The Incidence of Salmonella on the surface of Commercially Sachet Water sold in Abakaliki.

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The Incidence of Salmonella on the surface of Commercially Sachet Water sold in Abakaliki.

Salmonella is the genus name for a large number of bacteria. Each type is distinctly identifiable microscopically by its specific protein coating. Salmonella bacteria are rod shape, flagellated, grain stain negative, and known to cause diseases in humans, animals and birds (especially poultry) worldwide. Salmonella species are known to cause two major diseases; gastroenteritis (also known as non-typhoid salmonellosis or Salmonella poisoning) and typhoid fever in humans (Lima and Gueriant, 2009).

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Infections caused by these bacteria or their toxins are called salmonellosis. The bacterium known as salmonella belongs to the family Enterobacteriacea. They are typically anaerobic, unable to form spores, and motile, or able to move around spontaneously. An important characteristic of Salmonella bacteria is that they are able to grow and multiply outside twing host organisms, thus having greater survival chances than otherwise (Miller and Pegues, 2000).

The bacteria can have a combination of three antigens; the O antigen, H antigen and VI antigen. The O antigen is located in the cell wall of the bacterium, and each Salmonella bacillus may possess two or more O antigens on its surface. The H antigen is a flagella antigen that can be destroyed by heat and enables the motility of the Salmonella bacterium, the last antigen is known as the VI because it is related to the virulence of the bacterium. As a capsular antigen, its presence enhances the virulence of the bacterium that has it of all the sub-species of Salmonella. Only two, Salmonella typhi and Salmonella choleraesuis, have the VI antigen (Miller and Pegues, 2000).
The sachet drinking water was introduced into the Nigerian market as a less expensive means of accessing drinking water than bottled water (Ogundipe, 2008).

It also acts as an improvement over the former types of drinking water packaged for sale to consumers in hand filled, hand tied polythene bags. Today, the easy accessibility to drinking water in packaged forms has resulted in a big and thriving water industry with several hundreds of million litres of these water products consumed every year by Nigerians (Ogundipe, 2008).
Production, sales and consumption of packaged water is growing rapidly in most countries of the world especially in developing countries (Oyedeji et al., 2010; Mgbakor et al., 2011; Gangil et al., 2013). Many scientists all over the world have carried out researches on the microbial analysis of commercially vended packaged water using different laboratory techniques and reporting varying percentages of microbial isolation (Warburton et al., 1992; Bharath et al., 2003; Oladapoet al., 2009; Prasanna and Reddy, 2009; Kuitcha et al., 2010; Oludairo et al., 2013). There are tremendous public health hazards associated with the consumption of microbial contaminated packaged water; these include diseases and deaths (Oladapo et al., 2009).
Water is an essential part of human nutrition and is required for maintenance of personal hygiene, food production and prevention of diseases (Thliza et al., 2015) It is the most abundant substance in nature and occupies about 70% of the earth’s crust (Anyamene and Ojiagu, 2014; Thliza et al., 2015). Due to its natural abundance and also because the protoplasm of many living cells contain about 80% water and most biochemical reactions which occur in the metabolism and growth of living cells involves water medium it is considered a universal solvent (Nwosu and Ogueke, 2004). Water is a biological medium which exists as solid, liquid and gas (Thliza et al., 2015).

Aim
The aim of this study is to determine the incidence of salmonella on the surface of commercially sachet water sold in Abakaliki.

Objectives
1. To isolate Salmonella from the surface of sachet water sold in Abakaliki.
2. To carry out antibiogram of the isolates using conventional antibiotics.

CHAPTER TWO
LITERATURE REVIEW

Morphology of Salmonella
Scientific classification
Kingdom Bacteria
Phylum Proteobacteria
Class Gamma proteobacteria
Order Enterobacteria
Family Enterobacteriaceae
Genus Salmonella
Species typhimurium

Salmonella is a genus of rod-shaped, Gram-negative non-spore-forming, predominantly motile enterobacteria with diameters around 0.7 to 1.5µm lengths from 2 to 5µm, and flagella which grade in all directions (i.e. peritrichous). They are chemoorganotrophs obtaining their energy from oxidation and reduction reaction using organic sources, and are facultative anaerobics. Most species produce hydrogen sulfide (Townsend et al., 2001). Which can readily be detected by growing them on media containing ferrous sulfate, such as TSI most isolates exists in two phases: A motile phase I and a non motile phase II. Cultures that are non motile primary culture may be switched to the motile phase using cragie tube. Optimal growth between 35 – 370C and pH 7 – 7.5, can also survive refrigeration, freezing (much reduced growth at temperature < 150C and above 60C) and dry conditions, Sensitive to most disinfectants, Killed at high temperatures in general 600C for 2 – 6 minutes or 700C for 1 minute will kill the bacteria. Salmonella is closely related to the Escherichia genus and are found worldwide in cold and warm – blooded animal (including humans) and in the environment. Salmonella family belong to eh Enterobacteriaceae, trivially known as “enteric” bacteria they cause illnesses as typhoid fever, paratyphoid fever and food borne illness (Hohmann et al., 1996).

Disease Caused by Salmonella
Salmonella infection, or salmonellosis, is a bacterial disease of the intestinal tract. Salmonella is a group of bacteria that causes typhoid fever, food poisoning, gastroenteritis, enteric fever and other illnesses. People become infected mostly through contaminated water or foods, especially meat, poultry and eggs (Ansong et al., 2008).
Salmonellosis is an infection caused by Salmonella. According to the CDC (Centers for Disease Control and Prevention), approximately 1.4 million Americans are infected with salmonellosis every year, of which about 500 die. In 2004, US authorities announced that Salmonella was responsible for 42% of human bacterial infections, followed by Campylobacter 37%, Shigella 15%, E. coli O157:H7 2.6% (Ansong et al., 2008).
Typhoid fever is caused by Salmonella serotypes which are strictly adapted to humans or higher primates these include Salmonellatyphi, Paratyphi A, Paratyphi B and Paratyphi C. In the systemic form of the disease, salmonellae pass through the lymphatic system of the intestine into the blood of the patients (typhoid form) and are carried to various organs (liver, spleen, kidneys) to form secondary foci (septic form). Endotoxins first act on the vascular and nervous apparatus, resulting in increased permeability and decreased tone of the vessels, upset of thermal regulation, and vomiting and diarrhoea. In severe forms of the disease, enough liquid and electrolytes are lost to upset the water-salt metabolism, decrease the circulating blood volume and arterial pressure, and cause hypovolemic shock. Septic shock may also develop. Shock of mixed character (with signs of both hypovolemic and septic shock) is more common in severe salmonellosis. Oliguria and azotemia may develop in severe cases as a result of renal involvement due to hypoxia and toxemia (Baker et al., 2005).

Causes of salmonellosis
Salmonella live in the intestines of birds, animals and humans. Most human infections are caused by eating food or drinking water that has been contaminated by feces (excrement). Foods that are most commonly infected are:
i. Uncooked meat, seafood and poultry – contamination most commonly occurs during the slaughtering process. Harvesting sea foods in contaminated waters is also a common cause (Townsend et al., 2001).

ii. Uncooked eggs – the Salmonella are usually present in the eggs when laid if the chicken is infected. Raw eggs may be found in some types of mayonnaise and homemade sauces (Townsend et al., 2001).

iii. Fruits and vegetables – if fruit and vegetables have been watered or washed in contaminated water there is a much higher chance they will be contaminated. Some kitchen practices may contaminate fruits and vegetables – if the person preparing the food handles raw meat and then touches the fruit without washing his/her hands, for example.

iv. Lack of hygiene – kitchen surfaces that are not kept clean, lack of hand washing procedures during food preparation, and lack of hand washing after going to the toilet or changing a baby’s diapers, are common routes for contamination and infection. A person with contaminated hands can pass the infection on to other people by touching them, or touching surfaces which others then touch (Townsend et al., 2001).

v. Pet reptiles or amphibians – most reptiles and amphibians carry Salmonella in their gut without becoming ill. They shed the bacteria in their droppings, which can quickly spread onto their skin and then anything they come into contact with, including cages, toys, clothes, furniture and household surfaces. The Health Protection Agency (UK) advises families not to keep reptiles if there are children under 5, pregnant women, very elderly people, or people with weaker immune systems in the household (Boyd et al., 1996).

Tissue Invasion
Salmonella typhimurium are able to secrete small signaling molecules called autoinducers. The luxs gene is responsible for initiating a series of phosphate transfer reactions that produce these molecules and allow for cell to cell communication. Sugar compounds, preferably glucose activate LUXS and the resulting auto inducer concentration increase with the bacteria concentration till the substrate is depleted. At this point the auto inducer is degraded and can be recycled by the bacteria cell. This quorum sensing allows cell to determine the metabolic potential of the environment (Miller and Pegues, 2000).
Salmonella typhimurium infect by coming in direct contact with non-phagocytic cells. This contact induces the formation of appendages formed are shorter than flagella but thicker than both flagella and pili. They cause the host cytoskeleton to rearrange which allows the bacteria to enter the cell. This membrane ruffling system is due to 14 genes on the S. typhimurim chromosome. The inv genes are responsible for the assemblage and emission / withdrawal of these appendages. All of the inv genes must be intact for this system to work. One of these genes, invc, is responsible for ATPase which provides the energy needed to complete this reffling process (Miller and Pegues, 2000).

Toxin-Mediated Diseases
Salmonella typhimurium is a pathogenic Gram-negative predominately found in the intestinal lumen. Its toxicity is due to an outer membrane consisting largely of lipopolysaccharides (LPS) which protect the bacteria from the environment (Mills and Finlay, 1994). The lipopolysaccharide (LPS) is made up of an O-antigen, a polysaccharide core, and lipid A, which connect it to the outer membrane. Lipid A is made up of two phosphorylated glucosamines which are attached to fatty acid. These phosphate groups determine bacteria toxicity. Animals carry an enzyme that specifically removes these phosphate groups is an attempt to protect themselves from these pathogen (Taylor et al., 1983).
The O-antigen, being on the outermost part of the lipopolysccharide (LPS) complex is responsible for the host immune response. S. typhimurium has the ability to undergo acetylation of this O-antigen, which changes its conformation, and makes it difficult for anti-bodies to recognize (Taylor et al., 1983).

Pathogenesis of Salmonella Infection
Salmonella infections in humans vary with the serovar, the strain, the infectious dose, the nature of the contaminated food, and the host status. The infectious dose, the nature of the contaminated food and the host status (Hornick et al., 1970). Certain serovars are highly pathogenic for humans; the virulence of rarerserovar is unknown. Strains of the same serovars are also known to differ in their pathogenicity. An oral dose of at least 105salmonella typhi cells are needed to cause typhoid in 50% of human volunteers, whereas at least 109S. typhimuriumcells (oral dose) are needed to cause symptoms of a toxic infection. Infants, immunosuppressed patients, and those affected with blood disease are more susceptible to salmonella infection than healthy adults (Hornick et al., 1970).
In the pathogenesis of typhoid the bacteria enter the human digestive tract, penetrate the intestinal mucosa (causing no lesion), and are stopped in the mesenteric lymph nodes. There, bacterial multiplication occurs, and part of the mesenteric lymph nodes, viable bacteria and LPs (endotoxin) may be released into the blood stream resulting in septicemia release of endotoxin is responsible for cardiovascular, collapses and tuphosi (a stuporous state, origin of the name typhoid) due to action on the ventriculusneuri vegetative centers (Greisman and Ochman, 1996).
Salmonella excretion by human patients may continue long after clinical cure. Asymptomatic carriers are potentially dangerous unnoticed. About 5% of patients clinically cured from typhoid remain carriers for months or even years (Kidgell et al., 2002). Antibiotics are usually ineffective on salmonella carriage (even if salmonella are susceptible to them) because the site of carriage may not allow penetration by the antibiotic. Salmonella survive sewage treatments if suitable germicides are not used in sewage processing (Miller and Pegues, 2000). In a typical cycle of typhoid, sewage plant. Effluent from the sewage plant passes into a coastal river where edible shellfish (mussels, oysters) live. Shellfish concentrate bacteria as they filter several liters of water per hour. Ingestion by human of these sea foods (uncooked or superficially cooked) may cause typhoid or other salmonellosis. Salmonellae do not colonize or multiply in contaminated shellfish (Miller and Pegues, 2000). Typhoid is strictly a human disease.

The incidence of human disease decrease when the level of development of a country increases (i.e. controlled water sewage systems, pasteurization of milk and dairy products). Where these hygienic conditions are missing, the probability of fecal contamination of water and food remain high and so is the incidence of typhoid (Miller and Pegues, 2000).
Food borne Salmonella toxic infection are caused by ubiquitous salmonella serovars (e.g. typhimurium). About 12–24 hours following ingestion of contaminated food (containing a sufficient number of Salmonella), symptoms appear (diarrhea, vomiting fever) and last 2 – 5 days. Spontaneous cure usually occurs (Mills and Finlay, 1994). Salmonella may be associated with all kinds of food. Contamination of meat (cattle, pigs, goats, chicken etc) may originate from animal salmonellosis, but most often it results from contamination of muscles with the intestinal contents during evisceration of animals, washing, and transportation of carcasses. Surface contamination of meat is usually of little consequences as proper cooking will sterilize it (although handling of contaminated of hands, tables, kitchenware, towel, other foods, etc).However, when contaminated meat is ground, multiplication of Salmonella may occur within the ground meat and if cooking is superficial ingestion of this highly contaminated food may produce a salmonella infection. Infection may follow ingestion of any food that supports multiplication of salmonella such as eggs, cream, mayonnaise, creamed foods etc; as a large number of ingested Salmonellae are needed to give symptoms (Mills and Finlay, 1994).

Transmission of Salmonella
The source of organisms for Salmonella gastroenteristis include contaminated food or water. Most commonly persons acquire salmonella from contaminated poultry (turkeys and chickens). S. enteritidis or S. choleraesuis are the most commonly isolated species (Mills and Finlay, 1994). Enteric fever, in contrast, in generally transmitted from person to person and involves S. typhi (no animals’ reservoirs). Contamination of food or water with human feces and an asymptomatic human carrier state provide the reservoir.

Salmonella is transmitted to humans via the fecal-oral route. An infected individual sheds the bacteria in his feces, and the bacterium is viable for months in the environment in water, soil an manure (Miller, and Pegues, 2000). However, in people at risk such as infants, small children, the elderly, salmonella infections can because very serious, leading to complications. If these are not treated, HIV patients and those with suppressed immunity can become seriously ill. Children with sickle cell anaemia who are infected with salmonella may develop osteomyetlitis (Mills and Finlay, 1994).

Treatment for Salmonella
Even though Salmonella food poisoning is a bacterial infection, most practitioners do not treat simple cases with antibodies studies have shown that using antibodies does not usually reduce the length of time that the patient is ill (Townsend et al., 2001). Paradoxically, it appears that antibodies do, however, cause the patient to shed bacteria in their feces for a longer period of time. In order to decrease the length of time that a particular individual is a carrier who can generally not given. In situations where an individual has a more severe type of infection with Salmonella bacteria, a number of antibiotics may be used (Thong et al., 2000). Chloramphenicol was the first antibiotics successfully used to treat salmonella food poisoning. It is still a drug of choice in developing countries because it is not expensive, although some resistance has developed to it (Thong et al., 2000).
Ampicillin and trimethoprim-sulfonamide have been used successfully in the treatment of infection caused by chloramphenicol resistant strains. Newer types of antibiotics, such as cephalosporin or quinolone, are also effective. These drugs can be given by mouth or through a needle in the vein (intravenously) for very ill patients (Thong et al., 2000). With effective antibiotic therapy, patients feel better in 24 to 48 hours, the temperature returns to normal in three to five days, and the patient is generally recovered by ten to 14 days (Thong et al., 2000).

Prevention of Salmonella Infections
Salmonella contaminated the environment, poultry flocks and poultry products. An eradication program is unrealistic. The World Health Organization (WHO) recognizes that control of salmonella infection from poultry products can take place in three areas: Education of the public, Improvement in slaughter hygiene and technology, Control of infection in the chickens themselves (Thong et al., 2000).
Some other ways of prevention are as follow: Don’t eat foods containing row eggs, such as homemade Caesar salad dressing, cookie dough, and hollandaise sauce, or drink homemade eggnog made with row eggs, Handle raw eggs carefully, Keep eggs refrigerated, Throw away cracked or dirty eggs, Cook eggs thoroughly, Cook poultry products to an internal temperature of 170oF for breast meat and 180oF thigh meat, Wash hands immediately after handing raw poultry or raw egg, Wash hands immediately after handing reptiles or contact with pet feces (Thong et al., 2000).
These are some of the way to control Salmonella infection:
1. Start with chicks known to be Salmonella negative.
2. Monitor the flock of Salmonella infection
3. Take appropriate action in case of Salmonella outbreak.
4. Effective bio security.
5. Maximize the protective mechanism of the individual birds.
Vaccination and optimization of the intestinal flora (Taylor et al., 2001).
6. Sanitary: since Salmonella are acquired through ingestion of contaminated food stuffs, sanitary means of control are most important. Treatment of animal feeds reduces the overall level of organisms in the animal population, improved slaughtering practices prevent cross-contamination of animal products and proper hygienic by food-handlers for prevent contamination at the consumer level (Taylor et al., 2001).
7. Immunological: A vaccine for typhoid is available, since only one serotype is responsible for the disease. However, the vaccine is not very effective because the bacteremic stage (i.e. where bacteria contact the vaccine-induced antibody) is brief (Taylor et al., 2001)
8. Chemotherapeutic: Typhoid fever and salmonella septicemia may be treated using moderate to board spectrum antibiotics. Gastroenteritis should only be treated by replacing lost fluids, since antibiotic therapy does not affect the course of the disease and may not increase the number of resistant species (Taylor et al., 2001).

Sachet Water
Access to safe drinking water is important to health and development (Adetunde et al., 2014), but because of its inadequacy and government’s inability to provide enough, a number of small scale water producing industries are packaging and marketing factory filled sachet drinking water (Thliza et al., 2015). These are small nylon sachets containing 0.5L of water which are electrically heated and sealed at both ends (Adegoke et al., 2012). The sale and consumption of sachet water continue to grow astronomically and rapidly in most countries of the world (Mgbakor et al., 2011; Oyedeji et al., 2010; Gangil et al., 2013).
The quest for cheap and readily available source of potable water has led to the emergence of sachet water (Anyamene and Ojiagu, 2014) which is a locally sourced low cost alternative drinking water scheme providing sustainable access to safe water in rural and semi urban settings of developing nations (USEPA, 2012; Balogun et al., 2014). This is thought to be cheaper and more affordable than bottled water and also safer, more hygienic and better than hand filled, hand tied sachet polythene bag water initially popularly sold (Oyedeji et al., 2010; Akinde et al., 2011). Consequently, sachet water has gradually become the most consumed liquid for both the rich and poor (Akinde et al., 2011).
Current trends however unfortunately suggest that sachet water could be a route of transmission of enteric pathogens which raises issues of the problem of its purity and health concern (Oladapo et al., 2009; Mgbakor et al., 2011; Akinde et al., 2011).

Sachet Water Contamination
Sachet water, no matter their sources, are susceptible to microbial, toxic organic and inorganic contamination (Anyamene and Ojiagu, 2014; Sudhakar and Manatha, 2004; Gangil et al., 2013). The presence of coliforms in potable water is used as indicator of water contamination (Opara and Nnodim, 2014).
Salmonella typhiis used as indicator of possible recent sewage/ faecal contamination (Anyamene and Ojiagu, 2014; Onuh and Isaac, 2009; Opara and Nnodim, 2004) because this is one of the first bacteria present in water when contamination occurs and will present in larger quantities than some other pathogenic microbes. Other microbial indicators of possible faecal, soil and natural water contaminations are faecal Enterococci especially Enterobacterfaecalis, Clostridium perfringens spores, Clostridium sporanges, Salmonella typhi, Escherichia coli, Shigelladysenteriae, Vibrio cholera, Pseudomonas aeruginosa, Klebsiella, Aeromonas, Mycobacterium, Alcaligens, Actinetobacter, Chromobacterium, Serratia, Flavobacterium, Proteus, Bacillus subtilis, B. mycoides, Enterobacter cloaca, Enterobacteraeronenes, Nostocidafexibacter and Norcardia (WHO, 1993; Oyedeji et al., 2010).
These could cause different disease conditions and clinical signs such as Giardiasis, Cryptosporidiosis, Gastroenteritis, Diarrhea, Typhoid Fever, Cholera, Bacillary Dysentery, Hepatitis, Shigellosis (Hughes and Koplan, 2005). Water borne diseases are reported to account for 80% of illnesses in developing world, killing a child every 8 seconds. This is a global public health threat (Hughes and Koplan, 2005).

Predisposing Factors to Sachet Water Contamination
Various factors predispose Sachet water to contamination. These include contaminated sources of water ranging from rain water, shallow well water, rusty unwashed tanker to other contaminated sources (Dibua et al., 2007). Some contaminants enter Sachet water through seepage of sewage and rainfall runoffs into well water and exposed boreholes (Defives et al., 1999; Adegoke et al., 2012). Introduction of contamination during the process of production due to contaminated materials or external introduction of contamination from vending machines etc. (Omalu et al., 2010).
Prolonged storage of sachet water at favorable environmental conditions aiding total aerobic heterotrophic bacteria to grow to level that may be harmful to humans (Warburton et al., 1992). The unhygienic conditions and filthy environments in which some of the water packaging companies operate especially in developing countries could introduce contamination to sachet water. These companies are mostly not registered and are engaged in sharp, questionable practices which are encouraged by the irregular and ineffective monitoring of authorities that enforce standard. They therefore, do not follow international quality standards nor appropriate methods of water treatment (Oyedeji et al., 2010).
Improper handling by hawkers could be another reason why sachet water is contaminated (Adegoke et al., 2012). Absence of sterilization procedures such as pasteurization and thermal sterilization for the treatment of sachet water also increase susceptibility to contamination (Anyamene and Ojiagu, 2014). Ignorance on the implication of carelessness in the production line by producers with sole aim of making profit thereby disregarding safety and quality in producing water for the public (Ackah-Arthur et al., 2012). Use of non-feed grade sachets for water production and the release of sachet water to the public without production and expiry date markings could also enhance sachet water contamination (Adewoye et al., 2013).

Public Health Implications of Microbial Pollution of Commercially Sold Sachet Water
Water has always been associated with disease. The first reported case of polluted drinking water was that of Broad street water pump, London after which many other cases have been reported in different parts of the world (Gangil et al., 2013). Conditions resulting from microbial contamination of drinking water is said to affect a large part of the world’s population and continue to be one of the major health problems globally. It accounts for 80% of illnesses in developing world (Boubetra et al., 2011).
Microbial pollution of sachet water particularly in developing countries has grave implications on public health. It threatens the population’s existence causing diseases such as cholera (Njoku and Osondu, 2007; Ackah-Arthur et al., 2012), gastroenteritis (Grabow, 1996), hepatitis, typhoid fever, shigellosis (Anyamene and Ojiagu, 2014). WHO estimates that 88% of diarrhea cases is caused by unsafe water (CAWST, 2009; WHO, 2004). Mortalities due to water associated diseases and symptoms now exceeds 5 million people per year (Cabral, 2010).

Studies carried out on sachet water around the world revealed high level contamination of microorganisms which indicated that most of the brands were unfit for human consumption. They were found to be of doubtful quality and did not meet the WHO drinking water standards (Rutz, 1996). Consumption of these sachet water brands could result in public health hazard. This, therefore, requires concerted effort on the part of all stake holders to correct the anomaly.
Regulatory bodies should intensify effort at monitoring of sachet water manufacturers, periodic sanitary inspection of factories should be carried out.

Emphasis should be placed on good management practices (GMP) by producers of sachet water especially in terms of location of the factories and maintaining high level of hygiene within the premises of production. Regulatory agencies should ensure the enforcement of water quality regulations. Manufacturers should be compelled to use standardized equipment and water treatment procedures. Test and analysis of sachet water products at different stages of production; preproduction, production and postproduction including periodic market sample testing should be carried out by regulatory authorities.
Results of laboratory tests should be made available to sachet water producers to encourage them to improve on their production process. Hazard analysis and critical control point (HACCP) used in the food industry to prevent contamination could also be used in the water production industry. Manufacturers should be mandated to inscribe production and expiry dates on sachet water products, expiry date should not exceed 4 weeks from the date of production.
This is because storage at room temperature over a long period of time has been reported to increase the total heterotrophic bacteria to levels that can be harmful to human health. Manufacturers should be made to educate retailers on the proper handling of products to ensure product quality. There is need to create public awareness on the potential danger of consuming non certified, contaminated sachet water and the adverse effect of storage of products for long periods of time.

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