Vibrio, a gram negative bacterium consists of more than 70 species of which many are pathogenic. The pathogenic strains are usually associated with gastroenteritis and also infect open wounds that further leads to septicemia. The most important pathogenic vibrio species include V. cholerae, V. parahaemolyticus, V. vulnificus, V. fluvialis and V. mimicus. Cholera is an acute diarrheal illness caused by V. cholerae infecting the small intestine. According to Centers for Disease Control and Prevention (CDC), an estimated 3-5 million cases and over 100,000 deaths occur each year around the world due to cholera. Approximately one in 10 infected person will have profuse diarrhea and other characteristic symptoms (1).
Saltwater bodies and marine habitats serve as the natural reservoirs for these microorganisms as they prefer halophilic environment but they can be also found in freshwater (2). Thus, most of the vibrio infections are related to consumption of contaminated water and uncooked seafood (3). Many studies have revealed that there is association of these vibrios, especially Vibrio parahaemolyticus with many seafoods such as shrimps, crabs, fishes, molluscs etc (4). Consumption of these contaminated seafoods causes severe gastroenteritis in human beings and other animals. V. parahaemolyticus can also be associated with wound infections and septicemia. V. vulnificus is also present in marine environment, estuaries and brackish ponds. Infection by V. vilnificus leads to rapidly expanding cellulitis or septicemia. The microorganism enters the body through wounds and by eating undercooked or raw oysters. The bacteria spread into the bloodstream of people with compromised immune system and cause severe symptoms including blisters, skin lesions, septic shock and even death (5). Though hospitalization and deaths due to V. vulnificus infections are not very common in India, the incidence of V.vulnificus infections has increased tremendously in US and gulf countries. The same is the case with V. mimicus and V. fluvialis. V.mimicus is a non halophilic vibrio which causes sporadic episodes of gastroenteritis and ear infections whereas V. fluvialis is halophilic and causes symptoms similar to that of cholera. The symptoms include diarrhea, vomiting, abdominal pain, dehydration and fever. V.fluvialis also rarely causes wound infections and septicemia. In most cases the infections caused by vibrio can be prevented by proper sanitation and appropriate treatment. The commonly used drugs against non-cholera vibrio infections other gastroenteritis include combination of ceftazidime and doxycycline (6). Other alternative antibiotics include cefotaxime and/or fluoroquinolones. For diarrhea and gastroenteritis, additional oral replacement therapy or intravenous fluid replacement therapy is important to prevent rehydration (7).
Though there are this many antibiotic drugs against vibrio, reports of antibiotic resistance is increasing day by day. Resistance to tetracycline and other antimicrobial agents among V. cholerae has been demonstrated in both in vitro and in vivo experiments. Resistance can be acquired through the accumulation of selected mutations over time, or the acquisition of genetic elements such as plasmids. A likely risk factor for antimicrobial resistance is widespread use of antibiotics (8). Antibiotic resistance by bacteria can also be via structural mechanisms such as biofilm formation and drug efflux pump. Biofilms are surface adhered bacterial communities encased in self-produced extracellular matrix (ECM). Biofilm formation is dependent on the vibrio polysaccharide and other structures like flagella and pili (9). This biofilm prevents the drugs from reaching the microorganisms embedded in the extracellular matrix which is comprised of polysaccharides, proteins, lipids and extracellular nucleic acids. Therefore destruction of biofilm formation is one of the efficient mechanisms to overcome antimicrobial resistance to some extent.
Though the pathogenic vibrios form biofilm and the advantages of biofilm mode of life to the bacteria remains the same, the process of biofilm formation in each species differs greatly. Biofilm formation in Vibrio depends on various factors like expression of flagellin genes, pili genes, vibrio polysaccharide genes (vps), proteins in quorum sensing and c-di-GMP levels. The expression of these genes varies among different species of vibrio during biofilm formation. Hence there is possibility that the mode of action of antibiofilm compounds can also vary considerably. In many countries, herbal medicines and other natural compounds are used to treat diseases including diarrhea and skin infections. As these natural compounds are comparatively harmless, the complications and side effects due to the use of chemotherapeutic agents are avoided. This information has now changed the global research focus into finding novel, potent natural antimicrobial and antibiofilm agents that has activity against chronic infections.
Difference in vibrio biofilms
Among the pathogenic vibrios, V.cholerae has been credited with the most extensive studies as it is the perpetrator of hundreds and thousands of deaths in the developing countries due to severe watery diarrhea. Though V. cholerae biofilms have not been identified to play a major role in the development of diarrhea, it is identified to be important in disease transmission and pathogenicity. Studies on the relative infectivity of different forms of V. cholerae had suggested that the enhanced infectivity is for the V. cholerae shed in human stools. This is largely due to the presence of clumps of biofilm that disperse in vivo, providing a high dose of the pathogen (10).
Initiation of biofilm formation is when the bacterium get adhered to a surface. In V. cholerae, the preliminary attachment of the bacterium to the surface is facilitated by structures such as pili and flagella. Studies involving the flagellar and pili gene mutants showed decreased biofilm formation but did not completely inhibit it. Biofilm formation was re- achieved on prolonged incubation. In vibrios, mannose ‘sensitive haemagglutinin type IV pili (MSHA), toxin co-regulated pili (TCP) and chitin regulated pili (ChiPR) are the three types of pili identified to be playing crucial role in establishing biofilm. MSHA is understood to have an important function in the early attachment to surfaces. TCP have relevance in the virulence of V. cholerae and enhances microcolony formation in environmental substrates. Recent works revealed that MSHA and TCP are inversely controlled at various levels (11). The role of ChiRP is not fully understood but proposed to be important for attachment to chitinaceous substratum.
Unlike V.cholerae, V. parahaemolyticus employs ChiRP and MSHA for the establishment of biofilm. However, in V. vulnifcus MSHA contribute to attachment to human epithelial cells and also to other biotic surfaces (12).