Isolation and Antibiogram of Escherichia Coli Implicated With Domesticated Edible Snailachatinamarginata Sold in (Abakaliki Metropolis)

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Isolation and Antibiogram of Escherichia Coli Implicated With Domesticated Edible Snailachatinamarginata Sold in (Abakaliki Metropolis)

Foodborne pathogens pose a serious threat to food safety especially in developing countries where hygiene facilities and operational practices in abattoirs and retail shops are often properly implemented (WHO, 2007). Recently, the incidence of foodborne infections have greatly increased worldwide and it is estimated that nearly a quarter of the population is at risk (Ebenso and Ebenso, 2011; Vázquez and Sánchez, 2015).

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In most cases where foodborne infections and outbreaks occur in humans, meat and its related products are usually identified as the sources of contamination (Collins, 2007; WHO 2009; Ebenso and Ebenso, 2011; Agbonlahor  et al., 2014). Food borne diseases often follow the consumption of contaminated food-stuffs especially from animal products such as meat from infected animals or carcasses contaminated with pathogenic bacteria as Salmonella species, Shigella  species, Staphylococcus aureus, Listeria monocytogenes, Campylobacter species, and Escherichia coli (Nouichi and Hamdi, 2009).
The Giant African Land Snail (Archachatina  marginata) are one of the prominent snails species found abundantly in this part of the world are the edible giant land snails: Achatina achatina and Archachatina  marginata (Ajayi  et al., 2009). Land snail habitat ranges from the dense tropical high forest in southern Nigeria to the fringing riparian forests of the derived Guinea Savannah (Odaibo, 2007; Ajayi  et al., 2009). From November to March each year, Nigerian snails aestivate because of the hot dry weather.They are found extensively in the Southern parts of Nigeria and the entire West African coastal area, central and South Africa, where the weather is most favourable for their proliferation (Herbert and Kilburn, 2004). Snails are cold-blooded animals and therefore sensitive to changes in atmospheric humidity and temperature. When rainfalls the epiphragm breaks and very cold water stored inside before aestivation pours out of the aperture (Ajayi  et al.,2009), and the snails emerge to eat the new plant growth and the soft soil (Ajayi  et al.,2009; Odaibo, 2007).

Snails thrive best at temperature of about 10-23°C (Albequerque et al., 2009). Thus, it is important to note that the organism can cause infections to man when the snail meat is not properly cooked and when the processing is not done under sanitized condition (Fagbuaro  et al., 2006).
There is a very close association between snails and microbes because their habitat is filth, sewage, manure rotten materials and poor latrine system, it is therefore not suprising, the high level of microbial interaction with land snails, making them to become naturally contaminated with pathogens from the filth in which they live. The pathogens thus emain in their bodies throughout their further development and may finally be spread in the faeces and visceral fluid they produce (Fagbuaro  et al., 2006).

Aim of the Study
The aim of this research was isolation and antibiogram ofE. coli implicatedwith domesticated edible snail species (Archachatinamarginata) sold in Abakaliki metropolis, Ebonyi State.

Specific Objectives
The objectives of the study include:
i.    To determine the level of microbial load from the domesticated edible snails species (Archachatinamarginata) collected.
ii.    To isolate and characterize E. coli from domesticated edible snail species sold in Abakaliki metropolis.
iii.    To determine the prevalence of E. coliassociated with edible snails species sold in Abakaliki.
iv.    To investigate the antimicrobial susceptibility patterns of the isolates to different classes of antibiotics.
v.    To determine the multi-drug resistance pattern of the isolates against the different classes of antibiotics used.

CHAPTER TWO
LITERATURE REVIEW
Background of the Study
Most animals provide habitation to infectious microorganisms and subsequent transmission of many disease-producing microbes to man, this is because the basic language in ecosystem is that of interaction for transfer of energy through commensalism, symbiotic mutualism and parasitism. Such close association between animals and microbes have prompt many workers to investigate the role of animals in the dissemination of enteric pathogens(Odu  et al., 2010).
The Giant African land snails (Archachatina  marginata) are soft bodied animals that belong to the phylum Mollusca, class Gastropoda, family Achatinidae and order Pulmonata (Uwalaka and Ahaot, 2013). They are distributed throughout the world in the tropical and sub-tropical regions where most of the genera of the family are confined to Africa. They serve as wildlife dietary protein sources in Nigeria and some parts of Africa (Ohimain et al., 2014). They are found majorly in southern parts of Nigeria, North African coast area, central and South Africa where the weather is most favourable for their proliferation (Herbert et al., 2001). It has been observed that edible snails obtained from swamps in North African coast for consumption in North America carry with them Salmonella species (Andrews et al., 1975). Snail meat is a delicacy in diets of people in Southern Nigeria (Ebenso and Ebenso, 2011). Mollusc has been reported to implication as vehicles for human infections caused by E. coli. The E. coli have been reported to have long-term survival in manure, soil and pasture (Fenlon et al., 2000). Agbonlahor  et al. (2014) while investigating the bacteriology of edible African snails in the town of Ekpoma, Irrua, Iruekpen and Benin city all in Edo State, Nigeria isolated various Enterobacterceae organism thereby creating awareness on the possible public health risks that may result in the consumption of improperly processed snail meat. These organisms may remain in snails not as pathogens but as normal flora, but they can also cause diseases if eaten raw or improperly cooked. According to WHO (2009) estimates 200,000 deaths from food borne pathogens (especially Salmonella and E.coli). There is a very close association between snails and microbes because of their habit filth, sewage and rotten materials (Odu  et al., 2010). It is therefore not surprising the high level of microbial interaction with water snails, making them to become naturally contaminated with pathogens from filth in which they live (Fagburo  et al., 2006).

Brief Description and Biochemistry of E. coli
E. coli is Gram-negative (bacteria which do not retain Crystal violet dye), facultative anaerobic (that makes ATP by aerobic respiration if oxygen is present, but is capable of switching to fermentation or anaerobic respiration if oxygen is absent) and non-sporulating (Lecointre et al., 1998; Opperman, 2010). Cells are typically rod-shaped, and are about 2.0 micrometers (μm) long and 0.25–1.0 μm in diameter, with a cell volume of 0.6–0.7 μm. It can live on a wide variety of substrates (Moniri and Khorshid, 2003). E. coli uses mixed-acid fermentation in anaerobic conditions, producing lactate, succinate, ethanol, acetate and carbon dioxide. Since many pathways in mixed-acid fermentation produce hydrogen gas, these pathways require the levels of hydrogen to be low, as is the case when E. coli lives together with hydrogen-consuming organisms, such as methanogens or sulphate-reducing bacteria (Ling et al., 2000).
Optimal growth of E. coli occurs at 37 °C (98.6 °F) but some laboratory strains can multiply at temperatures of up to 49 °C (120 °F) (Lecointre et al., 1998).

Growth can be driven by aerobic or anaerobic respiration, using a large variety of redox pairs, including the oxidation of pyruvic acid, formic acid, hydrogen and amino acids, and the reduction of substrates such as oxygen, nitrate, fumarate, dimethyl sulfoxide and trimethylamine N-oxide (Feng and Weagant, 2009). Strains that possess flagella are motile. The flagella have a peritrichous arrangement (El Metwally et al., 2010).
E. coli and related bacteria possess the ability to transfer DNA via bacterial conjugation, transduction or transformation, which allows genetic material to spread horizontally through an existing population (El Metwally et al., 2010). This process led to the spread of the gene encoding shiga toxin from Shigella to E. coli O157:H7, carried by a bacteriophage (Ling et al., 2000).

Classification of Escherichia coli
According to scientific classification, E. coli belonged to Domain of Bacteria, Kingdom, Bacteria, Phylum, Proteobacteria, class, Gammaproteo bacteria, order, Enterobacteriales, family, Enterobacteriaceae, genus,Escherichia while specie name is  Escherichia coli (Arends and Weiss, 2004)

Pathogenesis and Role of E. coli in Disease
Most E. coli strains do not cause disease, but virulent strains can cause gastroenteritis, urinary tract infections, and neonatal meningitis. In rare cases, virulent strains are also responsible for hemolytic-uremic syndrome, peritonitis, mastitis, septicemia and Gram-negative pneumonia (Apun et al., 2011).
UPEC (uropathogenic E. coli) is one of the main causes of urinary tract infections (Warren et al., 2008). It is part of the normal flora in the gut and can be introduced in many ways.

In particular for females, the direction of wiping after defecation (wiping back to front) can lead to faecal contamination of the urogenital orifices. Anal intercourse can also introduce these bacteria into the male urethra, and in switching from anal to vaginal intercourse the male can also introduce UPEC to the female urogenital system (Salvatore et al., 2011).
In May 2011, one E. coli strain, Escherichia coli O104:H4, has been the subject of a bacterial outbreak that began in Germany. Certain strains of E. coli are a major cause of foodborne illness. The outbreak started when several people in Germany were infected with enterohemorrhagic E. coli (EHEC) bacteria, leading to hemolytic-uremic syndrome (HUS), a medical emergency that requires urgent treatment. The outbreak did not only concern Germany, but 11 other countries, including regions in North America (Omar et al., 2013). On 30 June 2011 the German Bundesinstitut für Risikobewertung (BfR) (Federal Institute for Risk Assessment, a federal, fully legal entity under public law of the Federal Republic of Germany, an institute within the German Federal Ministry of Food, Agriculture and Consumer Protection) announced that seeds of fenugreek from Egypt were likely the cause of the EHEC outbreak (Omar et al., 2013).
They are numerous works showing the role of E coli as a major enteric pathogen, particularly in developing countries.

However, the different types of E coli associated with enteric infections and which are classified into five groups according to their virulence properties are briefly described here: Enteropathogenic E. coli (EPEC) serotypes in the past were associated with serious outbreaks of diarrhea in newborn nurseries in the US (Ling  et al., 2000). They remain an important cause of acute infantile diarrhea in developing countries. Disease is rare in adults. Enteroinvasive E. coli (EIEC) types produce disease resembling shigellosis in adults and children (El Metwally et al., 2007). Enterotoxigenic E. coli (ETEC) types are a major cause of traveler’s diarrhea, and of infantile diarrhea in developing countries (Ling et al., 2000). Enterohemorrhagic E. coli (EHEC) occur largely as a single serotype (O157:H7) causing sporadic cases and outbreaks of hemorrhagic colitis characterized by bloody diarrhea. EHEC also may cause hemolytic uremic syndrome (HUS), an association of hemolytic anemia, thrombocytopenia, and acute renal failure. Enteroaggregative E. coli (EAEC) types exhibit a characteristic aggregative pattern of adherence and produce persistent gastroenteritis and diarrhea in infants and children in developing countries (Adzitey et al., 2011).
Transmission of pathogenic E. coli often occurs through faecal-oral transmission (Zinnah et al., 2007).

Common sources and routes of transmission includes: unhygienic food preparation and direct consumption of faecal /sewage-contaminated food or water (Zinnah et al., 2007). Moreover, dairy and beef are primary reservoirs of E. coli and they can carry it asymptomatically and shed it in their faeces to the environment (Sabota et al., 2008). Food products associated with E.coli outbreaks includes raw milk, raw ground beef, unpasteurized juice, unpasteurized cheese, raw seed sprouts, cucumber and foods contaminated by infected food workers via faecal-oral route. According to U.S. Food and Drug Administration, the faecal-oral cycle of transmission can be disrupted by cooking food properly, preventing cross-contamination, instituting barriers such as gloves by workers, instituting health care policies so that food industry employees seek treatment when they are ill. Shiga toxin-producing E. coli (STEC) can also be transmitted by flies as well as direct contact with farm animals, petting zoo animals, and air-bone particles found in animals-rearing environments (Okeke et al., 2000). Apart from gastrointestinal infections, E. coli have also been implicated in urinary tract infections, neonatal meningitis and cancer in man as well as animal diseases like; septicemia and diarrhea in newborn calves, acute mastitis in dairy cows and colibacillosis which is associated with chronic respiratory disease in poultry (Vogt and Dippold, 2005; Zhao et al., 2001).

Virulence Factors of E. coli Isolates
Escherichia coli strains isolated from extraintestinal infections often possess a number of properties not usually found in random faecal isolates (Jadhav et al., 2012). These include production of soluble and cell-bound hemolysins, the colicin V plasmid, production of the siderophores aerobactin and enterochelin, and special pilial antigens for adherence to target cells (Ryan and Ray, 2004). The hemolysin kills host cells and makes iron more available by releasing hemoglobin-bound iron from lysed red cells. To strip iron from the host iron-binding proteins (transferrin and lactoferrin), E. coli produces siderophores of both the hydroxamate (aerobactin) and phenolate (enterochelin) types (Podschun and Ullmann, 2008). Common or type 1 pili may mediate adherence to bladder cells; P-pili are virulence factors for strains causing pyelonephritis; S-pili, which recognize O-linked sialo-oligosaccharides of glycophorin A, are associated with meningitis and urinary tract infections. Certain afimbrial adhesions and outer membrane proteins also have been associated with urinary tract infections (Bingen, 2008).

The enzyme urease, produced by Proteus, and to a lesser extent by Klebsiella species, is thought to play a major role in the production of infection-induced urinary stones (Bingen, 2008). Urease hydrolyzes urea to ammonia and carbon dioxide. Alkalinization of the urine by ammonia can cause magnesium phosphate and calcium phosphate to become supersaturated and crystallize out of solution to form, respectively, struvite and apatite stones (Tambekar et al., 2006). Bacteria within the stones may be refractory to antimicrobial therapy. Large stones may interfere with renal function. The ammonia produced by urease activity may also damage the epithelium of the urinary tract (Omar et al., 2013).
Except in cases of bacteremia and other systemic infection, there is little evidence that endotoxin plays a role in most coliform and Proteus diseases. Humans with coliform bacteremia show many of the typical effects of endotoxin, including fever, depletion of complement, release of inflammatory mediators, lactic acidosis, hypotension, vital organ hypoperfusion, irreversible shock, and death (El Metwally  et al., 2007).

The Giant African Snail

Description of Archachatina  marginata
Archachatina  marginata, common name the giant West African snail or banana rasp snail, is a species of air-breathing tropicalland snail, a terrestrialpulmonategastropodmollusk in the family Achatinidae. They can grow up to 20 cm long, and live up to 10 years (Fagburo et al., 2006).
The snail has a bulbous protoconch that is large and broad, with a white or bluish-white columella, parietal wall and outer lip (Fagburo et al., 2006).

The shell of the snail can grow up to 21 centimeters in height, and 13 centimeters in diameter. The shell, when magnified, has the appearance of a woven texture (Odu  et al., 2010).

Scientific Classification of Archachatina  marginata
The Giant African land snails (Archachatina  marginata) are soft bodied animals that belong to the phylum Mollusca, class Gastropoda, super family Achatinoidae, family
Achatinidae, order Pulmonata, genus Archachatina and species Archachatina  marginata (Uwalaka and Ahaot, 2013).

They are distributed throughout the world in the tropical and sub-tropical regions where most of the genera of the family are confined to Africa (Pilsbry, 1904). They serve as wildlife dietary protein sources in Nigeria and some parts of Africa (Ohimain et al., 2014).
Bionomial name isArchachatina  marginata (Swainson, 1821) while the Sub-species of Archachatina  marginata include; Archachatina  marginata var. Ovum, Archachatina  marginata var. Stutralis, Archachatina  marginata var. Egregia, Archachatina  marginata var. Candefacta, Archachatina  marginata var. Eduradi, Archachatina  marginata var. Grevillei, Archachatina  marginata var. Icterica

Hosts of Archachatina  marginata
As a phytophagous gastropod, this species is not host-specific. It has been documented as causing economicdamage to various crop plants.

Invasive Species
The giant African snail is a macrophytophagous herbivore; it eats a wide range of plant material, fruit, and vegetables. It will sometimes eat sand, very small stones, bones from carcasses and even concrete as calcium sources for its shell. The giant West African snail is one of the worst invasive species in the world and is extremely devastating to any species that it affects. However, the more prevalent problem with the spread of the snail as an invasive species is that it is often a carrier of the disease rat lungworm (Nisbet, 20009). Within humans this causes the disease eosinophilic meningoencephalitis, which is what makes the snails’ spread to North America problematic. If the snails continues to spread it could potentially be a problem for the health of people all throughout North America from Cuba to the United States. Archachatina  marginata can live up to 10 years, and attain sexual maturity at 9–10 months under laboratory conditions. In addition to being an agricultural pest, they act as the reservoir host of rat lung parasites which cause eosinophilic meningoencephalitis in humans and are seen as a threat to public health because of this (Cowie  et al., 2009).

Current Distributionof Archachatina  marginata
Giant African Snails can be found in most southern hemisphere countries, eg. throughout Africa, Indian Ocean Islands, Australia, New Zealand, South America (Voss and Schmidt, 2001).  In addition, these snails have been identified in Southern, Southeastern and Eastern Asia, Polynesia and other Pacific Islands, West Indies and the United States.  Typically, Giant African Snails have been found along the coastline and in southern states of the USA.  However, sightings have also occurred in Wisconsin, Michigan and Ohio.  These snails thrive in humid, tropical climates (Nisbet, 20009).
The natural spread of this species is very slow; however, unintentional spread by individuals for food and as folk medicine is very common.

The USDA routinely checks for the species in the luggage of travelers from West Africa, Nigeria particularly, Ghana and Cameroon (Ogbu, 2011).

Ecology of Archachatina  marginata
Achatinids are nocturnal forest dwellers but can adapt to disturbed habitats. The snails prefer concealed habitats and if overcrowding occurs, they may colonize more open habitats. During periods of high humidity, Achatinids are more active but if the individuals are found during broad daylight it is most likely due to high population density (Ohimain  et al., 2014).
Eggs of Achatinids are normally laid in the soil, but can be found under leaves or rocks.

They produce as many as 40 eggs which are yellow in color with dark blotches and an incubation period of approximately 40 days.

Benefitsof Archachatina  marginata
Giant African Snails contribute to the degradation of animal matter.  In addition, the Giant African Snail provides nutrients to the India glowworm Beetle; specifically to the larvae (male larvae consume 20 to 40 Archachatinas; female larvae eat 40 to 60 Archachatinasduring their development).  Other beetle species consume the Achatina fulica, such as the lampyriad and the coprine beetle (Graczyk et al., 2001).
The hermit crab is one of the most dangerous predators to the Achatina fulica and has been known to use the shell as its home.

The coconut crab also views the Achatina fulica as a delicacy.  The domesticated duck along with a vast variety of other bird species forage on Giant African Snails.  Other mammals such as the wild pig prey on Achatina fulica (Fredericks, 2010).

Threatsof Archachatina  marginata
The Giant African Snails’ greatest lethal threat to humans is eosinophilic meningitis.  This condition is caused by the rat lungworm parasite, angiostrongylus cantonesnsis (Kweon, 2008).  Most often this parasite is transferred by eating the snail, as some humans consider snails a delicacy.  In addition the Giant African Snail can carry the gram-negative bacterium, aeromonas hydrophila, causing a wide variety of symptoms, especially in persons with a weak immune system.  These diseases can be transferred to humans by eating raw or undercooked infected snail meat or contaminated vegetables. Humans can also be infected by handling live GALS if the snails’ secretions contact mucous membranes of the eyes, nose, or mouth (Odu  et al., 2010).
Giant African Snails cause great economic peril to farmers due to their propensity in consuming large amounts of crops/plants.

Their diet consists of over 500 different plant species.  A wide variety of horticulture and medicinal plants are known to be attacked by this snail.  Not only does this decrease the income for agricultural producers, but it also impacts their living conditions (often requiring relocation) and decreases food and medical resources for humans, animals and other species (Kweon, 2008).
The economic consequences persist in eradicating these creatures, sometimes costing millions of dollars.  Another economic penalty involves the decrease in tourism.  As noted earlier, Giant African Snails thrive in warm, tropical conditions – often tourist destinations (Odaibo, 2007).

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