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In 2004 approximately 2.5 billion cases of diarrhea occurred worldwide, which resulted in 1.5 million deaths among children under the age of five. Greater than half of these were in Africa and South Asia (WHO, 2009) Children are more susceptible to the complications of diarrhea because a smaller amount of fluid loss leads toÂ dehydration, compared to adults.Â
Diarrhoea is a common symptom of gastrointestinal infections caused by a wide range of pathogens, including bacteria, viruses and protozoa. However, just a handful of organisms are responsible for most acute cases of childhood diarrhoea. (WHO, 1999)
The common causes of bacterial diarrhea are Salmonellae,Â Shigellae, CampylobacterÂ and some strains ofÂ Escherichia coli (Viswanathan, 2009). The mode of transmission of pathogen causing diarrhea is mostly by the fecal-oral route.
According to a WHO report, diarrhoea is more prevalent in the developing world due, in large part, to the lack of safe drinking water, sanitation and hygiene, as well as poorer overall health and nutritional status. According to the latest available figures, an estimated 2.5 billion people lack improved sanitation facilities, and nearly one billion people do not have access to safe drinking water. These unsanitary environments allow diarrhoea-causing pathogens to spread more easily. (UNICEF/WHO, 2009)
Diarrhea may be accompanied by cramping, abdominal pain, nausea, an urgent need to use the bathroom, or loss of bowel control. Some infections that cause diarrhea can also cause a fever and chills or bloody stools. (NDDIC, 2011)
The strategies recommended by WHO in prevention of diarrhea includes improved quality and quantity of water supply, promotion of hand washing with soap, safe storage of household water, community-wide sanitation promotion (UNICEF/WHO, 2009).
Improving sanitation facilities has been associated with an estimated median reduction in
diarrhoea incidence of 36 per cent across reviewed studies (Jamison et al, 2008)
A recent review of data from a number of studies showed that a 42%-47% reduction in diarrhea can occur when hand washing with soap and water is introduced into a community. Thus, hand washing promotion and interventions are estimated to have the potential to prevent one million deaths from diarrheal diseases (Curtis V & Cairncross S, 2003)
2.2 Salmonella species
Salmonella are Gram-negative, non-spore forming rod-shaped predominantly motile enterobacteria. The subspecies Salmonella enterica is facultative anaerobe with a size 0.7-1.5 by 2.0-5.0 Âµm in size (Bronze et al., 2005).Â Salmonella possess flagellae and are motile (Jawetz et al., 2007). In a clinical laboratory, they are usually isolated on MacConkey agar, Xylose Lysine Deoxycholate (XLD) agar or Desoxycholate-citrate agar (DCA) agar.
Salmonella characteristically ferments glucose coupled with the ability of to produce hydrogen sulphite when inoculated into triple sugar iron (TSI) agar. (Giannella et al. 1996)
SalmonellaÂ was named after Daniel Elmer Salmon (1850-1914), an American veterinary pathologist, who describedÂ Salmonella entericaÂ (formerlyÂ S. choleraesuis). However, it was his colleague and subordinate Theobald Smith who first discovered the bacterium in 1885, fromÂ pigs, in an investigation for the cause of hog cholera. Dr. Salmon was the administrator of theÂ USDAÂ research program, and thus the organism was named after him by Smith. (FDA/CFSAN, 2008)
2.3 Classification of Salmonella.
Salmonella entericaÂ is one of two Salmonella species (enterica and bongori) and a member of the Enterobacteriaceae family (Su, & Chiu, 2007).Â Salmonella entericaÂ species is subdivided into 6 subspecies entericaÂ (I),Â salamaeÂ (II),Â arizonaeÂ (IIIa),Â diarizonaeÂ (IIIb), houtenaeÂ (IV) andÂ indicaÂ (VI). The enteric (I) subspecies are usually found in warm-blooded animals, whereas for subspecies II, IIIa, IIIb, IV and VI, are found in cold-blooded animals (Murray et al., 2007).
Salmonella entericaÂ has 2610 different serotypes. The most well known serotypes are Typhi, Paratyphi, Enteriditis, Typhimurium and CholeraesuisÂ (Su, & Chiu, 2007). The serotypes are characterized by three surface antigens: the flagellar "H" antigen, the oligosaccharide "O" antigen and the polysaccharide "Vi" antigen (found in Typhi and Paratyphi serotypes) (Bronze et al., 2005).Â
SalmonellaÂ is a well-known genus because of its ability to cause disease. However, only a few of the more than 2,200 types serotypes ofÂ SalmonellaÂ cause infections in humans, with the majority of cases traced to only five to ten common forms, mostlyÂ S. typhimuriumÂ andÂ S. enteritidisÂ (Breslow, 2002).
2.4 Epidemiology of disease caused by Salmonella species
It is estimated that only 3% ofÂ SalmonellaÂ infections are laboratory confirmed and reported to the Centers for Disease Control and Prevention (CDC). This is probably due to inconsistent diagnosis and reporting techniques in the diagnosis of the infection. However, an estimated 1.4 million people in the United States are infected with nontyphoidÂ SalmonellaÂ (CDC, 2011)
An estimated 500 people are infected with typhoidÂ Salmonella annually in the United States. Most cases of documented typhoid disease in the United States are related to foreign travel to developing nations such as India (30%), Pakistan (13%), Mexico (12%), Bangladesh (8%), Philippines (8%), and Haiti (5%). (Linam et al, 2007)
Worldwide estimates of nontyphoidÂ SalmonellaÂ range from 200 million to 1.3 billion, with an estimated death toll of 3 million each year. (Coburn et al., 2007)
The serovars responsible for typhoid or enteric fever,Â typhiÂ andÂ paratyphi, that cause systemic illness lead to an estimated 21.7 cases and 217,000 deaths worldwide, of which paratyphoid fever accounts for 5.4 million cases (Harish at al., 2011).
According to Singh, the salient epidemiological risk features of typhoid fever are age group, occupation, socio economic factors, environmental factors and reservoir infection. (Singh, 2001)
2.4.1 Risk of typhoid fever among age groups.
The highest incidence rates of salmonella infection occur in children younger than 5 years, particularly those under one 1 year, and individuals older than 70 years (Gomez and Cleary 1998). In Thailand, 80% of cases classified as salmonella diarrhoea occurs in children under 2 years old (Sirinavin., et al 1988).
Salmonella infection is serious and life threatening in young infants, old age and immunocompromised patients, and the fatality rates are higher in these groups. (Sirinavin et al., 1998)
A study in 172 children with extra-intestinal salmonella infection in Thailand revealed that the overall case-fatality rate was 9.9%, with17% in immunocompromised patients and 3% in infants (Sirinavin., et al 1998).
2.4.2 Occupational risk
Chronic carriers of Salmonella infection especially food handlers are very important in the transmission; particularly in places with low environmental sanitation. (Wilson et al, 1975)
A study conducted in Accra by Mensah et al., had a prevalence rate of 3.2% of Salmonella infection among food sellers (Mensah et al., 2002). Another study done in Kumasi by Feglo et al had a carriage rate of 2.3% among 258 food vendors studied. (Feglo et al., 2004)
Handling of live cultures containing Salmonella species may serve as primary hazard through accidental parenteral inoculation and ingestion of pathogens. (Richmond, & McKinney, 1999)Â
2.4.3 Reservoir infection
The reservoir hosts for non-typhoidal salmonellosis are domestic and wild animals such as cattle, swine, poultry, wild birds, flies and pets as well as other humans with the chronic carrier state. (Kraus et al, 2003, Greenberg, 1964) Up to 90% of reptiles and amphibians harborÂ SalmonellaÂ in their gastrointestinal tracts and 6% of nontyphoid disease is related to direct contact with these animals. (Linam et al., 2007)
For serotype S. typhi, humans with the chronic carrier state are the only reservoir for the diseaseÂ (Ryan and Ray, 2004)
Salmonella carriers are people with salmonella in stool without diarrhoea. They are divided into groups: acute asymptomatic infection, transient or convalescent carriers, and chronic or persistent or permanent carriers. A study of Mexican infants showed that 74% of non-typhoidal salmonella infections were asymptomatic (Cravioto., et al 1990). Transient carriers are those who continue excreting salmonella in stool after infection or diarrhoea for less than a year, usually not more than 3 months. Chronic or persistent carriers are those who excrete salmonella in stool for more than one year. Incidence of chronic carriers of non-typhoidal salmonella is less than 1% (Buchwald., et al 1984).
2.4.4 Environmental and Socio Economic Factors.
Lack of sanitation, poor housing, limited water supply and poor personal hygiene were associated with highÂ ShigellaÂ rates in Guatemala (Beck, Muñoz and Scrimshaw, 1957)
In a study conducted in Panama, six representative types of dwellings were considered as an index of social and economic influences on the prevalence of enteric pathogens among infants with diarrheal disease. Some of the dwellings were considered standard and others substandard with their occupants of low socioeconomic status.
Infection rates for enteropathogenicÂ Escherichia coli,Â ShigellaÂ andÂ Salmonella among infants from the various groups of substandard dwellings ranged from 6.0 to 10.2%, in contrast to the zero infection rates observed in infants from the better-type housing (Kourany and Vasquez, 1969).
It is possible that safety improvement in the water supply consequent to economic development has diminished the importance of those factors in several countries especially in developed countries.
2.5 Transmission of Salmonella Infection.
Eggs and poultry are the most common sources of infection (Braden et al., 2006, Linam et al., 2007). Ingestion of contaminated water, milk, milk products, beef, fruit, vegetables, and dairy products are also common sources. Potential sources of infection for infants' with SalmonellaÂ are exposure to reptiles, riding in a shopping cart next to meat or poultry, or consuming liquid infant formula. (Jones et al., 2006)
In hospitals, the bacteria have been spread by personnel in pediatric wards, either on their hands or on inadequately disinfected scopes (Block, S. S. 2001). Flies can infect foods which can also be a risk for transmission to humansÂ (Greenberg, 1964).
Fecal-oral transmission from person to person in areas with poor sanitation and contaminated or non chlorinated water is the route for enteric or typhoid fever.
2.6 Pathogenicity of Salmonella infection
SalmonellaÂ infections most commonly begin with ingestion of bacteria in contaminated food or water. However, direct contact with animal and human carriers has also been implicated. (Linam et al., 2007) Studies involving healthy human volunteers required a median dose of 1 million bacteria to produce disease. However, point outbreaks suggest as few as 200 bacteria may produce nontyphoid gastroenteritis (Peques and Miller, 2009).
Once the bacteria survive the acidic stomach, they colonize the intestine and then move across the intestinal epithelium via 3 routes, i.e. by invasion of the enterocytes, invasion of epithelial cells and through dendritic cells that intercalate epithelial cells. Interaction with the epithelium and resident cells promote a proinflammatory response to include cytokines, chemokines, neutrophils, macrophages, dendritic cells, and T and B cells. This inflammatory host response can actually benefit the intestinal pathogens and contribute to the nature and severity of the infection by establishing a competitive advantage against the normal flora. (Grassl and Finlay, 2008) After crossing this epithelial layer, the bacteria replicate in macrophages in Peyer's patches, mesenteric lymph nodes, and the spleen. Once colonized, the bacteria may then potentially disseminate to the lungs, gallbladder, kidneys, or central nervous system. The nontyphoid species ofÂ SalmonellaÂ tend to produce a more localized response because they are believed to lack the human-specific virulence factors. However, theÂ typhiÂ serotype can develop the more invasive disease resulting in bacteremia. The severity of disease is related to the serotype, number of organisms, and host factors. (Grassl and Finlay, 2008)
Individual susceptibility toÂ SalmonellaÂ infection increases with extremes of age, immunodeficiency states, prior antibiotic use, neoplastic disease, achlorhydria or antacid use, recent bowel surgery, and malnutrition. (Parry et al, 2005)
2.7 Resistance of Salmonella infection to antibiotics.
The causative organism Salmonella typhi has rapidly gained resistance to antibiotics like
ampicillin, chloramphenicol and cortrimoxazole, and also to previously efficacious drugs like ciprofloxacin (Jesudason et al, 1992, Butt et al, 2003).
The incidence of multidrug resistant S. typhi was reported to be as high as 60% while there are reports noting a decline (Sanghavi et al., 1999; Chaude et al., 2002).
A US-based study noted an increase in the number of MDR strains and nalidixic acid resistant S. typhi, although overall, the isolates were sensitive to ciprofloxacin and cefriaxone. (Ackets et al., 2000)
Another study from Bangladesh reported a decrease in MDR isolates with no corresponding increase in sensitive strains (Rahman et al., 2002). A decreased susceptibility in ciprofloxacin resistance has been recorded in UK (Threlfall et al., 2001) as well as India (Baliga et al.,).
High antibiotic resistance of S. typhi strains to Ampicillin, Chloramphenicol, co-trimoxazole and Tetracycline have also been reported in various studies done in Ghana (Newman et al., 2006, Mills Robertsons et al., 2003)
2.8 Antibiotic susceptibility testing.
An important task of the clinical microbiology laboratory is the performance of antimicrobial susceptibility testing of significant bacterial isolates. The goals of testing are to detect possible drug resistance in common pathogens and to assure susceptibility to drugs of choice for particular infections.Â The testing methods mostly used include microdilution or rapid automated instrument, gradient diffusion method and the disk diffusion methods.
The disk diffusion susceptibility method is simple and practical and has been well-standardized. (Jorgensen et al, 2007, Bauer et al, 1966) The test is performed by applying a bacterial inoculum of approximately 1-2Ã-108CFU/mL to the surface of a large (150 mm diameter) Mueller-Hinton agar plate. Up to 12 commercially-prepared, fixed concentrations, paper antibiotic disks are placed on the inoculated agar surface. Plates are incubated for 16-24 h at 35Â°C prior to determination of results. The zones of growth inhibition around each of the antibiotic disks are measured to the nearest millimeter. The diameter of the zone is related to the susceptibility of the isolate and to the diffusion rate of the drug through the agar medium. The zone diameters of each drug are interpreted using the criteria published by the Clinical and Laboratory Standards Institute (CLSI, formerly the National Committee for Clinical Laboratory Standards or NCCLS) (CLSI, 2009)Â
2.9 Escherichia coli
Escherichia coli (commonly abbreviated E. coli) is Gram-negative, facultative anaerobes, non- sporulating and rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms. Most E. coli strains are harmless, but E. coli infection in children less than 1 year is considered pathogenic. Some serotypes can cause serious food poisoning in humans. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, and by preventing the establishment of pathogenic bacteria within the intestine (Hudault et al., 2001, Bentley et al., 1982)
2.9.1 History of Escherichia coli
Theodor Escherich, a German pediatrician, first discovered this species in the faeces of healthy individuals in 1885 and called it Bacterium coli commune due to the fact it is found in the colon and early classifications of Prokaryotes placed these in a handful of genera based on their shape and motility. Following a revision of Bacteria in 1895, Migula reclassified it as Bacillus coli (Migula et al., 1895). Later it was reclassified in the newly created genus Escherichia, named after its original discoverer (Castellani et al., 1919). The genus belongs in a group of bacteria informally known as "coliforms", and is a member of the Enterobacteriaceae family ("the enterics") of the Gammaproteobacteria. E. coli is one of the commonest causes of diarrhea in developing countries.
2.10 Classification and identification of Escherichia coli
E. coliÂ consists of a diverse group of bacteria. PathogenicÂ E.Â coliÂ strains are categorized into pathotypes. Six pathotypes are associated with diarrhea and collectively are referred to as diarrheagenicÂ E. coli. Shiga toxin-producingÂ E. coliÂ (STEC)-STEC may also be referred to as Verocytotoxin-producing E. coliÂ (VTEC) or enterohemorrhagicÂ E. coliÂ (EHEC). This pathotype is the one most commonly heard about in the news in association with foodborne outbreaks.
Other pathotypes includes the EnterotoxigenicÂ E.Â coliÂ (ETEC), EnteropathogenicÂ E.Â coliÂ (EPEC)
EnteroaggregativeÂ E.Â coliÂ (EAEC),EnteroinvasiveÂ E.Â coliÂ (EIEC) and Diffusely adherentÂ E. coliÂ (DAEC) (CDC, 2012)
In stool samples, microscopy will showÂ Gram-negativeÂ rods in clusters. On MacConkey agar, deep red colonies are produced, as the organism isÂ lactose-positive, and fermentation of this sugar will cause the medium'sÂ pHÂ to drop, leading to darkening of the medium.The organism is alsoÂ lysineÂ positive, and grows onÂ TSI slant. E. coli is alsoÂ indole-positive,Â methyl red-positive, Voges-Proskauer (VP) negative andÂ citrate-negative. There are other sensitive and specific methods for identification of E. coli such as ELISA, PCR, Tissue culture and immunological methods but unfortunately they are slow and expensive. (Paton and Paton, 1998)
2.11 Epidemiology of disease caused by Escherichia coli
EHEC/STEC strains are the most frequently identified diarrheagenicÂ E coliÂ serotypes in North America and Europe (Frenzen et al., 2005).
The EHEC/STEC pathotype was first recognized in 1982 in the United States following an outbreak of serotypeÂ E coliÂ O157:H7, which was associated with consumption of undercooked hamburgers from a national fast-food restaurant chain (Riley et al., 1983).Since that time, numerous outbreaks ofÂ E coliÂ O157:H7 have been recognized in the United States, and it is still the most common STEC serotype in this country.
EHEC/STEC incidence varies depending on the country. Rates range from 1.4 per 100,000 populations in Ireland to 2.6 per 100,000 in Australia. In Japan, the incidence was 2.74 per 100,000 populations, a rate higher than that of the United States (1.06 per 100,000 populations). Factors implicated in Japanese areas with higher rates included higher percentage of elderly, higher number of people in households, and higher percentage of children (Sakuma et al., 2006).
OtherÂ E coliÂ pathotypes (ETEC, EPEC, EAEC, EIEC and DAEC) are infrequently identified in the United States. While lack of available laboratory testing may play a role, incidence of infection caused by these organisms appears to be low (Clarke et al., 2003, Russo 2006). Acute infectious diarrhea is the second most common cause of death in children living in the developing countries, accounting for about 20% of the cases.
ETEC is the most frequently isolated enteropathogen in community-based studies in the developing world of children 5 years old or younger (Girard et al., 2006). ETEC strains account for about 280 million diarrhea episodes and about 400,000 deaths annually (Qadri et al., 2005).ETEC also is a major cause of travelers' diarrhea (Donnenberg 2005).
EPEC was originally recognized in the 1940s as a cause of nosocomial diarrhea in infants. Today, these organisms are still a leading cause of severe diarrhea in infants and young children (<6 months of age) in the developing world (Donnenberg 2005). A recent report found that atypical EPEC strains appear to be a cause of prolonged diarrhea in children in Australia (Nguyen et al., 2006).
EIEC is less common than ETEC or EPEC in the developing world and is associated with only a few characteristic serotypes (Nataro and Kapper., 1998).
EAEC strains were first recognized in 1987 and are most often associated with illness in developing countries. EAEC strains have been shown to cause acute and chronic diarrhea in the developing world (mostly in young children) and chronic diarrhea in HIV-infected persons (Donnenberg 2005). Asymptomatic infection can cause subclinical inflammatory enteritis and growth disturbances (AAP 2003).
DAEC infections have not been well studied but have been recognized as a cause of diarrhea in the developing world, particularly among children.
2.12 Transmission of Escherichia coli
An increasing number of outbreaks are associated with the consumption of fruits and vegetables (sprouts, spinach, lettuce, salad) whereby contamination may be due to contact with faeces from domestic or wild animals at some stage during cultivation or handling. Waterborne transmission has been reported, both from contaminated drinking water and from recreational waters.Â Person-to-person contact is an important mode of transmission through the oral-faecalÂ route. An asymptomatic carrier state has been reported, where individuals show no clinical signs of disease but are capable of infecting others. (WHO, 2013)
2.13 Pathogenicity of Escherichia coli
Most strains ofÂ E coliÂ live harmlessly in the colon and are poorly adapted to causing disease in healthy persons, but several pathogenic strains can cause specific illness in healthy and immunocompromised individuals (Qadri et al., 2005).
The pathogenic process starts from ingestion of organism in contaminated food and water. Bacteria attach the intestinal mucosa via pili, fimbriae or fibrils and later colonize the ileal mucosa. Expression of bacterial mechanisms allows evasion of host defenses resulting in rapid multiplication of bacteria. Certain strains cause damage to host organs. (Donnenberg 2005, Kaper et al., 2004)
Some pathogenicÂ E coliÂ strains produce cytotonic enterotoxins (encoded on plasmid or bacteriophage DNA) that induce watery diarrhea without causing substantial tissue damage.
Other strains harbor plasmid-encoded invasion factors that allow invasion of the mucosa or plasmid- or bacteriophage-encoded cytotoxic enterotoxins that can cause tissue damage. Either of these factors can induce a host inflammatory reaction and lead to dysentery (Evans et al., 1996, Mokady et al., 2005).
2.14 Antibiotic susceptibility testing
Antibiotics which may be used to treatÂ E. coliÂ infection includeÂ amoxicillin, as well as other semisynthetic penicillins, many cephalosporins,Â carbapenems,Â aztreonam,Â trimethoprim, sulfamethoxazole,Â ciprofloxacin,Â nitrofurantoinÂ and theÂ aminoglycosides.
Antibiotic resistanceÂ is a growing problem. Some of this is due toÂ overuse of antibioticsÂ by humans or due to the use of antibiotics as growth promoters in animal feeds (Johnson et al., 2006)
Stool carriage of drug-resistantÂ E coliÂ also was identified among a sample of household residents in rural Idaho. Resistance was clustered in households and consistent with either spread of organisms between persons in close contact or common-source acquisition (eg, shared contaminated food). Prevalence of intestinal carriage ofÂ E coliÂ resistant to nalidixic acid was 3%, to trimethoprim-sulfamethoxazole was 11%, and to extended spectrum cephalosporins was 1%. Nalidixic acid resistance was associated with recent use of antimicrobials in the household (Hanna et al., 2005)