Identification of Pathogenic Vibrio spp.

in Raw and Steamed Oyster Samples from Villa, Iloilo

A Special Problem Presented to Maam Adrienne Bugayong University of the Philippines in the Visayas Miag-ao, Iloilo

Abigail Puno Roy Dahildahil Mechelle Joy Poral Prem Patrick Parcon Nia Marie Sedayon Regine Marie Espaa John Christopher Luces Roena Nicole Villanueva Gertrude Mikee Caonero Maugri Grace Kristi Laluma

MARCH 2012

I. Introduction

A. Background of the Study

Bureau of Fisheries and Aquatic Resources (BFAR) and Food and Agriculture Organization (FAO) of the United Nation emphasizes that Philippines is one of the largest fish producer in the world ranking 8th among the oyster and mussel producing countries making it a great potential for expansion of suitable areas for shellfish seafarming . Oysters (Crassostrea spp) are molluscan bivalve filter feeders harvested in shallow, near-shore estuarine waters (WHO-FAO, 2005). Upon filtering the surrounding waters, accumulated microorganisms and other substances that are a natural microbiota of the marine and estuarine environment could contribute to the transmission of diseases to humans. Highly associated gastrointestinal diseases are clinically prominent in individuals who consume edible oysters that are raw and even partially steamed. According to the Department of Health in the Philippines, food and waterborne diseases with outbreaks due to typhoid, fever, cholera, food poisoning and Hepatitis A are the most common causes of diarrhea. Seventy percent of food and waterborne diseases resulting to diarrhea have resulted from ingestion of contaminated food or water. For the past 20 years, diarrheal disorders are the number one contributing factor of morbidity and mortality 1,997 and 6.7 per 100,000 populations, respectively. Various members of the family Vibrionaceae are opportunistic pathogens that are natural microbiota of estuarine and marine environments. The genus Vibrio belongs to the family Vibrionaceae having a microscopic characteristic of being a Gram-negative rod, non-spore forming and motile with a single flagellum. Ecological parameters influence the presence and persistence of different Vibrio species in the sea. These include nutrient availability, temperature, and salinity. Many of the pathogenic species constitute a considerable part of marine halophilic bacterial populations, requiring high concentrations of salt for growth and function. Vibrios are found less frequently when the temperature drops and when salinity increases making Vibrios to be abundant in tropical areas due to a favorable temperature (Sousa, et al., 2004). More than 35 Vibrio spp. have been identified (Flick, 2007), with 12 species made known to be pathogenic to humans. The most common pathogenic Vibrio spp. frequently isolated from seawater and shellfish are Vibrio cholerae, Vibrio mimicus, Vibrio parahaemolyticus, Vibrio vulnificus and the halophilic Vibrio alginolyticus, which are associated with the food-borne diseases of the gastrointestinal tract. They are associated with live seafood as they form part of the indigenous microbiota of the environment at the time of seafood capture or harvest. Capiz is bound by a 80-kilometer coastline, in possession of one of the richest fishing grounds. Being a major contributor in the aquamarine industry of the country, it is known as the Seafood Capital

of the Philippines. Because of such abundance, this place was chosen as the source of oysters to be used for the study. Furthermore, a number of seafood restaurants have been established in Villa, owing to the convenience of being located alongside the shoreline. A lot of consumers opt to eat a variety of seafood products from these restaurants making it a good source of analysis for presence of Vibrio spp In relation to this, it has been known that all Vibrio spp. are relatively susceptible to inactivation by cooking for at least 60C for several minutes. Most of the risk associated with relevant strains of Vibrio spp. in food specifically oysters comes from consuming it raw or from cross-contamination of other raw seafoods or contaminated water. (WHO Risk Assessment of Vibrio vulnificus in Raw Oysters, 2005).

B. Statement of the Problem

The special problem will look into the presence of Vibrio spp. in raw and steamed oyster (Crassostrea spp.) samples from different restaurants in Villa, Iloilo. It will also look into the possibility of isolating pathogenic Vibrio spp. (Vibrio cholerae, Vibrio mimicus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus) present in the samples.

C. Objectives

General

This study aims to detect the presence of pathogenic Vibrio spp. in oysters from the province of Capiz and different restaurants selling oysters in Villa, Iloilo.

Specific

This study aims to: 1. Determine the bacterial concentration in raw oysters from Capiz 2. Determine the bacterial concentration in the seawater sample gatheAkaline from Capiz 3. Determine the bacterial concentration in raw and steamed oysters from different restaurants in Villa, Iloilo. 4. Identify the five species of pathogenic Vibrio isolated in oysters (Crassostrea spp.). namely: Vibrio alginolyticus, Vibrio cholerae, Vibrio mimicus, Vibrio parahaemolyticus and Vibrio vulnificus

D. Significance of the Study

Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio mimicus, Vibrio alginolyticus are pathogenic species causing diarrheal diseases. Risk factors such as eating raw or undercooked seafood or contaminating food preparation will eventually lead to food borne diseases. These Vibrio species are natural inhabitants of the marine environment which are responsible for the contamination of seafood. Risk factors such as eating raw or undercooked seafood or contaminating food preparation will eventually lead to food borne diseases. Thus, determining the presence of Vibrio species requires the immediate recall and inspection from Department of Health on oysters and oysters selling restaurants. Containing the possible outbreak of food borne illness by Vibrio spp. specifically cholera in Panay is the main thrust of this study. Water borne diseases brought about by Vibrio cholerae could be traced to the consumption of seafood. This study could pioneer further researches pertaining to oysters and other seafood products as reservoir for Vibrio cholerae. This study will be beneficial people eating raw or steamed oysters and also restaurants selling oysters both in Capiz and Villa. Knowledge in the presence of pathogenic Vibrio spp. in oysters that are usually consumed would enable restaurants and individuals to consider a more careful manner of preparation of oysters. Oyster harvesters will also benefit from this study Also this study can also be able to hypothesize some other possible factors, excluding the source, involved in the presence of vibrio such as contamination Moreover, this study will be beneficial to restaurants selling oysters harvested in the shores of Capiz. Awareness of the presence of the pathogenic Vibrio spp. would thus allow restaurants to reconsider obtaining seafood supplies from Capiz and be more particular with their food preparation. Oyster harvesters would also benefit from this study. This study would also give more information on the efficacy of modern generation antibiotics and the resistance of Vibrio spp. gatheAkaline from the province of Capiz to modern generation antibiotics. Lastly, this study could be used as a baseline data for further researches.

E. Scope and Limitation

This study will isolate only the following vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio mimicus and Vibrio alginolyticus. Should the resulting isolate not be classified into the five target species, it will be assumed to belong to the vibrio genus given that it would be positive for all the presumptive tests.

The possible isolates will be obtained from Oysters (Crassostrea spp.) found in the province of Capiz which is known to be the major supplier in Western Visayas and from different restaurants located in Villa, Iloilo that obtain their oysters from Capiz. The restaurants are limited to Villa for the reason that it is one of the locations in Panay that are most popular for establishments that serve oysters.

Two sets will be involved in this experiment: raw and steamed homogenized oysters. However, steamed samples will only be tested for the oysters from the restaurants in Villa. This is because the manner of preparation done by the restaurants and that of the standardized method to be followed in this study may have great differences and inconsistencies that would lead to an inconclusive result. Also, water samples from the sea of Capiz will be collected will also be gatheAkaline in this experiment. This can be a source of possible baseline study in relation to the study. This study will be conducted on February up to March 2012 in the University of the Philippines Visayas Miag-ao Campus.

II. Review of Related Literature

A. Oyster (Crassostrea spp.) Oysters are bivalve mollusk that has a characteristic of having variety of sizes, shapes, shell texture and colors and vary in their mode of reproduction and sexual expression (Conte, University of California-Davis).Oysters strain water through their gills for breathing, feeding and removing waste products wherein particles of food from the minute algae and animals such as the planktons are retained and conveyed to the mouth through the movements of the palpi. Oysters have more than 100 living oyster species. There species that live in deep enough water to avoid being exposed to the air at low tide while there are those that settle in shallower areas. They have two-part shells clamp tightly together using their powerful adductor muscles to hold tight for moisture retention. The oyster is the most studied invertebrate organism, studying specifically on its biology in temperate waters specifically concentrating on the genus Ostrea, Crassostrea and Saccostrea. The oysters under the genus Crassostrea are consideAkaline the most important commercial species and have great potential for development due to their tolerance in estuarine conditions accompanied with abundant spatfall. The species of oyster under the genus Crassostrea must adapt themselves to the rapidly changing salinity and heavy silt loads due to the monsoonal climate in the tropics. Crassostra gigas and Crassostra virginica are the usual species being introduced in the tropical waters mostly in Southeast Asia having great commercial importance. Crassostrea iAkalinealei is commercially farmed and is widely distributed in the Philippines. Also Crassostrea belcheri is rapidly growing in the South China Sea covering the Philippines, Vietnam, Malaysian state and Indonesian islands. The elevated water temperature of the tropics can produce high oyster growth rates if adequate food is available. Due to the archipelagic nature of the Philippines having 7,100 islands with vast coastal waters, a lot of mariculture activities are being done. In the year 1988, the Philippines is ranked 11th largest fish producer in the world having fishing as the peoples main livelihood. Seafarming or mariculture was an alternative livelihood to sustain their daily life as they wait for the fishery resources to regenerate. It has three major commodities- seaweeds, shellfishes and fished. For the shellfish culture, only oysters (Crassostrea spp) and green mussel (Perna viridis) have been farmed since the 1940s. Furthermore, it has also been reported that the Philippines ranked 8th among the oyster and mussel producing countries in the world. There are four species of oysters being cultuAkaline in the Philippines according to Food and Agriculture Organization of the United Nations, the slipper-shaped oyster Crassostrea iAkalinealei, the subtrigonal oyster Crassostrea. malabonensis and the curly or palm rooted oysters Crassostrea palmipes and Saccostrea cucullata. These species are

abundant in Bacoor Bay and extend to the entire Manila Bay from Tarnate, Cavite to Mariveles, Bataan, along the coast of Northern Luzon, Lingayen Gulf, Tayabas and Sorsogon; and to some extent in Batangas Bay, Banate Bay in Iloilo, Binalbagan, Hinigaran and Himamaylan, Negros Occidental, and in areas around Catbalogan, Western Samar, Northern Leyte and Palawan. Commercial farming however tends to concentrate along the coast of various provinces largely in Bulacan, Capiz, Cavite, Pangasinan, Sorsogon and Negros Occidental. From the years 1980 to 1986, 1982 has the highest output with a production of 19,017 MT, but the production dropped to 11,469 MT the following year. An annual growth rate of 36% was recorded from the year 1983 to 1986 with a total are of 46 hectares unlike 707 hectares of the previous year which is due to rising production cost and poor sanitary quality of the product. Since oysters are edible organisms that are often ingested partially steamed or even raw, they provide a very high risk to the consumers health leading to highly associated gastrointestinal disorders (Sousa, et al., 2004). Mollusks are excellent transmission vehicle because of their being filter-feeders leading to concentration of bacteria present as a normal microbiota in the surrounding waters (Flick,GJ., 2007). The increasing consumption of raw mollusks especially oysters is a matter of great concern to the public health authorities especially in tropical countries. Bivalves are commonly associated with the transmission of diarrhea-related microbial agents such as enteropathogenic bacteria that may be present in the tissues of bivalves in concentrations higher than in the source water (Sousa, et al., 2004).

B. Vibrio spp.

Vibrio species are distributed in marine waters all over the world. They can adapt rapidly to environmental changes and comprises several human pathogens, which commonly cause outbreaks of severe diarrhea in tropical regions (Oberbeckmann, et. al., 2011). They tend to be most abundant during warmer periods of the year and in growing areas with moderate salinities, depending on the species. Vibrio spp. are among the fastest-growing bacteria in nature multiplying readily in oysters and in other molluscan shellfish (DePaola et al., 2010). Ingestion is the most common route of infection, whether of raw or undercooked seafood or by drinking contaminated water. Another major route of infection is through exposure of open wounds to seawater or brackish water or to fish or shellfish harvested from such waters (Nigro, et al., 2011).

Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio mimicus, and Vibrio alginolyticus are pathogenic species causing diarrheal diseases. These diseases are related to the consumption of raw or partially steamed shellfish and the occurrence of human intestinal and gastrointestinal diseases (Flick et al, 2007). V. cholerae, V. parahaemolyticus, and V. vulnificus, are the

most important vibrios associated with human illness (DePaola et al, 2010). They may cause gastrointestinal illnesses, either in isolated cases or in the form of outbreaks (de Sousa et al, 2004). Similar to the three, Vibrio mimicus has also been recognized as one of the causes of gastroenteritis. Vibrio cholerae and Vibrio parahaemolyticus both produce diarrhea. Vibrio parahaemolyticus, while being invasive, affects primarily the colon while the Vibrio cholerae although noninvasive, affects the small intestine through secretion of its enterotoxin. Vibrio vulnificus, on the other hand, causes wound infections, and gastroenteritis (DePaola et al, 2010). Vibrio parahaemolyticus infection is often selflimited, injection may cause septicemia that is life-threatening to people having underlying medical conditions, such as liver disease or immune disorders (Vieira,et al., 2010). Vibrio vulnificus is a bacterium that is rare but a serious cause of human illness. It causes fatal infections pAkalineominantly to persons with immunocompromising conditions, diabetes, and an elevated serum iron concentration due to chronic liver disease or alcohol abuse (Han ,et al., 2011). Vibrio alginolyticus was initially classified as a Vibrio parahaemolyticus biotype however, they have some phenotypic differences. Vibrio alginolyticus is a halophilic bacterium found naturally in temperate marine and estuarine environments. It is ubiquitous in seawater and tends to cause superficial wound and ear infections such as otitis media and otitis externa. Most reports of Vibrio alginolyticus wound infections result from exposure of cuts or abrasions to contaminated seawater. Vibrio alginolyticus-associated infections may rarely progress to bacteraemia and necrotizing fasciitis, particularly in the immunocompromised (Reilly, et al., 2011). Vibrio mimicus is the species closest to Vibrio cholerae. It shows similar phenotypes to Vibrio cholerae but differs in some biochemical characteristics. The molecular mechanisms underlying the differences in biochemical metabolism between Vibrio mimicus and Vibrio cholerae are currently unclear. Several Vibrio mimicus isolates have been found that carry cholera toxin genes and cause cholera-like diarrhea in humans. It sometimes causes diarrhea and internal infections in humans (Wang, 2011). Vibrio cholerae is the main cause of an acute intestinal disease called cholera. Often manifested as a constant diarrheal disease, cholera is associated with significant mortality as well as economic loss due to the strain on health care (Kelvin et al, 2011). Orally ingested Vibrio cholera colonized the intestinal epithelium through expression of toxin co-regulated pili which in turn is coordinately regulated with cholera toxin. This enterotoxin is responsible for the bulk of the secretory diarrhea observed during disease progression (Zheng et al, 2011) by stimulating the secretion of water and electrolytes in the intestinal lumen. Patients with cholera may suffer from acute watery diarrhea, vomiting, and dehydration but rarely present with fever (Mridha, 2011).

III. Methodology

A. COLLECTION OF OYSTERS (Crassostrea spp.) AND SEAWATER SAMPLES

There were four samples needed to be collected in this study. Three set of oyster sample were collected composing of two raw oyster samples and a steamed oyster sample. One set of raw and steamed oyster sample were collected from a restaurant in Villa, Iloilo and a set of raw sample was collected from the province of Capiz (which is harvest source of commercially sold oysters), acting as the negative control for the study. The raw oyster samples (negative control) were collected fresh along the shores in the province of Capiz. The oysters were harvested early in the morning during low tide periods where visible oysters was be picked either directly from the seabed or forcefully removed aseptically by a sterile knife from rocks where they attach. The raw and steamed oysters from Villa, Iloilo were bought on the day that fresh oysters were delivered to the restaurant. The fresh oyster that were bought was made sure that it is with at most the same batch of oysters that was collected in the province of Capiz being the source of harvested oysters sold in this restaurant in Villa, Iloilo. In addition to the samples that were collected in the study, seawater samples were collected from the province of Capiz. Other than the raw oysters that were collected in the province of Capiz, seawater sample in the location of the collected raw oysters were also collected and then subjected to identification of presence of Vibrio spp. It was collected properly using a sterile bottle then cover with carbon paper. Then preceding steps in identification of presence of Vibrio spp. being done in the oyster samples were also done with the water sample. For the raw oyster samples, the oysters collected were placed in a sterile bag without any ice to preserve the viability of possible Vibrio. For the steamed oyster samples, the procedure of its storage was done wherein it cant be contaminated. Samples were then transported from Roxas City, Capiz and Villa, Iloilo into the microbiology laboratory of University of the Philippines Visayas where the samples were immediately subjected to several bacterial analysis methods.

B. MICROBIAL CONTENT DETERMINATION

Both raw and steamed oysters and the seawater sample were analyzed using the heterotrophic plate count. The outside shells of the oyster samples were washed thoroughly with distilled water to get rid of unwanted debris. The shells were then opened by shucking, observing aseptic technique wherein a sterile knife was inserted between the shell about 2 cm from the hinge area and the shells was pushed open. The meat along with the liquor was removed and placed in a sterile beaker. The obtained sample was then macerated using sterile scissors until homogenous. In a separate sterile beaker 25 g of the homogenized sample was measuAkaline and added with 225 mL of 0.1% alkaline peptone water. The sample was then mixed using a blender in high speed rotation for two minutes. A ten-fold serial dilution of the sample was done for the raw sample from Capiz, the 101 to 103 dilutions were used for analysis using the pour plate method. This was also done to the water sample only that, there is no process of homogenizing of the sample. Further changes were done during the processing of the raw and steamed oysters collected from Villa having 104 to 106 dilutions as the concentration for analysis using the pour plate method. It was then incubated for 24 hours at 30C. After incubation, the number of colonies was then counted in a colony counter.

C. OBTAINING PRIMARY CULTURE FROM OYSTER SAMPLE AND SEAWATER SAMPLE

The methods to be used in this part were adapted from the study by Ottavianni, D. et al. (2003). First, the outer portion of the shell was washed thoroughly with distilled water and then, 25 g (approximately 12 oysters; include meat and liquor) of sample was weighed into a taAkaline jar. The weighed sample was then added with 225 ml of alkaline peptone water (creating a 1:10 ratio of sample to APW) and thoroughly mixed by blending it for 2 min at high speed rotation.

The Alkaline Peptone Water homogenate was incubated at 30C for 8-24 hours. A 3-mm loopful from the surface pellicle (top 1 cm) of the Alkaline Peptone Water homogenate culture was subcultuAkaline on the surface of a dried TCBS plate after the 8th hour of incubation and again after the 24th hour of incubation both using clock streak method and observing proper aseptic technique. The TCBS plates was then incubated overnight (18 to 24 h) at 30C. After incubation, morphologically distinct Acidic- and green-coloAkaline colonies from the TCBS agar were obtained and transferAkaline each to TSA (with 1% NaCl) plate observing proper aseptic technique at all times. The different plates having different isolates were labeled accordingly and were incubated at 30C for 24 hours.

This method was also done to the seawater sample except for the process of homogenizing the sample.

D. Presumptive Test for Genus Identification

The following procedures for the identification of Vibrio species was adapted from the standardization study for isolation and identification of Vibrio spp. done by Ottavianni D. et al. (2003). Each distinct colony isolated from the TCBS agar and cultuAkaline in the TSA was subjected to the four presumptive tests specific for the determination of Vibrio genus. The four biochemical tests were simultaneously for all of the isolates from the TSA, having duplicates for each isolate. The cultures were inoculated in a broth containing 3% NaCl and were incubated for 24 hours at 30C. Next, the culture samples were tested for their presence of cytochrome c by subjecting it to Oxidase test. Using aseptic technique, a loop full of the isolated culture was picked by a sterile wooden applicator stick and smeaAkaline on a strip of filter paper pre-treated with Kovacs reagent. A positive reaction was indicated by the formation of a purple color change in the filter paper. Positive results were evident 10 seconds after the addition of the bacterial culture. The third test for the identification of Vibrio spp. was based on the reaction of the bacterial cultures to TSI slant incorporated with 1% NaCl. Still following proper aseptic technique, isolates were inoculated into the modified TSI slant and were incubated at 30C for 24 hours. After incubation, the color changes the occurAkaline in the slant was examined. Last, culture samples were subjected to susceptibility test using 10 g of ampicillin following the disk diffusion method on Mueller-Hinton agar and observing 0.5 McFarland Turbidity Standard. Cultures that belong to the genus Vibrio were to have positive results for all four of the tests done.

E. SPECIES IDENTIFICATION:

The tests to be done for the identification of the specific species of Vibrio were adapted from the identification keys of Alsina and Blanch (1994) and modified to prioritize the identification of the five species that are of major concern in the study. All of the isolated cultures that were confirmed to belong to the Vibrio genus was then subjected to the following biochemical tests.

E.1. Primary Test

To determine which cluster of Vibrio species the positive samples belonged to, the following tests were done: Determination of arginine dihydrolase (ADH), lysine decarboxylase (LDC) and ornithine decarboxylase (ODC) metabolism were done by inoculating the samples into arginine dihydrolase agar, lysine decarboxylase slant and ornithine decarboxylase slant respectively and incubating for 48 hours at 30C. Aseptic technique was observed at all times in the inoculation of samples into the media. Depending on the results for these three tests, the cluster to which the species belong to were determined. Further biochemical tests were performed for species identification in samples that belong to the cluster ADH(-)/LDC(+)/ODC(+) for it is in this cluster that the five species of concern in this study belong.

E.2. Identification of Specific Vibrio Species:

Five different species namely Vibrio cholerae, Vibrio parahaemolyticus, Vibrio mimicus, Vibrio vulnificus and Vibrio alginolyticus were analyzed from the isolates if the isolated were positive for the five species stated above.

Vibrio cholera: Isolates that were Arginine dihydrolase negative and Ornithine decarboxylase and Lysine decrboxylase positive were inoculated into nutrient broths that have 0% NaCl and incubated at 30 degrees Celsius for 24 hours. After growth was observed in the media, the isolate was inoculated into sucrose broth incubated at 30 degrees Celsius for 24 hours and observed for fermentation. Isolates that were positive for growth were recorded to be under the species cholerae.

Vibrio mimicus: Isolates that were positive for growth in 0% NaCl media after incubation at 30 degrees Celsius for 24 hours were inoculated into sucrose fermentation media. Isolates that were observed to be negative for sucrose fermentation were recorded to be under the species mimicus.

Vibrio parahaemolyticus: Isolates that were observed to have negative growth on 0% NaCl broth incubated at 30 degrees Celsius for 24 hours were inoculated into media that contain 8% NaCl. Isolates that were positive for growth after incubation at 30 degrees Celsius for 24 hours were further inoculated into media containing 10% NaCl the media was then incubated at 30 degrees Celsius for 24 hours. Isolates that show growth in this media were recorded to be under the species parahaemolyticus.

Vibrio alginolyticus: Isolates that were observed to have negative growth on 0% NaCl broth after incubation at 30 degrees Celsius for 24 hours were inoculated into media that contain 8% NaCl. Isolates that were positive for growth were further inoculated into media containing 10% NaCl and incubated at 30 degrees Celsius for 24 hours. Isolates that did not show growth in this media were inoculated into MRVP broths and were tested after 24 hours. Isolates that had positive results were classified under the species alginolyticus

Vibrio vulnificus Isolates that were observed to have negative growth on 0% NaCl broth after incubation at 30 degrees Celsius for 24 hours were inoculated into media that contain 8% NaCl. Isolates that were negative for growth were further inoculated into lactose fermentation media and incubated at 30 degrees Celsius for 24 hours. Isolates that were observed to ferment the lactose were classified under the species vulnificus.

F.DISPOSAL OF BACTERIAL CULTURE All of the oyster shells, Petri plates, test tubes and other glass wares used for the bacterial isolation and identification tests will be soaked with Lysol disinfectant solution for 24 hours after which, the contents will be discarded in a waste bag labeled as infectious waste for proper disposal.

FLOW CHART FOR SPECIES IDENTIFICATION

Oyster and Seawater Samples

Growth from APW

Colonies from TCBS

Different Isolates from TSA

Oxidase Test

3% NaCl

Growth in TSI Slant

Susceptibility in Vibriostatic drug

Vibrio spp.

Arginine dihydrolase

Lysine decarboxylase

Ornithine decarboxylase

0% NaCl

Sucrose Fermentation

8% NaCl

V. cholerae

10% NaCl broth

Lactose Fermentation

V. mimcus

V. parahaemolyticus

MR-VP

V. vulnificus

V. alginolyticus

Legend:

+:

-:

IV. RESULTS

A. Microbial Content Determination: Heterotrophic Plate Count Dilution 1:10 1:100 1:1, 000 1:10, 000 1:100, 000 1:1,000,000 Raw Oyster (Capiz) TNTC TNTC TNTC Water Sample (Capiz) TNTC TNTC TNTC Cooked Oyster (Villa) Uncooked Oyster (Villa)

TNTC TNTC 142 37

TNTC 71 24 24

Dilution 1:10 1:100 1:1, 000 1:10, 000 1:100, 000 1:1,000,000

Serial dilution Calculation Raw Oyster Water Sample Cooked Oyster (Capiz) (Capiz) (Villa) TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC 14,200,000/ml 37,000,000/ml

Uncooked Oyster (Villa)

TNTC 710, 000/ml 2,400,000/ml 24,000,000/ml

B. Genus Identification SUMMARY OF RESULTS 8 Hours Growth on Culture Raw Oysters from Capiz Raw Oysters from Villa Cook Oysters from Villa + Presumptive Test Species Identification Growth on Culture + 24 Hours Presumptive Test Species Identification

Other Microorganism

+ Vibrio species

Other Microorganism

Other Microorganism

GENUS ANALYSIS OF RAW OYSTER FROM CAPIZ

TSI Replicate Replicate 1 2 Hydroge Hydroge n Sulfide n Sulfide Acidic Acidic Black Black X X Akaline Akaline w/ Acidic w/ Acidic Akaline Akaline X X Hydroge n Sulfide Acidic Black X Hydroge n Sulfide Akaline (little) Acidic X Akaline Akaline X Akaline Orange Hydroge n Sulfide Acidic Black X Hydroge n Sulfide Akaline (little) Acidic X Akaline Acidic X Akaline Orange 8 Hours NB Replicat Replicat Oxidase e1 e2 Sedimen t Turbid Sedimen t Turbid MHA Replicate 1 Replicate 2

Isolat e

Vibri o spp.

Negativ e

RESISTAN T

RESISTAN T

No growth

Pellicle Turbid

Negativ e

RESISTAN T

RESISTAN T

Pellicle Turbid Sedimen t

Pellicle Turbid Sedimen t

Negativ e

RESISTAN T

RESISTAN T

Turbid Sedimen t

Turbid Sedimen t

Negativ e

RESISTAN T

RESISTAN T

Pellicle Turbid Turbid Floculen t Floating Particles

Pellicle Turbid Turbid Floculen t Floating Particles

Negativ e

RESISTAN T

RESISTAN T

Negativ e

RESISTAN T

RESISTAN T

GENUS ANALYSIS OF RAW OYSTER FROM CAPIZ

TSI Isolates Replicate 1 Hydrogen Sulfide Akaline Acidic X Hydrogen Sulfide Akaline Acidic X Hydrogen Sulfide Akaline Acidic / Hydrogen Sulfide Akaline Acidic / Akaline Acidic X Hydrogen Sulfide Akaline Acidic Replicate 2 Hydrogen Sulfide Akaline Acidic X Hydrogen Sulfide Akaline Acidic X Hydrogen Sulfide Akaline Acidic / Hydrogen Sulfide Akaline Acidic / Akaline Acidic X Hydrogen Sulfide Akaline Acidic 24 Hours NA Replicate Replicate 1 2 MHA Oxidase Replicate 1 Replicate 2

Sediment Turbid

Sediment Turbid

Negative

RESISTANT RESISTANT

Sediment Turbid

Sediment Turbid

Positive

RESISTANT RESISTANT

Sediment Turbid

Sediment Turbid

Positive

RESISTANT RESISTANT

Turbid Sediment

Turbid Sediment

Positive

RESISTANT RESISTANT

Sediment Turbid

Sediment Turbid

Positive

30 mm

28 mm

No growth

Sediment Turbid

Negative

RESISTANT RESISTANT

GENUS ANALYSIS OF RAW OYSTER FROM VILLA

TSI Isolate Replicate Replicate 1 2 Not 1 Akaline Applicable Acidic 2 Acidic Acidic Acidic Acidic 3 Acidic Acidic Acidic Acidic 4 Acidic Acidic Acidic Acidic 5 Akaline Acidic Acidic Acidic 6 Acidic Acidic Acidic Acidic 7 Acidic Acidic Acidic Acidic 8 Acidic Acidic Acidic Acidic 9 Akaline Acidic Acidic Acidic 10 Akaline Acidic Acidic Acidic 8 Hours NB Oxidase Replicate Replicate Replicate Replicate 1 2 1 2 + + Sediment Sediment + + Sediment Sediment Pellicle + + Sediment Sediment Pellicle + Pellicle + Sediment Negative Negative MHA Replicate 1 8 mm Replicate 2 RESISTANT

Vibrio spp.

Negative

Negative Slightly Positive Negative

RESISTANT RESISTANT

Negative

RESISTANT RESISTANT

Negative

RESISTANT RESISTANT

+ + Sediment Sediment + + Sediment Sediment Pellicle Pellicle + + Sediment Sediment + + Sediment Sediment + + Sediment Sediment + + Sediment Sediment

Negative

Negative

RESISTANT RESISTANT

Negative

Negative

RESISTANT RESISTANT

Negative

Negative

25 mm

RESISTANT

Negative

Negative

RESISTANT RESISTANT

Negative

Negative

RESISTANT RESISTANT

Negative

Negative

RESISTANT RESISTANT

GENUS ANALYSIS OF RAW OYSTER FROM VILLA

TSI Replicate Replicate 1 2 Akaline Akaline Acidic Acidic + Acidic Acidic Acidic Acidic + Acidic Acidic Acidic Acidic + Acidic Acidic Acidic Acidic 24 Hours NB OResistantidase Replicate Replicate Replicate Replicate 1 2 1 2 + + Sediment Sediment Positive Positive Pellicle Pellicle + + Sediment Sediment Positive Positive Pellicle Pellicle + + Slightly Sediment Sediment Negative Positive Pellicle Pellicle + + Sediment Sediment Negative Negative Pellicle Pellicle MHA Replicate 1 17 mm Replicate 2 21 mm Vibrio spp.

Isolate

RESISTANT

20 mm

40 mm

RESISTANT -

58 mm

RESISTANT

GENUS ANALYSIS OF STEAMED OYSTER FROM VILLA

TSI Replicat Replicat e1 e2 Acidic Acidic Acidic Acidic 8 Hours NB OResistantidase Replicat Replicat Replicate Replicat e1 e2 1 e2 + + Sedime Sedimen nt t Negativ Negative Pellicle Pellicle e Flocule Floculen nt t + + Sedime Sedimen Negativ Negative nt t e Pellicle Pellicle + + Sedimen Negativ Sedime Negative t e nt + Sedime nt + Sedime nt + Sedime nt + Sedimen t + Sedimen t Pellicle + Sedimen t Pellicle + Sedimen t + Sedime nt Slightly Positive Not Applicabl e Negativ e MHA Replicate 1 Replicate 2 Vibri o spp.

Isolat e

RESISTAN T

RESISTAN T

Acidic Acidic Acidic Acidic Akaline Acidic Akaline Acidic Akaline Acidic Akaline Acidic Acidic Acidic

Acidic Acidic Acidic Acidic Acidic Acidic Acidic Acidic Akaline Acidic Akaline Acidic Acidic Acidic

RESISTAN T

RESISTAN T

RESISTAN T

38 mm

RESISTAN T

RESISTAN T

Negativ e

RESISTAN T

RESISTAN T

Negative

Positive

RESISTAN T RESISTAN T

RESISTAN T RESISTAN T

Negative

Negativ e

Not Applicab le

Negativ RESISTAN RESISTAN T T e

GENUS ANALYSIS OF STEAMED OYSTER FROM VILLA

24 Hours Isolat e TSI Replicat Replicat e1 e2 + Acidic Acidic Akaline Black + Akaline Acidic + Akaline Acidic w/ Black Acidic Acidic + Acidic Acidic Akaline slightly Acidic Akaline Acidic NB Replicat Replicat e1 e2 + + Sedimen Sedimen t t Pellicle Pellicle + + Sedimen Sedimen t t Pellicle Pellicle + + Sedimen Sedimen t t Pellicle Pellicle + + Sedimen Sedimen t t Pellicle Pellicle Oxidase Replicat Replicat e1 e2 Negative Negative MHA Replicate 1 RESISTAN T Replicate 2

77 mm

Negative

Positive

39 mm

RESISTAN T

Slightly Positive

Negative

RESISTAN T

RESISTAN T

Slightly Positive

Negative

28 mm

RESISTAN T

C. Species Identification on Amino Acid Assay Replicates 8h from raw oyster 24h from raw oyster (Plate E) 8 h from cooked oysters (Isolate 6) 24h from cooked oysters 1 (purple at the top) 1 2 1 2 + + Lysine Decarboxylase Ornithine Decarboxylase Arginine Dihydrolase

No Isolates + + (whitish purple) + (whitish in the bottom) No Isolates Lighter compaAkaline to uninoculated Lighter compaAkaline to uninoculated + + -

8 h from uncooked oysters (Isolate 4)

(purple at the top)

24h from uncooked oysters

No Isolates

V. Discussion Sapian is situated in the northern portion of Panay Island. It is the town next to Ivisan, northwest of Roxas City. As a coastal town, it is rich in fishery and aquatic resources. Mussel culture was developed in Sapian bay, wherein it is inter-mixed with oysters. Sapian had a total of 20,202 hectares of productive fishpond based on the record of the municipal assessor. Vast mangroves are found along the sea coast extending along the streams where water is brackish, thus serving as natural habitat and breeding ground of marine organisms.

Yet, among the estuaries where the Department of Agriculture obtains samples, it is in Sapian where the water was observed to be cloudy and dirty. Thus, the said municipality was the site chosen in this study, the source of one set of raw oyster samples serving as the negative control. A water sample where the raw oyster samples were collected was also obtained. This is to test any bacterial contamination brought about by the residents living within the vicinity of the estuarial waters. In the city, most seafood restaurants are established in Villa. Among those that are situated within the area, it is in (insert name of restaurant here) where the seafood products served are being commercially supplied by Sapian, Capiz. One set of raw and steamed oyster samples were obtained in the said restaurant.

HPC was performed to approximately determine the levels of heterotrophic bacteria in the water and oyster samples by counting the colony formation on the culture media. It should also be noted that the results acquired using an HPC test are not an accurate assessment of total heterotrophic concentrations

but, instead, are indications of culturable organisms present. Recovered bacteria can include those naturally found in the water environments. This may include species within the genera Pseudomonas, Aeromonas, , Klebsiella, Vibrio, and many others. The colonies are referred to as colony forming units (CFU).

Dilutions were done since the actual concentration of the sample is unknown. These samples should first be diluted in sterile media before transferring to plate media so that countable number of colonies may appear. Theoretically, the highest dilutions will produce the lowest number of CFUs and the lowest dilutions will produce the highest number of CFUs.

The approximation of the colony counts for the three dilutions of the raw oysters and water sample of Capiz were too numerous to count (TNTC). This is an indication of possible heterotrophic bacteria, including and possibly Vibrio from these samples.

Results show cooked samples from Villa also contained bacteria with an estimation ranging from TNTC to 37,000,000/ml. Likewise, uncooked samples from villa contained bacteria with an estimation range of TNTC to 24,000,000/ml.

These results do not only show the presence of the target Vibrio spp. Also, they indicate the presence of other microorganisms that thrive in the oyster samples and their surroundings. Furthermore, the heterotrophic plate count is limited to counting the bacterial species capable of growing on the specific media and temperature of incubation used in the study.

Even though heat is known to destroy some bacteria, the cooked samples from Villa showed presence of bacteria. The CFU counted from the cooked samples from Villa was even greater than the CFU counted from the uncooked samples. This result poses a question to the proper preparation of the oysters of that certain restaurant in Villa.

The APW homogenate of raw oyster samples from Sapian, Capiz was subcultured in a TCBS plate and incubated for 8 hours. This is the optimum growth for V. cholera. It subsequently gave a negative result. Another APW homogenate subcultured in a TCBS plate was incubated for 24 hours, since this is the optimum growth of other Vibrio species. There was a positive result in this presumptive test, but the findings for species identification were negative. The isolates are thus of Vibrio species in general, not V. parahaemolyticus, V. mimicus, V. alginolyticus, V. cholera and V.vulnificus. The TCBS subculture of the APW homogenate of raw oyster samples from Villa was incubated for 24 hours, giving a positive result for the presumptive test of Vibrio species. On the other hand, the result was negative for the 24-hour TCBS subculture of the APW homogenate of steamed oyster samples from Villa.Vibrio species were killed during the cooking process.

Species identification was performed to the isolates of the 24-hour TCBS subculture of the APW homogenate of the raw oyster samples from Villa. It was however unfortunate that the said isolates gave a negative result for the Vibrio species V. parahaemolyticus, V. mimicus, V. alginolyticus, V. cholera and V.vulnificus. This is an indication that the colony growth in TCBS may be some other Vibrio spp aside from those mention above.

The APW homogenates of both raw and steamed oysters from Villa were subcultured in a TCBS plate and incubated for 8 hours. The results for this presumptive test were negative. The isolates are thus not Vibrio species, but other bacterial microorganisms that can grow in TCBS. Furthermore, species identification was not performed with these results.

The presence of Vibrio species in raw oyster samples from Villa thus coincides with the positive result of the control, which is the raw oyster samples from Sapian, Capiz

Process: o o Nagkuha ta raw oyster and water sample from Sapian, Capiz Nagkuha ta sang raw and cooked oysters from a restaurant in Villa

Things to Discuss o Why did we choose Sapian o Among the estuaries that DA usually collects their samples, it is in Sapian where the water is somewhat cloudy and dirty There also some residents living within the vicinity of the estuarial waters which may contribute to any bacterial contamination of the water Why did we get raw oysters from Capiz when you have cooked and raw oysters from Villa? o o We used the oysters from Capiz as a control since it is the source of the oysters that are commercially sold in restaurants in Villa Why did we choose that restaurant? o among the restaurants in Villa, this restaurant obtained their oysters also from Sapian, Capiz

Why did we collect water sample from Sapian, Capiz to find out the bacterial concentration of the surrounding water which is the natural habitat of the oysters collected We incubated the samples from Sapian in TCBS for 8 hours which is the optimum growth for Vibrio cholera and for 24 hours since it is the optimum growth of other Vibrio.

Discussion of Results o Raw Oysters from Capiz (8 hours and 24 hours)

The raw oysters from Capiz that were incubated for 8 hours gave a negative result; on the other hand those that were incubated for 24 hours gave a positive result in presumptive test but negative for species identification. Thus these isolates are species of the genus Vibrio but not V. parahaemolyticus, V. mimicus, V. alginolyticus, V. cholera and V.vulnificus.

Raw Oysters and Cooked Oysters from Villa (24 hours)

The raw oyster collected from Villa that was incubated for 24 hours gave a positive result for the presumptive test of being a Vibrio species. While the cooked oysters from Villa that were also incubated

for 24 hours gave a negative result. The reason for this could be that the Vibrio microorganism was killed during the cooking process (Mangita study nag n prove ni)

Since the raw oysters from Villa that were incubated for 24 were confirmed to be a Vibrio spp, species identification process was done to the said isolate. Unfortunately, the isolate from the raw oyster collected from Villa gave a negative result for the V. parahaemolyticus, V. mimicus, V. alginolyticus, V. cholera and V.vulnificus species of Vibrio. This means that the colony that grow in TCBS may be some microorganism that may also grow in 8 hours after culturing in TCBS. Example of these microorganisms are: (Search: other microorganism na magrow sa 8 hours after culture sa TCBS kag TSA)

Both oysters from Villa that are raw and cook and were incubated for 8 hours gave negative results for the presumptive test thus the isolated colonies from these cultures are not Vibrio but other bacteria that can grow in TCBS. Furthermore with these results, species identification was not performed

The presence of Vibrio spp. in raw oysters from Villa coincides with the positivity of the control which were the raw oysters from Capiz.

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BUDGET (Estimated Expenses)

Laboratory Expenses Disposable petri plates (100 pcs) Distilled water (1 gallon) Extra Materials P 600.00 P 100.00 P 600.00

(Filter paper, tissue paper, applicator stick, Ziplock Plastic, gloves, trash bag, etc.) For Additional Reagents Needed P 1, 000.00

Field Expenses Transportation Lodging Fee Other Extra Materials (for emergency purpose) P 1, 000.00 P 1, 350.00 P 350.00 -----------------------TOTAL P 5, 000.00

GANTT CHART FEBRUARY 2012

8 wed 9 thu 10 fri 11 sat 12 sun 13 mon 14 tue 15 wed 16 thu 17 fri 18 Sat 19 sun 20 mon 21 tue 22 wed 23 thu 24 fri 25 sat 26 sun 27 mon 28 tue

Activity

Making Revisions on the Proposal

Buying of Materials

Autoclave of glass wares

Media Preparation

Go to sample site

Harvest Samples

Cook Samples

Inoculate Samples for Heterotrophic Plate Count Inoculate raw and steamed samples in APW Subculture samples from APW into another APW media Check growth on APW after 8 hours and clock streak on TCBS

Check growth on APW after 24 hours and clock streak on TCBS Counting of MPN from HPC Check and record growth in TCBS from 8 hours incubation and isolate into TSA Check and record growth in TCBS from 24 hours incubation and isolate into TSA Perform 4 Presumptive tests from 8 hours incubation Perform 4 Presumptive tests from 24 hours incubation Species Identification from 8 hours incubation Species Identification from 24 hours incubation

Analysis of Results

Paper Making Until Final Output

(to be continued