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Herpes: How To Live With It

drugtest — Thu, 28 Jan 2010 18:41:50 +0000

For the majority of people, the diagnosis of genital herpes (Herpes Simplex Virus 2 or HSV2) is a shock. For others, the diagnosis maybe a confirmation of suspicions they were having about their own health or their partner’s behavior. Seeking to answer the question of how the patient contracted the condition often leads to a search for blame and then self-recrimination. Living with herpes is something that initially may take some psychological adjustment for some patients. It need not mean the end of your sex life or that you will need to remain celibate for the rest of your life.

Firstly HSV2 and HSV1, better known as the cold sore virus, are just two of a related group of seven viruses that are known to infect humans. Others include the Varicella-Zoster virus, commonly known as chicken pox and shingles. Diagnosis of infection with either HSV1 or 2 can be established with a blood test known as the Western Blot test; the upside of this test is that a patient who does not have active lesions may be diagnosed through the presence of antibodies to either strain. Accuracy of this test is only 90-95% depending on the lab involved. Some instances have occurred where patients were diagnosed with either a false positive or a false negative. The most accurate diagnosis is with a physician taking the top off a fresh lesion, obtaining a swab from the base of the lesion and a lab growing a viral culture from it. Extracting a viable swab from the lesion can be quite painful for the patient.

HSV2 traditionally involved infections in genital areas, with the virus lying dormant in the sacral nerve at the base of the spine during periods when the patient is not experiencing lesions. HSV1 traditionally involves infections around the mouth and nose and lies dormant in the trigeminal nerve in the neck during non-active phases of the disease. Current epidemiology studies across the Western World indicate the incidence of HSV2 to be around one in eight people, or 12% of the population. Only one in five of those with antibodies have been diagnosed.

In real terms, in a room containing forty people, five have HSV2 but only one knows they have it. A further three of the five may have had an isolated symptom once or twice. This would have appeared so insignificant that they mistook it for a pimple, infected hair follicle or a boil. The final one in five is someone who has never had a symptom and may never do so. For this patient, and the other three undiagnosed patients, accusations of infection (generally followed by accusations of infidelity) from a partner are often met with counter accusations and disbelief. A conservative estimate of the world population with HSV1 antibodies and the ability to infect others is around 90%. Of these, roughly 45% are symptomatic. If you have been diagnosed with either infection, it is very possible you contracted it from someone who has no idea they have it themselves.

People have received the messages about safe sex and changed some of their practices, believing that only penetrative sex requires safe sex. Sexual health specialists now report that half the new HSV diagnoses in clinics have been microbiologically confirmed as HSV1 on the genitals, in the general community it is now estimated that 20% of all herpes infections in the genitals are in fact HSV1. On the plus side for the infected patient, when the HSV virus is not living in its ideal host environment (i.e. HSV1 infection of genitals, oral HSV2 infection) infections have been generally documented to be less severe and happen less frequently.

Another mistake many patients make, is assuming that they are not infectious during a dormant or asymptomatic phase of their disease. Studies have shown that even when a couple who are clinically discordant (i.e. one is positive and the other is negative) use what is recognized as gold standard treatment for reduction of risk to partners, the rate of transmission in a 12-month period is still 10%. This management of infection control involves the use of condoms during all sexual encounters and complete abstinence from sex during the positive partner’s symptomatic phases. Interestingly, sexual health experts report that if one partner has remained negative for 10 years in a clinically discordant partnership, it is very unlikely that they will contract the disease after this time. It is speculated that they have some immunity/protection either natural or acquired that science has not yet managed to identify.

A true primary infection of HSV2 can last for up to ten days, it involves a systemic response, where all the glands in the body are swollen, much as if the patient has influenza, as well as the obvious genital burning, itching, pain with urination or complete inability to urinate. Many patients think they are presenting with a primary infection, but, severity of symptoms indicates to the physician, this is in fact a recurrence. In these cases the patient’s primary infection would have been asymptomatic, but, for some reason, they have become run down and their immune system is not responding as it did when they were first infected. These and subsequent recurrences of HSV2 are usually around five days in duration, unless there is a serious immune system deficiency. In this case, the treating physician should refer the patient for further testing.

Because HSV transmission requires skin-to-skin contact and viral shedding to occur, typically an infection of HSV2 is specifically confined to the genitals. Affected areas include the vulva and labia in women and penis and scrotum in men, due to penetrative intercourse being quite localized. Where a patient has been infected with HSV1 on the genitals, the area is usually larger and vesicle distribution more extensive due to oral sex skin-to-skin contact covering a more extensive surface area of the genitals. Both viruses may be treated effectively with anti-viral drugs.

As stated earlier, each virus has its ideal host environment. For the patient infected with HSV1 on the genitals, this means subsequent infections are usually less virulent, and in some cases may only ever recur once or twice in their lifetime. For the patient infected with HSV2 on the genitals, the incidence of recurrence can vary greatly. Recurrences are related to the health of the immune system. Triggers may include stress, poor diet, lack of sleep, sunburn and in some women, their menstrual cycle. During the first year of infection, the number of recurrences may range from one to twelve, with an average being four to five. During subsequent years the immune system responds better, the patient learns what will trigger a recurrence and usually tries to avoid it. Eventually most patients can experience as few as one to two recurrences per year. Also, as the patient learns to better recognize the symptoms of an impending recurrence, they are able to administer anti-viral drugs earlier. This can minimize the length and duration of the attack, and possibly prevent lesions altogether. It is important for the patient to remember that despite avoiding a recurrence, they are still shedding the virus and they are still potentially infectious to their partner.

Maintenance doses of anti-virals may be taken daily to reduce the number of recurrences. Up to 50% of patients on these therapies report an absence of recurrences in a 12-month period. Where this therapy is discontinued, patients almost certainly will experience a recurrence within three weeks. This is generally followed by a reduction in the number of annual recurrences. There are a small number of female patients who have required this maintenance therapy with anti-viral drugs continuously since they first became available, over 15 years ago, in earlier forms. As recurrences reduce in frequency and severity, most patients eventually come to terms with their diagnosis. For some, this is never the case, sexual health physicians report that they need to refer between 10-20% of their patients for further psychological counseling. This is in spite the fact that they are very experienced with the disease counseling required for this diagnosis.

What is important, regardless of how well patients appear to cope with the initial diagnosis, is ensuring access to information. This can be obtained readily and anonymously from www.herpes.com, www.herpeshelp.com or www.genitalherpes.com these sites contain up to date facts and also links to other sites. These provide names and contact details of support groups, local clinics and sexual health specialists. Although HSV2 is a lifelong infection, with the right management and care it does not have to be symptomatic, nor should it hinder the patient from enjoying a loving and long-lasting, secure relationship.

Alcohol High School Testing in Schools, Should it Happen?

drugtest — Thu, 28 Jan 2010 08:52:32 +0000

When it comes to drug and alcohol high school testing , what is more important: Protecting constitutional rights or protecting students? The consensus from students is surprising. There are just as many students for random drug testing as against and in some schools, more students are for random testing than not. The same opinion varies among parents, counselors, coaches and teachers.

There is a big majority of some schools’ who believe there is a big alcohol and a drug problem affecting students. Recent high profile court decisions have determined that it is unconstitutional to randomly test students for drugs and alcohol. The schools that were randomly testing students were testing students who were involved in extracurricular activities- meaning these students had the choice to participate, unlike regular classes required by law. Private schools also fell into this category.

Because the school is private, different laws and regulations differ from public, government-run schools, so they were able to randomly test students. The schools who were randomly testing students all acted on “reasonable cause,” like obvious evidence of increased drug or alcohol use on campus. Now, with recent constitutional cases, schools who were about to begin random alcohol/drug testing have postponed their program until further decisions by state and federal courts.

How Are Students Tested Now?

Students were previously tested using two different methods; urine testing and mouth swab tests for saliva testing. Previously, using either urine or the swabbing tests would only measure very recent alcohol or drug use. Now with EtG alcohol and drug testing, ethyl glucuronide testing, a sample now is able to test several days of prior alcohol and drug use, where it wasn’t possible before. Both urine and swabbing testing are intrusive and cost money to perform. Under current alcohol high school testing rules, a student who tested positive would not face criminal charges, but face varying lengths of suspensions from sports or other extra curricular activities. The student would still attend classes but also be required to attend an approved treatment program.

New Testing Devices For Alcohol High School Testing

Hair alcohol testing is the latest and most accurate form of testing on the market today. With only an inch worth of scalp hair from a student, as much as 30 days of alcohol and drug use can be revealed using EtG testing. The instrument used for hair alcohol testing is called a liquid chromatography mass spectrometry, it is a sensitive machine used for testing the smallest of particles in a sample. Besides schools, lawyers, social workers and employers are finding that this type of hair alcohol testing is a far superior way to test someone for recent as well as past alcohol and drug abuse. Plus, hair alcohol testing is by far the least intrusive way to test someone’s metabolites.

Until there is a consensus in the U.S. courts over the rights of students with regards to random alcohol and drug testing, parents, teachers and coaches can only encourage students to abstain with the threat of other consequences or perhaps it will be a peer issue to threaten each other, “continue to abuse and we are all tested via police force, not just by a concerned counselor.”

Von Willebrand Disease in Dobermans

drugtest — Tue, 26 Jan 2010 08:44:07 +0000

brand Disease (VWD) is a common health issue in Dobermans. It is a disease like hemophilia, bleeding in humans, which can put lives at risk Dobermans from surgery or injury. Although it exists in other breeds such as poodles, Shelties, Scottish Terriers and the Pembroke Welsh Corgi, is more common in Dobermans. In a study of 15,000 Dobermans screened, 70% were carriers. Most of these dogs are not clinically affected.

Some of the symptoms of Von Willebrands Disease are excessive bleeding, nosebleeds, bleeding of the gums and bloody urine or feces. This requires special consideration prior to surgery and special attention to injuries. Physical and emotional stress can worsen the bleeding. The treatment for an episode of bleeding is a blood transfusion. Certain medicines should be avoided in dogs with von Willebrand’s disease. These include aspirin, antihistamines, sulfur-based antibiotics, ibuprofen and Amoxicillin. Your veterinarian will know how to treat your dog.

There are 3 types of VWD. Type I is the mildest form of the disease and is the most common in Dobermans. Type 2 is more severe and more common in German Shorthaired Pointers. Type 3 is the most severe form and is usually found in Scotland Terriers and Shelties, although as mentioned above, there are other breeds that can lead this gene.

In addition, surgery or injury, Dobermans are at risk of excessive bleeding during whelping and during the docking of the tails of puppies. It is so important to know that her doberman and her breeding line was tested and did not have Von Willebrand breeding affected dogs.

One way to test for Von Willebrand’s disease is a blood test as called Elisa. This test is not very accurate. We had one of our dogs in the test positive Elisa test, but was clear in the DNA test. The real way to test for this genetic disease is through a DNA test, done with a swab and it costs about $ 140.00. There are 3 levels of the results of DNA testing, of course, the carrier or affected. What this means in terms of breeding Dobermans is somewhat complicated. It is certain that a race with a clear doberman VWD VWD carrier. It is estimated that the bad genes would be eliminated over a period of 2-3 generations.

2 breeding Dobermans affected (in fact suffer from excessive bleeding) always produce 100% affected puppies. Breeding a dog affected with a carrier will result in half of the pups are affected, and half are carriers. 2 breeding VWD carriers will result in 25% of the pups were affected, 50% will be carriers and 25% will be normal. The rearing of a normal carrier of VWD a doberman that a carrier garbage half of the pups, and half normal puppies.

You may wonder why someone from a breeder or breed a doberman that has any indication of VWD. Why not just breed dogs without VWD affected or carries the disease? This would be the ideal situation, but only 1 / 3 of Dobermans are normal, which means they are not affected, or lead to disease. Using only the normal breeding dogs would greatly reduce the gene pool that have a negative impact on the race. Doberman breeders have worked so long to perfect the Dobermans temperament and health problems after 1970. To remove 2 / 3 of the breeding population would translate into the same problems that we have worked to correct.

It is important to buy a doberman puppy reputation of a breeder who has tested his dogs for Von Willebrand’s disease. Be an informed buyer.

Oral Fluid Drug Test

drugtest — Mon, 25 Jan 2010 02:00:58 +0000

Oral fluid drug tests have provided employers and employees with a more dignified, and safer, method of checking for drug use. Thanks to advances in technology, we have a safer and accurate method of checking for drugs in the workplace, particularly at short notice and in almost any workplace setting. Previously, urine samples would be required and this was not a time or cost efficient method due to the time constraints of collecting and sending samples, refrigeration and packing of the samples and the cost of the sample kits themselves. Then there was also the fact that some samples were difficult, if not impossible to collect, and the only alternative was to wait it out, this has been classified as the “bashful bladder syndrome”. Urine sample collection also was a time of awkwardness and embarrassment as well as outright mortification for some. And every employee would have to cooperate. Employers had to have restroom facilities available for the duration of the testing procedures or have the employees go off-site for additional testing which would always necessitate a higher cost to both employee and employer as then you would have to factor in another transportation cost. In addition, the privacy of the employees was non-existent, due to the nature of the test. Oral drug tests are able to provide quick and reliable results and have proven to be quite cost efficient. The cost of the kits is even cheaper than those for urine collection.

The drugs which can be tested for include THC, cocaine and heroin, alcohol, Ecstasy, barbiturates and amphetamines. These oral fluid collection tests may actually be superior at determining opiate usage than the urine test. This is the only test that shows whether a person is under the influence of a drug that he has just taken. This type of testing also has the added feature of being able to ascertain drug usage within the last 6 hours, something other tests could not do. The only single preparation a testee needs is to refrain from any eating and drinking for at least 1 hour prior to having the oral swab taken. Most employees prefer this test as it involves merely a swab between the cheek and lower gum which allows oral saliva to be collected onto the cotton padding of the swab. These tests can be used in any remote job locations. Employers can always choose to hire someone on the spot, thanks to the efficiency and accuracy of these tests, if they wish. There do not have to be any restroom facilities available at the test site. No private rooms are ever needed and no private monitor is necessary to document the authenticity of the oral saliva collection procedure. There are some of these oral fluid collection test kits which will allow the testing of multiple people at one time. Technology, time management and cost effectiveness rolled up into one efficient kit. These tests are also available for home use.

The reliability and accuracy of these tests are in the 98-99% range and they are quite easy to read by those who are charged with administering and documenting the tests. No longer is there any worry about being splashed with bodily fluids and there is no cleaner method available for collecting a body fluid specimen. The employee is able to maintain his composure and dignity during the entire testing procedure and he does not feel violated, as many did under the constant observation during the urine tests. One of the better features of this test is the ability to isolate any of the target drugs that have just been taken. In the past, there were many stories about people that would actually take urine tests while high and yet the urine test would be clean because the drug was too new within the body to show up. With the use of the urine collection tests, the collection could only be done after the drugs had sufficient time to metabolize in the body. It was only after the drug metabolization had begun and the body had begun to excrete that the older tests were able to isolate traces of them. This is not the case any longer thanks to the advent of the new oral fluid collection technology. There is no loss of man hours due to waiting for the completion of a test or to get the results, it takes only a minute ,or so ,to do an oral test, and 10 minutes to see the results. There is no way of adulterating, falsifying or confusing this test. For many employers it is a wonderful innovation, for which there truly was a need.

Oral fluid tests can sometimes be hampered by a lack of oral saliva, and recent food or drink that has been ingested can also have an effect on the drug quantification. But these are quite small negatives. These tests are only geared, unfortunately, for the major types of abused drugs: opiates, cocaine and heroin, barbituates, alcohol and THC. They can only tell recent drug use. Which is one reason, their scope includes the more prevalent, illegal drugs, and the tests can not give any indications of any past drug histories. These tests can and do clearly show how greatly improved today’s drug testing method truly is.

Although the number of drugs that an oral fluids test can check for is relatively small, this one small negative is offset by the numerous positive factors that oral fluid collecting kits have brought to the table. In the past, the procedures surrounding a urine drug screen did often cause humiliation and anger or resentment. An oral test manages to get the job done and over with almost as soon as the person can blink. And any employer would far rather know if an employee is actually using drugs at work, as opposed to knowing that an employee had drank some beer at his home during his time off. This test makes a great deal sense when it is applied and used in a proper setting. These tests may soon become the gold standard in the majority of workplace settings, particularly in settings where it is imperative that the employees are sober and alert, such as in the trucking or heavy machinery fields.

Advantages of Saliva Drug Testing

drugtest — Sun, 24 Jan 2010 12:33:44 +0000

Saliva drug testing is a convenient method of drug screening and an effective alternative for urine drug testing. Collecting sample for saliva drug test is a straightforward process and can be done in less time. As the collection of sample can be done under supervision, there are minimum chances of tampering it. This prevents tampering and increases drug test results accuracy. There are many other advantages of saliva drug testing, which makes it effective for drug screening.

Advantages of Saliva Drug Testing:

Simple and discreet

The sample collection for saliva drug test is a simple procedure. It does not have the issues of invasiveness as in the case of urine drug testing. A typical sample collection process involves placing a swab into the mouth of the person for two to five minutes.

Accuracy of results

Saliva drug testing is less prone to manipulation. The sample collection can be performed under supervision, thus decreasing the chances of adulteration. The integrated specimen collection methods in the saliva drug screening will instantly detect any external adulteration of the sample. The accuracy of the saliva drug test is deemed by the United States court system.

Drug detection

Saliva drug test can detect many commonly abused drugs, effectively. Saliva drug test can identify abuse of barbiturates, cannabinoid, cocaine, opiates, PCP, and amphetamines.

Drug detection times

Saliva drug tests are considered better at detecting drug abuse within a period of 1 or 2 days. There are certain ultra-sensitive saliva drug test kits that can even detect if a person is currently high on drugs. This is not possible in case of many other drug tests.

Easy to administer

The main hassle during drug tests will be with collection of sample. As it can be ignored in case of saliva drug testing, the process of administering the drug screening will be hassle-free. Saliva drug testing is also easy to administer and does not consume much time. Organizations can conduct it in the workplace itself, causing minimal disruption in the work environment.

Quicker detection rates

Saliva drug tests have quicker detection rates compared to blood and urine drug testing. The analysis of the drug screening can be done at the site of specimen collection. Saliva drug testing is an effective tool to monitor drug abuse in organizations.

Saliva drug testing is an effective drug screening method, especially in sensitive places. Many organizations are presently using saliva drug test kits for monitoring their employees for drug-free workplace. Saliva drug tests are hard to adulterate and provide results within minimum efforts.

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

drugtest — Fri, 22 Jan 2010 03:05:12 +0000

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.

REFERENCES

1. Richard J., and Bennett N T.1993.Infections Caused by Halophilic

Marine Vibrio Bacteria. From the Department of Surgery, University of Florida, Gainesville, Florida Vol. 217, No. 5, 525-531

2. Colwell, R. R., and W. M. Spira. 1992. The ecology of V. cholerae, p. 107–127.

In D. Barua and W. B. Greenough III (ed.), Cholera. Plenum Medical Book

Co., New York, N.Y.

3. West, P. A., and J. V. Lee. 1982. Ecology of Vibrio spp. including V. cholerae

in natural waters of Kent, England. J. Appl. Bacteriol. 52:435–448.

4. Xu, H. S., N. C. Roberts, F. L. Singleton, R. W. Attwell, D. J. Grimes, and

R. R. Colwell. 1982. Survival and viability of nonculturable Escherichia coli

and Vibrio cholerae in the estuarine and marine environment. Microb. Ecol.

8:313–323.

5. Baumann, P., and R. H. W. Schubert. 1984. Family II. Vibrionaceae, p.

516–550. In N. R. Krieg and J. G. Holt (ed.), Bergey’s manual of systematic

Bacteriology, vol. 1. Williams & Wilkins, Baltimore, Md.

6. Farmer, J. J., III, F. W. Hickman-Brenner, and M. T. Kelly. 1985. Vibrio, p.

282–301. In E. H. Lennette, A. Balows, W. J. Hausler, and H. J. Shadomy

(ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology,

Washington, D.C.

7. Kay, B. A., C. A. Bopp, and J. G. Wells. 1994. Isolation and identification of

Vibrio cholerae O1 from fecal specimens, p. 3–20. In I. K. Wachsmuth, P. A.

Blake, and O. Olsvik (ed.), Vibrio cholerae and cholera: molecular to global

perspectives. ASM Press, Washington, D.C.

8. West, P. A., and R. R. Colwell. 1983. Identification and classification of

Vibrionaceae–-an overview, p. 285–341. In R. R. Colwell and M. B. Hatem

(ed.), Vibrios in the environment. John Wiley & Sons, New York, N.Y.

9. Chun, J., A. Huq, and R. R. Colwell. 1999. Analysis of 16S-23S rRNA

intergenic spacer regions of Vibrio cholerae and Vibrio mimicus. Appl. Environ.

Microbiol. 65:2202–2208

10. Nandi, B., R. K. Nandy, S. Mukhopadhyay, G. B. Nair, T. Shimada, and A. C.

Ghose. 2000. Rapid method for species-specific identification of Vibrio cholerae

using primers targeted to the gene of outer membrane protein W. J.

Clin. Microbiol. 38:4145–4151.

11.Gabriella caldini, Angela neri, Stefania cresti, Vieri boddi,

Gian maria rossolini, and Eudes lanciotti high prevalence of vibrio cholerae non-o1 carrying heat-stable enterotoxin-encoding genes among vibrio isolates from a temperate-climate river basin of central Italy. Applied and environmental microbiology July 1997, Vol. 63, No. 7 p. 2934–2939

12. Abbott, S.L., Cheung, W.W.K.W. and Janda, J.M. (1993) Evaluation of a new

selective agar, thiosulfate-chloride-iodide (TCI), for the growth of pathogenic

Vibrio species. Medical Microbiology Letters 2, 362–370.

13 .Morris, G.K. (1982) Media for Vibrio species. In Quality Assurance and Quality Control of Microbiological Culture Media ed. Corry, J.E.L. and Verlay, G.I.T. pp. 169–174. Germany: Darmstadt.

14. Oliver, J.D. (2003) Culture media for the isolation and enumeration of pathogenic

Vibrio species in foods and environmental samples. In Culture Media for Food

Microbiology, 2nd Edn eds Corry, J.E., Curtis, G.D.W. and Baird, R.M.

Amsterdam: Elsevier Science, in press.

15. Ferguson, G. E., C. W. Lingham, S. K. Love, and R. 0. Vernon. 1947. Springs of Florida, p. 196. Fla. Geol. Bull. no. 31. E. 0. Printing Co., DeLand, Fla.

16. Lee, J. S. 1973. What seafood processors should know about Vibrio parahemolyticus. J. Milk Food Technol. 36:405-408.

17.Gopal, S, SK Otta, I Karunasagar, M Nishibuchi, and I Karunasagar. “The occurrence of Vibrio species in tropical shrimp culture environments; implications for food safety.” International Journal of Food Microbiology. 2005 Jul 15;102(2):151-9.

18.Graf, J. “The Light-Organ Symbiosis of Vibrio fischeri and the Hawaiian Squid, Euprymna scolopes.” February 2005. Accessed 8 July 2005.

19.Ho, Hoi. “Vibrio infections.” eMedicine. 22 May 2005. Accessed 8 July 2005.

20.Oliver, JD. “Wound infections caused by Vibrio vulnificus and other marine bacteria.” Epidemiology and infection. 2005 Jun;133(3):383-91.

21.Sarkar, M, S Das, A Bandyopadhaya, K Ray, and K Chaudhuri. “Upregulation of human mitochondrial NADH dehydrogenase subunit 5 in intestinal epithelial cells is modulated by Vibrio cholerae pathogenesis.” FEBS letters. 2005 Jun 20;579(16):3449-60.

22.Senoh, Mistutoshi, Shin-Ichi Miyoshi, Keinosuke Okamoto, Belen Fouz, Carmen Amaro, and Sumio Shinoda. “The Cytotoxin-Hemolysin Genes of Human and Eel Pathogenic Vibrio vulificus Strains: Comparison of Nucelotide Sequences and Application to the Genetic Grouping. Microbiology and immunology. 2005;49(6):513-9.

23.Gerhard,P.,R.G.E.Murray,R.N.Costilow,E.W.Nester,W.A.Wood,N.R.Krieg,andG.B.phillips.1981.Manual of method for generalbacteriology.American SOCIETY FOR microbiology, Washington, D.C.

24. Mary. L. G and rita r. colwell, Enumeration, isolation and characterization of N2 fixing bacteria from sea water. Department of microbiology, University of Maryland. Vol 50 no .2. 1985

Other References

1.Dennis Kunkel Microscopy, Inc.

2.Fix, Douglas F. “Vibrio.” Accessed 8 July 2005.

3.Todar, Kenneth. Todar’s Online Textbook of Bacteriology. 2005. Accessed 8 July 2005.

4.Vibrio fischeri Genome Project. 19 September 2002. Accessed 8 July 2005.

5.http://microbewiki.kenyon.edu/index.php/Vibrio

6.Center for Disease Control. 1973. Vibrio parahaemolyticus gastroenteritis. Morbid. Mortal. Weekly Rep. 22:231-232.

To Test or not to Test for Drugs and Alcohol in Schools

drugtest — Tue, 19 Jan 2010 13:37:06 +0000

Although the Supreme Court ruling is a set back for schools who are attempting to help students, reduce crime or improve a school’s image, the fight is far from over and concerned students, teachers, district heads and counselors are looking into alternatives. How could students encourage other students to participate in an all volunteer drug and alcohol high school testing program?

Perhaps the “how” students are tested will make the difference. Previously, urine alcohol testing and swab testing were used to quickly test whether or not a student was using alcohol or drugs. Now, with new technology, there are other less intrusive ways to test students. The procedure is called Hair Alcohol Testing. It is the least intrusive alcohol high school test on the market. Hair alcohol testing is becoming the new preferred method for testing a person for drugs and alcohol for several reasons including:

•    Accuracy

•    Long term results- revealing a student’s consumption record up to 12 months with just 1 inch of hair!

•    Least intrusive method on the market: 1 small inch of scalp hair cut underneath the head

•    A trusted and respected method for detecting drugs and alcohol

If schools were able to offer an almost fun way to test themselves then perhaps the curiosity alone would temp them to volunteer? With new drug and alcohol testing devices, perhaps states don’t need to get involved at all- just leave it up to good ol peer pressure to change things.

It’s not that most schools were having huge problems with teens coming to class intoxicated or under the influence; in fact even before the Supreme Court ruling; most students were volunteering to be tested for drugs and alcohol.

In order to avoid mandatory random drug testing, several student bodies took it upon themselves to test each other for alcohol and drug use. It wasn’t as challenging as you would think to get students to monitor themselves, to avoid the latter. Possibly it was because most school’s consequences for students with “positive results” were minor with no arrests, just counseling, parent involvement and sports and other privileges revoked.

A high school in Washington State attempted a two-year voluntary program but only received a 65 percent participation rate and those were only students in extra curricular activities. Although the turn out was low, a noticeable decline of alcohol and drug use could be detected.

Just because there is a couple bad apples in the group doesn’t mean the entire crop is bad. Students know this and they are eager to prove they are clean. What is more powerful than a “mandatory test” given by the state? A test backed by peer pressure.

Interference With Cell-to-cell Signaling: a Potential Therapeutic Approach Against Vibrio Species

drugtest — Sun, 17 Jan 2010 04:49:20 +0000

Vijaya baskar .P 1* AND Veera ravi.A 2

1. Department of biotechnology Dr. G.R.D.C.S, Coimbatore.

2. Department of biotechnology, Alagappa University, karaikudi.

ABSTRACT

Bacterial biofilms are sessile communities with high cell density that are ubiquitous in natural, medical, and engineering environments; they are fascinating since they are primitive tissues with an advanced chemical communications network. Currently, there is an explosive amount of biofilm research, most of it with the ultimate aims of biofilm prevention, control, or eradication.The present investigation was aimed to study the cell signaling principle among seaweed epibiotic bacterial organisms , totally 54 bacterial isolates were made out of 20 seaweed species. Among 54 bacterial isolates 17 of them producer, another 17 of them inducer strains rest of 20 isolates were normal that have not showing any signs of activity. The 17 producers and 17 inducer strains were subjected to the cross species induction by quorum sensing principle and found that only 3 of them have responded to the cross species induction in production of antibacterial compound against the respective inducer strains. All the 3 producers and inducer were identified by biochemical analysis and surprisingly that all the 3 producer strain belongs to Genus Pseudomonas and inducer strains were belongs to Genus Vibrio. The supernatant obtain from mixed culture of Pseudomonas & Vibrio shows the antibiotic activity against the various Vibrio species which were isolated from other sources. Such as Vibrio from primary film, Vibrio from sediment Vibrio from seaweeds epibiotics. The obtain results clearly shows the particular Pseudomonas from seaweed epibionts has the capability of producing potential antibiotic compound against wide range of Vibrio species through quorum sensing.

*Corresponding author e-mail: vijay10bas@yahoo.co.in

INTRODUCTION

We are living in a microbial planet. About 71 % of the surface of this planet is covered by sea water. A typical milliliter of seawater contains 103 fungal cells, 106 bacteria, and 107 viruses, including pathogens that cause widespread mortalities and microbes that initiate fouling of host surfaces (Rheinheimer, 1992). Thus, marine plants and animals are continually exposed to high concentrations of potentially harmful microbes. These microorganisms in nature exists as free living planktonic mode of life in sea water or it may exist as epibiotic organisms in various living and nonliving surfaces. Among living organisms, seaweeds and invertebrates act as suitable substrate for the establishment of epibiotic organisms Seaweeds are known to release a large amount of organic carbon into the surrounding environment providing a nutrient rich habitat for microorganisms like bacteria. Bacteria are generally considered to be independent unicellular organisms. One cell accomplishes all of the tasks of feeding, locomotion, ‘reproduction, respiration and all other processes necessary to keep an organism alive. There are several classes of bacteria such as primary film forming bacteria, sediment bacteria, symbiotic bacteria, and epibiotic bacteria in various aquatic organisms. The marine surface environment is a site of intense composition for living space by a wide variety of organisms. Bacteria are generally recognized as primary colonizers of this habitat and are able to form biofilm on marine surface such as invertebrates and algae (Bryers, et al., 1982). Bacteria may also be abundant on the surfaces of some algae as an important epibiotic organism. In many cases, the bacterial population found to be specific, with changes occurring throughout the year or life span of the algal surface. This algal-bacterial relationship is symbiotic in most cases; the epibiotic bacteria in seaweed play a protective role by releasing secondary metabolites into the surrounding seawater that help preventing extensive fouling of the surface. Epibiotic bacteria are therefore attracting attention as a source of new natural products. Bacteria from the larvae of some crustaceans protect them from fungal infection by the production of simple antimicrobial compounds. Bacteria isolated from the surface of a tunicate prevented the settlement of barnacle and tunicate larvae exposed to the bacteria as biofilm in petridishes (Evelyn et al., 2001).

Seaweeds itself secretes secondary metabolites to prevent fouling and grazing. In addition to that epibiotic bacteria on macro algae can also produce antifouling compounds that work in concert with the seaweed derived compounds to protect the seaweed surface. Recent studies have highlighted important roles of epibiotic bacteria colonizing the surface of seaweeds and releasing antifouling compounds. For the past 50 years antibiotics have revolutionized medicine by providing cures for formerly life threatening diseases. However, strains of bacteria have recently emerged that are virtually unresponsive to antibiotics such multidrug resistance, arising mainly through antibiotic misuse, is now recognized as a global health problem. In this situation, it is clear that new classes of antibiotics are urgently needed. Many marine bacteria have been shown to produce secondary metabolites that display antibacterial properties. The first antibiotic from a marine bacterium was identified and characterized in 1966. In addition, bacteria in biofilm on the surface of marine organisms have been documented to contain a higher proportion of antibiotic producing bacteria than some other marine environments (Burgess, et al., 1999). Marine epibiotic bacteria, associated with nutrient-rich algal surfaces have also been shown to produce antibacterial secondary metabolites which inhibit the settlement of potential competitors. Recently a lot new novel antibiotics such as Phenazine, thiomarinol, phenazine-1-carboxylic acid, 1-hydroxyphenazine 2-n-heptylquinol-4-one, 2-n-nonylquinol-4-one pyolipic, loloatins, agrochelin, sesbanimides, pelagiomicins, indomycione and indomycione have been identified from various marine epibiotic bacterial organisms. In particular, some species of the genus Pseudomonas produce both antibiotics and several other bioactive substances. For example, Pseudoalteromonas rubra and Pseudoalteromonas aurantia have been reported to be antibiotic producing bacteria. The phenomenon of higher organisms utilizing their associated microflora for the production of beneficial secondary metabolites is common in the marine environment (Yotsu, et al., 1987). A study of bacteria isolated from marine algae surfaces indicated that the incidence of antibiotic producing strains from this habitat was 20% whereas that from sea water was only a few percent. In addition, some bacteria that previously did not produce any active compounds have been found to be producing such metabolites when they are exposed to other bacterial species or extra cellular chemical from other bacteria. Bacteria may also produce antimicrobial compounds when they sense the presence of competing organisms. However, few attempts have been made to study such chemical communication between different bacterial species or how this might affect. The secretion of antimicrobial compounds (Mearns-Spragg, et al., 1998). Bacterial communication by the chemical signals for specific function is simply known as Quorum sensing. In which a bacterial population receives input from the environment and elicits an appropriate response (Hiroaki and Kristina. 2003). The term “quorum sensing” describes the ability of a microorganism to perceive and response to diffusible signal molecules. Bacterial cells sense their population density through a sophisticated cell to cell communication system and trigger expression of particular genes. Tne first system of density-dependent regulation was studied in detail with the luminescence of Photobacterium fischeri (formerly known as Vibrio fischeri) by Bassler et al., 1997. Eventually, they discovered that 3-oxo-N-(tetrahydro-2-oxo-3-furanyl) hexanamid or N-3-(oxohexanoyl) homoserine lactone (OHHL) was responsible the agent in the broth that induced luminescence. Followed by this many researchers have confirmed that in Gram negative bacteria acyl-homoserine lactone is responsible for the cell to cell communication system.

In gram positive bacteria peptide and derivative peptide based signaling molecules seem to be the predominant mode of communication. During high cell density the marine bacteria can produce enzymes, surfactants, toxins, and antibiotics by the chemical signal communication. Marine epibiotic bacteria are also known to produce compounds active against drug resistant hospital pathogen by the cross species induction method. Building on assays described by Austin (Billaud and Austin 1990) a screening procedure has been developed in which marine bacteria are challenged by exposing them to terrestrial bacteria prior to assay of antimicrobial compounds. Hence in this present investigation it is proposed to find out the ability of sea weed epibiotic bacterial organism to produce antibacterial compounds through quorum sensing.

MATERIALS AND METHODS:

SAMPLE COLLECTION:

Seaweed samples were collected from Gulf of Mannar Marine Biosphere Reserve and identified up to species level by using CMFRI bulletin (14) as follows:

Table 1. List of Seaweeds species collected for the present study

SPECIES NAME FAMILY

Halimeda gracilis Chlorophyceae

Ulva lactuca Chlorophyceae

Microdictyon tenunis Chlorophyceae

Chondrococcus hornemonii Chlorophyceae

Enteromorpha intestinalis Chlorophyceae

Caulerpa cupressoides Chlorophyceae

Caulerpa racemosa Chlorophyceae

Dictyota dichotoma Phaeophyceae

Turbinaria ornata Phaeophyceae

Padina gymnospora Phaeophyceae

Sargassum cinearifolium Phaeophyceae

Dictyota batryensis Phaeophyceae

Sargassum sps Phaeophyceae

Hypnea musciformis Rhodophyceae

Acanthophora dendroides Rhodophyceae

Jania rubens Rhodophyceae

Hypnea valentiae Rhodophyceae

Hypnea pannose Rhodophyceae

Hypnea esperi Rhodophyceae

Acanthophora spicifera Rhodophyceae

ISOLATION OF EPIPHYTIC BACTERIA

The collected seaweed samples were thoroughly washed with sterile seawater to removes the loosely attached bacteria/particles. Seaweed fronds were scrubbed with sterile cotton swabs to obtain epiphytic bacteria. Epiphytic bacterial organism in the swab were inoculated in sterile peptone broth (50% sea water) and incubated at 28°C in an incubated shaker (220 rpm / min) for overnight. After the incubation period the enriched cultures were serially diluted up to 10-8 concentration and 200 microlitre of each diluted samples were transferred into the nutrient agar plate (50% sea water). The plates were incubated at 28°C for 5 days and the plates with crowded colonies were selected. In the crowded plates those colonies, which showed the sign of inhibition zone around its margin to the neighboring colony, were selected and considered as producer strain. The neighboring sensitive colonies were treated as inducer strain. Both producer and inducer strains were streaked repeatedly until to get pure culture. The pure culture were properly labeled and subjected to the quorum sensing analysis.

QUORUM SENSING

EXPERIMENT NUMBER 1

In this present study, the producer and inducer strains were cross reacted to find out the production of antibiotic compound through quorum sensing. Totally three set of cultures were maintained as follows (along with one as control).

A. Live cells of producer and inducer strains

B. Live cells of producer strain alone

C. Live cells of inducer strain alone

In culture system A 200ul of 16 hours old broth culture of both producer and inducer strains were added to the 15 ml of nutrient broth.

In culture system B 200ul of 16 hours old producer strain alone was inoculated.

In culture system C 200ul of 16 hours old inducer strain alone was inoculated.

All the cultures were incubated at 28°C for 5 days. After the incubation period the cultures were centrifuged at 10,000 rpm for 15mins. The supernatant was collected and subjected to antibacterial assay with respective inducer strain.

EXPERIMENT NUMBER 2

In this experiment, culture supernatant was obtained as per the procedure given in the experiment 1. 50ml of supernatant was mixed with equal volume of 80% methanol and 1% acetic acid mixture and it was shaked thoroughly in a separating funnel. Finally the methanol and acetic acid fractions were collected and concentrated by evaporation using water bath at 55°C. The viscous colloidal residues were resuspended in 600 microlitre of 50% methanol and it was used for antibacterial assay against different test organism.

TEST ORGANISMS:

1. Epiphytic Vibrio from seaweeds

2. Vibrio from primary film

3. Vibrio from Sediments

4. Pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella sp. and Proteus sp

The test organisms Vibrio species were isolated from seaweed as epiphyles, biofilm, sediment and puffer fish by using TCBS medium (Hi media) The pathogenic bacteria were collected from clinical laboratories.

ANTIBIOTIC ASSAYS

Antibiotic activity was performed in duplicate using a standard paper disc diffusion method as well as well assay. In well assay 10mm in diameter wells were made in marine agar plates and the plates were swabbed with 16 hours old inducer strain. To these wells 200ul of cell free supernatant were added to each well. In paper disc assay the Watmann no.1 filter paper discs (6mm in diameter) were saturated with 200ul of cell free supernatant. The impregnant discs were Dlaced in the centre of the plates swabbed with test organisms. The plates were Incubated at 37°C overnight and observed for inhibition zone. The zone of inhibition was measured as the distance from the border of paper disc to the edge of the clear zone and expressed in mm.

BACTERIAL IDENTIFICATION

The organisms responded to the quorum sensing process alone were identified by the following biochemical analysis.

Colony morphology, Gram staining, Motility test, Oxidase test, Catalase test, Indole Production, Methyl red test, Voges Proskauer test, Citrate Utilization test, Triple sugar Iron test, Nitrate reduction test, Lactose fermentation, Urease test

Starch hydrolysis test, Protein hydrolysis test, Lipid hydrolysis test, Oxidative / Fermentative test, Salt concentration (0%, 3%, 5%, 7%, 10%), TCBS, Growth in Temperature, 42°C and 47°C

All the above mentioned biochemical tests were performed by following standard methodology given in the Microbiological Laboratory Manual by James 3.Cappuccino (1999).

RESULTS AND DISCUSSION:

QUORUM SENSING/CROSS SPECIES INDUCTION ANALYSIS

In the present investigation totally 54 isolates were collected out of seaweed species. Among 54 isolates, 17 of them are producer strain, another 17 are the inducer strain rest of 20 isolates is normal and not showing any signs of activity (Table.2).

a) Among these 17 producers strain 6 strains were isolated from Hypnea musiformis. 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from Sediment.

b) Among these 17-inducer strain 6 strains were isolated from Hypnea musiformis, 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from sediment.

All the 17 strains were named as

PRODUCERS STAINS

BrA+, BrB+, BrC+, BrD+, BrE+, BrF+ Hypnea musiformis

GcA+, GcB+, GcC+, GcD+, GcE+, GcF+ Gracillaria edulis

U1+, U2+, U3+, U4+ Ulva lactuca

SA+ Sediment

INDUCER STRAIN

BrA-, BrB-, BrC-, BrD-, BrE-, BrF- Hypnea musiformis

GcA-, GcB-, GcC-, GcD- GcE-, GcF- Gracillaria edulis

U1-, U2-, U3-, U4- Ulva lactuca

SA- Sediment

In this experiment among 17 Producer and Inducer strains only 3 of them have responded to the quorum sensing principle. (BrB+/BrB-), (GcC+/GcC) and (SA+/SA-)

Table 2: The results of Quorum Sensing analysis of epibiotic bacterial isolates from seaweeds.

Seaweed sample Producer organism Inducer organism Cross-species producer with inducer Cross-species supernatant test with inducer Zone of clearance (mm)

Hypnea musiformis

1. BrA+

2. BrB+

3. BrC+

4. BrD+

5. BrE+

6. BrF+ BrA-

BrB-

BrC-

BrD-

BrE-

BrF- Br A+ /Br A-

Br B+ /Br B-

Br C+/Br C-

Br D+/Br D-

Br E+/Br E-

Br F+/Br F- BrA-

Br B-

BrC-

BrD-

BrE-

BrF- NIL

39

NIL

NIL

NIL

NIL

Gracillaria

edulis 7. GcA+

8. GcB+

9. GcC+

10. GcD+

11. GcE+

12. GcF+ GcA-

GcB-

GcC-

GcD-

GcE-

GcF- Gc A+/GcA-

Gc B+/GcB-

Gc C+/GcC-

Gc D+/GcD-

Gc E+ /GcE-

Gc F+/GcF- GcA-

GcB-

GcC-

GcD-

GcE-

GcF- NIL

NIL

26

NIL

NIL

NIL

Ulva lactuca

13. U1+

14: U2+

15. U3+

16. U4+ U1-

U2-

U3-

U4- U1+/U1-

U2+/U2-

U3+/U3-

U4+/U4- U1-

U2-

U3-

U4- NIL

NIL

NIL

NIL

Sediment 17. SA+ SA- SA+/SA- SA- 28

c) The normal 20 bacterial strains isolated from 20 algal species were crossed with terrestrial bacteria such as E-coli, Staphylococcus aureus separately

This experiment does not showed any inhibition zones

Bacterial identification

The 3 producer and 3 inducer strains which were responded the quorum sensing principles alone were subjected to biochemical analysis for identification. The obtained results revealed that all the producer strains showed sings of Pseudomonas sps and the inducer strains showed signs of Vibrio sps. So, based on the obtained result all the producer strains seems to be a Pseudomonas sps where as all the Inducer strain belongs to the genus vibrio.

In the present investigation, it was aimed to produce the antibiotics from the seaweed epibionts through quorum sensing principle. The bacterial isolates of seaweed epibionts were identified as species of Pseudomonas and Vibrio from seaweeds Hypnea musiformis and Gracillaria edulis. In this study the Pseudomonas acts as a producer strain and Vibrio as inducer strain. The recent finding says that the seaweed epibionts having potential to control the metabolic activity of competitor organisms. Allison et al., 1998 reported that many bacterial strains up on attaching to a surface produce exopolysaccharides or exopolypeptides. In addition, it has been postulated that exopolysaccharides could mediate the attachment of the bacteria to the surface and induce metabolic changes.

The results of Vanderivere and Kirchman 1993 suggest that the addition of increased surface by adding sand will induce the exopolymer synthesis through the high cell density dependent system. In the same way the bacterial organisms attached to the surface of seaweed shows alteration of genes expression may be due to the response to the high competitive environment. When cell density increases the competition for space and nutrients are also increased. So the existing bacteria were forced to protect themselves in this competitive environment. Normally in this condition the bacteria will be activated to induce the expression of certain hidden genes in genetic material through quorum sensing. The quorum sensing is principles were active compounds (autoinducer) from bacterial cell will promote the expression of a particular hidden gene of other bacterial organism in a stressed condition.

Quorum sensing usually focused on the bacteria growing in homogeneous environment. However few studies have attempted to a study this principle in heterogeneous environment also. In this present investigation we have attempted to study both homogeneous as well as heterogeneous environment. In former one we have isolated producer strain in seaweed eipbionts and it shows inhibitory activity against the inducer organism at the same seaweed epibionts. Later producer strains from seaweed epibionts, were treated with various Vibrio organisms from different environment. The obtain result of this study shows that the producer strain are capable of secreting antibiotic compounds not only to their natural competitors in its own habitate but also to the pathogen inhabiting in a distant related environment.

In the gram negative bacteria AHSL is an active principle of quorum sensing. Our producer strain is also been identified as Pseudomonas sps. So in these organisms also active principle must falls under the AHSL. The cell-cell signaling mechanism can either require import of the signal and subsequent interaction with intracellular effectors or a two-component signaling system that transducers the information across the membrane. In V. harveyi genetic analysis of the density sensing apparatus has two independent density-sensing systems, and each is composed of a sensor-auto inducer pair; system one is composed of sensor I and Al -1, and system two is composed of sensor 2 and AI-2. The two densities – sensing system are redundant, because a null mutation in either system alone results in expression of hidden genes (Bassler, et al.,1999.).

The earlier genetic analysis in Pseudomonas reveals the Pseudomonas consist of two quorum sensing systems as Las R1-I and Rh1R-l and have linked with R and I genes, in addition recently a third Lux R homolog that is advanced to a cluster of quorum sensing – controlled (qsc) genes were detected. Las R is a transcriptional regulator that responses primarily to the Las I – generated signal and Rh1R is a transcriptional regularly that responses best to the Rhl -generated signal. In Pseudomonas auriginosa, at low population densities Las I produce a basel level of 3-O-C12-HSL. As density increases, 3-0-C12-HSL builds to a critical concentration, at which point interacts with LasR. This Las R -3-0-012-HSL complex that activates transcription of a number of genes [Whileley, et al., 1999].

We suggest that the above said mechanisms in Pseudomonas with quorum sensing principle might have occurred in the present study also. This induces the bacteria Pseudomonas in epibiotic seaweeds to secrete certain active compound against to the competitor Vibrio species.

In this present work the totally 54 isolates were screened from 20 different seaweed species out of which 34 species were showed the signs of quorum sensing i.e. 17 producer and 17 inducer strains, but when these organisms where subjected to quorum sensing principle in mixed culture only 3 them have responded. So the present study reveals around 17% of bacterial species isolated from seaweeds and sediment were responded to quorum sensing. According to the results of bacteria isolated from marine algae surfaces indicated that the incidence of antibiotic producing strains from this habitat was 20% where as that from sea water was only a few percent. In the present study also reveals more or less the same ratio in Pseudomonas spp. was observed. Our results also reveals a results of Kell et al., 1995; Stead et al., 1996; they have said the culture supernatant of Pseudomonas sps known to contain AHLS which induces the production of phenazine antibiotics. In this investigation due to time constraint, It was not attempted to identify the active compound secreted by Pseudomonas through quorum sensing, which may leave the space for the further intensive research in future.

In concluding this discussion, the quorum sensing is wider spread among bacterial population then was previously thought, (In Gram positive, Gram negative bacterial communication). Current assays for antimicrobial activities are inadequate because some antibiotic producing bacteria may require the presence of another bacterial species. These findings have important implication for the discovery of novel antimicrobial compounds from marine bacteria and may allow the development of new methods for screening novel compounds active against multidrug resistant bacteria.

CONCLUSION:

The present investigation was aimed to study the quorum sensing principle among seaweed epibiotic bacterial organisms. In the past few decades there was no findings of new novel antibacterial class compounds were identified. But, the pathogenic microorganisms show much higher rate of resistant development even to the potential antibiotics. So, there is an urgent need to discover new novel antibiotic compounds. The marine inhabitants such as microorganisms, seaweeds, invertebrates, etc., act as an undepleted source of wide range of natural products among which the seaweeds act as a potential source of antibiotic compounds. Currently the cross species induction / quorum sensing attracts the total attention of researchers in finding new novel drugs against multidrug resistant pathogenic microorganisms. So, the present study aim to find out the capability of seaweed epibiotic bacterial organisms to produce novel drugs against the animal and plant pathogens

REFERENCES

Alim, I., and K. Yuto. 2003. Mc21-A, a Bacteriocidal antibiotic produced by a new marine bacterium, Pseudoalteromonas phenotica sp. nov. O-Bc 30T, against Methicillin -Resistant Staphylococcus aureus. Antimicro. Agents Chemoth. 47: 480 – 488.

Allison, D. G., B. Ruiz, C. Sanjose, A. Jaspe, and P.Gilbert. 1998. Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms. FEMS Microbial Lett. 167: 179-184.

Armstrong, E., J. D. Mckenzie, and G.T. G. Worthy. 1999. Aquaculture of sponges on scallops for natural products research and antifouling. J. Biotechnol. 70: 163-174. 70. 21-

Bainton, N. J., P. Stead, and S.R. Chhabra. 1992. N-(3-oxohexanoyl)-L-homoserine Lactone regulated carbapenem antiobiotic production in Erwinia carotovora. Biochem. J. 288: 997-1004.

Bassler, B. L., E. P. Greenberg, and A. M. Sterens. 1997. Cross species induction of luminescence in the quorum – sensing bacterium Vibrio harveyi. J. Bacteriology. 179: 4043-4045.

Bassler, B.L.1999. Curr.Opin.Microbiol.2: 582-587.*

Bernan, V. S., M. Greenstein, and W. M. Maiese. 1997. Marine microorganisms as a source of new natural products. Adv. Appl. Microbiol. 43: 57 – 89.

illaud, A. C, and B. Austin. 1990. Inhibition of the fish pathogen, Serratia liquefaciens by an antibiotic producing isolates of planococcus recovered from sea water. Journal of

Boyd, K.G., A. Mearns -Soragg, G. Briedley, K. Hatzidimitriou, A. Rennie, M. Bregu, M. 0. Hubble, and J. G. Burgess. 1998. Antifouling potential of epiphytic marine bacteria from the surface of marine algae. Plouzane, France: 128 -136.

Bryers, J. D., and W. G. Characklis. 1982. Processes governing primary biofilm formation. Bioeng. 24: 2451-2476

Burgess, J. G., K. Boyd, E. Armstrong, T. Pisacane, and D. R. Adams. 2003. The development of a marine natural product-based antifouling paint. Biofouling. 19: 197 -205.

Burgess, J. G., E. M. Jordan, M. Bregu, and A. Mearns-spragg. 1999. Microbial antagonism: a neglected avenue of natural products research. J. Biotechnology. 70: 27-32.

Burgess, J. G., H. Miyashita, H. Sudo, and T. Matsunaga.1991. Antibiotic production by marine photosynthetic bacterium Chromatium purpuratum NKPB 031704: Localization of activity to the chromatophores. FEMS Microbiol. Lett. 84: 301 – 306.

Cappuccino J. G., and N. Shermann. 1999. Microbiology, A Laboratory manual, Fourth edition, Addison Wesley, New York.

Davies, D. G., M. R. Parsek, J. P. Pearson, B. H. Iglewski, J. W. Costerton, and E. P. Greenberg. 1998. The involvement of cell to cell signals in the development of a bacterial biofilm. Science. 280: 295-298.

Dekievit, T. R., and B. H. Iglewski. 2000. Bacterial quorum sensing in pathogenic relationships. Infect. Immun. 66: 4839-4849.

Dimango, E., H. J. Zar, R. Bryan, and A. Prince. 1995. Diverse Pseudomonas aerugniosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J. Clin-lnvestig. 96: 2204-2210.

Evelyn, A., Y. Liming, K.G. Boyd, P. C. Wright, and J. G. Burgess. 2001. The symbiotic role of marine microbes on living surfaces. Hydrobiologia. 461: 37 – 40

Burgess, J. G., M. Elizabeth, M. Bregu, A. Meams-Sprugg, and K. G. Boyd. 1999 Microbial antagonism: a neglected avenue of natural products research. J. Biotechnology. 70. 27-32.

Hiroaki, S., and K. M. Smith. 2003. Molecular mechanisms of bacterial quorum sensing as a new drug target. Curr. Opin. Chem. Biol. 7: 586-591.

Hoang, T., Y. Ma, R. J. Stern, M. R. Meneil, and H. P. Schwezer. 1999. Construction and use of low-copy number T7 expression vectors for purification of problem proteins: purification of Mycobacterium tuberculosis RmID and Pseudomonas aeruginosa Lasl and Rhll proteins, and functional analysis of purified Rhll. Gene. 237: 361 – 371.

Holmstrom, C, and S. Kjelleberg. 1994. The effect of external biological factors on, settlement of marine invertebrates and new antifouling technologies. Biofouling. 8: 147-160.

Holmstrom, C, D. Rittschof, and S. Kjelleberg. 1992. Inhibition of settlement by larvae of Balanus amphitrite by a surface – colonizing marine bacterium. Appl. Env. Microbiol. 58: 2111 -2115.

Ines, M. M., B. Karaigher, U. Cepon, and I. Mahne. 2003. Variability of the Quorum sensing system in natural isolates of Bacillus sp., Food technol. Biotechnol. 41: 23 – 28.

Kell, D. B., A. S. Kaprelyants, and A. Grafen. 1995. Pheromones, Social behaviour and the functions of secondary metabolism in bacteria. Trends in Ecol. and Evol. 10: 126-129.

Korber, D. R., J. R.Lawrence, H. M. Lappin-scott, and J. W. Costerton. 1995. Growth of microorganisms on surfaces. In. Microbial. Biofilm, Lapp in – Scott. Cambridge, UK: Cambridge university press.

Lemos, M. L, A. E. Toranzod, and L. J. Barja. 1986. Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds. Microbial Ecology. 11: 149-163.

Liming, Y., G. K. Boyd, and J. G. Burgess. 2002. Surface induced attachment induced production of Antimicrobial compounds by marine epibiotic Bacteria using modified Roller Bottle. Mar. Biotechnology. 4: 356 – 366.

Liming, Y., K. G. Boyd, and D. R. Adams. 2003. Biofilm specific cross species induction of antimicrobial compounds in Bacilli. AppI.Envi.Microbiol, 69:3719-3727.

Mckenney, D., K. E. Brown, and D. G. Allison. 1995. Influence of Pseudomonas aeruginosa exoproducts on virulence factor production in Burkholderia cepacia: evidence of interspecies communication. J. Bacterial 177: 6989 – 6992.

Mearns – Spragg, A., K. G. Boyd, and M. O. Hubble. 1997. Antibiotics from surface associated marine bacteria. Proceedings of the fourth underwater science symposium. The society for underwater Technology, London: 147 – 157.

Meains – Spragg, A., M. Bregu, K. G. Boyd, and J. G. Burgess. 1998. Cross species induction and enhancement of antimicrobial activity produced by epibiotic bacteria from marine algae and invertebrates, after exposure to terrestrial bacteria. Letters in Applied Microbiology. 27: 142-146.

Miller, M. B., and B. L. Bassler. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55: 165-199.

Msadek, T. 1999. When the going gets tough: survival strategies and environmental signaling network in Bacillus subtilis. Trends Microbiol. 7: 201-207.

Patterson, G. L., and C. Ml. Bolis. 1997. Fungal cell – wall poly sacchraides elicit an antifungal secondary metabolite in the Cyanobacterium Scytonema ocellatum. J. Phycology. 33: 54 – 60.

Rheinheimer, G.1992.Aquatic microbiology (Wiley, New York), 3rd Ed.

Stead, P., B. A. M. Rudd, H. Bradshaw, D. Nobel, and M. J. Dawson. 1996. Induction of phenazine biosynthesis in cultures Pseudomonas aeruginosa by L-N-(3-oxohexanoyl) homoserine lactone. FEMS Microbiology Letters 140: 15-22.

Trischman, J.A., D.M.Tapiolas, P.R.Jensen, R.Dwight, W.Fenical, T.C.Mckee, C.M.Ireland, T.J.Stout, J.CIarely. 1994. Salinamide – A and salinamide – B – anti -inflammatory depsipeptides from a marine streptomycete. J.Am.Chem.Soc. 116: 757 -758.

Vandevivere, P., and D. L. Kirchman.1993. Attachment stimulates exopolysaccharide synthesis by a bacterium. Appl. Environ. Microbiol. 59: 3280-3286.

Wagner, V. E., D. Bushneil, and L. Passador. 2003. Micro array analysis of Pseudomonas aeruginosa quorum sensing regulons. J. Bacteriol, 185: 2080 – 2095.

Whiteley, M., K.M.Lee, and E.P.Greenberg.1999. Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc.Natl.Acad.Sci.USA.96:13904-13909.

Yotsu, T., Y.Meguro, A. Endo, M. Murate, H. Naold, and T. Yasumoto.1987.

Production of tetrodotoxin and its derivatives by Pseudomonas sp. Toxicon 25: 225 – 228

Zhang, L., P. J. Murphy, and A. Kerr. 1993. Agrobacterium conjugation and gene regulation by /V-acyl-L-homoserine lactones. Nature 362: 446 – 448.

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Studies on Quorum Sensing Principle Among Seaweed Epibiotic Bacterial Organisms

drugtest — Thu, 14 Jan 2010 17:23:10 +0000

Introduction

We are living in a microbial planet. About 71 % of the surface of this planet is covered by sea water. A typical milliliter of seawater contains 103 fungal cells, 106 bacteria, and 107 viruses, including pathogens that cause widespread -ortalities and microbes that initiate fouling of host surfaces ‘Rheinheimer, 1992). Thus, marine plants and animals are continually exposed to high concentrations of potentially harmful microbes. These microorganisms in nature exists as free living planktonic mode of life in sea water or it may exist as epibiotic organisms in various living and nonliving surfaces. Among living organisms, seaweeds and invertebrates act as suitable substrate for the establishment of epibiotic organisms Seaweeds are known to release a large amount of organic carbon into the surrounding environment providing a nutrient rich habitat for microorganisms like bacteria. Bacteria are generally considered to be independent unicellular organisms. One cell accomplishes all of the tasks of feeding, locomotion, ‘reproduction, respiration and all other processes necessary to keep an organism alive. There are several classes of bacteria such as primary film forming bacteria, sediment bacteria, symbiotic bacteria, and epibiotic bacteria in various aquatic organisms. The marine surface environment is a site of intense composition for living space by a wide variety of organisms. Bacteria are generally recognized as primary colonizers of this habitat and are able to form biofilm on marine surface such as invertebrates and algae (Bryers, et al., 1982). Bacteria may also be abundant on the surfaces of some algae as an important epibiotic organism. In many cases, the bacterial population found to be specific, with changes occurring throughout the year or life span of the algal surface. This algal-bacterial relationship is symbiotic in most cases; the epibiotic bacteria in seaweed play a protective role by releasing secondary metabolites into the surrounding seawater that help preventing extensive fouling of the surface. Epibiotic bacteria are therefore attracting attention as a source of new natural products. Bacteria from the larvae of some crustaceans protect them from fungal infection by the production of simple antimicrobial compounds. Bacteria isolated from the surface of a tunicate prevented the settlement of barnacle and tunicate larvae exposed to the bacteria as biofilm in petridishes (Evelyn et al., 2001). Seaweeds itself secretes secondary metabolites to prevent fouling and grazing. In addition to that epibiotic bacteria on macro algae can also produce antifouling compounds that work in concert with the seaweed derived compounds to protect the seaweed surface. Recent studies have highlighted important roles of epibiotic bacteria colonizing the surface of seaweeds and releasing antifouling compounds. For the past 50 years antibiotics have revolutionized medicine by providing cures for formerly life threatening diseases. However, strains of bacteria have recently emerged that are virtually unresponsive to antibiotics such multidrug resistance, arising mainly through antibiotic misuse, is now recognized as a global health problem. In this situation, it is clear that new classes of antibiotics are urgently needed. Many marine bacteria have been shown to produce secondary metabolites that display antibacterial properties. The first antibiotic from a marine bacterium was identified and characterized in 1966. In addition, bacteria in biofilm on the surface of marine organisms have been documented to contain a higher proportion of antibiotic producing bacteria than some other marine environments (Burgess, et al., 1999). Marine epibiotic bacteria, associated with nutrient-rich algal surfaces have also been shown to produce antibacterial secondary metabolites which inhibit the settlement of potential competitors. Recently a lot new novel antibiotics such as Phenazine, thiomarinol, phenazine-1-carboxylic acid, 1-hydroxyphenazine 2-n-heptylquinol-4-one, 2-n-nonylquinol-4-one pyolipic, loloatins, agrochelin, sesbanimides, pelagiomicins, indomycione and indomycione have been identified from various marine epibiotic bacterial organisms. In particular, some species of the genus Pseudomonas produce both antibiotics and several other bioactive substances. For example, Pseudoalteromonas rubra and Pseudoalteromonas aurantia have been reported to be antibiotic producing bacteria. The phenomenon of higher organisms utilizing their associated microflora for the production of beneficial secondary metabolites is common in the marine environment (Yotsu, et al., 1987). A study of bacteria isolated from marine algae surfaces indicated that the incidence of antibiotic producing strains from this habitat was 20% whereas that from sea water was only a few percent. In addition, some bacteria that previously did not produce any active compounds have been found to be producing such metabolites when they are exposed to other bacterial species or extra cellular chemical from other bacteria. Bacteria may also produce antimicrobial compounds when they sense the presence of competing organisms. However, few attempts have been made to study such chemical communication between different bacterial species or how this might affect. The secretion of antimicrobial compounds (Mearns-Spragg, et al., 1998). Bacterial communication by the chemical signals for specific function is simply known as Quorum sensing. In which a bacterial population receives input from the environment and elicits an appropriate response (Hiroaki and Kristina. 2003). The term “quorum sensing” describes the ability of a microorganism to perceive and response to diffusible signal molecules. Bacterial cells sense their population density through a sophisticated cell to cell communication system and trigger expression of particular genes. Tne first system of density-dependent regulation was studied in detail with the luminescence of Photobacterium fischeri (formerly known as Vibrio fischeri) by Bassler et al., 1997. Eventually, they discovered that 3-oxo-N-(tetrahydro-2-oxo-3-furanyl) hexanamid or N-3-(oxohexanoyl) homoserine lactone (OHHL) was responsible the agent in the broth that induced luminescence. Followed by this many researchers have confirmed that in Gram negative bacteria acyl-homoserine lactone is responsible for the cell to cell communication system.

In gram positive bacteria peptide and derivative peptide based signaling molecules seem to be the predominant mode of communication. During high cell density the marine bacteria can produce enzymes, surfactants, toxins, and antibiotics by the chemical signal communication. Marine epibiotic bacteria are also known to produce compounds active against drug resistant hospital pathogen by the cross species induction method. Building on assays described by Austin (Billaud and Austin 1990) a screening procedure has been developed in which marine bacteria are challenged by exposing them to terrestrial bacteria prior to assay of antimicrobial compounds. Hence in this present investigation it is proposed to find out the ability of sea weed epibiotic bacterial organism to produce antibacterial compounds through quorum sensing.

MATERIALS AND METHODS

SAMPLE COLLECTION

Seaweed samples were collected from Gulf of Mannar Marine Biosphere Reserve and identified up to species level by using CMFRI bulletin (14) as follows:

Table 1. List of Seaweeds species collected for the present study

SPECIES NAME FAMILY

Halimeda gracilis Chlorophyceae

Ulva lactuca Chlorophyceae

Microdictyon tenunis Chlorophyceae

Chondrococcus hornemonii Chlorophyceae

Enteromorpha intestinalis Chlorophyceae

Caulerpa cupressoides Chlorophyceae

Caulerpa racemosa Chlorophyceae

Dictyota dichotoma Phaeophyceae

Turbinaria ornata Phaeophyceae

Padina gymnospora Phaeophyceae

Sargassum cinearifolium Phaeophyceae

Dictyota batryensis Phaeophyceae

Sargassum sps Phaeophyceae

Hypnea musciformis Rhodophyceae

Acanthophora dendroides Rhodophyceae

Jania rubens Rhodophyceae

Hypnea valentiae Rhodophyceae

Hypnea pannose Rhodophyceae

Hypnea esperi Rhodophyceae

Acanthophora spicifera Rhodophyceae

ISOLATION OF EPIPHYTIC BACTERIA

The collected seaweed samples were thoroughly washed with sterile seawater to removes the loosely attached bacteria/particles. Seaweed fronds were scrubbed with sterile cotton swabs to obtain epiphytic bacteria. Epiphytic bacterial organism in the swab were inoculated in sterile peptone broth (50% sea water) and incubated at 28°C in an incubated shaker (220 rpm / min) for overnight. After the incubation period the enriched cultures were serially diluted up to 10-8 concentration and 200 microlitre of each diluted samples were transferred into the nutrient agar plate (50% sea water). The plates were incubated at 28°C for 5 days and the plates with crowded colonies were selected. In the crowded plates those colonies, which showed the sign of inhibition zone around its margin to the neighboring colony, were selected and considered as producer strain. The neighboring sensitive colonies were treated as inducer strain. Both producer and inducer strains were streaked repeatedly until to get pure culture. The pure culture were properly labeled and subjected to the quorum sensing analysis.

QUORUM SENSING

EXPERIMENT NUMBER 1

In this present study, the producer and inducer strains were cross reacted to find out the production of antibiotic compound through quorum sensing. Totally three set of cultures were maintained as follows (along with one as control).

A. Live cells of producer and inducer strains

B. Live cells of producer strain alone

C. Live cells of inducer strain alone

In culture system A 200ul of 16 hours old broth culture of both producer and inducer strains were added to the 15 ml of nutrient broth.

In culture system B 200ul of 16 hours old producer strain alone was inoculated.

In culture system C 200ul of 16 hours old inducer strain alone was inoculated.

All the cultures were incubated at 28°C for 5 days. After the incubation period the cultures were centrifuged at 10,000 rpm for 15mins. The supernatant was collected and subjected to antibacterial assay with respective inducer strain.

EXPERIMENT NUMBER 2

In this experiment, culture supernatant was obtained as per the procedure given in the experiment 1. 50ml of supernatant was mixed with equal volume of 80% methanol and 1% acetic acid mixture and it was shaked thoroughly in a separating funnel. Finally the methanol and acetic acid fractions were collected and concentrated by evaporation using water bath at 55°C. The viscous colloidal residues were resuspended in 600 microlitre of 50% methanol and it was used for antibacterial assay against different test organism.

TEST ORGANISMS:

1. Epiphytic Vibrio from seaweeds

2. Vibrio from primary film

3. Vibrio from Sediments

4. Pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella sp. and Proteus sp

The test organisms Vibrio species were isolated from seaweed as epiphyles, biofilm, sediment and puffer fish by using TCBS medium (Hi media) The pathogenic bacteria were collected from clinical laboratories.

ANTIBIOTIC ASSAYS

Antibiotic activity was performed in duplicate using a standard paper disc diffusion method as well as well assay. In well assay 10mm in diameter wells were made in marine agar plates and the plates were swabbed with 16 hours old inducer strain. To these wells 200ul of cell free supernatant were added to each well. In paper disc assay the Watmann no.1 filter paper discs (6mm in diameter) were saturated with 200ul of cell free supernatant. The impregnant discs were Dlaced in the centre of the plates swabbed with test organisms. The plates were Incubated at 37°C overnight and observed for inhibition zone. The zone of inhibition was measured as the distance from the border of paper disc to the edge of the clear zone and expressed in mm.

BACTERIAL IDENTIFICATION

The organisms responded to the quorum sensing process alone were identified by the following biochemical analysis.

Colony morphology, Gram staining, Motility test, Oxidase test, Catalase test, Indole Production, Methyl red test, Voges Proskauer test, Citrate Utilization test, Triple sugar Iron test, Nitrate reduction test, Lactose fermentation, Urease test

Starch hydrolysis test, Protein hydrolysis test, Lipid hydrolysis test, Oxidative / Fermentative test, Salt concentration (0%, 3%, 5%, 7%, 10%), TCBS, Growth in Temperature, 42°C and 47°C

All the above mentioned biochemical tests were performed by following standard methodology given in the Microbiological Laboratory Manual by James 3.Cappuccino (1999).

RESULTS AND DISCUSSION:

QUORUM SENSING/CROSS SPECIES INDUCTION ANALYSIS

In the present investigation totally 54 isolates were collected out of seaweed species. Among 54 isolates, 17 of them are producer strain, another 17 are the inducer strain rest of 20 isolates is normal and not showing any signs of activity (Table.2).

a) Among these 17 producers strain 6 strains were isolated from Hypnea musiformis. 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from Sediment.

b) Among these 17-inducer strain 6 strains were isolated from Hypnea musiformis, 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from sediment.

All the 17 strains were named as

PRODUCERS STAINS

BrA+, BrB+, BrC+, BrD+, BrE+, BrF+ Hypnea musiformis

GcA+, GcB+, GcC+, GcD+, GcE+, GcF+ Gracillaria edulis

U1+, U2+, U3+, U4+ Ulva lactuca

SA+ Sediment

INDUCER STRAIN

BrA-, BrB-, BrC-, BrD-, BrE-, BrF- Hypnea musiformis

GcA-, GcB-, GcC-, GcD- GcE-, GcF- Gracillaria edulis

U1-, U2-, U3-, U4- Ulva lactuca

SA- Sediment

In this experiment among 17 Producer and Inducer strains only 3 of them have responded to the quorum sensing principle. (BrB+/Bo-?, (GcC+/GcC) and (SA+/SA-)

Table 2: The results of Quorum Sensing analysis of epibiotic bacterial isolates from seaweeds.

Seaweed sample Producer organism Inducer organism Cross-species producer with inducer Cross-species supernatant test with inducer Zone of clearance (mm)

Hypnea musiformis 1. BrA+2. BrB+3. BrC+4. BrD+5. BrE+6. BrF+ BrA-BrB-BrC-BrD-BrE-BrF- Br A+ /Br A-Br B+ /Br B-Br C+/Br C-Br D+/Br D-Br E+/Br E-Br F+/Br F- BrA-Br B-BrC-BrD-BrE-BrF- NIL39NILNILNILNIL

Gracillariaedulis 7. GcA+8. GcB+9. GcC+10. GcD+11. GcE+12. GcF+ GcA-GcB-GcC-GcD-GcE-GcF- Gc A+/Gc-Gc B+/Gc-Gc C+/Gc-Gc D+/Gc-Gc E+ /GcE-Gc F+/Gc- GcA-GcB-GcC-GcD-GcE-GcF- NILNIL26NILNILNIL

Ulva lactuca 13. U1+14: U2+15. U3+16. U4+ U1-U2-U3-U4- U1+/U0-U2+/U0-U3+/U0-U4+/U4- U1-U2-U3-U4- NILNILNILNIL

Sediment 17. SA+ SA- SA+/SA- SA- 28

c) The normal 20 bacterial strains isolated from 20 algal species were crossed with terrestrial bacteria such as E-coli, Staphylococcus aureus separately

This experiment does not showed any inhibition zones

Bacterial identification

The 3 producer and 3 inducer strains which were responded the quorum sensing principles alone were subjected to biochemical analysis for identification. The obtained results revealed that all the producer strains showed sings of Pseudomonas sps and the inducer strains showed signs of Vibrio sps. So, based on the obtained result all the producer strains seems to be a Pseudomonas sps where as all the Inducer strain belongs to the genus vibrio.

In the present investigation, it was aimed to produce the antibiotics from the seaweed epibionts through quorum sensing principle. The bacterial isolates of seaweed epibionts were identified as species of Pseudomonas and Vibrio from seaweeds Hypnea musiformis and Gracillaria edulis. In this study the Pseudomonas acts as a producer strain and Vibrio as inducer strain. The recent finding says that the seaweed epibionts having potential to control the metabolic activity of competitor organisms. Allison et al., 1998 reported that many bacterial strains up on attaching to a surface produce exopolysaccharides or exopolypeptides. In addition, it has been postulated that exopolysaccharides could mediate the attachment of the bacteria to the surface and induce metabolic changes.

The results of Vanderivere and Kirchman 1993 suggest that the addition of increased surface by adding sand will induce the exopolymer synthesis through the high cell density dependent system. In the same way the bacterial organisms attached to the surface of seaweed shows alteration of genes expression may be due to the response to the high competitive environment. When cell density increases the competition for space and nutrients are also increased. So the existing bacteria were forced to protect themselves in this competitive environment. Normally in this condition the bacteria will be activated to induce the expression of certain hidden genes in genetic material through quorum sensing. The quorum sensing is principles were active compounds (autoinducer) from bacterial cell will promote the expression of a particular hidden gene of other bacterial organism in a stressed condition.

Quorum sensing usually focused on the bacteria growing in homogeneous environment. However few studies have attempted to a study this principle in heterogeneous environment also. In this present investigation we have attempted to study both homogeneous as well as heterogeneous environment. In former one we have isolated producer strain in seaweed eipbionts and it shows inhibitory activity against the inducer organism at the same seaweed epibionts. Later producer strains from seaweed epibionts, were treated with various Vibrio organisms from different environment. The obtain result of this study shows that the producer strain are capable of secreting antibiotic compounds not only to their natural competitors in its own habitate but also to the pathogen inhabiting in a distant related environment.

In the gram negative bacteria AHSL is an active principle of quorum sensing. Our producer strain is also been identified as Pseudomonas sps. So in these organisms also active principle must falls under the AHSL. The cell-cell signaling mechanism can either require import of the signal and subsequent interaction with intracellular effectors or a two-component signaling system that transducers the information across the membrane. In V. harveyi genetic analysis of the density sensing apparatus has two independent density-sensing systems, and each is composed of a sensor-auto inducer pair; system one is composed of sensor I and Al -1, and system two is composed of sensor 2 and AI-2. The two densities – sensing system are redundant, because a null mutation in either system alone results in expression of hidden genes (Bassler, et al.,1999.).

The earlier genetic analysis in Pseudomonas reveals the Pseudomonas consist of two quorum sensing systems as Las R1-I and Rh1R-l and have linked with R and I genes, in addition recently a third Lux R homolog that is advanced to a cluster of quorum sensing – controlled (qsc) genes were detected. Las R is a transcriptional regulator that responses primarily to the Las I – generated signal and Rh1R is a transcriptional regularly that responses best to the Rhl -generated signal. In Pseudomonas auriginosa, at low population densities Las I produce a basel level of 3-O-C12-HSL. As density increases, 3-0-C12-HSL builds to a critical concentration, at which point interacts with LasR. This Las R -3-0-012-HSL complex that activates transcription of a number of genes [Whileley, et al., 1999].

We suggest that the above said mechanisms in Pseudomonas with quorum sensing principle might have occurred in the present study also. This induces the bacteria Pseudomonas in epibiotic seaweeds to secrete certain active compound against to the competitor Vibrio species.

In this present work the totally 54 isolates were screened from 20 different seaweed species out of which 34 species were showed the signs of quorum sensing i.e. 17 producer and 17 inducer strains, but when these organisms where subjected to quorum sensing principle in mixed culture only 3 them have responded. So the present study reveals around 17% of bacterial species isolated from seaweeds and sediment were responded to quorum sensing. According to the results of bacteria isolated from marine algae surfaces indicated that the incidence of antibiotic producing strains from this habitat was 20% where as that from sea water was only a few percent. In the present study also reveals more or less the same ratio in Pseudomonas spp. was observed. Our results also reveals a results of Kell et al., 1995; Stead et al., 1996; they have said the culture supernatant of Pseudomonas sps known to contain AHLS which induces the production of phenazine antibiotics. In this investigation due to time constraint, It was not attempted to identify the active compound secreted by Pseudomonas through quorum sensing, which may leave the space for the further intensive research in future.

In concluding this discussion, the quorum sensing is wider spread among bacterial population then was previously thought, (In Gram positive, Gram negative bacterial communication). Current assays for antimicrobial activities are inadequate because some antibiotic producing bacteria may require the presence of another bacterial species. These findings have important implication for the discovery of novel antimicrobial compounds from marine bacteria and may allow the development of new methods for screening novel compounds active against multidrug resistant bacteria.

REFERENCES

1. Alim, I., and K. Yuto. 2003. Mc21-A, a Bacteriocidal antibiotic produced by a new marine bacterium, Pseudoalteromonas phenotica sp. nov. O-Bc 30T, against Methicillin -Resistant Staphylococcus aureus. Antimicro. Agents Chemoth. 47: 480 – 488.

2. Allison, D. G., B. Ruiz, C. Sanjose, A. Jaspe, and P.Gilbert. 1998. Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms. FEMS Microbial Lett. 167: 179-184.

3. Armstrong, E., J. D. Mckenzie, and G.T. G. Worthy. 1999. Aquaculture of sponges on scallops for natural products research and antifouling. J. Biotechnol. 70: 163-174. 70. 21-

4. Bainton, N. J., P. Stead, and S.R. Chhabra. 1992. N-(3-oxohexanoyl)-L-homoserine Lactone regulated carbapenem antiobiotic production in Erwinia carotovora. Biochem. J. 288: 997-1004.

5. Bassler, B. L., E. P. Greenberg, and A. M. Sterens. 1997. Cross species induction of luminescence in the quorum – sensing bacterium Vibrio harveyi. J. Bacteriology. 179: 4043-4045.

6. Bassler, B.L.1999. Curr.Opin.Microbiol.2: 582-587.*

7. Bernan, V. S., M. Greenstein, and W. M. Maiese. 1997. Marine microorganisms as a source of new natural products. Adv. Appl. Microbiol. 43: 57 – 89.

8. Billaud, A. C, and B. Austin. 1990. Inhibition of the fish pathogen, Serratia liquefaciens by an antibiotic producing isolates of planococcus recovered from sea water. Journal of

9. Boyd, K.G., A. Mearns -Soragg, G. Briedley, K. Hatzidimitriou, A. Rennie, M. Bregu, M. 0. Hubble, and J. G. Burgess. 1998. Antifouling potential of epiphytic marine bacteria from the surface of marine algae. Plouzane, France: 128 -136.

10. Bryers, J. D., and W. G. Characklis. 1982. Processes governing primary biofilm formation. Bioeng. 24: 2451-2476

11. Burgess, J. G., K. Boyd, E. Armstrong, T. Pisacane, and D. R. Adams. 2003. The development of a marine natural product-based antifouling paint. Biofouling. 19: 197 -205.

12. Burgess, J. G., E. M. Jordan, M. Bregu, and A. Mearns-spragg. 1999. Microbial antagonism: a neglected avenue of natural products research. J. Biotechnology. 70: 27-32.

13. Burgess, J. G., H. Miyashita, H. Sudo, and T. Matsunaga.1991. Antibiotic production by marine photosynthetic bacterium Chromatium purpuratum NKPB 031704: Localization of activity to the chromatophores. FEMS Microbiol. Lett. 84: 301 – 306.

14. Cappuccino J. G., and N. Shermann. 1999. Microbiology, A Laboratory manual, Fourth edition, Addison Wesley, New York.

15. Davies, D. G., M. R. Parsek, J. P. Pearson, B. H. Iglewski, J. W. Costerton, and E. P. Greenberg. 1998. The involvement of cell to cell signals in the development of a bacterial biofilm. Science. 280: 295-298.

16. Dekievit, T. R., and B. H. Iglewski. 2000. Bacterial quorum sensing in pathogenic relationships. Infect. Immun. 66: 4839-4849.

17. Dimango, E., H. J. Zar, R. Bryan, and A. Prince. 1995. Diverse Pseudomonas aerugniosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J. Clin-lnvestig. 96: 2204-2210.

18. Evelyn, A., Y. Liming, K.G. Boyd, P. C. Wright, and J. G. Burgess. 2001. The symbiotic role of marine microbes on living surfaces. Hydrobiologia. 461: 37 – 40

19. Burgess, J. G., M. Elizabeth, M. Bregu, A. Meams-Sprugg, and K. G. Boyd. 1999 Microbial antagonism: a neglected avenue of natural products research. J. Biotechnology. 70. 27-32.

20. Hiroaki, S., and K. M. Smith. 2003. Molecular mechanisms of bacterial quorum sensing as a new drug target. Curr. Opin. Chem. Biol. 7: 586-591.

21. Hoang, T., Y. Ma, R. J. Stern, M. R. Meneil, and H. P. Schwezer. 1999. Construction and use of low-copy number T7 expression vectors for purification of problem proteins: purification of Mycobacterium tuberculosis RmID and Pseudomonas aeruginosa Lasl and Rhll proteins, and functional analysis of purified Rhll. Gene. 237: 361 – 371.

22. Holmstrom, C, and S. Kjelleberg. 1994. The effect of external biological factors on, settlement of marine invertebrates and new antifouling technologies. Biofouling. 8: 147-160.

23. Holmstrom, C, D. Rittschof, and S. Kjelleberg. 1992. Inhibition of settlement by larvae of Balanus amphitrite by a surface – colonizing marine bacterium. Appl. Env. Microbiol. 58: 2111 -2115.

24. Ines, M. M., B. Karaigher, U. Cepon, and I. Mahne. 2003. Variability of the Quorum sensing system in natural isolates of Bacillus sp., Food technol. Biotechnol. 41: 23 – 28.

25. Kell, D. B., A. S. Kaprelyants, and A. Grafen. 1995. Pheromones, Social behaviour and the functions of secondary metabolism in bacteria. Trends in Ecol. and Evol. 10: 126-129.

26. Korber, D. R., J. R.Lawrence, H. M. Lappin-scott, and J. W. Costerton. 1995. Growth of microorganisms on surfaces. In. Microbial. Biofilm, Lapp in – Scott. Cambridge, UK: Cambridge university press.

27. Lemos, M. L, A. E. Toranzod, and L. J. Barja. 1986. Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds. Microbial Ecology. 11: 149-163.

28. Liming, Y., G. K. Boyd, and J. G. Burgess. 2002. Surface induced attachment induced production of Antimicrobial compounds by marine epibiotic Bacteria using modified Roller Bottle. Mar. Biotechnology. 4: 356 – 366.

29. Liming, Y., K. G. Boyd, and D. R. Adams. 2003. Biofilm specific cross species induction of antimicrobial compounds in Bacilli. AppI.Envi.Microbiol, 69:3719-3727.

30. Mckenney, D., K. E. Brown, and D. G. Allison. 1995. Influence of Pseudomonas aeruginosa exoproducts on virulence factor production in Burkholderia cepacia: evidence of interspecies communication. J. Bacterial 177: 6989 – 6992.

31. Mearns – Spragg, A., K. G. Boyd, and M. O. Hubble. 1997. Antibiotics from surface associated marine bacteria. Proceedings of the fourth underwater science symposium. The society for underwater Technology, London: 147 – 157.

32. Meains – Spragg, A., M. Bregu, K. G. Boyd, and J. G. Burgess. 1998. Cross species induction and enhancement of antimicrobial activity produced by epibiotic bacteria from marine algae and invertebrates, after exposure to terrestrial bacteria. Letters in Applied Microbiology. 27: 142-146.

33. Miller, M. B., and B. L. Bassler. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55: 165-199.

34. Msadek, T. 1999. When the going gets tough: survival strategies and environmental signaling network in Bacillus subtilis. Trends Microbiol. 7: 201-207.

35. Patterson, G. L., and C. Ml. Bolis. 1997. Fungal cell – wall poly sacchraides elicit an antifungal secondary metabolite in the Cyanobacterium Scytonema ocellatum. J. Phycology. 33: 54 – 60.

36. Rheinheimer, G.1992.Aquatic microbiology (Wiley, New York), 3rd Ed.

37. Stead, P., B. A. M. Rudd, H. Bradshaw, D. Nobel, and M. J. Dawson. 1996. Induction of phenazine biosynthesis in cultures Pseudomonas aeruginosa by L-N-(3-oxohexanoyl) homoserine lactone. FEMS Microbiology Letters 140: 15-22.

38. Trischman, J.A., D.M.Tapiolas, P.R.Jensen, R.Dwight, W.Fenical, T.C.Mckee, C.M.Ireland, T.J.Stout, J.CIarely. 1994. Salinamide – A and salinamide – B – anti -inflammatory depsipeptides from a marine streptomycete. J.Am.Chem.Soc. 116: 757 -758.

39. Vandevivere, P., and D. L. Kirchman.1993. Attachment stimulates exopolysaccharide synthesis by a bacterium. Appl. Environ. Microbiol. 59: 3280-3286.

40. Wagner, V. E., D. Bushneil, and L. Passador. 2003. Micro array analysis of Pseudomonas aeruginosa quorum sensing regulons. J. Bacteriol, 185: 2080 – 2095.

41. Whiteley, M., K.M.Lee, and E.P.Greenberg.1999. Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc.Natl.Acad.Sci.USA.96:13904-13909.

42. Yotsu, T., Y.Meguro, A. Endo, M. Murate, H. Naold, and T. Yasumoto.1987.

43. Production of tetrodotoxin and its derivatives by Pseudomonas sp. Toxicon 25: 225 – 228

44. Zhang, L., P. J. Murphy, and A. Kerr. 1993. Agrobacterium conjugation and gene regulation by /V-acyl-L-homoserine lactones. Nature 362: 446 – 44