Characterization and Antimicrobial Susceptibility of Vibrio Spp. Isolated From Different Environments

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INTRODUCTION

The genus Vibrio consists of Gram-negative straight or curved rods, motile by means of a single polar flagellum. Vibrios are capable of both respiratory and fermentative metabolism. O2 is a universal electron acceptor; they do not denitrify. Most species are oxidase-positive. In most ways vibrios are related to enteric bacteria, but they share some properties with pseudomonads a well. The Family Vibrionaceae is found in the “Facultatively Anaerobic Gram-negative Rods” in Bergey’s Manual (1986), on the level with the Family Enterobacteriaceae. In the revisionist taxonomy of 2001 (Bergey’s Manual), based on phylogenetic analysis, Vibrionaceae, Pseudomonadaceae and Enterobacteriaceae are all landed in the Gammaproteobacteria. Vibrios are distinguished from enterics by being oxidase-positive and motile by means of polar flagella. Vibrios are distinguished from pseudomonads by being fermentative as well as oxidative in their metabolism. Of the vibrios that are clinically significant to humans, Vibrio cholerae,the agent of cholera, is the most important.

Most vibrios have relatively simple growth factor requirements and will grow in synthetic media with glucose as a sole source of carbon and energy. However, since vibrios are typically marine organisms, most species require 2-3% NaCl or a sea water base for optimal growth. Vibrios vary in their nutritional versatility, but some species will grow on more than 150 different organic compounds as carbon and energy sources, occupying the same level of metabolic versatility as Pseudomonas. In liquid media vibrios are motile by polar flagella that are enclosed in a sheath continuous with the outer membrane of the cell wall. On solid media they may synthesize numerous lateral flagella which are not sheathed.

Vibrios are one of the most common organisms in surface waters of the world. They occur in both marine and freshwater habitats and in associations with aquatic animals. Some species are bioluminescent and live in mutualistic associations with fish and other marine life. Other species are pathogenic for fish, eels, and frogs, as well as other vertebrates and invertebrates. V. cholerae and V. parahaemolyticus are pathogens of humans. Both produce diarrhea, but in ways that are entirely different. V. parahaemolyticus is an invasive organism affecting primarily the colon; Cholerae is noninvasive, affecting the small intestine through secretion of an enterotoxin. Vibrio vulnificus is an emerging pathogen of humans. This organism causes wound infections, gastroenteritis, or a syndrome known as “primary septicemia.”

METHODOLOGY

EXPERIMENT: 1

Collection of Samples (Collins et al, 1973):

Marine Samples were collected in different locations of Rameshwaram marine region (Palk bay) at the depth of 1 – 2 m at various locations. Sewage and soil samples were collected in and around G.R.D. College campus.

EXPERIMENT:2

2.1. Bacterial Enumeration (Mary et al, 1985):

Number of culturable, aerobic, heterotrophic bacteria present in water and sediment samples was determined by plating on marine agar and nutrient agar. For marine isolates, the media were prepared by the 3.5% sodium chloride (NaCl). Then the plated were incubated at appropriate time and temperature.

EXPERIMENT: 3

3.1. Isolation of vibrio spp from water and sediment samples (Mary et al, 1985):

Different selective media were used for the isolation of vibrio sp from marine source. For marine isolates, the media were prepared by the 3.5% sodium chloride (NaCl). Media used for the isolation of vibrio sp are 1. Thiosulfate Citrate Bile Sucrose Agar (with 3.5% NaCl) 2. Marine agar medium;

EXPERIMENT: 4

4.1 Identification

Microscopy: Gram-staining characteristics and cell morphologies were determined by standard methods (Gerhardt et al., 1981). Motility was observed in wet mount using microscope.

Cultural characteristics : Colony morphology of various isolates of vibrio sp were observed on Nutrient agar, Thiosulfate Citrate Bile Sucrose Agar (TCBS), Blood agar, Mac conkey agar, Mannitol salt agar and results were tabulated.

Physiological characterization: Preliminary physiological characterization such as catalase test, starch hydrolysis test, indole test, MRVP test, citrate test, oxidase test, string test, carbohydrate fermentation test (sugars used-sucrose, lactose, glucose, maltose, Mannitol) , cholera red reaction were carried out and results were tabulated.

EXPERIMENT: 5

Anti microbial susceptibility test Kirby-Bauer method

Procedure:

1. Make a suspension at an appropriate turbidity of the bacterial culture to be tested.

2. Place a sterile cotton swab in the bacterial suspension and remove the excess fluid by pressing and rotating the cotton against the inside of the tube above the fluid level. The swab is streaked in at least three directions over the surface of the Mueller-Hinton agar and TCBS agars obtain uniform growth. A final sweep is made around the rim of the agar. Be sure to streak for confluency.

3. Allow the plates to dry for five minutes.

4. Using sterile forceps, place disks containing the following antibiotics on the plate: penicillin G, ampicillin, cephalothin, erythromycin, tetracycline, methicillin, streptomycin or other appropriate antibiotic disks.

5. Incubate the plates within 15 minutes after applying the disks. The plates should be incubated soon after placing the disks since the test is standardized under conditions where diffusion of the antibiotic and bacterial growth commence at approximately the same time.

6. Following overnight incubation, measure the diameter of the zone of growth inhibition around each disk to the nearest whole mm. Examine the plates carefully for well-developed colonies within the zone of inhibition.

7. Using a standard table of antibiotic susceptibilities, determine if the strain is resistant, intermediate, or susceptible to the antibiotics tested.

RESULT AND DISCUSSION

Totally 35 samples were collected in different locations of marine region, garden soil, sewage water and domestic water.Vibrio bacteria are gram-negative and largely halophilic. Vibrio is rod-shaped, and can be straight or curved. They are motile organisms, using a single polar flagellum to travel. Vibrios are one of the most common organisms in surface waters of the world. They occur in both marine and freshwater habitats and in associations with aquatic animals. Some species are bioluminescent and live in mutualistic associations with fish and other marine life. These samples were processed through the commonly used procedures such as selective media (listed below), Gram’s staining, wet mount observation for motility and bio chemical tests includes catalase test, starch hydrolysis test, indole test, MRVP test, citrate test, oxidase test, string test, carbohydrate fermentation test (sugars used-sucrose, lactose, glucose, maltose, Mannitol) , cholera red reaction were carried out and results were tabulated for identification of vibrio sp from the above samples, and that can be processed, the details of the description as shown

COLONY MORPHOLOGY OF vibrio sp.

MEDIA DETAILS

Nutrient agar Moist, translucent, regular, disc shaped, 1-2mm in size, bluish tinge can be seen in transmitted light as distinctive colour

Mac Conkey agar Colorless colonies after, prolonged incubation pink colour colonies were seen. (plate:2)

Blood agar Colonies were surrounded by a zone of hemolysis

Thiosulphate citrate bile salt agar Colonies are yellow in colour due to fermentation.

Mannitol salt agar No prominent growth was observed.

The colony morphology of vibrio strains was varying during the isolation in the selective media. The colonies were very clear, moist, disc shaped, yellow and pink colonies were observed from various sources. The mother culture was sub cultured in the same media for culture maintenance. All the isolated vibrio strains were numbered for the easy identification and convenience. Gram staining reaction was recorded from heat fixed smears of vibrio culture. Motility and cell shape were determined by direct observation of wet mounts of fresh broth culture using microscopy. The chacteristics of Vibrio on TCBS medium shows that most of isolates were motile and capable of producing yellow and green pigmentation. Thiosulphate-citrate-bile salts-sucrose (TCBS) agar (Difco) is a selective medium commonly used to isolate members of the genus Vibrio from estuarine environments. The high concentration of thiosulphate and citrate and the strong alkalinity of the medium largely inhibit the growth of Enterbacteriaceas. Oxbile and cholate suppress primary enterococci .Any coliform bacteria, which may grow, cannot metabolize sucrose. Only a few sucrose Protease strains can grow to from yellow, vibriod like colonies. The mixed indicator thymol blue, bromothymol blue changes its color to yellow, when acid is formed even in this strongly alkaline medium

Out of 35 different samples collected, only 17 samples were showing the presence of vibrio sp, most of the marine samples collected from various marine region shows positive results for vibrio sp, when compared to other samples such as sewage water and soil samples. And in case of domestic water sample (tap water and mineral water) shows absence of vibrio strain. Vibrios are inhabitants of aquatic environments. They occasionally infect humans, causing intestinal or extraintestinal diseases. The most prevalent diseases caused by vibrios are those that are well reported, including cholera and other forms of acute gastroenteritis. In addition, there may be many other vibrio-caused diseases that go unreported some of the virulence determinants of Vibrio spp. are well characterized.

ENUMERATION OF BACTERIAL POPULATION

The total bacterial population were observed and enumerated on the marine agar plates and nutrient agar plates. The vibrio colonies were isolated and enumerated in a TCBS medium. Both populations were counted and shown

Showing the total bacterial population and vibrio count.

S.NO LOCATION Total bacterial Population Vibrio Population

1 Marine region 108 × 105 65 × 102

2 Garden soil 142 × 106 21 × 102

3 Sewage water 161 × 106 42 × 102

4 Tap water TFTC 0

5 Mineral water TFTC 0

Physiological characteristics of vibrio sp

BIO CHEMICAL TEST NO OF

POSITIVES NO OF

NEGATIVES

OXIDASE TEST 16 1

NITRATE TEST 16 1

CATALASE TEST 8 9

INDOLE TEST 17 0

METHYL RED TEST 17 0

VOGES PROSKEUAR 5 12

STRING TEST 17 0

CHOLERA RED RXN 4 13

CITRATE TEST 7 10

STARCH HYDROLYSIS 0 17

HEAMOLYSIS 5 12

SUCROSE 15 2

MANNITOL, ACID 16 1

LACTOSE 0 17

GLUCOSE, ACID 17 0

GLUCOSE, GAS 1 16

MALTOSE 16 1

By observing the results of physiological tests, most of the tests were found to be positive for vibrio cholerae.The isolates which showed positive results for vibrio cholerae were mainly isolated from marine environment when compared to other samples such as sewage water and soil samples. Through this result we observed that Vibrios are inhabitants of aquatic environments. Marine animals can injure humans in several ways. Some animals cause injury by inducing infection. These infections result when oral bacteria are introduced in to the tissues of victims who are bitten. Bacteria present within the tissues of marine animals can cause infection when they are ingested. In addition, seawater itself contains bacteria, so that skin and soft tissue injuries exposed to seawater may become secondarily injected.

Kirby-Bauer Disk-Diffusion Method: Antibiotic Disk Susceptibilities

Most of the isolates from marine source were highly incidence of antibiotic resistance was evident against Amoxycillin, Ampicillin, Carbencillin and Cefuroxime followed by Rifampin and Streptomycin over soil and sewage samples. However, antibiotic resistance was lower against Chloramphenicol, Tetracycline, penicillin G, Nalidixicacid, Gentamycin Sulphafurazole, Trimethoprim, Neomycin and Amikacin. This may be due to the fact that terrestrial bacteria entering into seawater with antibiotic resistant plasmids may be responsible for the prevalence of the resistance in genes in the marine environment. However, there are few reports available on acquired antibiotic resistance against ampicillin (68%), cefuroxime (66.7%), amikacin (55%), kanamycin (58.8%) and trimethoprim (76.7%) in Sparus sarba in China. It can be presumed that anthropogenic factors (hospital effluents) might have influenced in acquiring resistance in Vibrio spp due to these antibiotics, as there are no reports available on the use of these drugs for aquaculture in India. However, the results of our present study serve as a baseline data for future research on the extent of antibiotic resistance, which may be revealed through isolation of plasmids, their transformation efficiency and conjugation experiments. Results of incidence of multiple antibiotic resistance in Vibrios may improve our knowledge on drug resistant strains and its effect on future therapy of shrimp as well as human diseases. Therefore, unscrupulous use of antibiotics against diseases should be avoided and restrictions for the use of antibiotics may be implemented by a nationwide antibiotic policy for India.

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Characterization and Antimicrobial Susceptibility of Vibrio Spp. Isolated From Different Environments