Non Specific Response Of The Intestine Biology Essay

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Bacterial gastroenteritis is a very common health disorder. Gastroenteritis-causing pathogens are the second leading cause of morbidity and mortality worldwide. In developing countries, where sanitation is suboptimal, epidemics of bacterial gastroenteritis can develop and cause significant mortality. (Marks, 1994; Hamer and Gorbach, 1998). This high mortality rate may be due to inadequate or lack of resources for proper treatment of diarrhea associated with bacterial gastroenteritis in the developing countries.

Diarrhea is a non-specific response of the intestine to several different conditions, including infections, drugs and inflammatory bowel disease (Swati and Fasano, 2005). It could also be define as increase in volume or fluidity of stools, changes in consistency, and increased frequency of defecation (Nikhil and Sanderson, 2004).

The annual child mortality rate reported by the World Health Organization is 120 million, of which 5 million are associated with diarrhoeal disease. (WHO/CDD, 1993). Although mortality rates among these children have declined globally from 146 per 1,000 in 1970 to 79 per 1,000 in 2003 (WHO, 2005), the situation in Africa is strikingly different as compared with other regions of the world. In order to give a balanced picture of childhood morbidity and mortality pattern in Nigeria, Onyiuruka (2005), suggested that data from private health institutions are required. This, he said is because private health centers contribute significantly to the provision of health care to the populace.

Children are thought to be more prone to parasitic or bacterial infections probably because of their unawareness to pathogens and their effect in the environment or extremely low level of sanitation standard. Their pica behaviors including nose picking, sand eating or even ingestion of feces are part of the predisposing factors to gastroenteritis.

The incidence of diarrheal disease in children has been reduced because of improved public health measures, management, hygiene, increased used of oral dehydration therapy (ORT) and a better understanding of nutrition. However, diarrheal diseases continue to be an important cause of morbidity and mortality in children worldwide. Also it is a particular problem where young children come into close contact with other children, such as in child day care centers (Chen, 2010).

Some predisposing factors to diarrheal diseases include little or no access to safe water and sanitation, as well as poor hygiene and feces disposal practices at home (Daniels et al., 1990; Haggerty et al., 1994; LaFond 1995; MacDougall and McGahey 2003).

A WHO report on global water supply provides worrisome figures of current and future scenarios for Africa (WHO, 2000). According to the report, only African regions of all the regions in the world show a decline in the proportion of the population that had access to sanitation between 1990 and the year 2000. These and many other factors, such as poor housing and overcrowding, are basically associated with poverty. Though there has been progress towards better water and sanitation observed in other regions, it has not yielded any reduction in diarrhea morbidity, suggesting that poor hygiene practices (Yeager et al., 1999) and the ingestion of contaminated food (Lanata, 2003) may be the most important factors and where preventive interventions, like hand-washing (Curtis and Cairncross, 2003), should be promoted. In addition, poverty limits access to health care and restricts appropriate and balanced diets. Inequities in exposure and resistance add up to inequities in coverage of available preventive interventions, access to an appropriate health provider, and care, making poor children more likely to become sick than the better-off children (Victora et al., 2003).

HIV infection has added considerably to the burden of diarrheal diseases among adults and children. This is of particular importance in African countries that show high HIV prevalence. However, the scarcity of data makes it difficult to quantify its synergy in morbidity and its contribution to the mortality burden.

Aeromonas and E.coli are part of the major etiological agent implicated for causing gastroenteritis in children. According to Miguel et al., E.coli and Aeromonas species are leading cause of acute diarrhea in children. Isolation rates for Aeromonas has ranged from <1% to more than 60% in diarrheic populations in various geographic locations (Brenner et al., 2005). Although a study by Ashiru et al. (1993) has shown that a small percentage of gastroenteritis can be attributed to Aeromonas hydrophilia in Nigeria.

Aeromonas

Aeromonas species are Gram negative, non-spore forming; rod shaped with straight ends but sometimes can appear as cocco-bacilli or with filamentous forms. They belong to the order Aeromonadales which contains a single family aeromonadacea. They include motile (single, polar flagellum) and non-motile species as well as mesophilic and psychrophilic species. Some species are either primary or opportunistic pathogens in humans as well as a variety of other warm-blooded and cold-blooded animals and invertebrates.

They are facultative anaerobes that occur ubiquitously but predominant in aquatic environments. Cells are 0.3-1.0 x 1.0-3.5um and can occur singly, in pairs, or even as short chains (Altwegg, 1999).

The early interest of von Graevenitz in Aeromonas stimulated others to begin to study this aquatic-borne organism, which seemed to be more common in clinical samples than previously realized (von Graevenitz and Mensch, 1968). While initially believed to be an opportunistic organism capable of infecting only immune-compromised individuals, a body of evidence now indicates that Aeromonas is a primary cause of extra-intestinal illness and is strongly associated with gastrointestinal disease (Kelly et al., 1993; Janda and Abott, 1996, 1998; Joseph, 1996).

Aeromonas-associated diarrhea is a worldwide phenomenon seen in both industrialized and developing nations spanning all age groups. While principally observed in healthy persons, it can also be found in those suffering from underlying maladies, including immune disorders such as HIV infection (Figueras, 2005). Recently the occurrence of haemolytic-uremic syndrome following an Aeromonas gastroenteric disease has been described. This syndrome is very similar to the one brought on by E. coli O157:H7 and is caused by an Aeromonas cytotoxin which is genetically and antigenically different from the E. coli cytotoxin (Bottarelli and Ossiprandi, 1999).

Holmberg and Farmer (1984) described Aeromonas gastroenteritis as a mild, self-limiting infection. A number of factors, including age, immune-competence, infection dose, underlying illness, and expression of sufficient virulence factors by the infecting organism, affect the ability of Aeromonas spp to cause diseases (Nichols et al., 1996). Since Aeromonas species are ubiquitous in aquatic environment and readily isolated from both nutrient rich and nutrient-poor environments (Holmes et al., 1996) water is suggested as their main route of transmission (Ewing et al., 1961). The presence of Aeromonas species in foods most likely reflects contact of these foods with contaminated water, as reflected in the name of the species, A. hydrophila, which means 'water loving'. However, a conclusive link between the consumption of Aeromonas-containing food and diarrheal disease has not yet been identified.

Aeromonas share many biochemical characteristics with members of the Enterobacteriaceae, from which they are primarily differentiated by being oxidase positive. In the early 1980s, DNA relatedness studies of the motile aeromonads resulted in the establishment of three phenotypic species, namely Aeromonas hydrophila, A. sobria and A. caviae, from among the eight reported genomospecies or DNA-RNA hybridization groups (Popoff et al., 1981). The mesophilic species such as A.hydrophila, A. caviae, A. sobria, among others has been associated with a wide range of gastric infections in humans (Janda and Abbott, 1996).

There is a frank periodicity associated with the isolation of Aeromonas species from the human gastrointestinal tract. Since these bacteria are not normal inhabitants of the gut, most studies have found the recovery of Aeromonas from fecal specimens to increase coincidentally with the warmer months of the year (Khardori, 1988). This rise in numbers no doubt occurs because mesophilic Aeromonas grow optimally at elevated water temperatures, thus leading to increased concentrations of bacteria in freshwater environments as well as in domestic water supplies (Edberg, 2007).

Although they have been commonly isolated from healthy patients (Janda et al., 1995) and gastroenteric patients, their role in disease etiology remains unclear. There has been considerable debate as to whether the mesophilic Aeromonas are primary enteropathogens, prompted largely by failure to establish significant infection in volunteer studies (Morgan et al., 1985). However, there have been reports of laboratory acquired infections in microbiologists who unintentionally ingested significant doses of Aeromonas and developed self-limiting diarrhea (Joseph, 1996).

No animal model has ever been established that can faithfully reproduce the Aeromonas-associated diarrheal syndrome, although many attempts have been made (Kelleher, 2000). This is a stumbling block to confirm Aeromonas as an etiological agent of diarrhea (Evans, 1976). Koch's postulate (3) requires that the proposed pathogen be fully isolated from the body and grown in pure culture, and it must be shown that it can induce the disease anew. Falkow (2004) proposed a supplement to Koch's postulates, in a molecular format relying on the use of genetic mutations that is more in line with today's research methodologies. Even with the use of this newer set of standards, Koch's postulate (1) 'The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms' has not been fulfilled, since the phenotype is not associated exclusively with pathogenic members or strains of the genus and the same traits can be found in what are assumed to be nonpathogenic varieties (Falkow, 2004; von Graevenitz, 2007).

On MacConkey agar, the majority of the mesophilic species grow as non-lactose fermenters, but a fair number of A. caviae isolates can ferment lactose. Colonies of the psychrophilic, nonmotile A. salmonicida subspecies are pinpoint in size after 18-24hrs at 20-220c, but after 4 days of incubation they are circular, convex, entire, friable, and 1-2mm in diameter (Griffin et al., 1953). The aeromonads grow over a wide temperature range (0-450c), with the mesophilic strains growing between 10 and 420c (Hanninen, 1994). The optimum temperature range is 22-370c depending on the strain. While earlier studies have reported 280c as the optimum temperature for motile aeromonads (Popoff, 1984), at least one study on the effect of incubation temperature on growth and soluble protein profiles suggests that in some cases 370c may be the optimum growth temperature (Statner et al., 1988). Psychrophilic strains in the A. salmonicidia subspecies grow at temperature generally ranging from 2-300c

E. coli O157:H7

Theodor Escherich first described E. coli in 1885, as Bacterium coli commune, which he isolated from the feces of newborns. It was later renamed Escherichia coli, and for many years the bacterium was simply considered to be a commensal organism of the large intestine. It was not until 1935 that a strain of E. coli was shown to be the cause of an outbreak of diarrhea among infants. The over 700 serotypes are identified by antigenic drift of their surface "O" antigens (lipopolysaccharides or molecules on the bacterial surface of gram-negative bacteria). From the evolutionary point of view, the members of genus Shigella (dysenteriae, flexneri, boydii, sonnei) are actually E. coli strains "in disguise" (i.e. E.coli is paraphyletic to the genus). (Lan, 2002).

Most of the E. coli are normal flora of the small intestine and colon and do not cause disease in the intestines (non-pathogenic). Todar (2007) refers to them as the primary facultative anaerobe of the human gastrointestinal tract. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, (Bentley, 1982) and by preventing the establishment of pathogenic bacteria within the intestine. (Hudault, 2001; Reid, 2001). E. coli are not always confined to the intestine, and their ability to survive for brief periods outside the body makes them an ideal indicator organism to test environmental samples for fecal contamination. (Feng, 2002; Thompson, 2007).

Transmission of pathogenic E. coli often occurs via fecal-oral transmission (Gehlbach, 1973; USDHHS and FDA, 2006; Evans, 2007). According to the U.S. Food and Drug Administration, the fecal-oral cycle of transmission can be disrupted by cooking food properly, preventing cross-contamination, instituting barriers such as gloves for food workers, instituting health care policies so food industry employees seek treatment when they are ill, pasteurization of juice or dairy products and proper hand washing requirements (USDHHS and FDA, 2006).

E. coli O157:H7 was first recognized as a food-borne pathogen in 1982 during an investigation into an outbreak of hemorrhagic colitis (bloody diarrhea) associated with the consumption of contaminated hamburgers (Riley et al., 1983). CDC estimated that 85% of E. coli O157:H7 infections are food-borne in origin (Mead et al., 1999) in line with Riley's contribution as explained above.

In August of 2000, a daycare in California was identified as the source of an E. coli O157:H7 outbreak.  Health department officials who investigated the outbreak determined that the probable "index case"-a child who unknowingly brought the bacteria into the facility-experienced "explosive diarrhea at the daycare on the afternoon of 8-3-00." (Marler, 2010).

The first reported outbreak of E. coli O157 infection in the developing world, which occurred in southern Africa in 1992 (Effler et al., 2001) has been followed up with outbreaks in Central African Republic in 1996 and Cameroon in 1997 (Cunin et al., 1999). E. coli O157 illness has been reported in Nigeria since 1994 but as not constituted an outbreak (Ogunsanya et al., 1994; Olorunshola et al., 2000).

A better understanding of bacterial enteric pathogenesis has grown increasingly important because of the emergence of new pathogens and growing problems of resistance among enteric pathogens and other enteric flora.

Antibiotic susceptibility

Use of antibiotics has treatment for gastroenteritis has been on the high side in developing countries where prevention of enteric illness by virtue of improved hygiene and provision of sanitation and water treatment is unfeasible. The ripple effect is that high resistance to readily available antibiotics such as tetracycline, ampicillin, among others, set in. The prevalence of antibiotics resistance in bacterial isolates worldwide may be due to the selection and spread of resistant organisms in developing countries which can often be traced to complex socioeconomic and behavioral antecedents (Lamikanra and Okeke, 1997; Hart and Kariuki 1998).

Ingestion of antibiotics is known to provide selective pressure ultimately leading to a higher prevalence of resistant bacteria, even among persons who have not taken antibiotics (Levin, 1997; Levy, 1997). According to Rodriguez (2007) the use of acid-suppressing drugs may increase the risk of gastroenteritis as Gastric acid in the stomach serves as a defense mechanism against gastrointestinal infections caused by ingested bacteria.

Bacterial resistance could be acquired and is common in isolates from healthy persons and patients with community-acquired infections in developing countries, where the need for antibiotics is driven by the high incidence of infectious disease (Kunin, 1993). Residents of developing countries often carry antibiotic-resistant fecal commensal organisms (Lamikanra 1989; Calva et al., 1996; Woolfson 1997). Several factors, such as urban migration, crowding and improper sewage disposal encourages the exchange of antibiotic-resistant organisms between people and the exchange of resistance genes among bacteria, thereby increasing the prevalence of resistant strains. A broth conjugation experiments by Adams et al. (1998), showed the transfer of the oxytetracycline-resistant phenotype from A. salmonicida to E.coli and found this resistant phenotype to be encoded by high-molecular weight R-plasmids that could be characterized by restriction digest profiles.

Bacterial gastroenteritis is a problem in many developing countries, especially in their rural areas, in that well-trained health personnel are scarce and cannot serve the entire population facilitating community health workers and others with minimal training to treat minor ailments (Pearson, 1995). Also, unqualified drug sellers offer alternative drugs when the prescribed drugs are out of stock or refill prescriptions without consulting the prescriber (Dua, 1994; Kigotho, 1997) increasing the rate of antibiotic resistance in gastroenteric bacteria.

Information from routine susceptibility testing of bacterial isolates and surveillance of antibiotic resistance, which provides information on resistance trends, including emerging antibiotic resistance, is essential for clinical practice and for rational policies against antibiotic resistance.

The recommendations of WHO for ensuring proper drug use (Couper, 1997) can be adapted to combat the escalation of community-acquired antibiotic resistance in developing countries. The misuse of antibiotics by health-care professionals, unskilled practitioners, and patients can be alleviated by auditing antibiotics, limiting antibiotic choice, developing prescription guidelines, and emphasizing continuing medical and public education.

1.2 Aims and Objective of Study

Aim

This present work is directed at monitoring the prevalence of Aeromonas spp and E.coli amongst primary school children at Obafemi Owode L.G.A, Mowe, Ogun State, Nigeria (St. David Anglican Nursery and Basic School, Imedu Nla Nursery and Primary School).

Objective of Study

To confirm E.coli as the dominant etiological agent of gastroenteritis in children at Obafemi- Owode Local Government Area.

To determine the frequency of Aeromonas infection amongst children in the study population.

To determine the effective antibiotics that could be used in treating E.coli and Aeromonas spp infection.

To establish the relationship between age, sex and socioeconomic status on bacterial (E.coli and Aeromonas) infection amongst children.

CHAPTER TWO

MATERIALS AND METHOD

2.1 Study Population

The study population was drawn from randomly selected students in two primary schools at Obafemi Owode L.G.A, Mowe, Ogun State, Nigeria (St. David Anglican Nursery and Basic School, Imedu Nla Nursery and Primary School). A total of 104 (58 female and 46 male) stool samples were collected from both symptomatic and asymptomatic students. Informed consent was obtained from the students, head teacher and medical personnel involved in the management of the students.

2.2 Sampling technique

Pupils were chosen randomly in each class with almost equal number of males and females.

2.3 Determination of Sample Size

Using formula: - n = Pq

(E / Z)2

Where; P = prevalence of previous studies = 15% (Abdullahi et al., 2010)

q =100-P =100-15= 85 E = allowable error = 5% CI= 1.96

Z= standard normal distribution at 95% n= number of samples to be collected= 195.

Therefore: n = 15 x 85 = 1275 = 195 samples.

(5/1.96)2 (2.55)2

2.4 Sample Collection

Early morning stool samples were collected from students in sterile universal containers. The bottles have screw caps with attached spoon for easy sample collection and reduction of contamination by other microorganism in the environment. The stool donors were properly instructed on how to collect the sample and making sure the sample were not contaminated with urine or water. The samples were transported in a container containing ice bags and were processed less than 2 hours on arrival to the Laboratory. The basic information obtained from each student includes age and sex. An assumption on the socio-economic status of the students was made based on the type of school attended.

2.5 Preparation of Media

The media used in this study include MacConkey agar, Kliggler Ion agar, Simon Citrate agar, Motility agar, Nutrient agar, Mueller Hinton agar, Peptone water. The required gram of each medium was weighed using a weighing balance and weighing boat. The measured gram of each medium was dispensed into a sterile dry conical flask to which appropriate amount of distilled water was added. The mixture was heated and stirred using a magnetic stirrer and sterilized according to the manufacturer's instruction. 40g of Urease salt was suspended into 100ml of sterile distilled water and allowed to dissolve.

2.6 Sterilization of Materials

Glass wares such as conical flasks, test tubes, bijou bottles, pipette, among others, forceps, were sterilized in hot air oven at a temperature of 1600c for about two hours. Petri dishes and media used were autoclaved at 1210c for 15 minutes. Also, Inoculating loops were flamed until red hot using a spirit lamp before use.

2.7 Processing of specimens

The specimens were processed according to guidelines provided by Cheesbrough (2007) for the laboratory diagnosis of enteric pathogens. These include macroscopy, Gram's stain, motility testing, culture, biochemical testing and antimicrobial sensitivity testing. A loopful of specimen was inoculated into 10ml of peptone water, well mixed and incubated at 370c for 18-24 hours. The inoculated plates were then subcultured on MacConkey agar which was also incubated at 370c for 18-24 hours. The plates were observed for growth and distinctive cultural characteristics. Resultant colonies were identified using standard biochemical tests.

2.7.1 Gram Staining Test

The different colonies observed on the plates were Gram stained. The purpose of Gram's staining is to identify the pure culture as either gram positive or gram negative organism. This is indicated by color differences (purple or pink) due to the ability of the bacteria to retain the dyes. Gram positive bacteria would show a characteristic purple color and Gram negative bacteria a pink color.

A smear of the pure culture was made on a clean grease-free slide, air dried and heat fixed by passing it through flame for about 3-4 times. One to two drops of crystal violet was added to the smear, allowed to stand for 30 seconds and rinsed under slow running water. Two drops of Lugol's iodine was added and left for 60 seconds. This was then rinsed with 70% ethanol solution and immediately under slow running water. The slides were flooded with safranin, left for 30 seconds and rinsed off under slow running water. The slides were blotted dry and viewed under the microscope.

2.7.2 Biochemical Test

Identification of the isolates (Gram negative bacteria) were done using different biochemical test such as Oxidase test, Indole test, Urease test, Citrate utilization test, Motility test, Gas production and Sugar fermentation test. All tests were done using the methods described by Collee and Miles (1989); Porter and Duguid (1989).

2.7.3 Citrate Test

The Simon citrate test was done to identify bacteria that could utilize citrate as their sole source of carbon. The agar contains an indicator and its normal color is green (neutral pH- 6.9). Pure cultures picked from the plates were inoculated into already prepared citrate agar in slanting position inside bijou bottles. The bottles were incubated for 24hours and observed for color change. A color change to blue indicates acidic pH while a yellow coloration indicates an alkali pH (Bello, 2002).

2.7.4 Oxidase Test

An oxidase reagent was used for this biochemical procedure. A piece of filter paper was placed on a sterile petri dish; the filter paper was soaked with few drops of the prepared oxidase solution. Each pure culture were picked and placed on the soaked filter paper and observed for color change. A blue/purple coloration indicates a positive result and a negative result is characterized by no coloration.

2.7.5 Motility, Indole, Urease Test

Motility indole urease agar is a multitest agar used to test for indole production while simultaneously determining other characteristics of the bacterium such as urease metabolism and indole production. This test was done using a motility agar to which urease solution was added (MIU agar). An isolated colony was picked with an inoculating needle and stabbed approximately two-thirds of the way into the agar and then removed following the same path as the entry.  This was then incubated at 37°C for 24 to 48 hours or until growth is evident (Baron and Finegold, 1990).

Motility test

The MIU agar stabbed with the pure culture was observed for cloudiness in the medium (growth away from the stab line). For a non-motile organism, growth may be seen along cracks in the medium caused by gas production, but there will be clear pockets of no growth (Cheesbrough, 2002; Perilla, 2003). The use of motility agar require experience as organisms may be weakly motile, or the flagella may be damaged due to heating, shaking, or other trauma giving a false-negative motility tests reaction.

Indole test

The indole test screens for the ability of an organism to degrade the amino acid tryptophan and produce indole (Maria, 2010). Certain microorganisms can metabolize tryptophan by tryptophanase. The enzymatic degradation leads to the formation of pyruvic acid, indole and ammonia. The presence of indole is detected by addition of Kovac's reagent into the incubated, inoculated motility agar. The inoculated MIU agar was incubated at 37°C for 24 hours. After incubation interval, 1 ml Kovacs reagent was added and the tube was shaken gently and read immediately. A bright pink color in the top layer indicates the presence of indole while the absence of color means that indole was not produced i.e. indole negative

Urease

The motility agar contains urea and phenol red. Any color change of the inoculated medium such as from yellow to pink indicates a positive test.

2.7.6 Sugar fermentation and Acid Production Test

This was done using Kliggler Ion Agar. From the degree of acid produced during fermentation, differentiation can be made between non-fermenters, glucose-fermenters (which produce a relatively small amount of acid) and those which ferment both glucose and lactose (producing a relatively large amount of acid which diffuses throughout the medium and easily over neutralizes the aerobic deamination reaction in the slant). The medium was inoculated with the needle, first stabbing down the center to the bottom of the tube and then streaking up the slant. Incubation was done for 24 hours at 37°C. Gas production was interpreted as presence of cracks or bubbles in the medium. Hydrogen sulfide producing organisms may produce a black precipitate to such a degree that the reaction in the butt is completely masked (MacFaddin, 1985).

2.7.7 Antimicrobial susceptibility testing

Sensitivity of isolates to antimicrobial agents was determined on Mueller-Hinton agar plates using the disc diffusion method of Scott (1989). Interpretation of results was done using the zone sizes. Any zone diameter above or equal to 12mm shows susceptibility to the antibiotics while that below 12mm shows resistance to the antibiotics. All isolates were tested for sensitivity to the following antibiotics: Ampicillin (10µg), Tetracycline (10µg), Gentamicin (10µg), Cotrimoxazole (25µg), Streptomycin (10µg), Nalidixis Acid (30µg), Nitrofurantoin (200µg), Colistin (25µg) all of Abtek Biologicals Ltd, UK.

CHAPTER THREE

RESULTS

3.1 Age and Sex Distribution of Pupils

A total of one hundred and four (104) fecal samples were analyzed. The pupils age in the study population range between four to eighteen years. A total of forty-five and fifty-nine samples were obtained from st. David Nursery and Basic school (School A) and Imedu Nla Nursery and Primary School (School B) respectively. In school A, there were twenty-four males and twenty-one females while in school B, there were twenty-seven males and thirty-two females.

The highest numbers of participant in the study population were between age 6-10yrs (Males-32, Females-30) (Figure I). Age predominance was observed in both the number of stool samples collected (Table 1) and the total number of isolates identified (Table 2).

Table 3 shows that in general, there were fifty-one males and fifty-three females. No sex predominance was observed (Male to female ratio is 1:1).

3.2 Major Findings within Sex and Age Distribution

Table 2 shows that Group 6-10 has the highest number of isolates (50%) and group ≤5 has the lowest number of isolates (10%). No isolate was obtained in ages >15 and this may be as a result of the limited amount of sample obtained from the group. In school A both age 6-10 and 11-15 has equal number of isolates. In school B, group 6-10 has highest number of isolates. There were no isolated enteropathogens in ages ≤5 and >15 (the two extremes).

In Table 3, Males in age range 6-10 and 11-15 show highest infection with E.coli O157:H7. Age group ≤5 and >15 showed no E.coli infection. Furthermore, Females in age 11-15 shows highest number of E.coli infection and there was no infection in ages between 6-10 and > 15.

Table 4 shows the age distribution of pupils involved in the study. In respect to the number of samples analyzed, age group ≤5 has the highest number of E.coli infection (16.67%) while age range 6-10 has the lowest number of isolates (3.23%).

3.3 Enteric Pathogens Isolated from the study

Aeromonas spp. was not isolated from any of the 104 fecal samples analyzed. However, Seven E.coli (6.73%) of the total number of samples analyzed) and three other gastroenteric bacteria which include Salmonella species (0.96%), Proteus vulgaris (0.96%), Shigella species (0.96%) were isolated (Table 5). The biochemical tests performed for characterization of the gastroenteric bacteria isolated from the samples is shown in Table 6.

The macroscopic appearance of the stool samples are shown in Table 7. There was no considerable difference in the total number of isolates from diarrheic (watery or mucoid stool) and non-diarrheic (formed or semi-formed) sample. The watery and mucoid stool yielded the lowest number of isolates out of which none was from school B and only one from school A. Both schools show the same proportion of isolates. The proportion of the isolates in school A is E.coli (6.78%), Shigella (2.2%) while in school B, E.coli (6.67%), Salmonella (1.6%), Proteus vulgaris (1.6%) respectively (Table 5).

Equal numbers of isolates were obtained from both schools in the sampled population (Table 2). The frequency of E.coli was obtained at different proportions from the different sampled populations (Table 5) - though their ratio is 1:1.

Figure II shows that E.coli is more predominant in ages 11-15 and absent in ages above 15 years. Also, E.coli is predominantly found in both males and females about age 11-15 and at equal proportions (Table 3). As implicated in Figure III, frequency of E.coli increases as age increases showing a correlation between age and frequency of E.coli isolated.

3.4 Antimicrobial Susceptibility Patterns

Table 8 shows antimicrobial susceptibility test result of the four different bacterial species tested: E.coli, Salmonella spp, Shigella spp and Proteus vulgaris against 8 antibiotics. Isolates for Aeromonas spp could not be recovered and were thus not available for antimicrobial susceptibility. Tetracycline and Amoxicillin were the least effective antibiotics, with an overall susceptibility less than or equal to 30%. Gentamicin (overall susceptibility of 100%) was the most effective antibiotic against all the organisms tested. Generally, Ofloxacin and Nalidixis acid were also effective on all the bacterial pathogen both with overall susceptibility of 90%. Nitrofurantoin and Augmentin are also effective against most of the bacterial pathogen except Salmonella spp. Cotrimazole is only very effective against Salmonella spp and Proteus vulgaris. Resistance to more than two antibiotics was common and was observed in four (57.1%) E.coli isolates, one (100%) Shigella isolate and one (100%) Salmonella isolates.

3.5 TABLES AND FIGURES SHOWING RESULTS

Figure I. Bar Chart Showing gender variance with age distribution in the study population

Table 1. Age and Proportion of Stool Samples Collected from the Sampled Population

Age group

Number of stool samples collected

Percent

≤ 5

6

5.77

6-10

62

59.62

11-15

35

33.65

> 15

1

0.96

Total Number of samples

104

100

Table 2. Age Distribution and Frequency of Gastroenteric Bacteria isolated at Imedu Nla and st. David Nursery and Primary School

School A

School B

No. of Isolates

No. of Isolates

Total No. of Isolates

Percentage Total

Age group

≤ 5

1

-

1

10

6-10

2

3

5

50

11-15

2

2

4

40

> 15

-

-

-

-

Total No. of Isolates

5

5

10

aSchool A is st. David Nursery and Basic school, bSchool B is Imedu Nla Nursery and Primary School.

Table 3. Frequency of Escherichia coli Isolated from Male and Female Students in the Sampled Population

Age Group

No. of Males

No. of Females

E.coli Positives

Male

Female

≤5

2

4

-

1

6-10

32

30

2

-

11-15

17

18

2

2

>15

-

1

-

-

Total

51

53

4

3

Table 4. Frequency of Isolated Escherichia coli and Sample Size According to the Pupils' Age

Age Group

No. of cases evaluated

No. of E.coli positive cases

Percentage per cases evaluated

≤5

6

1

16.67

6-10

62

2

3.23

11-15

35

4

11.43

>15

1

-

-

Total

104

7

6.73

Table 5. Enteropathogens identified in the 104 specimens studied

Names of Pathogen

School A

n=45

School B

n=59

Total number infected

Percentage

N=104

Frequency

Percent

Frequency

Percent

E.coli

4

6.78

3

6.67

7

6.73

Salmonella

-

-

1

-

1

0.96

Shigella

1

2.2

-

1.6

1

0.96

Proteus vulgaris

-

-

1

1.6

1

0.96

Aeromonas

-

-

-

-

-

-

Total

10

9.62

aSchool A is st. David Nursery and Basic school, bSchool B is Imedu Nla Nursery and Primary School.

Table 6. Biochemical Test Performed On The Isolated Gastroentric Bacteria

Organism

Indole

Motility

Urease

Gas

Lactose

Glucose

Hydrogen Sulfide

Citrate

Oxidase

E.coli

+

+

-

+

A

A

-

-

-

Others

Salmonella

-

-

-

+

K

A

-

+

+

Shigella

-

-

-

-

K

+

-

-

Proteus vulgaris

+

-

+

-

A

K

-

-

-

aA means acid production (Lactose or Glucose positive) bK means alkaline production (Lactose or Glucose negative)

Table 7. Appearance of Faecal Samples and Frequency of Isolation of Gastroenteric Bacteria in Imedu Nla and st. David Nursery and Primary School Pupils' Stool

School A

School B

No. of Isolates

No. of Isolates

Total No. of Isolates

Appearance of Stool

Watery Stool

1

2

3

Watery and Mucoid Stool

1

-

1

Formed Stool

2

1

3

Semi-formed Stool

1

2

3

Total No. of Samples

5

5

10

aSchool A is st. David Nursery and Basic school, bSchool B is Imedu Nla Nursery and Primary School.

Figure II. Frequency of Escherichia coli Strains Isolated from Faecal Specimens According To The Student Age. There were no E.coli strains isolated in ages above fifteen

Figure III. Bar Chart Showing The Relationship Between The Frequency of Escherichia coli and Age Distribution In The Sampled Population

Table 8. Percentage Antimicrobial Susceptibility Patterns of Enteric Bacteria from Sampled Population

Organism

OFL

AUG

COT

NIT

NAL

GEN

AMX

TET

Escherichia coli (n=7)

6 (85.7)

5 (71.4)

3 (42.9)

4 (57.1)

6 (85.7)

7 (100)

2 (28.6)

1 (14.3)

Salmonella (n=1)

1 (100)

0

0

0

1 (100)

1 (100)

0

0

Shigella (n=1)

1 (100)

1 (100)

1 (100)

1 (100)

1 (100)

1 (100)

0

0

Proteus vulgaris (n=1)

1 (100)

1 (100)

0

1 (100)

1 (100)

1 (100)

1 (100)

0

Overall Susceptibilty (n=10)

9 (90)

7 (70)

4 (40)

6 (60)

9 (90)

10

(100)

3 (30)

1 (10)

Where TET= Tetracycline (10µg), GEN= Gentamicin (10µg), COT= Cotrimoxazole (25µg), NAL= Nalidixis Acid (30µg), NIT= Nitrofurantoin (200µg), OFL= Ofloxacin (5µG) AUG= Augmentin (30 µg), AMX=Ampixicillin (10µg).

CHAPTER FOUR

DISCUSSION

The aim of the study is to determine the prevalence of Aeromonas spp. and E.coli infection amongst primary school children in relation to gastroenteritis and to determine the antimicrobial susceptibility profiles of the bacterial isolates in two population groups in Obafemi-Owode local government area, Ogun state, Nigeria.

4.1 Aeromonas

The use of enrichment broths in clinical laboratories has been exclusively used for the recovery of Aeromonas spp. from stool (fecal) samples though in low concentration. This view wasn't widely accepted by researchers who suggested that this low concentration might be expected in convalescent patients, carriers, and those with subclinical infections. It was later suggested that enrichment should not be routinely used since it would interfere with the interpretation of epidemiological studies when trying to interpret the relationship between the Aeromonas spp. and acute diarrhea (Robinson et al., 1984, 1986).

Aeromonas was not isolated from this study despite the use of standard isolation procedures. This is in line with Ashiru et al. (1993) who found out that Aeromonas is rarely associated with human infections in Nigeria but in contrast to studies by Kandakai-Olukemi et al. (2007) in Nasarrawa and Obi et al. (1997) in Edo state. This may result because they have only been incriminated as being transmitted by water and no conclusive link in their transmission by food or the time the research was conducted (December through February). Ashish et al. (2008) complemented our findings that Aeromonas infection occurs at higher frequency during warmer months (May through October).

It is well known that Nigerians with low socio-economic status though fetch water from different sources but allow the water to settle, a technique for ensuring water purity and safety before drinking. Thus, the inability to detect any Aeromonas infection in the pupils could also be attributed to this fact.

Khardori and Fainstein (1988) reported that Aeromonas hydrophila could be responsible for infections localized on the skin and soft tissues. Skin infection reveals the poor hygienic behavior of the pupils which is one of the predisposing factors to Aeromonas infection.

4.2 E. coli O157:H7

The study by Su and Brandt (1995); Smith et al. (2003); Prescott et al. (2008) confirms E.coli as the major etiology of gastroenteritis as observed in this study table 4. Also table 7 shows that males are more susceptible than females which agree with Garvey et al. (2003) but negate the work of Akinjogunla et al. (2008), and Lothar et al. (1998). Furthermore, ages between eleven and fifteen showed higher infection rate than other age groups. This findings contrast that of Garvey et al. (2003) who found highest prevalence in young children (<4yrs) but agrees with Nzeako's work on Aeromonas in Nsukka, Nigeria who proposed that children between aged five to fifteen are in their primary or secondary school age where they move freely on their way to- and- from school and may likely drink or eat contaminated water or food respectively (Nzeako et al., 2002).

This study has, thus, revealed that E.coli were significantly associated with diarrhea in the region. According to the U.S Food and Drug Administration, the fecal-oral cycle of it transmission could be destroyed by cooking food properly. The high prevalence of E.coli observed in this study could be as a result of the pica behavior of children, general low hygiene level found among children of this age (11-15) or cross-contamination of already prepared food.

4.3 Antibiotic susceptibility

The application of appropriate antimicrobial therapy which reduces the risk of resistance should be employed for treatment of E.coli infection. The percentage susceptibility patterns are as presented in Table 8. The susceptibility pattern of E.coli isolated from this study (Table 8), shows that among the popular drugs, gentamicin is about the only drug that the organisms are still largely sensitive to. Unlike the report by Okeke et al. (2000) and Akinjogunla et al. (2009) that recorded E.coli resistance to gentamycin. Ofloxacin, augmentin and nalidixis could serve as alternatives to gentamicin in the area.

Antibiotic resistance is now considered as a major health threat (Anon, 1997) and has reached alarming proportions worldwide in recent years. Yah and Eghafon (2007); Okeke et al. (1999) confirmed that the major selecting force in bacterial antibiotic resistance is the abuse/misuse of antibiotics. The ease at which one can obtain tetracycline and ampicillin may play a role in the high resistance exhibited by E.coli to the drugs.

The increasing resistance of E.coli to Tetracycline and Ampicillin in Obafemi-Owode L.G.A. calls for the authorities to conduct a detailed research works on antibiotic susceptibility pattern of gastroenteric bacteria in the region in order to provide effective treatment for gastroenteritis in the region. Great emphasis must be placed on preventive hygiene practices as opposed to an increasing reliance on antibiotic therapy in most countries and complacency about home hygiene should no longer be acceptable.

In this study three other bacterial genera were also isolated, namely Salmonella, Shigella species and Proteus vulgaris. All together, these pathogens can be transmitted through contaminated water or food, or poor hygiene. In addition, social and environmental factors such as inappropriate disposal of fecal samples and over-crowding in classes, as observed in one of the schools sampled in this research, are major risk factor for the presence of gastroenteric bacteria isolated from the student's stool samples. This agrees with the risk factors described by Daniels et al. 1990; Haggerty et al. 1994; LaFond 1995; MacDougall and McGahey 2003. The risk factors listed above reflect the living conditions, lifestyle, and environmental conditions of the local population. Albright et al. (2005) suggested that systematic and sustained teachings on how to avoid certain types of behavior that favors infection and good personal hygiene are the best approaches to significant and enduring reduction of the scourge of intestinal parasitism in children.

4.4 Limitations to the Study

Cost was a very strong limiting factor. Also it was hard getting consent from school management and the inability of some of the students to remember bringing their samples to school the next day.

4.5 Recommendation

More research should be carried out on the relationship between Aeromonas and skin infection because most of the pupils had skin infection but were not chosen preferentially since random sampling was intended and that was not the aim of the study. Further studies should be done on other etiologies of gastroenteritis in the area to aid concise and effective treatment of diarrheic pupils. This was born out of an observation in one of the schools where the school nurse administered flagyl to a girl suffering from diarrhea without any laboratory diagnosis. Simple strategies, such as better water treatment and hygiene education could decrease the level of gastroenteritis in the region.

4.6 Conclusion

In conclusion, this study shows that E.coli not Aeromonas is the predominant causative agent of gastroenteritis in Obafemi-Owode L.G.A, Ogun State. Also, E.coli infection is more predominant among males than females and in ages between six and eleven.

The high prevalence observed amongst the male pupils might be because females in the age range 6-10 and 11-15 are more hygienic conscious than their male counterpart. This study has shown that a small percentage of gastroenteritis can be attributed to Aeromonas spp. Absence of Aeromonas infection could suggest other bacterial pathogens as identified in this study or parasites as the causative agent of gastroenteritis in primary school pupils. Improved access to safe water, basic sanitation and hygiene, and cubing pica behaviors could protect children from gastroenteritis. The development of new antibiotics may offer a short term solution to the problem of resistance among gastroenteric bacteria especially E.coli but more effective measures, such as health education and further research on the prevention of infections through quality sanitation, should be encouraged.

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