 5 pathogenic vibrios/ml and 560 total vibrios/ml were obtained. In this sample, v. alginolyficus (ca. 60%) and V. @vialis (ca. 40%) were the most abundant pathogenic vibrios isolated from the 41°C membrane. V. campellii (25%) and V. anguillarum (27%) were also recovered from the membrane incubated at 35°C.
For total Vibrios
For pathogenic Vibrios
4 Filtrate 0.1 ml
i Filtrate 10 ml.
k Incubate 7h at 35.5”C
& Incubate 7h at 41.5%
-+
COUflt
,
i
colonies
1 Transfer each colony to 200 /A of Marine Broth 1 Oxidase test
Incubate 18h at 35.5%
t----.
.&
Gram stain
I Transfer 1 O’pl aliquotes to each biochemical test i 1 I jOj3(6[8
I
I ]lOlO]
0.3.6,8,10 L 0 G V
Fig. I. Scheme of complete presumptive identification.
method
for both
total
I L = = = = =
2
G
NaCl concentration (%). Lyslne decarboxylation Ornithine decarboxylation Gas production of glucose Voges-Proscauer
and
pathogenic
vibrio
quantification
and
their
For aquaculture, this rapid methodology could avoid dissemination and permit rapid treatment with bactericides, or by modifying the physicochemical conditions (temperature, salinity, etc.) of the pond. Of course, for a more definitive identification, other tests must be performed, and the methodology proposed here has not been proven for clinical samples. Nevertheless, for samples of Vibrio affecting humans or for microbiological research proposes, this methodology could be used as a rapid technique for Vibrio recovery and presumptive identification.
Acknowledgements The authors are grateful to Dr. J.L. Ochoa for criticism of the manuscript and to Roy Bowers for clarifying the English. This research was partially supported by the (Secretaria de Pesca. Mexican government) project in tile pond technology.
J. Hernandez-Lopez
et al. I Journal of Microbiological
Methods 21 (1995)
143-149
149
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