Bacteria: Vibrio cholerae

BACTERIA

Vibrio cholerae T Ramamurthy, National Institute of Cholera and Enteric Diseases, Kolkata, India GB Nair, Translational Health Science and Technology Institute, Haryana, India r 2014 Elsevier Inc. All rights reserved.

Glossary Case fatality rate (CFR) CFR (case fatality rate or case fatality ratio) is the proportion of fatalities within a selected population over the course of the disease. CFR is typically stated as a percentage and signifies a measure of risk. Cholera toxin (CT) The cholera toxin (CT) secreted by Vibrio cholerae is an oligomeric complex composed of six protein subunits: a single enzymatic A subunit and five receptor binding B subunits. GM-1 ganglioside GM1 is a prototype ganglioside (monosialotetrahexosylganglioside) and its oligosaccharide groups extend beyond the surfaces of the intestinal cell membrane. These carbohydrates act as specific receptors for cholera toxin secreted by V. cholerae. Horizontal gene transfer Transfer of genes (also known as lateral gene transfer) between bacterial cells through transformation, conjugation, transduction etc., which can be detected by analysing G þ C contents, codon usage, amino acid usage, and gene position. Lateral flow system A type of capillary based sandwich immunochromatographic assay with built-in controls for the rapid detection of a pathogen or antibodies from liquid clinical specimens used for medical diagnostics.

Introduction Toxigenic Vibrio cholerae that produces cholera toxin is responsible for the deadly disease ‘cholera,’ which is coded as 001 in the International Classification of the Diseases. Vibrio cholerae is well-known as a waterborne pathogen and is also increasingly recognized as the cause of foodborne infections. Its close association with marine fauna and flora leads to transmission of diarrhea through seafood. Like other foodborne pathogens, transmission of V. cholerae occurs through secondary contamination coupled with its long-term survival in foods. In the US, the estimated illness caused by toxigenic V. cholerae is approximately 54 per year and the foodborne transmission is 90%. The ecological fitness and change in the genomic constitution by horizontal gene transfer makes the organism robust to meet the challenges posed by the environment as well the host’s defense mechanisms. Successive appearance of classical and El Tor biotypes, emergence of a novel serogroup O139, and acquisition of

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Ligated rabbit ileal loop An in vivo technique used in the confirmation of enterotoxins produced by diarrheagenic bacteria. The sterile culture supernatant from the test organisms is inoculated into a ligated segment of ileum may induce the onset of intestinal fluid accumulation as early as 4–18 h due to elevated cyclic adenosine monophosphate. Quorum sensing A system of stimulus and response that can be directly correlated to the population density of V. cholerae or any other bacteria. V. cholerae generally use quorum sensing to coordinate gene expression according to the cell density either in the gut or in the aquatic environment. Rugose V. cholerae The rugose variant forms of V. cholerae have corrugated colonies, well-developed biofilms and exhibit increased levels of resistance to osmotic and oxidative stresses. V. cholerae can undergo phenotypic variation in response to environmental stresses, resulting in rugose and smooth colonial variants phase. Toxigenic V. cholerae V. cholerae strains that produce cholerae toxin and the infection caused by these strains are characterized by a severe watery diarrhea due to the effect of this toxin. Generally, V. cholerae O1, O139 and some of the non-O1 and non-O139 strains belongs to this group.

some of the classical biotype features by the recent El Tor strains are the important events in the changing epidemiology of cholera. Improper control measures and deteriorating status of drinking water, sanitation, and hygiene in many developing countries will perpetuate the disease cholera. This article will focus on the clinical microbiology, epidemiology, pathogenesis, and molecular aspects of V. cholerae.

Historical Outline Toxigenic V. cholerae has been associated with cholera, a devastating diarrheal disease which occurs in the form of epidemics and pandemics. There is no clear indication when and where the disease cholera originated. Based on the disease symptoms it appears that cholera was rampant in many parts of the world around the sixteenth century. The period from 1817–1961 was described as the history of the seven pandemics (Table 1) and each pandemic affected almost all the countries in the world. The duration of each pandemic was

Encyclopedia of Food Safety, Volume 1

doi:10.1016/B978-0-12-378612-8.00117-7

Bacteria: Vibrio cholerae

Table 1

Details of seven cholera pandemics

Pandemic

Period

Number of years prevailed

Approximate number of affected countries

1 2 3 4 5 6 7

1816–26 1829–51 1852–60 1863–75 1881–96 1899–23 1961–till date

11 23 9 13 16 25 449

21 36 37 46 24 24 Ongoing

not uniform, as the pandemicity was based on the intensity rather than the extent of time. The classical biotype was involved in sixth and seventh pandemics. Although strains of the El Tor biotype caused sporadic infections and cholera epidemics as early as 1910, it was not until 1961 that this biotype was recognized as the causative agent of seventh cholera pandemic. Though V. cholerae was first visualized by Pacini in 1854 and then by Koch in 1884, the virulence properties of this pathogen remained unknown for many years. In 1959, S.N. De first showed that the enterotoxin produced by V. cholerae evokes fluid accumulation when cell-free filtrates are tested in ligated rabbit ileal loops. Later this toxin was purified to homogeneity and named as choler toxin (CT). A novel V. cholerae serogroup O139, synonym Bengal, was identified in 1992 from the Indian subcontinent which spread to other Asian countries in the following years. Emergence of O139 serogroup was considered as the beginning of eighth pandemic, but due to its slow disappearance from late-1990s in many countries, this view was not considered. Extension of El Tor biotype from the Indian subcontinent and Haiti to the continents of Africa and subsequently South America makes the seventh pandemic both temporally and spatially the longest and most widely spread pandemic of cholera.

Taxonomy and Characteristics of V. cholerae Vibrios belong to the large bacterial class Gammaproteobacteria within the phylum Proteobacteria. The family Vibrionaceae consist of seven genera and the genus Vibrio has more than 50 species of which 11 are of clinical importance including V. cholerae. The somatic O antigen is heat-stable and is composed of an amino acid sugar D-perosamine (4-amino-4,6dideoxy-D-mannose) in which the amino groups are acylated by 3-deoxy L-glycero-tetronic acid. The different O groups are referred to as serogroups. For serogrouping, only the somatic (O) antigen is used, as the flagellar (H) antigen is homogenous in all the Vibrio species. The two serogroups, O1 and O139, are associated with epidemic cholera and linked to its ability to produce CT, the main toxin responsible for the disease. Based on the three antigenic factors designated A, B, and C, the O1 serogroup is divided into three serotypes; Inaba, Ogawa, and Hikojima. Ogawa strains produce the A and B antigens and a small amount of C, whereas Inaba strains produce only the A and C antigens. The rare Hikojima subtype

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contains all three factors, thereby reacting with both Inaba and Ogawa antisera. Inaba strains are mutant of the Ogawa O antigen encoding gene rfbT, and the conversion from Ogawa to Inaba may be due to the antiOgawa immune selective pressure. Vibrio cholerae O1 is classified into two biotypes, classical and El Tor. The characteristics used to distinguish the biotypes are hemolysis, agglutination of chicken erythrocytes, Voges– Proskauer reaction, inhibition by polymyxin B (50-U), susceptibility to classical IV, and El Tor V phages. After the emergence of the O139 serogroup, the isolates that were identified as V. cholerae on the basis of biochemical tests but that were negative for O1 and O139 serogroups are referred to as non-O1, non-O139 strains. The non-O1 V. cholerae was previously referred to as noncholera vibrios or nonagglutinable vibrios. On the basis of the differences in lipopolysaccharide somatic antigens, this large group has been divided into more than 200 serogroups. Phage typing of V. cholerae O1 and O139 are not much in use as this assay is confined only to the Reference centers.

Clinical Symptoms of V. cholerae Infection Human volunteer studies demonstrated that 103–104 of V. cholerae O1 administered with sodium bicarbonate buffer (to neutralize the stomach acidity) develop diarrhea and the lower inocula correlated with a longer incubation period and decreased stool volumes. Food has a buffering capacity comparable to that of sodium bicarbonate, thereby increasing the chances of infection when contaminated food/water is consumed. Patients infected with CT-producing V. cholerae (mainly O1/O139 serogroups) develop the most severe clinical manifestations of the disease, termed cholera gravis. Depending on the inoculum size, the incubation period of the infection varies between few hours up to 5 days. The main symptoms include vomiting, profuse effortless watery diarrhea (500–1000 ml h1), anorexia and abdominal cramps due to hypokalemia at the acute stage. This stage may rapidly lead to tachycardia, hypotension, and vascular collapse due to dehydration. The other external symptoms include weak peripheral pulses, poor skin turgor, sunken eyes, wrinkled hands and feet. Signs of dehydration can be detected with higher plasma protein concentration, hematocrit, serum creatinine, urea nitrogen, plasma specific gravity, severe acidosis manifested by depression of blood pH and plasma bicarbonate and an increased serum anion gap. Owing to prolonged circulatory collapse, ischemic renal tubular necrosis may also be seen. Children may develop hypoglycemia with coma and convulsions. The nontoxigenic V. cholerae, mostly belong to the serogroups other than O1 and O139, but cause milder diarrhea commonly known as gastroenteritis. The non-O1 non-O139 strains are also associated with invasive extraintestinal disease, septicemia, formation of ascites with generalized abdominal tenderness and high white blood cell count (WBC) with polymorphonucleocytes. Compared with patients with nonbacteremic infections, patients with non-O1, nonO139 bacteremia are more likely to have cirrhosis and thrombocytopenia.

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Bacteria: Vibrio cholerae

Food sample Enrichment in alkaline peptone water (pH 8.0) (37 °C, 6−8 hrs.)

Plating in TCBS medium

Cell pellet from 1 ml culture BSA/MEA medium

(37 °C, 16−18 hrs) Typical colony

Klinger agar Nutrient agar (37 °C, 16−18 hrs) KIA

Oxidase test +ve

Buoyant gradient centrifugation

Oxidase test +ve

Boiling to lyse the cell/DNA denaturation

Filtration Serology with V. cholerae 01/0139 antisera (provisional test)

Serology with V. cholerae 01/0139 antisera If 01, serology with Ogawa/lnaba antisera

ompw PCR multiplex with ctxA +rtb 01/0139

Low/high centrifugation

RNAase free DNAase treatment

RT-PCR

RT-PCR

Molecular typing of confirmed V. cholerae strains Figure 1 Flow chart showing the isolation and identification of V. cholerae from foods using culture and molecualr methods. BSA, bile salt agar medium; KIA, Klinger iron agar; MEA, meat-extract agar; PCR, polymerase chain reaction; RT-PCR, real-time polymerase chain reaction; TCBS, thiosulphate-citrate-bile salts-sucrose.

Detection Methods Culture Methods A stepwise method for the isolation and identification of V. cholerae is shown in Figure 1. Unlike clinical stool specimens, the concentration of V. cholerae in food samples may not be very high and hence an enrichment step is essential. Alkaline peptone water (APW) is the most commonly used enrichment broth with alkaline pH (8.4–9.2). Usually, enrichment lasts for 6–8 h at 37 1C, as excess incubation in APW may result in overgrowth of other organisms. If the plating cannot be done within this stipulated time, a secondary enrichment in APW is necessary to inhibit the growth of background organisms. The most commonly used plating medium for V. cholerae is thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is available from several commercial sources. The sucrose fermenting V. cholerae isolates are readily detected on this medium as large, golden yellow, smooth colonies (Figure 2). For the presumptive identification, nonselective media such as bile-salt agar (BSA) and meat-extract agar (MEA) can be used in parallel with the TCBS agar. But these nonselective media are not commercially available. Selective media that are being used for the isolation of members of the family Enterobacteriaceae are not suitable for isolation of V. cholerae. For routine identification of suspected colonies, few biochemical tests are mandatory. Either conventional tests or commercial systems can be adopted for identification and the

Figure 2 Typical sucrose fermenting colonies of V. cholerae in TCBS medium after 16 h of growth at 37 1C.

tests should be very simple, rapid, and specific. A crucial test for distinguishing V. cholerae from members of the Enterobacteriaceae is the oxidase test, in which, V. cholerae gives a positive response. Fresh colonies obtained directly from BSA or MEA agar can be used for oxidase test and growth from TCBS agar should not be used. Suspected V. cholerae colonies from the isolation plate can also be tested in the Kligler iron agar (KIA)

Bacteria: Vibrio cholerae

medium for confirmation. Typical V. cholerae yielding an alkaline slant over acid but with no gas or H2S are then tested for oxidase activity. The key confirmation of V. cholerae O1/O139 is agglutination with antisera raised against the respective serogroup. Confirmed O1 strains should be tested with Ogawa and Inaba antisera for completion of the serological testing. Satisfactory serological results can be obtained from colonies picked from nonselective media or KIA medium.

Immunological Methods Several direct methods are available based on antigen– antibody reactions for the detection of V. cholerae O1 and O139 either by using stool specimens or enriched cultures of food samples. Fluorescent antibody technique, coagglutination tests, and lateral flow-based detection systems for the detection of O1 or O139 are now commercially available. Cholera SMART kit (New Horizons Diagnostics Corp, USA) and Crystal VC (Span Diagnostics, India) are colloidal gold-based lateral flow immunoassay for the detection of V. cholerae O1/O139 strain within 10–20 min. At times, it is crucial to confirm production of CT by V. cholerae. With tissue culture assay employing Chinese hamster ovary or Y-1 adrenal cells elongation cells caused by the action of CT which can then be confirmed by neutralization of the activity using antiCT antibody. Enzyme-linked immunosorbent assay (ELISA) using purified ganglioside M-1 (GM1) as the capture molecule or a highly sensitive bead ELISA, which uses polystyrene beads coated with antiCT antibody as the solid phase are being used in many reference laboratories. A latex agglutination for the detection of CT is less time-consuming than the ELISA, with excellent sensitivity and specificity. For the detection of V. cholerae O1 from foods, an enzyme immunoassay has been formulated with specific rabbit antiserum, immobilized target bacterial cells and beta-D-galactosidase-labeled goat antirabbit immunoglobulin G as tracer. Animal models are also available for confirmation of CT-producing strains of V. cholerae, but these models are prohibitive as one cannot accommodate many strains in the assay in addition to the animal ethical issues.

Molecular Methods For screening larger number of isolates, deoxyribonucleic acid (DNA) and oligonucleotide probes are useful as it reduces the labor and screening time. The molecular approach confirms V. cholerae as pathogenic strains if the DNA probes are targeted toward virulence genes such as CT encoding gene (ctx). Molecular methods are routinely used in food industry because majority of the V. cholerae strains isolated from environmental and food are nonpathogens as they lack recommended virulence marker genes. Polymerase chain reaction (PCR) technique has also been used to detect ctx or other virulence gene sequences. Several PCR methods are now available using multiplex format with virulence genes along with speciesspecific targets such as ompW and biotype-specific tcpA or hlyA. Detection of these genes directly from the stool or food samples is not advisable as the sample may contain substances that can be inhibitory to the PCR. To overcome this hindrance, extraction of DNA/RNA (ribonucleic acid) from the samples is

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recommended following quick extraction protocols, which are now available in the form of silica column or quick spin purification kits. PCR can amplify target DNA from both viable and nonviable cells of V. cholerae. Detection of pathogens in clinical and food samples is important to ensure that positive test results are associated with viable bacteria. Positive results caused by dead cells may lead to misguided decisions concerning the effectiveness of treatment and destruction of the suspected foods. Combination of multiplex PCR with a colorimetric microwell plate sandwich hybridization assay using phosphorylated and biotinlylated oligonucleotide probes are specific and sensitive for the detection of the microbial pathogens in shellfish. Immobilized oligonucleotide array that targets mutation regions of the 23S rRNA gene or amplified PCR products are being used for the identification of bacteria causing foodborne infections including V. cholerae. Similarly, DNA microarray-based identification system in combination with multiplex PCR targeting ompU, toxR, tcpI, and hlyA is also available for the detection of V. cholerae. Real-time PCR (qPCR) assay that identify the ctxA or any other marker gene from viable toxigenic V. cholerae can also be adopted as an alternative method for standard culture methods. Before real-time quantitative PCR, buoyant density gradient centrifugation followed by filtration and low- and high-speed centrifugation is recommended to separate bacteria from complex food materials as well as to remove compounds that inhibit rapid detection methods. Loop mediated isothermal amplification PCR assay that was designed with five primers targeting ompW seems rapid and specific in detection of V. cholerae. Unlike normal PCR, there is no cycling process involved in this technique. Detection of viable but nonculturable (VBNC) state of V. cholerae O1 is difficult with the existing molecular methods. The transcriptome-based RT-PCR analysis detects increase in the expression mRNA of VC0230 (iron(III) adenosine-triphosphate-binding cassette (ABC) transporter), VC1212 (polB), VC2132 (fliG), and VC2187 (flaC) in the VBNC state. Thus, these genes appear to be suitable markers for the detection of V. cholerae VBNC.

Virulence Features and Pathogenicity of V. cholerae CT is directly responsible for the characteristic symptoms of cholera. The pathogenesis of cholera begins with colonization of toxigenic V. cholerae in the upper intestine and secretion of CT. This toxin is composed of two types of subunits, a 56-kDa oligomer composed of five identical ‘light’ B subunits (‘B’ for binding) responsible for receptor binding and a single ‘heavy’ 28-kDa toxic active A subunit (‘A’ for active toxin). Monosialoganglioside GM1: (Gal(b1–3)GalNac(b1–4)(NeuAc(a2–3) Gal(b1–4)Glc)-ceramide acts as a cell membrane receptor for CT. First, the CT binds to the GM1 receptors on host cells through its pentamer B subunit followed by translocation of A subunit to the cytosol of the target cell. The A subunit catalyzes the adenosine diphosphate (ADP)-ribosylation of the host G protein Gsa, which in turn activates host cell adenylate cyclase. This is accomplished by elevating cAMP levels in intestinal cells through the activation of a G-protein (Gsa) that controls host cell adenylate cyclase activity. The cyclic adenosine

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Bacteria: Vibrio cholerae

monophosphate (cAMP) that accumulates in target cells activates protein kinases, which in turn phosphorylate membrane proteins one of which is the cystic fibrosis transmembrane conductance regulator chlorine and bicarbonate conductance channel. Active Cl and HCO3 transport into the lumen of the intestine produces an osmotic movement of water out of the tissues resulting profuse secretory diarrhea. V. cholerae non-O1, non-O139 serogroups (otherwise known as noncholera vibrios are ubiquitous in the aquatic environment and fauna living therein. The reported asymptomatic carriage rate is approximately 4% among persons involved in high-risk activities and their contribution in several outbreaks have been published in many findings. Unlike O1 and O139, the non-O1, non-O139 serogroups does not appear to be a single virulence mechanism similar to the heterogeneity seen among diarrheagenic Escherichia coli. The pathogenic mechanisms of non-O1, non-O139 serogroups are different from that of O1 and O139 serogroups as they lack the ctx gene cassettes but heat-stable enterotoxin (Stn), El Tor-like hemolysin (Hly) plays a role in human pathogenesis. Clinical strains of V. cholerae rarely carry the heat-stable enterotoxin encoding genes (stn/sto) but sequence type (ST) positive strains are detected in high proportions with environmental V. cholerae O1, O139, and non-O1/non-O139 strains. Type three secretion system is one of the other possible virulence factors commonly found in non-O1, non-O139 serogroups (30–40%). Prevalence of putative accessory virulence genes (mshA, hlyA, and RTX) both in the clinical O1/O139 serogroups as well as non-O1, non-O139 serogroups supports a hypothesis that these genes impart increased environmental fitness. Hemagglutinin/protease and mannose-sensitive hemagglutinin are the other possible virulence factors among non-O1, non-O139 vibrios. In several instances, it was shown that the non-O1, non-O139 serogroups carry ctx and associated with cholera-like diarrhea. Considering the importance of non-O1/non-O139 V. cholerae isolated from the aquatic environment or food samples they should be screened for the presence of ctxA, stn/sto, tcpA, and other virulence marker genes for the human health risk assessment.

Risk Factors and Reservoirs Several risk factors have been identified for the prevalence and spread of cholera, which include population displacement and refugee crisis, heavy rain and floods, traditional funeral rituals and feasts, water storage in large mouthed containers, ice made up of contaminated water, lack of previous disease exposure in endemic regions, asymptomatic carriers/food handlebars, etc. Vibrio cholerae is ubiquitous in the water and sediments of various aquatic water bodies including costal, estuarine, and freshwater systems. Because of its widespread distribution, foods are easily contaminated at various levels of food preparation. The distribution of non-O1, non-O139 vibrios is very common in these environments. Isolation of O1 and O139 from the environments is reported during epidemic and interepidemic periods due to fecal contamination. Overall, the most commonly noted risk factor for cholera outbreaks was transmission through foods that account for 32 and 71% in South America and East Asia, respectively. In several reports, seafood is responsible for many sporadic cases and epidemics of V. cholerae infections and strains are generally devoid of ctx. The

exoskeleton chitin of copepods, shrimps, and crabs are made up of mucopolysaccharide, a preferential substrate of V. cholerae that enhances adherence through their production of chitinase.

Survival Strategies Quorum sensing helps the bacteria for cross communication and alters their gene expression in concordance with several ecological factors. In several findings it was shown that quorum sensing is important in the infectious cycle of V. cholerae in humans and passage through acid barriers in the stomach. The colonized cells of V. cholerae O1 on shrimp carapace showed remarkable resistance to the effects of high temperatures, low pH, and desiccation conditions. This increased resistance to extreme environmental conditions of V. cholerae O1 may have significant implications on food safety and contamination of edible parts of shrimps. It has been suggested that its environmental persistence is associated with conversion of V. cholerae into VBNC state or rugose type and these forms of V. cholerae enhance their survival in many adverse conditions. It is noteworthy to mention here that the rugose variants can survive in the presence of high concentrations of chlorine and other disinfectants.

Epidemiology Cholera with Reference to Contamination of Foods In cholera endemic countries, the case fatality rate (CFR) remains o5% but in some African countries the CFR is approximately 50% during peak outbreak periods. In many cholera outbreak investigations, water was recognized as the primary source for transmission. In the past 60 years, outbreaks of cholera have been documented with consumption of contaminated food. Transmission of cholera may vary from place to place, influenced by local customs and practices. Molluscan and crustacean seafood are generally contaminated in its natural environment at the time of harvest or during preparation. Food items initially free from V. cholerae may become contaminated when mixed with water, other contaminated food, or through foodhandlers. In Guatemala, the 1991 cholera epidemic was significantly associated with contaminated street-vended food items. Foodborne outbreaks of cholera occur mostly in developing countries and have potential cause of large morbidity and mortality that require considerable public health and acute care resources. Storing contaminated meals at ambient temperatures, the common practice in most of the developing countries, allows the growth of V. cholerae. Many epidemiological studies have shown that food plays an important role in the transmission of V. cholerae, and different foods have been incriminated in many epidemic outbreaks of cholera. The Guinea-Bissau cholera epidemic in 1994 that resulted in 15 878 reported cases and 306 deaths was strongly associated with eating at a funeral with a nondisinfected corpse. A huge cholera epidemic in western Kenya in 1997 with 14 275 cholera admissions and 547 deaths was due to use of contaminated water from Lake Victoria or from a stream, sharing food with a person with watery diarrhea, and

Bacteria: Vibrio cholerae

attending funeral feasts. Ingestion of undercooked, contaminated fish has long been known to be associated with cholera transmission. Household epidemiological studies indicate that cholera infection is more likely to occur through patient’s/carrier’s hands rather than by consumption of contaminated foods. Undercooked seafood continues to account for most US cholera cases. In majority of the cases, V. cholerae non-O1, non-O139 is involved in the seafood associated cholera-like diarrheal infection and asymptomatically infected foodhandlers play a great role in the transmission of cholera. Travelers to epidemic countries may be at increased risk of contracting cholera if they ingest contaminated food or water. It has been estimated that approximately 0.2 cases per 100 000 European and North American travelers suffer from cholera without any fatalities. Violations of retail food establishment rules and regulations, and underutilization of safer, postharvest processed shellfish resulted in significant increase in the incidence of V. cholerae mediated infections in the USA.

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parameters such as concentrations of the template DNA, Taq polymerase enzyme, annealing temperature, and number of PCR cycles.

Mobile Genetic Elements Mobile genetic elements (MGE) such as plasmids, integrons, and integrating conjugative elements are used in molecular typing of V. cholerae. This is not a routine fingerprinting method, but it gives information on movement of antimicrobial resistance genes in V. cholerae strains as they are mostly located in MGEs. Conjugative plasmid IncC was found to be responsible for multidrug resistance in V. cholerae O1 associated with many cholera outbreaks in Africa. Integrons, the gene capture and expression systems and integrating conjugative elements which are chromosomal selftransmissible MGEs are specifically detected in El Tor strains indicating they may be considered as a recent evolutionary trend.

Molecular Typing Methods CTX Prophage and CT Genotypes Molecular typing has become an essential component to compliment epidemiological data, and hence many methods have been established for the identification of DNA finger prints of V. cholerae. Following are some of the currently used methods in the molecular epidemiology of cholera.

PCR-Based Typing Though PCR is used for the detection of several virulence encoding genes, this assay is also employed for strain typing in which DNA fragments can be amplified (100–435 kb) even if the template DNA is in minute quantity. Several PCR methods such as random amplification of polymorphic DNA (RAPD) with the use of single oligo, amplified fragment length polymorphism (AFLP) technique with two sets of restriction enzyme-primer combinations, enterobacterial repetitive intergenic consensus sequence that targets arrangements of conserved sequences in most of the bacteria are used in typing V. cholerae. The advantage of such PCRs is that they can be used in laboratories with minimal facility. Several studies performed with RAPD-PCR using V. cholerae O1 has shown the genetic dissimilarity in many Asian and African countries. The AFLP analysis supported that a single clone of pathogenic V. cholerae has caused several cholera outbreaks in Asia, Africa, and Latin America during the seventh pandemic. Generally, PCR assays are rapid and simple means of typing strains for epidemiological studies. However, consistency of the results of PCR is subject to various experimental

Analysis of CT prophage is important to determine the evolution of toxigenic strains of V. cholerae. The CT encoding gene (ctxAB) reside in the genome of a lysogenic filamentous phage known as CTXF, which is located on a 4.5 Kb ‘core region’ of the CTX element (Figure 3). Adjacent to the core is the RS2 region encoding open reading frame (ORF) rstR. Based on the allelic types, the rstR is classified as rstRclass, rstRET, and rstRcalc, respectively for classical, El Tor, and O139 strains. Using ctx restriction fragment length polymorphism (RFLP), the structure, organization, and location of the CTX prophages can be determined. Identification of distinct strains of V. cholerae O1 belongs to biotype classical and El Tor, and O139 demonstrates the evolutionary significance and clonal dynamicity of the pathogen (Figure 3). RFLP, the structure, organization, and location of the CTX prophages were identified in many V. cholerae O1 as well O139 strains isolated from different geographical regions. The unique clonal nature of the US Gulf Coast V. cholerae O1 was identified with 6–7 kb HindIII restriction fragments that contained ctx gene. Unlike classical or El Tor vibrios, the assembly of ctx in O139 strains differed with three or more copies of this gene. The CTX genetic element was different in V. cholerae O139 strains that resurged in Calcutta and Bangladesh in 1996, China, from 1993–99. Of recent V. cholerae O1 Inaba from India, presence of CTX prophage was detected in a single site of the chromosome with at least two RS elements. Interestingly, V. cholerae O1 El Tor isolated from Mozambique carried a classical type (CTXclass) prophage and the genomic RS2 (2.4 kb)

TLC

ig1

rstR

ig2

rstA

RS1 (2.7 kb)

rstB

rstC

ig1

rstR

ig2

rstA

Core (4.5 kb) rstB

cep

OrfU

ace

zot

ctxAB

CTX prophage

Figure 3 Typical genetic structure and arrays of CTX prophage in V. cholerae. Small black triangles in between the genome sequence indicate the repeat regions flanking the integrated phage DNA.

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Bacteria: Vibrio cholerae

analysis of CTX prophage together with chromosomal phage integration sites showed that these strains carried two copies of prophages located in the small chromosome in tandem. DNA sequencing of ctxB of V. cholerae O1 and O139 strains identified several CT genotypes. The CT genotype 1 was found in classical biotype worldwide and El Tor biotype strains associated with the US Gulf Coast. The El Tor strains from Australia belong to CT genotype 2 and 3 was common in the seventh pandemic El Tor strains including the Latin American epidemic strains. Emergence of El Tor strains having CT genotype 1 was reported from India, Bangladesh, Vietnam, and also from few African countries. The epidemiological significance of this trend is related to several recent cholera outbreaks with increased severity of the illness and also for a tendency for the outbreak to become protracted. Identification of new CT genotypes such as 4–6 in V. cholerae O139 strains in also considered as a novel genetic trend.

Ribotyping This method exploits the DNA polymorphism of rRNA genes (rrn) in the chromosome of V. cholerae after digestion with BglI. Universal probe generated from the E. coli (pkK3535) RNA is used to screen the restriction patterns of bacterial DNA. The rrn operons and their flanking regions cause ribotype variation in V. cholerae O1 due to recombinational events. This typing method has identified 7 and 20 ribotypes among classical and El Tor biotypes, respectively. Analysis of V. cholerae O139 strains isolated in India and Bangladesh revealed four different ribotypes. Vibrio cholerae O1strains isolated after the emergence of O139 and reemerged O139 strains in India and Bangladesh showed newer ribotypes. This change in the ribotype profiles was correlated well with the new antimicrobial resistance patterns, which indicated successive replacement of different clones of V. cholerae O1/O139 in these regions.

Multilocus Sequence Typing In multilocus sequence typing (MLST), the genetic variations of V. cholerae at several housekeeping genes are indexed after nucleotide amplification and sequencing. MLST data is highly suitable for software based analysis and hence can be adopted in the long-term epidemiological studies. However, standardized methods are not adopted in the MLST for the universal use, as the target genes varies from 3 to 26. Results of split decomposition analysis of three housekeeping genes, mdh, dnaE, and recA showed that widespread recombination plays an important role in the emergence of toxigenic strains of V. cholerae O1. With the Argentinean V. cholerae O1 isolates, six distinct genetic lineages identified among seven housekeeping loci. The gyrB, pgm, and recAbased MLST analysis performed better than pulse-field gel electrophoresis (PFGE) as there was clear clustering of epidemic serogroups. MLST analysis using nine genetic loci showed that the Mozambique isolates that harbored classical CTX prophage had the ST identical to that of El Tor N16961, a representative of the current seventh cholera pandemic.

However, MLST of V. cholerae is incoherent as many investigations did not follow the specific set of genes in the analysis.

Variable Number of Tandem Repeat (VNTR) Loci In many housekeeping genes, the DNA regions known as VNTR are cataloged on the basis of their repeat unit. VNTR is otherwise called simple sequence repeats . This repetitive DNA contains monomeric sequences and arranged in a head-to-tail configuration. These repeat loci are highly conserved and hence the discrimination power is more compared with that of MLST. Five VNTR loci of V. cholerae strains collected between 1992 and 2007 from different areas in India showed that each VNTR locus was highly variable, with 5–19 alleles. The eBURST (based upon related sequence types) analysis of sequence types revealed four large groups of genetically related isolates with two groups containing O139 serogroup and the other two groups including O1strains. VNTR also helped to track the spread of specific genotypes across time and space. Genetic relatedness of V. cholerae collected from 2004–05 from Bangladesh showed minimal overlap in VNTR patterns between the two communities that was consistent with sequential, small outbreaks from local sources. VNTR-analysis of nontoxigenic V. cholerae that caused outbreak in Rostov region in 2005 showed that they differed from previous outbreaks and formed separate group with strains isolated from patients, carriers, and environment. Phylogenetic analysis of the combined VNTR data also showed a clear discrimination between the clinical O1 and O139 strains and the environmental isolates.

PFGE PFGE has proven to be highly effective molecular typing technique for different foodborne bacterial pathogens. Database for PFGE patterns for various foodborne pathogens have been established, and being used by many PulseNet groups all over the world. PFGE was shown to be useful for the identification of spread of specific clones of many pathogens. In the PFGE, suspected sources of the pathogens have been identified, followed by implementation of timely interventions to prevent further spread of the pathogens. In the US and other countries, considerable reduction of foodborne infections was observed after the inception of PulseNet program. International PFGE typing protocol for V. cholerae was established for generation and submission of subtype patterns to the database. Most of the published works shows in-house PFGE typing scheme and cannot be compared with data generated by others due to variation in several parameters. However, in the PFGE, various parameters are controllable including the electrophoretic programs. PFGE patterns of representative V. cholerae O1, El Tor strains from Australia, Peru, Romania, and the US were different from Asian countries, such as Bangladesh, India, and Thailand, indicating a close genetic relationship or clonal origin of the isolates in the same geographical region. In India, Thailand, Iran, South Africa, clonality of V. cholerae O1 tends to differ during each cholera outbreak.

Bacteria: Vibrio cholerae

In Italy and Albania, cholera epidemic occurred during 1994 after more than a decade. PFGE analysis indicated that the strains from both the countries belonged to the same clone that was part of the larger global spread of epidemic ribotype 6 strains, which started in southern Asia in 1990. PFGE-type patterns of Peruvian V. cholerae O1 strains isolated during 1991–95 suggest that genetic changes are occurring in Latin American cholera epidemic, more frequently than previously reported. Vibrio cholerae O1 strains that appeared in India after the O139 appearance had new pulsotypes in which the H type was the predominant one. Pulsotypes A–C dominated before 1992 and F type was common among the O139 serogroup. Pulsotype H was stable for a long time in India and was associated with several cholera outbreaks since 1993. This trend was the same in Bangladesh and Thailand, though the pulsotype nomenclature was different. Pulsotype IV, which was a new clone introduced after 1993 from overseas was frequently present in both domestic and imported cases from 1994 to 1997 in Aichi, Japan. PFGE was used to identify the clonality and spread of V. cholerae O1 in Kenya. The PFGE profiles of Iranian V. cholerae O1strains were similar to that of Pakistan, Nepal, and India, suggesting the dissemination of common clones in this region. Nontoxigenic V. cholerae O1 strains isolated between 1998 and 2000 from Mexico differed in the PFGE patterns compared with Latin America and US Gulf Coast clones. The outbreak associated nontoxigenic V. cholerae O1 strains from India had more resemblance with O139 serogroup rather than classical or El Tor V. cholerae. The El Tor hybrid strains from Mozambique that appeared during 2004–05 are different from the Bangladeshi hybrid strains and overall the El Tor hybrid strains differed markedly from conventional classical and El Tor strains. The PFGE patterns of toxigenic O139 strains isolated from turtles in Sichuan, China, during 2004 were identical with the patterns of strains that appeared in the outbreaks, thereby indicating the sources of infection causing these outbreaks.

Table 2

Prevention and Control Measures As death occurs in 50–70% of the untreated severe cholera cases, adequate rehydration therapy in mandatory. In severe cases, rehydration can be accomplished by intravenous infusion of fluid followed by oral rehydration with oral rehydration solution (ORS). Various modifications to the standard ORS have been successfully made that include hypoosmolar or hyperosmolar solutions, rice-based ORS, zinc supplementation, and the use of amino acids, including glycine, alanine, and glutamine. The precise rates of fluid administration should be adjusted according to the patient’s state of hydration and volume of the stool. Antimicrobial agents such as tetracycline and fluoroquinolones are effective in reducing the volume of the stool and duration of the diarrhea. Preservation of foods from contamination of V. cholerae without spoiling their natural flavor, esthetic, and nutritive values is always a big challenge. Some of the normal practices such as addition of lime juice to foods thereby reducing pH toward acidic kills most V. cholerae. Many modern techniques such as high pressure processing, cold temperatures, ionizing irradiation, exposure to chlorine or iodophor, etc. decontaminates most of the vibrios. Preventive measure such as restricting the consumption of oysters during summer months, cleanliness of cutting boards, microbial monitoring of shellfish/shrimp growing areas, postharvest practices including depuration and relaying in offshore waters can reduce V. cholerae mediated infections. WHO’s Strategic Advisory Group of Experts on immunization recently recommended that cholera vaccination should be considered in endemic areas targeting the higher-risk population to reduce the disease burden. However, this vaccination should not interrupt the provision of other priority health interventions to control and prevent cholera outbreaks. Two oral cholera vaccines are now available i.e., Dukoral and Shanchol and their characteristics and other details are described in Table 2. mORCVAX vaccine is basically identical to that of Shanchol, except it was made by a different

Available oral cholera vaccines and their characteristics

Cholera vaccine a

Dukoral

Shanchol/mORCVAXb

a

Characteristics

Vaccine schedule

Vaccine: Monovalant vaccine with formalin and heat-killed whole cells of (1  1011 bacteria). Vibrio cholerae O1 consisting of both Ogawa and Inaba serotypes and classical and El Tor biotypes along with recombinant cholera toxin B subunit (rCTB) 1 mg Usage: Given with bicarbonate buffer, 150 ml for adults and 75 ml for children aged 2–5 years to protect the CTB Expected protection: After 7 days Vaccine: Bi vaccine with formalin and heat-killed whole cells of V. cholerae O1 consisting of both Ogawa and Inaba serotypes and classical and El Tor biotypes along with the O139 serogroup, without cholera toxin B subunit Usage: Can be given directly without bicarbonate including children Z1 year Expected protection: After 7 days

2 doses Z7 days apart 1 booster dose for every 2 years for adults and children Z6 years. For children 2–5 years, 1 booster is recommended for every 6 months

Not licensed for children aged o2 years. Identical vaccines in terms of V. cholerae strains different manufacturers using different methods.

b

553

Two liquid doses 14 days apart for individuals aged Z1 year with booster dose after 2 years 1 booster dose for every 2 years for adults and children Z6 years. Booster dose for children for every 6 months is not required

554

Bacteria: Vibrio cholerae

manufacturer with different methods. The protective efficacy (PE) of Dukoral cholera vaccine was more than 80% with two doses of vaccine irrespective of the age groups. Because CTB is structurally and functionally similar to the heat-labile toxin produced by enterotoxigenic E. coli (ETEC), Dukoral vaccine gave 67% protection against ETEC, which is another enteric pathogen commonly found in developing countries. The PE of Shancol vaccine for all age groups after two doses was 66% and the overall effectiveness after 3–5 years was 50%. Overall, vaccination can be considered as an additional preventive tool by the health authorities to prevent cholera outbreaks and its spread to newer areas. Information regarding cholera outbreaks and characteristics of the strains would help many clinicians and laboratory works to prevent its spread and proper management of cholera. Though official notification of cholera outbreaks by WHO Member States is mandatory under the International Health Regulations, the reporting by many countries is incomplete due to political and economical reasons that also include tourism and food export. The online forum, Program for Monitoring Emerging Diseases supported by the International Society for Infectious Diseases complements the WHO cholera reports and provides detailed information on subnational, monthly, and temporal distribution of cholera cases.

Further Reading Asakura H, Ishiwa A, Arakawa E, et al. (2007) Gene expression profile of Vibrio cholerae in the cold stress-induced viable but non-culturable state. Environmental Microbiology 9: 869–879. Cooper KL, Luey CK, Bird M, et al. (2006) Development and validation of a PulseNet standardized pulsed-field gel electrophoresis protocol for subtyping of Vibrio cholerae. Foodborne Pathogens and Disease 3: 51–58. De K, Nandy RK, and Nair GB (2005) Microbiological and molecular methods to identify and characterize toxigenic Vibrio cholerae from food samples. In: CA Catherine (ed.) Methods in Biotechnology: Food-Borne Pathogens: Methods and Protocols, vol. 21, pp. 99–124. Totowa, NJ, USA: Humana Press Inc.

Fukushima H, Katsube K, Hata Y, Kishi R, and Fujiwara S (2007) Rapid separation and concentration of food-borne pathogens in food samples prior to quantification by viable-cell counting and real-time PCR. Applied and Environmental Microbiology 73: 92–100. Hong BX, Jiang LF, Hu YS, Fang DY, and Guo HY (2004) Application of oligonucleotide array technology for the rapid detection of pathogenic bacteria of foodborne infections. Journal of Microbiological Methods 58: 403–411. Kaper JB, Morris Jr. JG, and Levine MM (1995) Cholera. Clinical Microbiological Review 8: 48–86. Levine MM, Black RE, Clements ML, et al. (1981) Volunteer studies in development of vaccines against cholera and enterotoxigenic Escherichia coli: A review. In: Holme T, Holmgren J, Merson MH, and Mollby R (eds.) Acute Enteric Infections in Children. New Prospects for Treatment and Prevention, pp. 443–459. Amsterdam: Elsevier/North-Holland Biomedical Press. Morris Jr. JG (1990) Non-O group 1 Vibrio cholerae: A look at the epidemiology of an occasional pathogen. Epidemiological Reviews 12: 179–191. Morris Jr JG, Wilson R, Davis BR, et al. (1981) Non-O group 1 Vibrio cholerae gastroenteritis in the United States: Clinical, epidemiologic, and laboratory characteristics of sporadic cases. Annals of International Medicine 94: 656–658. Nair GB, Mukhopadhyay AK, Safa A, and Takeda Y (2008) Emerging hybrid variants of Vibrio cholerae O1. In: Faruque SM and Nair GB (eds.) Vibrio Cholerae Genomics and Molecular Biology, pp. 179–190. Norfolk, UK: Caister Academic Press. Panicker G, Call DR, Krug MJ, and Bej AK (2004) Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays. Applied and Environmental Microbiology 70: 7436–7444. Ramamurthy T and Nair GB (2006) Foodborne pathogenic Vibrios. In: Simjee S (ed.) Infectious Disease: Foodborne Diseases, pp. 113–154. Totowa, NJ: Humana Press, Inc. Srisuk C, Chaivisuthangkura P, Rukpratanporn S, et al. (2010) Rapid and sensitive detection of Vibrio cholerae by loop-mediated isothermal amplification targeted to the gene of outer membrane protein OmpW. Letters in Applied Microbiology 50: 36–42.

Relevant Websites www.promedical.org Program for Monitoring Emerging Diseases (ProMED). http://pulsenetinternational.org/ Pulse-Field Gel Electrophoresis Protocol (PFGE).