i

NWONAH, ROSEMARY.C PG/M.Sc./05/39799

RISK ASSESSMENT OF CASSAVA WASTEWATER FOR SURVIVAL, GROWTH AND DISSEMINATION OF Salmonella AND Shigella ORGANISMS IN

THE ENVIRONMENT.

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A THESIS SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY, FACULTY OF

BIOLOGICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA

Webmaster

Digitally Signed by Webmaster’s Name DN : CN = Webmaster’s name O= University of Nigeria, Nsukka OU = Innovation Centre

MARCH, 2011

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TITLE PAGE

RISK ASSESSMENT OF CASSAVA WASTEWATER FOR SURVIVAL, GROWTH AND DISSEMINATION OF Salmonella AND Shigella ORGANISMS IN THE ENVIRONMENT.

BY

NWONAH, ROSEMARY.C PG/M.Sc./05/39799

TO THE SCHOOL OF POST GRADUATE STUDIES UNIVERSITY OF NIGERIA, NSUKKA

TO THE SCHOOL OF POST GRADUATE STUDIES UNIVERSITY OF NIGERIA, NSUKKA

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER’S DEGREE (M.Sc.)

IN PUBLIC HEALTH MICROBIOLOGY.

SUPERVISOR: PROF. C.U. IROEGBU

DATE: MARCH, 2011.

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CERTIFICATION

Miss Nwonah, Rosemary Chikamnayo, a postgraduate student in the

Department of Microbiology, majoring in Public Health Microbiology,

has satisfactorily completed the requirements for course work and

research for the degree of Master in Science (M.Sc.) in Microbiology.

The work embodied in her dissertation is original and has not been

submitted in part or full for either diploma or degree of this University or

any other University.

Dr. (Mrs.) I.M. Ezeonu Prof. C.U. Iroegbu

Head Supervisor

Department of Microbiology Department of Microbiology

University of Nigeria, Nsukka University of Nigeria, Nsukka

iv

DEDICATION

To My Best Pal and Beautifier,

The Holy Spirit…..

and to all the good and loving People

whose inspiration, love, prayers and support

blessed and sustained me in the years of this program.

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ACKNOWLEDGEMENT

I thank God Almighty for His grace upon me and for making me His Best, and a

partaker of His Divinity.

My special gratitude goes to my supervisor, Prof. C.U. Iroegbu for patiently guiding

through this work

I am highly indebted to my parents and siblings for their moral and financial support

and all the love constantly showered on me. I love you all.

All my lecturers in microbiology, I am grateful. Other lecturers who also taught me,

Prof. I.U. Obi of Crop Science and Prof. J.C. Agunwamba of Civil Engineering, I

appreciate all your efforts.

My deepest and sincere gratitude goes to the staff of National Root and Crop

Research Institute, Umudike, Abia State especially Mr. Solomon Azoke of the

Cassava unit who helped with the two known varieties of cassava.

I also appreciate the efforts of Mr. Sly and Dr. Egbuji of the Federal College of

Veterinary and Medical laboratory Technology, NVRI, Vom, Plateau State. They

gave me the strains of Salmonella typhi and Shigella dysenteriae used for this study.

The technical staff of the departments of Microbiology, Crop Science and Animal

Science, UNN, I say thank you for your assistance.

Ifeanyi Okoro, a pal in deed, who took and made all my orders for purchases in

Lagos, thank you.

I am also grateful to my brethren, friends and colleagues.

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TABLE OF CONTENTS Title page - - - - - - - - - i Certification - - - - - - - - - ii Dedication - - - - - - - - - iii Acknowledgement - - - - - - - - iv Table of Contents - - - - - - - - v List of Tables - - - - - - - - vii List of Figures - - - - - - - - viii Abstract - - - - - - - - - ix 1.0 INTRODUCTION - - - - - - - 1 1.1 Objectives of the Study - - - - - - 3 2.0 LITERATURE REVIEW - - - - - - 4 2.1 Bacteriology of Salmonella and Shigella - - - - 4 2.2 Physiology - - - - - - - - 4 2.3 Pathogenic Enterobacteria - - - - - - 5 2.4 Pathogenesis and Pathology of Salmonella and Shigella infections - - - - - - - - - - 8 2.5 Epidemiology and Mode of Transmission - - - 11 2.6 Survival and Sources in the Environment - - - - 14 2.7 Modes of acquisition of new genetic factors by these Pathogen - - - - - - - - - 17 2.8 Cassava - - - - - - - - 18 2.8.1 Composition of Cassava - - - - - - 18 2.8.2 Cassava Wastes - - - - - - - 18 2.8.3 Cassava Wastewater and Survival of Microorganisms - - 21 3.0 MATERIALS AND METHODS - - - - - 24 3.1 Media and Media Preparation - - - - - 24 3.2 Sample Collection - - - - - - - 24 3.3 Isolation of Organisms - - - - - - 24 3.4 Isolation of Microorganisms from the Cassava Processing Soil 25 3.5 Determination of Microbial Succession during the fermentation for Garri production - - - - - - - - 25 3.6 Isolation of Microorganisms from Cassava Processing soil using acidic medium - - - - - - - - - 26 3.7 Identification Tests - - - - - - - 26 3.7.1 Morphological characterization - - - - - 26 3.7.2 Motility Test - - - - - - - - 27

vii

3.7.3 Indole Test - - - - - - - - 27 3.7.4 Catalase test - - - - - - - - 27 3.7.5 Citrate Utilization Test - - - - - - 28 3.7.6 Urease Test - - - - - - - - 28 3.7.7 Potassium Cyanide Test - - - - - - 28 3.7.8 Triple Sugar Iron (TSI) Test - - - - - 29 3.7.9 Sugar Fermentation Test - - - - - - 29 3.8 Characterization of Fungal Isolates - - - - 29 3.9 Survival and Multiplication of Salmonella and Shigella organisms in Cassava Waste water - - - - - - - 30 3.10 Proximate Composition of Samples - - - - 31 4.0 RESULTS - - - - - - - - 32 4.1 Isolation of Salmonella and Shigella organisms from the different Processing sites - - - - - - - - 32 4.2 Proximate Analyses, pH and Microbial population of the Cassava Waste Water - - - - - - - - - - 32 4.3 Soil microorganisms with capacity to utilize cyanide isolated from the cassava processing sites - - - - - - - 32 4.4 Determination of Microbial Succession during the fermentation for Garri production - - - - - - - - 40 4.5 Proximate Analysis of Cassava Variety Used for Experimental determination of Salmonella and Shigella in fresh Waste water Sample - - - - - - - - - - - 40 4.6 Survival and Amplification of Salmonella and Shigella organisms in the Cassava Waste Water - - - - - - - 40 5.0 DISCUSSION - - - - - - - 55 Conclusion - - - - - - - - - 61 References - - - - - - - - - 62 Appendices - - - - - - - - - 77

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LIST OF TABLES

Table 1. Biochemical Reactions of the Different Bacterial Contaminants 34 Table 2. The pH Readings and Proximate composition of the processing sites’ waste water - - - - - - - - - 37 Table 3. Frequency of Isolation of Microorganisms from Ibagwa 1 Cassava Processing Sites - - - - - - - - 38 Table 4. Frequency of Isolation of Microorganisms from Ibagwa 2 Cassava Processing Sites - - - - - - - - 39 Table 5. Microbial Population in the Soil from Ibagwa 1 Cassava Processing Site - - - - - - - - - - 42 Table 6. Microbial Population in the Soil from Ibagwa 2 Cassava Processing Site - - - - - - - - - - 43 Table 7. pH changes of the medium at various time interval - - 44 Table 8. Microbial Population in the Soil from Ibagwa 1 Cassava Processing Site after acidic pH treatment - - - - - - 45 Table 9. Microbial Population in the Soil from Ibagwa 2 Cassava Processing Site after acidic pH treatment - - - - - - 46 Table 10. pH changes and the microorganisms associated with the fermentation of the waste water - - - - - - 48 Table 11. Proximate composition and Analysis of the cyanide content of the resultant waste water - - - - - - - 49

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LIST OF FIGURES

Figure 1. The Reaction of linamarin with linamarinase in Cassava - - - - - - - - 20 Figure 2. Distribution of Bacterial Isolates according to Cassava Processing Sites - - - - - - - 35 Figure 3. Frequency of Isolation of Bacterial Strains from Cassava Processing Sites - - - - - 36 Figure 4. Changes in pH of the fermenting cassava (local variety) with time - - - - - - 47 Figure 5. Changes in the pH of the two fermenting cassava (known varieties) with time - - - - - 50 Figure 6. Growth curve of Salmonella typhi in high cyanide Cassava waste water (NR8082) - - - - - 51 Figure 7. Growth curve of Shigella dysenteriae in high cyanide Cassava waste water (NR8082) - - - - - 52 Figure 8. Growth curve of Salmonella typhi in low cyanide Cassava waste water (TMS92/0326) - - - - 53 Figure 9. Growth curve of Shigella dysenteriae in low cyanide Cassava waste water (TMS92/0326) - - - - 54

x

ABSTRACT

Waste water samples from six cassava processing plants around Nsukka were cultured

in MacConkey and Xylose lysine Deoxycholate Agar, after enrichment in Selenite F

broth for isolation of Salmonella and Shigella species. Isolates were characterized

using standard bacteriological techniques. Subsequently, soil samples were made

acidic and/or supplemented with cyanide-containing waste water, to simulate

conditions imposed by cassava waste water pollution, and then tested for support of

growth of isolates. Salmonella (n=5) and Shigella (n=2) pathogens as well as other

Gram negative bacterial pathogens (n=41) were identified among the bacterial isolates

obtained. Further investigation from the cassava waste water and soil confirmed the

presence of other microorganisms such as Bacillus spp., Pseudomonas spp.,

Corynebacterium spp., Aspergillus spp. and a lot of yeast cells. A microbial

succession trend of the fermentation of a local variety of cassava was found with yeast

strains, Candida pelliculosa, Candida tropicalis, Candida krusei, beginning and

giving way to Bacillus spp. and then Corynebacterium spp. The pH of the fermenting

cassava waste water decreased from 6.84 to 3.70. The isolates obtained from a

medium of pH 3.00 included Arthrobacter spp., Bacillus spp., E.coli, Candida

tropicalis and Candida pelliculosa. The survival and amplification of Salmonella

typhi and Shigella dysenteriae strains in the two known variety of cassava (NR8082

and TMS 92/0326) were monitored by spectrophotometric method. The results show

that cassava waste water, which is allowed to flood the environment, is a potential

medium for growth and dissemination of Salmonella typhi and Shigella, and thus a

potential risk for spread of typhoid and shigellosis in the community.

1

1.0 INTRODUCTION

A wide variety of enteric pathogenic bacteria have been occurring in water

supplies and the environment. This is because the water sources and the land continue

to receive agricultural, industrial, and municipal wastes. Most of these pathogenic

organisms found in waste water may be excreted by infected humans and animals,

including carriers of the particular pathogens (Tchobanoglous et al., 2003). Some of

these organisms which are transmissible by the fecal-oral route can be transmitted

through water and foods contaminated particularly through unhygienic practices

(Curtis et al., 2000).

Pathogenic bacteria that have been transmitted by waste water include

Salmonella, Shigella, Campylobacter, enteropathogenic Escherichia coli, Vibrio

cholerae, Leptospira, Yersinia, Clostridium and Mycobacterium species (A.P.H.A.,

1992). However, the extent to which it survives in the environment and the

occurrence at an infectious dose are the critical characteristics of an outbreak of

disease.

In most families and commercial cassava processing plants, the waste water is

allowed to run as surface water and get contaminated by organisms already existing in

the soil environment including pathogens from feacal materials littered around. Such

waste water has been known to constitute a nuisance with reference to the stench that

comes from the putrefying effluent. As the cassava waste, which is mostly

carbohydrate, is degraded in the environment, there is likelihood that it would provide

a medium for amplification of the population of the pathogens mentioned above.

Ribas and Barana (2003) have already noted that minerals such as nitrogen,

carbon, phosphorus, potassium, calcium, magnesium, sulfur, zinc, manganese, copper,

iron, and sodium, which can be utilized as substrate for microbial growth, are

contained in the waste water.

Although, there are studies on the microorganisms involved in fermentation of

cassava products (Obadina et al., 2006); the microbiological safety of these cassava

products is yet to be evaluated. All that most bacteria need to multiply in the

environment would be appropriate nutrients and warmth. Cassava and its products

like other food materials have potentials for supporting the growth of both pathogenic

and spoilage microorganisms (Obadina et al., 2006).

2

Cassava waste water may be high in cyanide and a high positive correlation

has been found between coliform or E.coli and free cyanide (Agunwamba, 2004).

This indicates that the presence of cyanide may enhance bacterial growth and that

cyanide may not be toxic to this group of bacteria. Cyanide is very toxic to a number

of cellular processes, and occurs as a common metabolite in various plants, fungi, and

microorganisms (Meyers et al., 1993). The interactions between microorganisms and

cyanide, however, remain of interest, since the mechanisms of tolerance and

assimilation are poorly understood.

The survival of Salmonella and Shigella in cyanide containing cassava waste

water could also be enhanced by other factors. These include the viable but non-

culturable (VBNC) state and the ability of Salmonella to use a secretion system to

protect itself inside amoeba (University of Liverpool, 2009). Its ability to survive in

amoeba is a huge advantage to its continued development as it may be more resistant

to disinfectants and water treatment. The existence of the viable but non-culturable

state for both pathogens is also significant since this state cannot be detected in the

natural environment by routine bacteriological methods (Rowan, 2004). The presence

of virulence genes in these pathogens might be involved in its survival and infection

mechanisms.

A virulence factor in Salmonella, known as the siderophore has also been

reported in other cyanotrophic organisms and there is a correlation between

siderophore production and bacterial cyanide degradation (Huertas et al., 2006). The

production of siderophore is also required to chelate iron since cyanide binds it

strongly. An iron forms very stable complex with cyanide and it is not available for

the organism in the presence of cyanide. The 669Da catecholate siderophore

enterobactin has been isolated from E.coli and Salmonella typhimurium (Huertas et

al., 2006).

The cassava waste water is being investigated as an environmental source of

infection a medium where Salmonella and Shigella contaminating the environment

could replicate to infective dose, thus placing the human and livestock population at

risk of infection and disease.

STATEMENT OF PROBLEM

Salmonella and Shigella organisms are enteric pathogens that cause

typhoid/paratyphoid and bacillary dysentery, respectively. These organisms are highly

infectious, and are responsible for many thousands of morbidity and mortality each

3

year particularly in the developing regions of the world with poor environmental

sanitation (Tchobanoglous et al., 2003). Salmonella and Shigella species have been

implicated in many diarrheal diseases and studies have shown their predominance as

major causes of diarrheal cases in various countries of the world (Okeke et al., 2003;

Brooks et al., 2003 and Cho et al., 2006). Both pathogens have been the cause of

morbidity and mortality in children and the elderly especially in developing countries

(Vargas et al., 2004). Salmonella and Shigella species have been isolated in other

environmental specimens, e.g. drinking water, flies, seafood, cooking utensils (Bai et

al., 2004). Outbreaks of salmonellosis and shigellosis which are multidrug resistant

have also complicated treatment and increased the threat these pathogens pose to life

(Brooks et al., 2003; Iwalokun et al., 2001; Wang et al., 2005 and CDC, 2006). A

significant amount of wastewater is released during garri processing and this

wastewater contains a huge amount of organic matter which pathogens such as

Salmonella and Shigella species can utilize as substrate, and multiply rapidly to large

numbers in the environment. The possibility of preventing Salmonella and Shigella

organisms’ gaining access into garri processing wastewater, probably as a result of

sewage sludge mixing with the wastewater, is an environmental risk reduction

strategy to be assessed in this research.

1.1 OBJECTIVES OF THE STUDY

1. To survey different cassava waste water disposal points in Nsukka for isolation of

Salmonella and Shigella contaminants.

2. To evaluate cassava waste water after fermentation for survival and amplification

of Salmonella and Shigella cell count onto infective doses.

4

2.0 LITERATURE REVIEW

2.1 Bacteriology of Salmonella and Shigella organisms.

Salmonella and Shigella belong to the family of Enterobacteriaceae. This family has

the largest and most heterogeneous collection of medically important Gram-negative

bacilli. A total of thirty genera and more than one hundred and twenty species have

been described and these have been classified based on biochemical properties,

antigenic structure, and nucleic acid hybridization and sequencing. Despite the

complexity of this family, more than 95% of the medically important isolates belong

to only ten genera and constitute fewer than twenty-five species (Murray et al., 1998).

The genera in the family include Arsenophonus, Budvicia, Buttiauxella, Cedecea,

Citrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia, Ewingella, Hafnia,

Klebsiella, Kluyvera, Leclercia, Leminorella, Moellerella, Morganella,

Obesumbacterium, Pantoea, Pragia, Proteus, Providencia, Rahnella, Salmonella,

Serratia, Shigella, Tatumella, Xenorhabdus, Yersinia, Yokenella (Holt et al., 1994).

Enterobacteriaceae are ubiquitous organisms that are found worldwide in soil, water,

and vegetation and are part of the normal intestinal flora of most animals including

humans (Chessbrough, 2002).

2.2 Physiology

Members of this family are moderately sized, Gram-negative bacilli. All

members can grow rapidly aerobically and anaerobically (facultative anaerobes) on a

variety of non-selective agar media e.g. blood agar and selective agar media e.g.

MacConkey agar. The Enterobacteriaceae have simple nutritional requirements,

ferment glucose, reduce nitrate, (except Arsenophonus, a number of Erwinia spp,

most Xenorhabdus spp, and some strains of Klebsiella pneumonia sub sp. ozanae,

Pantoea, and Yersinia) and are oxidase negative and catalase positive, except for

Shigella dysenteriae O group 1 and Xenorhabdus spp. other than X. luminescens. The

absence of cytochrome oxidase activity is an important characteristic because it can

be measured rapidly and is used to distinguish Enterobacteriaceae from many other

fermentative and non-fermentative Gram-negative bacilli (Holt et al., 1994).

5

2.3 Pathogenic Enterobacteria.

Numerous virulence factors have been identified in the members of the family

Enterobacteriaceae. Some are common to all genera, and others are unique to specific

virulent strains. The virulence factors include;

(i) Endotoxin: This is a virulence factor shared among all aerobic and some

anaerobic Gram-negative bacteria. The activity of this toxin depends on the lipid A

component of Lipopolysaccharide, which is released at cell lysis. Many of the

systemic manifestations of Gram-negative bacterial infections, including the

activation of complement, the release of cytokines, leukocytosis, thrombocytopenia,

disseminated intravascular coagulation, fever, decreased peripheral circulation, shock

and death, are initiated by endotoxin. Klebsiella pneumonia produces an endotoxin

that appears to be independent of factors that determine receptor and capsular

characteristics.

(ii) Exotoxins: An Example of this is the type 111 secretion system. The yop

exotoxins of Yersinia pestis drastically affect the actin cytoskeleton, interfering with

integrin-mediated phagocytosis and allowing uptake of the facultative intracellular

bacterium. The ipa proteins of Shigella flexneri contribute to the killing of neutrophils

by necrosis, thus allowing the pathogen to enter the host cells via disruption of the

epithelial barrier.

(iii) Antigenic phase variation: The expression of capsular K and flagellar H

antigens is under the genetic control of the organisms. Each of these antigens can be

alternatively expressed or not expressed (phase variation) which can protect from

antibody-mediated cell death. This can be seen in Salmonella spp.

(iv) Capsule: This helps to protect the bacteria from phagocytosis and these

capsular antigens interfere with the binding of antibodies to the bacteria and are poor

immunogens or activators of complement. In Klebsiella pneumonia, the invasion of

the host cell is also facilitated by the large polysaccharide capsule surrounding the

bacterial cell. In addition, this capsule acts as barrier and protects the bacteria from

phagocytosis. The Vi antigen of Salmonella typhi can inhibit adsorption.

(v) Cell wall receptors: Their presence enables Klebsiella pneumonia to attach

to the host cell, thereby altering the bacterial surface so that phagocytosis by

polymorphonuclear leukocytes and macrophages is impaired and invasion of the non-

phagocytic host cell is facilitated.

6

(vi) Sequestration of Growth Factors: The siderophores are used by these

bacteria to scavenge for iron that is bound in hemeproteins (e.g. transferrin,

lactoferrin). Iron can also be released from host cells by hemolysins produced by the

bacteria. For instance, enterochelin from Escherichia coli and Salmonella spp.

scavenge bound iron from the host. Yersinia pestis can also absorb organic iron as a

result of a siderophore-independent mechanism (Murray et al., 1998).

2.3.1. Shigella

Shigellae are Gram-negative, straight rods. They are non-motile, facultative

anaerobes of the family Enterobacteriaceae. They are chemoorganotrophic, which is,

having both a respiratory and a fermentative type of metabolism. Shigellae are able to

grow at optimal temperature of 37oC when cultured on appropriate media such as

MacConkey Agar, Deoxycholate Citrate agar and Xylose lysine Deoxycholate Agar.

Biochemically, they catabolize D-Glucose and other carbohydrates, producing acid

and a few strains form gas. They are oxidase-negative, catalase positive, methyl red

positive and the production of indole varies.

Shigellae are classified into four species: Shigella dysenteriae, Sh. flexneri, Sh.

boydii, and Sh. sonnei, also designated Groups A, B, C, and D, respectively. Group A

has 13 serotypes, Group B has 6 serotypes with 15 subtypes, and Group C has 18

serotypes while Group D contains only a single serotype. Shigellae are differentiated

from the closely related Escherichia coli on the basis of pathogenecity, physiology

(i.e. failure to ferment lactose, or decarboxylate lysine) and serology.

The infectivity dose (ID) is extremely low. As few as ten Shigella dysenteriae

bacilli can cause clinical disease whereas 100 to 200 bacilli are needed for Shigella

sonnei or S. flexneri infection (Abuhammour, 2002).The incubation period of the

disease is 1 to 4 days which is usually followed by sudden onset of acute symptoms.

Extra intestinal manifestations

Central nervous system: Symptoms include severe headache, lethargy,

meningismus, delirium, and seizures lasting less than 15 minutes, especially with Sh.

dysenteriae. Severe toxic encephalopathy is rare, but lethal complications occur when

initial symptoms are followed by sensory obtundation, seizures, coma, and death in 6

to 48 hours. The pathogenesis of neurologic manifestations during shigellosis is

unclear; but data now clearly show that Shiga toxin is not responsible.

7

Hemolytic uremic syndrome (HUS): Microangiopathic hemolytic anemia,

thrombocytopenia and renal failure have been reported with Sh. dysenteriae and occur

because of vasculopathy mediated by Shiga toxin.

Septicemia is rare except in malnourished children with Sh. dysenteriae infection.

Septicemia sometimes is caused by other gram-negative organisms and is related to

loss of mucosal integrity during Shigella infection.

Shigella sepsis may be complicated with disseminated intravascular coagulation

(DIC), bronchopneumonia, and multiple organ failure in lethal cases. Reiter syndrome

which is a triad of arthritis, conjunctivitis, and uretritis is commonly observed in

adults carrying HLA-B27 histocompatibility antigen. Hepatitis, if present, is usually

mild. Myocarditis is identified with cardiogenic shock, arrhythmias, and heart block

(Abuhammour, 2002).

2.3.2. Salmonella

Salmonellae are Gram-negative, straight rods which are motile by peritrichous

flagella. Like Shigella, they are facultative anaerobes belonging to the family

Enterobacteriaceae. They are chemoorganotrophic, having both a respiratory and a

fermentative type of metabolism. Optimal temperature for growth is 37oC and they

are able to catabolize D-Glucose and other carbohydrates with the production of acid

and usually gas. They are characterized by somatic or O antigen, flagellar or H

antigen, and Vi antigen (This is possessed by only a few serovars).

The Salmonella species that are known to be commonly pathogenic in humans

include S. typhi, S. paratyphi, S. enteritidis, S. typhimurium, and S. choleraesuis (Holt

et al., 1994; Baker, 1997). The infectivity dose for symptomatic salmonellosis to

develop would be a large inoculum (106 to 108 bacteria) or reduced for people at

increased risk for disease because of age, immunosuppression, or underlying disease

(e.g. leukemia) or reduced gastric acidity (Murray et al., 1998).

The complications of salmonellosis may include vascular infection, focal

infection including osteomyelitis and a chronic carrier state (S. typhi) (Baker, 1997).

Individuals who have underlying disease involving defects in cell-mediated immunity

or phagocytic function are particularly vulnerable to salmonellosis and its

complications. Specifically, this group include individuals with AIDS,

lymphoproliferative disease, a history of transplantation, hemoglobinopathies, chronic

granulomatous disease, bartonellosis, malaria, histoplasmosis, and schistosomiasis,

8

meningitis (almost exclusively in infants), endocarditis, possible involvement from

many other sites (Barrali, 2006).

2.4 Pathogenesis and Pathology of Shigella and Salmonella infections.

2.4.1. Shigella

In the colonic mucosa, Shigella species appear unable to attach to

differentiated mucosal cells, rather they are proposed to cross the epithelial layer by

invading M (micro fold) cells located in Peyer’s patches which are also overlaying the

lymphoid follicles. This allows them reach the basolateral pole of epithelial cells

where they induce their uptake. It should be noted that Shigella species are able to

lyse phagocytic vacuole and replicate in the host cell cytoplasm (unlike Salmonella

that replicate in the vacuole) (Murray et al., 1998).

Entry into the epithelial cells involves re-arrangements of the cell cytoplasm

that extend beyond the zone of contact between the bacterium and the cell membrane,

leading to membrane ruffling and engulfment of the bacterium within a vacuole. Once

internalized by epithelial cells, bacteria rapidly lyse the membrane of the entry

vacuole and gain access to the cell cytoplasm where they multiply with a generation

time of approximately 40 minutes. By inducing actin polymerization at one of their

poles, intracellular bacteria move within the cytoplasm of infected cells. This

movement generates the formation of protrusions that contain one bacterium at their

tip and are engulfed by adjacent epithelial cells, thereby allowing bacteria to

disseminate from cell to cell without being exposed to the external milieu (Parsot,

2005).

Peptidoglycan fragments released by intracellular bacteria are detected by the

Nod I pathway, leading to phosphorylation and degradation of I Kappa B, trans-

location of NF-Kappa B to the nucleus and activation of NF- Kappa B regulated

genes. Analysis of the transcriptome of infected epithelial cell showed, in particular,

increased expression of the gene encoding IL-8, a potent chemo attractant for

neutrophils. Thus, epithelial cells actively participate in the detection and signaling of

invasive bacteria to host defenses. Bacteria released from M cells (after their initial

uptake) or epithelial cells (after intracellular multiplication) interact with

macrophages, escape from the phagocytic vacuole and induce apoptosis of infected

cells. Apoptotic macrophages release pro-inflammatory cytokines, including IL-I and

IL-18, which together with IL-8 released from infected epithelial cells, leads to

9

recruitment of polymorphonuclear cells (PMN) at the site of infection. Transmigration

of PMN destabilizes the epithelial barrier and facilitates further invasion by luminal

bacteria (Parsot, 2005).

Although the molecular basis of shigellosis is complex, the initial step in

pathogenesis is clearing bacterial invasion or penetration of the colonic mucosa, the

resulting focus of Shigella infection is characterized by degeneration of the epithelium

and by an acute inflammatory elements, and dependent upon the ileocecal flow. As a

result, the patient will pass frequent, scanty, dysenteric stools (Hale, 1991).

The virulence factors include;

a) Toxins: Sh. dysenteriae produce an exotoxin, Shiga toxin. Also enterotoxins

designated shET1 and shET2 have been identified, and the genetic loci encoding these

toxins have been localized to the chromosome and plasmid respectively. The

shET1locus is present on the chromosome of Sh. flexneri 2a, but it is only

occasionally found in other serotypes. In contrast, shET2 is more widespread and

detectable in 80% Shigellae representing all four species. These enterotoxins may

elicit the diarrheal prodrome that often precedes bacillary dysentery.

b) Siderophores: Ability to secrete iron from chelating compounds,

‘siderophores’ , which chelate iron from intestinal fluids and then are taken up to

release iron inside the bacterium for its metabolic needs. These are under control of

plasmids and are regulated tightly by genes such that, under low iron conditions,

expression of the siderophore system is high.

c) Lipopolysaccharide (LPS): LPS plays an important role in resistance to

nonspecific host defense encountered during tissue invasion. These genes help in

invasion, multiplication, and resistance to phagocytosis by tissue macrophages. LPS

enhances the cytotoxicity of Sh ET on human vascular endothelial cells.

d) Intestinal Adherence Factor: This favors colonization in vivo and in animal

models. This is 97kd outer membrane protein (OMP) encoded by each gene on

chromosomes. This codes for intimin protein and anti-intimin response is observed in

children with hemolytic uremic syndrome (HUS). (Abuhammour, 2002; Murray et al.,

1998; Schmidt and Hensel, 2004).

10

2.4.2. Salmonella

Serovars of Salmonella show a strong propensity to produce syndromes

peculiar to them, for instance, S. typhi, S. paratyphi A, and S. schottmuelleri produce

enteric fevers; S. choleraesuis produce septicemia or focal infections. S. typhimurium

and S. enteritidis produce gastroenteritis, etc; however, on occasion, any serotype can

produce any of the syndromes.

Salmonella, after ingestion colonize the ileum and colon, they invade the

intestinal epithelium, and proliferate within the epithelium and lymphoid follicles.

The mechanism by which it invades the epithelium involves an initial binding to

specific receptors on the epithelial cell surface followed by invasion. Invasion is

dependent on rearrangement of the cell cytoskeleton and probably involves increases

in cellular inositol phosphate and calcium. Attachment and invasion are under distinct

genetic control and involve multiple genes in both chromosomes and plasmids

(Giannella et al., 1975).

After the organism has invaded the epithelium, it multiples intracellular and

spreads to mesenteric lymph nodes and throughout the body through the systemic

circulation. The reticulo-endothelial cells take up the organism, then confines and

controls spread of the organism. Depending on the serotype and the effectiveness of

the host defenses against that serotype, some organisms may infect the liver, spleen,

gall bladder, bones, meninges, and other organs. Fortunately, most serovars are killed

promptly in extra intestinal sites, and the most common human Salmonella infections,

remains confined to the intestine where they cause gastroenteritis.

After the intestine has been invaded, most salmonellae induce an acute

inflammatory response, which can cause ulceration. The organisms may elaborate

cytotoxins that inhibit protein synthesis, this is brought about by the invasion of the

mucosa which causes the epithelial cells to synthesize and release the various pro-

inflammatory cytokines such as IL-I, IL-6, IL-8, TNF-2, IFN-U, MCP-I, and GM-

CSF. These evoke an acute inflammatory response and may also be responsible for

damage to the intestine. As a sequale to the intestinal inflammatory reaction,

symptoms of inflammation such as fever, chills, abdominal pain, leukocytosis, and

diarrhea are common. The stools may contain polymorphonuclear leukocytes, blood,

and mucus (Boyd, 1985).

In the pathogenesis of Salmonella gastroenteritis and diarrhea, only the strains

that penetrate the intestinal mucosa are associated with the appearance of an acute

11

inflammatory reaction and diarrhea. This diarrhea is due to secretion of fluid and

electrolytes by the small and large intestines. The mechanisms of secretion are

unclear, but the secretion is not merely a manifestation of tissue destruction, and

ulceration. Salmonella, however, can penetrate the intestinal epithelial cells, but

unlike Shigella and invasive E.coli, do not escape the phagosome. Therefore, the

extent of intercellular spread and ulceration of the epithelium is minimal. Salmonella

escape from the basal side of epithelial cells into the lamina propria and the systemic

spread of the organism can occur, giving rise to enteric fever. Invasion of the

intestinal mucosa is followed by activation of mucosal adenylate cyclase; the resultant

increase in cyclic AMP induces secretion (Koo et al., 1984).

The mechanism by which adenylate cyclase is stimulated is not understood, it

may involve local production of prostaglandins or other components of the

inflammatory reaction. In addition, Salmonella strains elaborate one or more

enterotoxin- like substances which may stimulate intestinal secretion. However, the

precise role of these toxins in the pathogenesis of Salmonella enterocolitis and

diarrhea has not been established. (Boyd, 1985; Giannella et al., 1975; Giannella et

al., 1973 and Koo et al., 1984).

The virulence factors of Salmonella include;

1. Ability to invade cells

2. A complete lipopolysaccharide coat.

3. The V1 antigen which protects the organism from antibody mediated

destruction.

4. Ability to replicate intracellularly.

5. Presence of Plasmids.

6. The elaboration of toxins

Most virulence factors of Salmonella are determined by chromosomal genes, and

many of these are located within the pathogenecity island (PAI).

2.5 Epidemiology and Mode of Transmission.

2.5.1 Epidemiology and mode of Transmission of shigellosis.

A review of available literatures show that Sh. flexneri is the main serogroup

found in developing countries (Agtini et al., 2005; Khalil et al., 1998; Iwalokun et al.,

2001) with Sh. sonnei being the next most common (Rosenberg et al., 1997; Wang et

al., 2005). Sh. dysenteriae and Sh. boydii occurs with equal frequency. Reports have

12

shown that Sh. sonnei is the most common serogroup found in industrialized

countries, followed by Sh. flexneri (McCombie et al., 1988; CDC, 2005; CDC, 2006).

The proportions of each species vary from country to country. The annual

number of Shigella episodes world wide is estimated to be 165 million, of which more

than 100 million occur in developing countries with more than 1 million deaths

(Dupont, 2005). Although, Shigella is endemic worldwide, it affects certain

populations more than others. In developing countries, high rates of morbidity and

mortality are known to occur among displaced populations.

Burden of shigellosis in developing countries:

Proper estimation of the burden of Shigella disease in developing countries has two

important dimensions: a clinical dimension that provides the magnitude of morbidity

and mortality attributable to Shigella and a biological dimension that provides the

distribution of Shigella serotypes in different settings.

(i) The clinical dimension: Morbidity and mortality. Sh. dysenteriae type 1, also

referred to as Shiga bacillus, has been recognized as the major cause of epidemic

dysentery for nearly 100years. The pandemic that began in Central Africa in 1979

progressed to East Africa and has since become particularly problematic among

refugee populations (CDC, 1994).

In general, both the incidence and the fatality rates are highest among the very

young and the elderly (Wang et al., 2005). Although epidemic Shigella dysentery is

the most dramatic manifestation of Shigella infection in developing countries, the

majority of Shigella infections are due to endemic shigellosis. Endemic Shigella is

responsible for approximately 10% of all diarrheal episodes among children younger

than five years living in developing countries and up to 75% of diarrheal deaths

(Kotloff et al., 1999).

From available literature, it can be concluded that, of the estimated 165

million cases of Shigella diarrhea that occur annually, 99% occur in developing

countries, and in these developing countries 69% of episodes occur in children under

five years of age. Also, of the approximately 1.1 million deaths attributed to Shigella

infections in developing countries, 60% deaths occur in the under- five age group

(Clemens et al.,1999; Kotloff et al.,1999).

These conclusions are, however, limited by several features which include,

estimates of the burden are based on multiple studies for children, because data are

limited for adults. Also, it is to be appreciated that in developing countries, a

13

substantial fraction of Shigella deaths occur in persons who never seek medical care

or who die after discharge from the hospital (Clemens et al., 1999).

(ii) The biological dimension: distribution of serogroups and serotypes. Due to the

fact that immunity to Shigella is serotype-specific, vaccine protection will therefore

depend on the representation of the serotypes in the vaccine. Therefore, knowledge of

the distribution of serotypes in addition to serogroups of clinical isolates is of crucial

importance in designing new vaccines.

Mode of Transmission of Shigella infection:

Most transmission of the organism is by fecal/oral transmission, this is by

person –to –person spread and ingestion of contaminated food or water. These modes

of transmission are most common in situations in which hygiene is limited, e.g. child

care centers and other institutional setting (CDC, 1986; CDC, 1992; Rosenberg et al.,

1997).

Flies may be important in the transmission of bacillary dysentery, especially in

the tropical climates. Bacteriologic surveys of fly populations indicate that flies can

occasionally be shown to be positive for Shigella bacteria. The low dose required for

infection at least partially explains the potential for fly transmission of shigellosis

(Dupont, 2005).

Zoonotic transmissions have also been reported. A small cluster of dysenteric

illness, due to Shigella flexneri, was identified among technical assistants of a primate

research unit. All of the affected individuals had been in regular contact with a colony

of cynomolgus macaque monkeys, one of which was known to have suffered from

acute hemorrhagic colitis in the preceding few weeks. Four monkeys were found to be

excreting Sh. flexneri bacilli of identical antigen type (1b) to that isolated from the

human cases. Investigation of working practices revealed the potential for

inadvertment faecal-oral spread and the need to improve existing control methods. It

was concluded that this small outbreak of shigellosis represents a primate-associated

occupational zoonosis (Kennedy et al., 1993).

14

2.5.2. Epidemiology and Mode of Transmission of Salmonella infection.

First, an asymptomatic human carrier state may last from many weeks to

years. Thus, human as well as animal reservoirs exist. Interestingly, children rarely

become chronic typhoid carriers. Second, use of antibiotics in animal feeds and

indiscriminant use of antibiotics in humans increase antibiotic resistance in

salmonellae by promoting transfer of R factors (Gianella, 2007). In many countries,

the incidence of human Salmonella infection has increased markedly over the years.

Salmonellosis is a zoonosis and has an enormous animal reservoir. The most

common animals are chickens, turkeys, pigs, cows, reptiles, various domestic and

wild animals. Salmonella can survive in meat, and animal products, that are not

thoroughly cooked, therefore, animal products, are the main vehicle of transmission

(Lewis, 1998; Murray et al, 1998).Recent outbreaks of salmonellosis have being

linked to poultry meat products, eggs, ice cream, alfalfa sprouts, milk and cereal

(W.H.O., 2005).

There is also person-to-person transmission via the fecal –oral route (Barrali,

2006). Many other foods, including green vegetables contaminated from manure,

have been implicated (Kariuki et al., 2006). Contacts with infected animals including

domestic animals such as cats and dogs are other means of spread. Domestic animals

probably acquired the infection in the same way as humans i.e. through consumption

of contaminated raw meat, poultry, or poultry- derived products (W.H.O, 2005).

2.6 Survival and Sources of these pathogens in the Environment.

2.6.1 Shigella.

Shigella can survive best at low temperature (subzero and refrigeration) and

better in low moisture foods. It can survive heating to 63°c for 2 to 3 min. Despite its

relatively high minimum pH for growth, Shigella is among the most acid resistant of

food borne pathogens. Some strains can survive exposure to pH 2.5 or 3.0 for 2 hours,

and for a few hours to a day in fruit juices of various pH values. The minimum

temperature for growth is 6 to 7°c while the maximum is 45 to 47°c. However,

Shigella is rapidly inactivated at temperatures above 65°c and at pH less than 4.0 but

can persist for some time (ESR, 2001).

A study by Islam et al., (1996) demonstrated that the survival of Sh. flexneri in the

aquatic environment is greatly influenced by the physicochemical factors such as the

temperature, pH and salinity. When the average temperature, pH and salinity in fresh

15

water environment were 25°c, pH 7.0, and 0% salinity respectively, Sh. flexneri

survived more than two weeks. This probably explains the outbreaks of shigellosis

attributed to swimming in contaminated water in the United States (Rosenberg et al.,

1976) as well as their presence in surface water e.g. ponds, lakes, wells, and rivers,

which could act as sources of infection (Islam et al., 1996).

Nakamura (1962) who studied the transmission of Shigella sonnei among

school children found that the organism could be isolated from toilet seats, toilet

floors, clothes, bedding, toys and floors of homes. Also contamination of children’s

hands was correlated with contamination of toy balls and other inanimate objects.

The capability of pathogenic microorganisms to exist in the viable but non-culturable

(VBNC) state also contributes to the potential health hazard of Shigella existing in

VBNC state. The significant problem in elucidating the potential hazard of non-

culturable pathogenic bacteria is the inability to detect such cells in the natural

environment by routine bacteriological culture methods (Rowan, 2004). A study

performed by Rahman et al., (1996), on Shigella dysenteriae type1 demonstrated that

VBNC Shigella dysenteriae type1 remains potentially virulent, on the basis of the

experimental evidence that maintained the production of the ShT toxin and the

adherence to Henle 407 cells.

Although, inanimate objects play an important role in the transmission of

shigellae, little information on the survival of Shigella on contaminated inanimate

objects is available. According to Islam et al., (2001), in a study using conventional

culture, fluorescent antibody (FA) technique, and Polymerase Chain Reaction (PCR)

techniques, to determine how long Sh. dysenteriae type1 could survive on various

inanimate objects. The Sh. dysenteriae type1 remained culturable longest on cloth,

followed by wood; aluminium, glass, and lastly on plastic. It was viable for significant

periods of time on the different fomites, with VBNC cells detectable on cloth, wood,

plastic, aluminium and glass surfaces five days after inoculation. Therefore, in places

where poor hygienic conditions prevail and contact with contaminated household

surfaces occurs frequently, Sh. dysenteriae type1 in the VBNC state on fomites may

be a potentiator in the transmission of shigellosis.

Despite all these other means of transmission, humans still remain the principal

reservoir of infection (Sur et al., 2004).

16

2.6.2 Salmonella

Salmonella can survive at an optimal temperature of 42°C and an optimal pH

of 5.5, though it has a minimum pH of 4.5 and maximum pH of 8.0. Salmonella can

certainly survive but may not actively grow in many environmental waters. As

Salmonella bacteria are present in the faeces of humans and birds, they are often

present in faecally-polluted waters. Domestic fowls are considered the greatest single

reservoir of salmonellae; other sources include raw milk, raw milk products, meat and

meat products, undercooked or raw eggs and egg products, and contaminated water

(Barrali, 2006). Other foods that have been implicated are fish, shell fish and

vegetables (Kariuki et al., 2006). The potential reservoirs include pets such as turtles,

tortoises, chicks, dogs, cats as well as swine, cattle, rodents.

Otokunefor et al. (2003) found that Salmonella survived in the droppings of

lizard for 4 weeks in tap water and wet sand, 6 weeks when in direct contact with air,

and up to 8 weeks when mixed with dry sand. This actually shows that reptiles are

proven carriers and sources of environmental contamination with Salmonella.

Therefore, water may be contaminated directly or indirectly by reptile droppings and

serve as a vehicle of transmission. Another study showed that the survival of

Salmonella typhi and Shigella flexneri in different water samples and at different

temperatures, in 0.9% NaCl (physiological saline) and at room temperature, S. typhi

survived for 29 days while Sh. flexneri was for 57 days which was longer than their

survival in distilled water at the same temperature ( Uyanik et al.,2008 ).

Salmonella typhimurium and Shigella can multiply in the gut of housefly and can be

excreted for weeks or longer (Levine and Levine, 1991). There is, therefore, a risk of

contamination associated with the exposure of food to flies.

Outside the host, Salmonella can survive in ashes for 130days; rabbit carcass

for 17days, dust for up to 30days, feaces for up to 62 days, linoleum floor for 10

hours, ice for 240 days and skin for 10 to 20 minutes. (Chan,2000). Moreover,

Salmonella uses a secretion system to protect itself inside amoeba, a unicellular

organism capable of living both on land and in water. The system is called SP12 type

111. Its ability to survive in amoeba is a huge advantage to its continued development

as it renders the organism more resistant to disinfectants and water treatment

(University of Liverpool, 2009). Salmonella can also exist in the viable but non-

culturable (VBNC) state (Rowan, 2004). This is another survival mechanism used by

this pathogen in the environment.

17

2.7 Salmonella and Shigella as agents of genetic change in the environment.

1. Virulence Factors

Acquisition of pathogenicity islands (PAIs) by horizontal gene transfer is an

important mechanism in development of disease-causing capability and the evolution

of bacterial pathogenesis. Pathogenicity islands are a subset of horizontally-acquired

genomic islands (GIs) that are present in various microbial pathogens and contain

genes associated with virulence. Although PAIs are loosely defined entities, many of

them can be identified by features such as the presence and association with tRNA

genes, insertion sequence (IS) elements or repeated sequences at their boundaries.

Bacterial pathogenicity/virulence determinants that can be found in PAIs include type

111 secretion system (e.g. LEE PAI in pathogenic E. coli and Hrp PAI in

Pseudomonas syringae), super-antigen, colonization factor, iron uptake system (e.g.

SH1-2 in Shigella flexneri) and enterotoxin (e.g. espc PAI in E.coli and she PAI in Sh.

flexneri). The widespread presence of PAIs in these pathogens is due to their efficient

mechanisms of horizontal transfer (Yoon et al., 2006).

The horizontally acquired pathogenecity islands are major contributors to the

virulent nature of many pathogenic bacteria, these chromosomally encoded regions

typically contain large clusters of virulence genes and can upon incorporation,

transform a benign organism into a pathogen. Many PAIs are situated at tRNA and

tRNA-like loci, which appear to be sites for integration of foreign sequences. For

example, the tRNA sel C has repeatedly served as the integration site of pathogenecity

islands in enteric bacteria, including the 70 kb PAI-1 of uropathogenic E.coli, the 35-

kb LEE island of enteropathogenic E.coli and the 24-kb SH1-2 island of S. enterica.

The sequences flanking pathogenecity islands frequently contain short direct repeats.

These are reminiscent of those generated upon integration of mobile genetic elements

and ORFs (Open Reading Frames) within certain PAIs which display sequence

similarity to bacteriophage integrases. Several phages, including Ø R73 and P4 of

E.coli, P22 of S. enterica, insert at or near tRNA genes, suggesting that pathogenecity

islands are transferred and acquired through phage-mediated events, or that their

incorporation involves the action of conserved integrases (Ochman et al., 2000).

2. Antibiotic Resistance

Presence of plasmids, transposable elements and integrons promote the

transfer of genes for drug resistance from one bacterial genome to another. Resistance

to antimicrobials often spread by transfer of DNA between bacterial strains. Some

18

variants of Salmonella have developed multi-drug resistance as an integral part of the

genetic material of the organism and are, therefore, likely to retain their drug-resistant

genes even when antimicrobial drugs are no longer used, a situation where other

resistant strains would typically lose their resistance. These drug-resistant Salmonella

emerge in response to antimicrobial usage in food animals (W.H.O., 2005).

Several enteric pathogens (e.g. Salmonella) are capable of generating different

types of hybrid plasmids. This consists of the resistant genes that are responsible for

resistance to conventional antibiotics (Chiu et al., 2004). Resistance to broad-

spectrum cephalosporins is due to the production of extended-spectrum ß- lactamases.

A variety of such ß-lactamases have been described in Salmonella. The genes

encoding extended-spectrum ß-lactamases could be carried by conjugative plasmids,

transposons, or integrons (Chiu et al., 2004). These mobile genetic elements could

spread under selective antibiotic pressure between bacterial species (W.H.O., 2005;

Chiu et al., 2004).

2.8 CASSAVA

2.8.1 Composition of Cassava

There are a number of varieties of cassava that range from low cyanide

content (also called the ‘sweet cassava’) to higher cyanide content (referred to as

‘bitter cassava’). Raw cassava usually has an iron content of 1 to 2 mg/ 100g of dry

matter. The potassium content is greater than that of calcium, phosphorus, and iron,

Cassava roots have high vitamin C content, but it can be destroyed in factory

processing or cooking.

Cassava juice is milky, smells of cyanide, and consists of 91.00% water,

0.13% essential oils containing sulfur, 2.3% gum, 1.14% saponins, 1.66% glycosides

and 3.80% nonspecified components. Cassava lipids range from 0.1% to 1.0%.

Carbohydrate is the highest fraction of cassava root composition with starch

constituting the largest part (Cereda and Takahashi, 2006).

2.8.2 Cassava waste

These are the residues generated by processing. The processing of cassava

roots generates both solid and liquid residues. Among the liquid wastes is

manipueira, which is formed during flour making and starch extraction, when the

19

water present in the roots is pressed out. Also the water from washing the roots is

considered as liquid waste (Cereda and Takahashi, 2006).

The liquid waste considered here is the waste water extracted from grated and pressed

root tubers. This is the type obtained from garri processing. Garri is the most popular

form in which cassava is consumed in Nigeria. Traditionally, garri is prepared from

cassava roots by fermenting peeled and mashed cassava pulp in jute bags for a period

of about 3 to 5 days (Odoemelam, 2005). This waste water generated is usually

allowed to flow into rivers, or percolate into the soil, causing serious environmental

pollution problem. It has been reported that this liquid residue contains minerals such

as nitrogen, carbon, phosphorus, potassium, calcium, magnesium, sulphur, zinc,

manganese, cooper, iron and sodium (Ribas and Barana, 2003).

The mashing of cassava roots and subsequent dewatering encourage the

leaching out of cyanogen (Odoemelam, 2005). It has been observed that the number

of processing steps involved in the production of cassava foods influence the level of

residual cyanide in the processing. The integrated accelerated cassava processing

method also results in detoxification of the product. Detoxification of cassava

involves hydrolysis of the cyanogenic glycosides by the linamarase enzyme into

acetone cyanohydrins and glucose and further dissociation of the acetone cyanohydrin

at pH above 5.0 to yield hydrogen cyanide and acetone (Obilie et al., 2004). The

hydrogen cyanide produced is removed during processing by either volatilization or

solubilisation (Mkpong et al., 1990). Other reports attribute detoxification of grated

cassava during fermentation to endogenous linamarase, which are able to attack the

previously compartmentalized cyanogenic glycosides following loss of cellular

integrity during grating (Maduagwu, 1983; Vasconcelos et al., 1990; Obilie et al.,

2004).

It should be noted that the major cyanogenic glycoside in cassava is linamarin,

while a small amount of lotaustralin (methyl linamarin) is also present. There is also

the enzyme, linamarinase, which catalyses the reaction that rapidly hydrolyses

lotaustralin to a related cyanohydrin and glucose. While under neutral conditions,

acetone cyanohydrin decomposes to acetone and hydrogen.

20

H3C Hydrogen Acetone Cyanide

Figure 1.The reaction of linamarin with linamarinase in cassava.

CH3 OH C C = N CH3

Acetone cyanohydrin

CH2OH CH3 O C C = N CH3

HO HO

HO

Linaminarase

CH2OH

HO HO

HO

+ HO

Linamarin

Glucose

Spontaneous Hydroxynitrate H3C Lyase C = O + HC=N

21

2.8.3. Cassava Wastewater and Survival of Microorganisms.

Cassava waste water contains cyanide. This cyanide may have adverse effects

on animals, higher plants and microorganisms. In microorganisms, it interferes with

the oxidative phosphorylation pathway, combining cytochrome-oxidase to inhibit

electron transport and hence inhibit the formation of adenosine triphosphate (ATP).

However, microorganisms can also utilize substrates that contain cyanide if they are

capable of anaerobic metabolism or if they can split it into carbon and nitrogen

(Cereda and Takahashi, 2006).

Numerous microorganisms have been discovered in both prokaryotic and

eukaryotic taxa which degrade cyanide-containing compounds. What is known so far

is that growth on cyanide requires that it be enzymatically converted to ammonia.

Once formed, it can then be readily incorporated into cellular macromolecules by

established mechanisms. The pathways used include hydrolytic, oxidative, reductive,

substitutive and transfer reactions. The microorganisms which are involved in

substitutive reaction include Thiobacillus denitrificans, Bacillus subtilis, Bacillus

stearothermophilus, Bacillus megaterium etc. The hydrolytic conversion has been

reported for Alcaligenes oxylosooxidans subspecies denitrificans, Bacillus pumilus,

and Pseudomonas species (White et al., 1988). The oxidative conversion has been

described in Pseudomonas fluorescens NCIMB11764 only (Kunz et al., 1998). Other

bacteria that have shown ability to degrade cyanide to ammonia and carbon dioxide

include Bacillus pumilus, Pseudomonas paucimobilis (Meyers et al., 1993).

Escherichia coli and Klebsiella oxytoca follow pathways which utilize the enzyme

cyanide dioxygenase and nitrogenase, respectively, while fungi such as Fusarium

lateritium, F. solani and several Trichoderma strains metabolise cyanide by various

other pathways (Ingvorsen et al., 1991; Meyers et al., 1991; Watanabe et al., 1998).

Cyanide degrading enzymes are generally produced by mesophilic

microorganisms, often isolated from soil, with temperature optima typically ranging

between 20°C and 40°C, reflecting the growth optima of the source organisms. Soil

pH may be a particularly important factor in the bioremediation of cyanide

contaminated soils. The pH optima for bacterial growth are typically 6 to 8 while that

of fungi growth is 4 to 5. Cyanide–degrading enzymes generally have pH optima

between 6 and 9. Therefore, extremes of pH may have a significant effect on

biodegradation. However, Fusarium solani and a mixed cultures of fungi including F.

22

solani, F. oxysporum, Trichodermma polysprum, Scytalidium thermophilium and

Penicillium miczynski were capable of degrading iron-cyanide at pH 4 (Barclay et al.,

1998).

Microorganisms degrade cyanide, either to detoxify or use it as a source of

nitrogen for growth. This is because cyanide as a salt, KCN or NaCN, is toxic for

bacterial growth. The bacteria have to grow with cyanide as a limiting factor. It

should be noted that in the cyanide molecule, the oxidation state of C (+2, like that in

CO) and N (-3, like that in NH4 +) makes this compound a bad carbon source but a

good nitrogen source for bacterial growth (Kunz et al.,1998; Huertas et al.,2006). A

microorganism can metabolize cyanide only when, in addition to a biodegradative

pathway to convert cyanide into an assimilable product (NH4+), it also harbors a

cyanide-resistant mechanism (generally a cyanide insensitive oxidase). Moreover, the

microorganism thriving in cyanide containing media will need a system for taking up

iron from the medium since iron forms very stable complexes with cyanide making it

unavailable to the organism (Igeno et al., 2007).

Further experiments showed that a reaction between cyanide and a metabolite

excreted into the medium was responsible for cyanide removal. Since cyanide

removing activity in culture fluids consistently co-purifies with iron-chelating

activity, it was concluded that the responsible metabolite was a siderophore (Kunz et

al., 1998). Ferric siderophores chelate iron from transferrin or lactoferrin or other host

proteins and are transported through outer membrane receptors. These receptors can

be classified into two general types depending on the structure of the siderophore

molecule: the catecholate and the hydroxamate types (Huertas et al., 2006). The

structure of siderophores is variable. The only feature that they share is the presence

of functional groups that can provide a high-affinity set of ligands, which are

generally oxygenated, for co-ordination of ferric ions. In addition, there are many

other compounds which combine these types of siderophore or present other chelating

compounds such as hydroxyacids.

Thus, in Escherichia coli and Salmonella typhimurium, the 669Da catechotate

siderophore enterobactin has been isolated. Ferrichrome is a 740 Da hydroxamate

type siderophore produced by E. coli and fungi. Pyoverdins are the most frequent

siderophores produced by Pseudomonas strains. The biosynthetic pathways of

siderophores are diverse. Often, siderophores are transported to the cytoplasm through

an ATP-dependent ABC-type transporter. For cyanotrophic organisms, production of

23

siderophores is also required to chelate iron since cyanide binds it strongly (Huertas et

al., 2006).

Mante et al., (2003) showed that in fermenting cassava dough, enteric

pathogens survived to different extents depending on pH and their sensitivity to acids.

Raising the pH of the fermented cassava dough prolonged the survival of these

enteropathogens, with Shigella dysenteriae and E.coli surviving longer than

Salmonella typhimurium and Salmonella enteritidis. This actually showed that low pH

/high acidity was responsible for the reduction in the number of the enteropathogens.

The acid nature of the cassava waste water probably will favor the growth of

microorganisms which are acid tolerant. Through the activities of these acid-tolerant

organisms, the pH could be raised, thereby encouraging the survival of other

microorganisms. The growth and survival of the enteric pathogens may not be

unconnected with the fact that the waste water contained substances that can be

utilized by them. The presence of other microorganisms originating from the soil,

water and materials used during processing of cassava and the prevailing

environmental conditions are all contributing factors. Therefore, all these make the

enteric pathogens to be transient microorganisms surviving in the absence or low

presence of cyanide.

It is, therefore, possible that the cassava waste water could be a source of

transmission of Salmonella and Shigella pathogens, since it is usually not treated

before being discharged into the environment. There is also a high likelihood of it

coming in contact with faeces from humans and animals, the potential reservoirs of

these pathogens. With the nutrients available in the cassava waste water and activities

of the cyanotrophic and acid tolerant microorganisms, Salmonella and Shigella could

be amplified in the cassava waste water at the appropriate pH and temperature. It is

against this background that the project was conceived to search cassava waste-water

discharged into the environment for Salmonella and Shigella; and to study the factors

that favour survival of these organisms in cassava waste-water.

24

3.0 MATERIALS AND METHOD

3.1 Media Preparation

Different media were used in the course of this study. They included

MacConkey agar (Fluka), Nutrient agar (Fluka), Xylose lysine Deoxycholate agar

(Oxoid CM0469B), Sabouraud Dextrose agar (Fluka), Selenite F broth (Oxoid

CM0395B&LP0121A), Triple sugar iron agar (Oxoid CM0277B), Sulphide Indole

Motility (SIM) medium (Oxoid CM0435B). These media were prepared according to

the manufacturers’ instructions.

3.2 Sample Collection

The samples of cassava waste water and soil were collected from cassava

processing sites in Nsukka and its environs. The samples of waste water were

collected using sterile syringes and sterile screw-capped bottles and the samples were

obtained from four different spots at each processing site while the soil was collected

with sterile spatula into sterile screw-capped bottles. They were taken to the

laboratory for immediate processing and analysis.

Origin of the Plant Material: Cassava tubers of two different varieties; TMS92/0326

(low cyanide) and NR8082 (high cyanide) were obtained from the National Root and

Crop Research Institute (NRCRI), Umudike, Abia State.

Source of bacterial isolates: The two strains of bacteria, Salmonella typhi and Shigella

dysenteriae were obtained from the Federal College of Veterinary and Medical

Laboratory Technology, NVRI, Vom, Plateau State. These were used as standard

culture.

3.3 Isolation of Organisms

The cassava waste water collected was used for various microbiological

analyses including isolation of Salmonella and Shigella pathogens carried out as

follows: 10ml of the waste water was centrifuged and both the sediment and

supernatants were used to inoculate separate sterile test tubes containing 5ml of

Selenite F broth. The above Selenite F cultures were incubated at 370C for 24hrs;

thereafter subcultured on duplicate MacConkey agar plates and incubation continued

at 370C for further 24hrs. The emergent colonies were purified by re-plating on

MacConkey agar plates before plating on Xylose lysine Deoxycholate agar plates in

duplicates and incubated at 370C for 24hrs. The resulting colonies were purified on

25

the same media before they were stored in Nutrient agar slant at 40C for further use.

The colonies were characterized into their respective bacterial genus using Gram-stain

reaction, catalase reaction and other relevant biochemical tests.

For isolation of other microorganisms, the cassava waste water was plated

directly on Nutrient agar, MacConkey agar and Sabouraud Dextrose agar plates in

duplicates by using a standard wire loop. The plates were incubated at 370C for 24hr

for bacteria and at room temperature for fungi. The resulting colonies were sub

cultured on the respective media and these were further characterized by Gram

reaction and other relevant biochemical tests. The isolates were stored on Nutrient

agar slants (bacteria) or Sabouraud Dextrose agar slants (fungi).

3.4 Isolation of Microorganisms from the Cassava processing Soil

A basal medium containing (NH4)2 SO4 (2.0g), MgSO4.7H2O (0.2g), 0.05g

CaCO3 (0.05g), FeSO4.7H2O (1.0g) and MnSO4 (10mg) dissolved in 1 litre of distilled

water was prepared. A 200ml portion of the solution was put into each of two

different conical flasks; these were autoclaved at 1210C for 15min and allowed to

cool. To each of the flasks were added 10ml of sterile (membrane filtered) high

cyanide NR8082 cassava waste water and 10g of soil from a cassava processing site

(Ibagwa site 1 or Ibagwa site 2). The initial pH of the flask was noted before

incubating at room temperature with shaking at intervals for 48hrs. The pH was also

noted at the end of the incubation period. At 24hr interval, 0.1ml of the samples was

aseptically aspirated and spread on MacConkey agar, Nutrient agar and Sabouraud

Dextrose agar plates and incubated at 370C (for bacteria) and room temperature (for

fungi) for 24hrs. The isolates obtained were re-plated by three successive subcultures

in nutrient agar (NA), MacConkey agar and Sabouraud Dextrose agar (SDA). They

were characterized following standard bacteriological and mycological procedures

and stored on Nutrient agar slants (bacteria) or SDA slants (fungi).

3.5 Determination of Microbial Succession during fermentation for Garri

Production

The cassava used here is the local variety. Four tubers of cassava were peeled,

washed and grated by means of a sterile hand grater and the resultant pulp tied in a

sterile sieve bag. This was kept in a clean bucket with a lid. The pH was monitored at

every 24hr interval for four weeks. A 0.1ml of the cassava waste water was

26

aseptically aspirated and cultured on Nutrient agar, MacConkey agar and Sabouraud

Dextrose agar plates respectively. The plates were incubated at 370C (bacteria) and

room temperature (fungi) for 24hrs. This procedure was repeated at every 24hr

interval for four weeks. The resulting colonies were sub-cultured and characterized

using standard microbiological procedures.

3.6 Isolation of Microorganisms from Cassava processing Soil using an acidic

medium

A basal solution containing KH2PO4 (10mg), FeCl2. 6H2O (100mg), MgSO4.

7H2O (100mg), CaCl2.2H2O (100mg), MnSO4. 4H2O (50mg), ZnSO4. 7H2O (50mg),

CoCl2. 2H2O (100mg) and Na2 MO4. 2H2O (10mg) dissolved in 1 litre of distilled

water was prepared (Dumestre et al., 1997). The basal medium was poured into two

flasks containing 200ml per flask and was adjusted to pH 3.0 using 1N HCl. It was

sterilized at 1210C for 15min. After cooling, 50mg/litre filter-sterilized KCN was

added to each of the flasks. 10g of soil samples (Ibagwa site 1 or site 2) were also

added to each flask. The culture was incubated at room temperature and monitored for

3 days. The pH of the culture was noted every 24 hr within this period. At every 24hr

interval, 0.1ml was aseptically aspirated from each flask and plated on Nutrient agar,

MacConkey agar and Sabouraud Dextrose agar plates respectively. These plates were

subsequently incubated at 370C (bacteria) or room temperature (fungi) for 24hr. The

resulting colonies were sub-cultured and characterized using standard bacteriological

or mycological techniques. The isolates were stored in slants for further use.

3.7 Identification of Isolates

Various types of bacteriological techniques were used in identifying the isolates

obtained.

3.7.1 Morphological characterization:

Each bacterial isolate was Grain-stained and examined microscopically. A thin

smear of the bacterial growth was made on normal saline dropped on a clean glass

slide. After air-drying, the bacteria were fixed by passing the slide over a Bunsen

flame and left to cool. The slides were flooded with crystal violet, left for 30sec, and

then rinsed with water, shaking off excess. They were flooded with iodine and left for

60sees before rinsing with water. A decolorizer (acetone alcohol) was added until the

27

blue dye was no longer running off the slides, and left for 10secs, they were

immediately rinsed with water. The slides were flooded with a counterstain, safranine,

and allowed to remain without drying for 30secs. Then, rinsed with water and allowed

to air dry. They were examined under oil immersion lens at 100 x for bacterial cells

using the microscope.

3.7.2 Motility Test:

Sulphide, Indole and Motility (SIM) medium (Oxoid) was used for the

motility test. This was to demonstrate the ability of the isolates to move away from

the point of inoculation. The medium was prepared according to the manufacturers’

instruction. It was then dispersed into clean test tubes in 10ml aliquots and sterilized

by autoclaving at 1210C for 15mins. The tubes were allowed to cool and dry after

which the isolates were inoculated by stabbing to a depth of 2cm by means of an

inoculating needle. There were incubated at 370C for 48hr. Positive motility was

shown by turbidity away from the line of inoculation while growth confined at the

point of inoculation was a negative result.

3.7.3 Indole Test:

Preparation of Kovac’s Reagent: The individual components are p-

Dimethylaminobenz-aldehyde (10g), Isobutyl alcohol (150ml) and concentrated

hydrochloric acid (HCl) (50ml). The first component was dissolved in the alcohol, the

conc. HCl was slowly added with constant stirring of the mixture. The prepared

reagent was pale-colored and stored in a brown bottle in a refrigerator until when

needed.

A 48hr culture in the SIM medium was used for indole test. Six drops of the

Kovac’s reagent were added to each tube and gently shaken. The appearance of a

pinkish color in the tube was taken as positive for indole.

3.7.4 Catalase Test.

The test demonstrates the ability of bacteria to produce the enzyme, catalase

that breaks down hydrogen peroxide to water and oxygen. A drop of hydrogen

peroxide (3%w/v) was placed on an emulsified isolate on a clean glass slide. A

positive result was indicated by effervescence.

28

3.7.5 Citrate Utilization Test

Simmon’s citrate agar was used to determine the utilization of citrate as a sole

carbon source. This was composed of Magnesium sulphate (0.2g), Monoammonium

phosphate (1.0g), Dipotassium phosphate (1.0g), Sodium citrate (2.0g), Sodium

chloride (5.0g), Bacteriological agar (15.0g), Bromothymol blue as indicator and

Distilled water (1000mg). The individual components were dissolved in the distilled

water and the agar added and stirred to dissolve completely before dispensing in a

10ml aliquot into test tubes with lids. These were sterilized by autoclaving at 1210C

for 15mins. They were allowed to cool by slanting. The isolates were streaked on the

slants and incubated at 370C for 48hrs. A positive result was indicated by observation

of a color change from green to prussian blue.

3.7.6 Urease Test

The urea agar slants was used to differentiate Proteus spp. and Yersinia

enterocolitica from other Enterobacteriaceae by their ability to rapidly hydrolyse urea,

a reaction that is catalyzed by the enzyme, urease. The components were yeast extract

(0.1g), Monopotassium phosphate (0.091g), Disodium phosphate (0.095g], Urea

(20g), Phenol red (0.01g) as indicator, distilled water (1000ml) and bacteriological

agar (15.0g). The individual components were mixed together and the distilled water

added and stirred to homogenize the mixture. This was dispensed into test tubes in

10ml aliquots and sterilized by autoclaving at 1210C for 15 min. The tubes were

allowed to form slants while cooling. The isolates were streaked on the slants and

incubated at 370C for 24hrs. A positive result was indicated by the observation of a

color change from orange to magenta (purplish red).

3.7.7 Potassium Cyanide (KCN) Test:

The potassium cyanide broth was prepared with peptone (0.6g), Sodium

chloride (1.0g), Potassium dihydrogen phosphate (0.05g), disodium hydrogen

phosphate Na2HPO4.2H2O (1.13g) dissolved in 200ml of distilled water and sterilized

by autoclaving at 12101C for 15mins. A 0.5% w/v filter-sterilized potassium cyanide

(KCN) was added to the medium after it had cooled. The broth was dispensed into

different tubes in 10ml aliquots. The KCN medium was inoculated with an isolate and

incubated at 370C for 48hr. A positive result was indicated by the observation of

turbidity in the tubes.

29

3.7.8 Triple Sugar Iron (TSI) Test:

These are carbohydrate-containing screening media used to identify the ability

of gram-negative bacilli to ferment these carbohydrates (glucose, sucrose and lactose)

and to produce hydrogen sulphide (H2S). This was prepared according to the

manufacturer’s instructions by weighing 6.46g in 100ml distilled water. It was heated

to homogenize the solution. After cooling, it was dispensed into test tubes such that

the volume of the medium was sufficient to give a deep butt and a long slant. These

were autoclaved at 1210C for 15min. There were left in a slanted position to cool. The

slants were inoculated by streaking the surface with an isolated colony from a plate,

and without removing the inoculating loop; the butt was stabbed from top to bottom.

These tubes were incubated at 370C with caps slightly loosed and the reactions were

read within 24hours. The results were noted by observing the different colors of the

butt and slant, production of hydrogen sulphide (H2S) by black precipitate and

formation of gas by cracks or bubbles.

3.7.9 Sugar Fermentation Test:

The tests were done to determine the ability of the isolates to metabolize

sugars with the production of acid with or without gas. Phenol red peptone water was

prepared, this consisted of peptone 10g, sodium chloride 5g, and Phenol Red indicator

0.025g, to make up to 1000ml. 15g of each sugar was introduced into 1000ml of the

phenol Red peptone water. These were dispensed into tubes with inverted Durham’s

tubes. The tubes were autoclaved at 1210C for 15min, and allowed to cool. The

isolates were introduced into the different types of sugar and incubated at 370C for

5days. Acid production was indicated by change in color (i.e. from red to yellow),

while gas production was observed by the downward displacement of the liquid in the

Durham’s tubes.

N.B: The sugars used for this test are Lactose, Sucrose, D-Sorbitol, Raffinose,

Mannitol, Inositol and Inulin.

3.8 Characterization of Fungal Isolates:

A slide culture of the isolates was prepared by stab-inoculating the isolate on

the sides of a cut out piece of Sabouraud Dextrose agar which was later incubated at

300C for 24hrs. The isolates were stained using lactophenol cotton blue as the staining

agent and viewed using both 10 x and 40 x eye piece lens of the microscope. The

30

colonial morphology was taken note of and compared to known representative fungal

groups according to Pitt and Hocking (1997).

Yeast Identification Tests: The isolates identified as yeast cells were further

characterized using the fermentation and assimilation patterns of these sugars:

Dextrose, Maltose, Sucrose, Lactose, Galactose, as well as the assimilation of

Potassium nitrate (KNO3). This was according to Collins and Lyne (1970).

3.9 Survival and Multiplication of Salmonella and Shigella organisms in Cassava

Waste water.

3.9.1 Preparation of the Plant Materials:

The two varieties of cassava, NR8082 and TMS92/0326 were peeled, washed

and grated. The resultant pulp was put into sterile sieve bags and kept in clean buckets

with lids. These were left to ferment for five days.

3.9.2 Purification of the Cassava Waste Water:

The cassava waste water was collected aseptically and centrifuged at 3,000

rpm for 1hour. It was filtered using Whatman’s paper filters to remove as much solute

as possible. It was further purified using Whatman membrane filters (0.45 �).

3.9.3. Sample Preparation for bacterial enumeration:

i. McFarland Nephelometer standards: A 0.5 McFarland was prepared by

reacting 0.05ml of 1% aqueous solution of Barium chloride and 9.95ml of 1%

chemically pure Sulphuric acid. Slowly and with constant agitation, these solutions

were poured one after the other into a clean test tube and covered with a lid. It was

labeled 0.5 McFarland. The density of the turbidity standard of the tube was verified

using a spectrophotometer to determine absorbance at 625nm.

ii. Preparation of bacterial Isolates: The bacterial isolates, Salmonella typhi and

Shigella dysenteriae were plated out on Xylose lysine Deoxycholate agar (Oxoid),

and incubated at 370C for 24hrs. The isolated colonies were taken and emulsified into

sterile normal saline in test tubes until they matched the 0.5McFarland standard.

31

3.9.4 Sample Preparation for Absorbance Readings:

The purified cassava wastewater was aseptically dispensed into sterile bijou

bottles in 5ml aliquots. The 0.5McFarland standard inoculum of the test organism was

added in a 5ml aliquot into each of these test tubes and they were labeled NRST

(containing NR8082 cassava waste water & Salmonella typhi), NRSHD (containing

NR8082 cassava waste water & Shigella dysenteriae), TST (containing TMS92/0326

cassava wastewater & Salmonella typhi) and TSHD ( containing TMS92/0326

cassava wastewater & Shigella dysenteriae). These test tubes were incubated at 370C

for 5 days. The initial absorbance reading was taken at 0hr and the rest at 24, 48, 72,

96 and 120hr at 625nm wavelength.

3.10 Proximate Composition of Samples:

The proximate composition of the two varieties of cassava waste water

(NR8082 and TMS 92/0326) were determined as well as that of wastewater obtained

from Ibagwa site 1 and site 2. This is actually the percentage concentrations of the

crude protein, crude lipid, crude carbohydrate, crude fibre, moisture and ash and these

were determined according to A.O.A.C. (1990).

Statistical Analysis: One way ANOVA was carried out to compare the means of

different treatments where significant F values at p<0.05 were obtained. Differences

between individual means were tested using Fischer’s Least Significant difference

(F_LSD) test.

32

4.0. RESULTS

4.1. Isolation of Salmonella and Shigella organisms from the different

processing sites.

From the waste water in the processing sites, various bacterial contaminants

were isolated and these are distributed as shown in Table 1. The frequency of

occurrence of the isolates in each processing site is shown in Figure 2, the highest

being from Ibagwa 2 (31.25%) and Ibagwa 1 (27.08%) being the next while the least

occurrence were from Orba 1 (6.25%) and Ugwunkwo (6.25%). The distribution of

each isolate is shown in Figure 3 where the highest occurrence is in the group of other

Citrobacter spp. (20.83%) and the lowest occurrence in the Yersinia spp. (2.08%)

group. Salmonella spp. has an occurrence of 10.42% while that of Shigella spp. is

4.17%.

4.2. Proximate Analyses, pH and Microbial Population of Cassava Waste

water.

From the waste water collected in the cassava processing sites (Ibagwa site1& Ibagwa

site 2), the following parameters were checked for; pH, proximate composition and

the presence of microorganisms. The pH readings of the waste water and the

proximate composition are shown in Table 2 while the predominant microorganisms

isolated from the two processing sites are shown in Tables 3 and 4 respectively. The

most frequently isolated microorganism from Ibagwa site 1 is Bacillus spp. (31.25%)

while that from Ibagwa site 2 is Corynebacterium spp. (40.38%).

4.3. Soil microorganisms with capacity to utilize cyanide isolated from the

cassava processing sites.

This experiment was carried out in two sections.

Section 1: Soil Microorganisms isolated using a medium containing high cyanide

(NR8082) cassava wastewater.

Cassava species can be categorized into those with high cyanide and those with low

cyanide content. Cyanide is a respiratory poison for most organisms including

bacteria and it is assumed that only those that can metabolise cyanide would be able

to utilize cassava with high cyanide content. So, inocula were drawn from soil sample

cultured in basal medium supplemented with high cyanide cassava NR8082 after

incubation at 37oC for 24hr and 48hr successively. Inoculum taken at 24hr interval

33

yielded 4 x 102 cfu of Citrobacter diversus and 3.9 x 102cfu of Candida pelliculosa

and no other organism. The inoculum taken at 48hr interval from the same experiment

yielded 3.0 x 102cfu of Candida pelliculosa only (Table 5). Similar experiment with

soil sample from Ibagwa 2 Cassava Processing Site yielded 3 organisms, namely

E.coli (60cfu), Bacillus spp. (70cfu) and Candida pelliculosa (130cfu) at 24hr

interval. At 48hr interval, only Candida pelliculosa (250cfu) was present (Table 6).

34

Table 1. Biochemical Reactions of the Different Bacterial Contaminants.

Bacterial Motility Indole Citrate Hydrogen Urease Lysine Growth D-Glucose Lactose Sucrose D-Mannitol D-Sorbitol Raffinose Inositol Total Contaminants (36C) production (Simmons) sulphide (TSI) hyd. decarb. in KCN (acid) fermentation showing characteristics Arizona spp. + - + + - + - + + - + + - - 4

Salmonella spp. + - + + - + - + - - + + - + 5

Edwardsiella spp. + + - + - + - + - - - - - - 5

Citrobacter fruendii + - + + w - + + + + + + + - 4

Other Citrobacter + + + - + - + + + + + + - - 10

Proteus spp. + -/+ + + +/- - + + - + +/- - - - 2

Escherichia coli + + - - - + - + + + + + + - 3

Shigella spp. - -/+ - - - - - - - - + + + - 2

Klebsiella spp. - -/+ + - - - + + + + + + + + 2

Enterobacter spp. + - + - w w + + + + + + + + 5

Serratia spp. + - + - w w + + +/- + + + -/+ + 3

Yersinia spp. - - - - +/- - - - - - + + - - 1

KEY: + means 90% or more positive within 48hr - means less than 10% positive within 48hr w means weak reaction +/- means some strains may be positive while some be negative Source: Bergey’s Manual of Determinative Bacteriology, 1974.

35

36

37

Table 2. pH and Proximate Composition of the waste water

Proximate Analysis Component Ibagwa1 (%) Ibagwa 2 (%) pH 7.60 5.10 Moisture 91.7 90.5 Ash 0.6 0.8 Fat trace trace Fibre trace trace Protein 0.91 0.38 Carbohydrate 6.79 8.32

38

Table 3. Frequency of Isolation of Microorganisms from Ibagwa 1 Cassava Processing Site.

Microbial population Number of isolates

Bacillus spp. 35 Pseudomonas spp. 25 Aspergillus spp. 20 Candida pelliculosa 18 Citrobacter spp. 8 Morganella morganii 5 Edwardsiella tarda 4 Salmonella spp. 1 Arizona spp. 2 Citrobacter fruendii 1 Proteus spp. 2 Klebsiella spp. 1 Enterobacter spp. 1

39

Table 4. Frequency of Isolation of Microorganisms from Ibagwa 2 Cassava Processing Site.

Microbial population Number of isolates Corynebacterium spp. 42 Aspergillus spp. 30 Torulopsis glabrata 16 Enterobacter spp. 11 Proteus spp. 6 Hafnei alvei 3 Edwardsiella tarda 1 Salmonella spp. 3 Arizona spp. 2 E. coli 1 Shigella spp. 1 Klebsiella spp. 1 Serratia spp. 1

40

Section 2: Soil Microorganisms isolated using a medium whose pH was adjusted

to 3.0.

When the experiment was repeated in a medium set at an initial p H of 3.0 and inocula

drawn at intervals of 24, 48 and 72 hrs, the succession of organisms are as shown in

Table 8 (for Ibagwa 1 Soil Sample) and Table 9 (for Ibagwa 2 Soil Sample) with the

given number of organisms for each time interval.

4.4. Determination of Microbial Succession during the fermentation for Garri

production.

The pH of fermenting cassava was monitored for 28 days to observe the changes. The

pH reduced gradually until the 7th day (pH 3.70) and this remained constant until the

20th day before it slightly increased to pH 4.00 which also remained constant until the

end of the 28th day. The results are plotted in Figure 4. During the fermentation

process, the waste water was plated out at every 24 hr interval to determine the

microbial population with the various pH changes. The results as presented in Table

10 shows Candida spp., as being constantly isolated until it yielded Bacillus spp. and

Corynebacterium spp.

4.5. Proximate Analysis of Cassava Variety Used for Experimental

determination of Salmonella and Shigella in fresh Waste water Sample.

The cyanide content of the two varieties of cassava (NR8082 and TMS 92/0326)

obtained from National Root and Crop Research Institute, Umudike, was analyzed

and the result is shown in Table 11, with NR8082 having the higher proportion of

HCN content. The percentage proximate composition of the two cassava varieties are

also in Table 11. During the fermentation process, the pH was observed to decline as

the time increases and these changes are plotted in Figure 5.

4.6. Survival and Amplification of Salmonella and Shigella organisms in Cassava

Waste water.

The changes in the survival and amplification of known strains of Salmonella

typhi and Shigella dysenteriae in the waste water of these two varieties of cassava

were monitored using a spectrophotometer at a wavelength of 620nm. The results as

plotted in Figures 6 to 9 showed a rapid increase before steady decline with the

41

exception of Salmonella in TMS 92/0326 which had a different growth pattern

showing a continuous growth by the end of the 120th hour period.

42

Table 5. Microbial population in the Soil from Ibagwa 1 Cassava Processing Site.

Time Interval Microorganisms Colony count (cfu) at which inoculum detected of Isolates obtained was tested(hr) 24 Citrobacter diversus 4.0 x 102 Candida pelliculosa 3.9 x 102

48 Candida pelliculosa 3.0 x 102

43

Table 6. Microbial population in the Soil from Ibagwa 2 Cassava Processing Site. Time Interval Microorganisms Colony count (cfu) at which inoculum detected of Isolates obtained was tested(hr)

24 E.coli 60 Bacillus spp. 70 Candida pelliculosa 130 48 Candida pelliculosa 25

44

Table 7. pH changes at the different time interval.

Sample Source pH

Time intervals (hr) 0 24 48 72

Ibagwa 1 3.00 4.80 7.10 8.00 Ibagwa 2 3.00 5.20 7.60 8.40

45

Table 8. Microbial population in the Soil Sample of Ibagwa 1 After acidic pH Treatment.

Time Interval Microorganisms Colony Counts(cfu) at which inoculum detected of Isolates obtained was tested(hr) 24 Arthrobacter spp. 3.5 x 102

Bacillus spp. 4.3 x 102

Candida pelliculosa 3.0 x 102

Candida tropicalis 2.6 x 102

48 E. coli 3.1 x 102

Staphylococcus spp. 1.8 x 102

Candida guillermondii 2.1 x 102

72 Corynebacterium spp. 2.7 x 102

Trichosporon cutaneum 1.3 x 102

Torulopsis glabrata 1.6 x 102

46

Table 9. Microbial population in the Soil Sample of Ibagwa 2 after acidic pH Treatment.

Time Interval Microorganisms Colony count (cfu) at which inoculum detected of Isolates obtained was tested(hr) 24 Bacillus spp. 4.8 x 102

E.coli 4.1 x 102

Salmonella spp. 50 Candida pelliculosa 1.1 x 102

Candida tropicalis 1.4 x 102

48 Candida pelliculosa 4.8 x 102

72 Micrococcus spp. 3.3 x102

47

48

Table 10. pH Changes and the microorganisms associated with the Fermentation of the waste water.

Fermentation period (Days) p H Organisms detected

1 6.84 Candida pelliculosa Candida tropicalis 2 5.10 Candida pelliculosa Candida tropicalis 3 4.61 Candida pelliculosa 4 4.36 Candida pelliculosa Candida tropicalis 5 4.10 Candida krusei 6 4.00 Bacillus spp. 7 3.70 0 8-19 3.70 0 20 4.00 Corynebacterium spp. 21-28 4.00 Corynebacterium spp.

49

Table 11. Proximate Composition and Cyanide content of the Resultant waste water.

Component Proximate Analysis NR8082 (%) TMS 92/0326 (%) Moisture 92.20 93.91 Ash 0.52 0.21 Fat 0.30 0.50 Fibre Nil Nil Protein 0.70 0.79 Carbohydrates 6.28 4.78 Cyanide content

( % HCN/100ml) 0.0766 0.0183

50

51

52

53

54

55

5.0. DISCUSSION

The processing of garri from cassava generates a huge amount of waste water,

which is usually discharged into the environment with little or no treatment. The site

where the processing is done is often polluted by putrid waste water that later gives

concern to the neighborhood. The offensive odor results from the fermentation, that

is, the microbial breakdown of the waste water. This instantly suggests that

microorganisms would survive or thrive in the waste water. When this waste water is

left in pools in the environment, there would be contamination as a result of the

unhygienic lifestyle of the people around, the type of toilet facilities used and even the

rainfall which washes feacal materials from man and animals into the waste water

pool.

Evaluation of the different cassava processing sites (Table 1) showed that

Salmonella, Shigella and other pathogens were present as contaminants. The presence

of E.coli shows that there was feacal contamination in some of these cassava

processing sites. Surface run-off water during the rains in these areas is presumably

responsible for washing materials into the pits meant for the cassava waste water.

Therefore, the microorganisms, Salmonella and Shigella, may be contaminants from

poorly disposed human faeces. The results in Figure 2 show that most of the

pathogens were isolated from Ibagwa sites 1 and 2. Analysis of waste water from

these two cassava processing sites show the pH of Ibagwa site 1 to be 7.60 and that of

Ibagwa site 2 was 5.10. The acid nature of Ibagwa site 2 could be due to the higher

level of activities taking place there, hence continuous introduction of large volumes

of new waste water into the pool.

The microbial flora encountered from waste water at these sites show that the

Enterobacteria were surviving alongside others. The microbial population, as shown

in Tables 3 and 4, indicate that some isolates were present in one site but absent in the

other. Determination of pH at different points in each pool showed that some spots

tested alkaline and some neutral or acid depending on the charge of new effluent of

the cassava waste water that got to that spot. The spots that tested alkaline were areas

that the Salmonella and Shigella contaminants would possibly thrive. According to

Jay (1986), the minimum pH tolerance for Salmonella and Shigella spp. is 4.5 while

the maximum is 8.0. The pH of the cassava processing sites was found to be within

56

this range. The Salmonella and Shigella would grow within the neutral to alkaline pH

range.

The proximate analysis of waste water in this study showed presence of

carbohydrates, lipids, and proteins and these are good sources of carbon and nitrogen

for microbial growth. The limiting factors to the growth of microorganisms in cassava

waste water presumably include the presence of cyanide and the acidic pH of the

waste water among other things. From these results, Salmonella and Shigella will

survive when a) other microorganisms start the fermentation and incidentally raise the

pH. b) the apparent toxicity of the cyanide content is overcome by Salmonella,

Shigella and other microorganisms which are capable of breaking down this substrate.

c) the pH of 7.60 observed with waste water in Ibagwa site 1 may not result only from

microbial breakdown activity but may show that extraneous substances flowing into

the waste water pool provided a buffering effect.

Cassava species contain varying amounts of cyanide (15 to 400 mg HCN/I

kg); and cyanide is known to exert effects on the respiratory processes of some living

things including microorganisms. For any organism including Salmonella and

Shigella to survive this effect, it has to possess a capacity to utilize cyanide. From the

results in Tables 5 and 6, not only Salmonella and Shigella but other microorganisms

which are capable of utilizing cyanide were present in the waste water samples. So,

cyanide would not constitute a hindrance to the survival of Salmonella and Shigella in

the cassava waste water because they have the capacity to breakdown and utilize it.

Bacillus spp., Pseudomonas spp., Aspergillus spp., Candida pelliculosa, Citrobacter

spp. and Morganella morganii were present in Ibagwa site 1 while Ibagwa site 2 had

Corynebacterium spp., Aspergillus spp., Torulopsis glabrata, Enterobacter spp.,

Proteus spp. and Hafnei alvei, all of which have variously been cited to utilize

cyanide (Baxter and Cumming, 2006). Although these microorganisms were not

among those encountered in the laboratory other studies have shown that Bacillus

spp., Pseudomonas spp., Corynebacterium spp., Proteus spp., Citrobacter spp.,

Enterobacter spp., Aspergillus spp., Candida pelliculosa, Torulopsis glabrata have all

been associated with fermentation of cassava (Roger et al., 2007; Amoa-Awua et al.,

1997; Okafor, 1977; Anike and Okafor, 2008; Oyewole and Odunfa, 1988;

Oguntoyinbo, 2007; Oyewole, 2001). The presence of Morganella morganii and

Hafnei alvei, which were detected in this work have not been recorded in any other

57

study unless they were among the unspecified enterobacteria and coliforms

documented by Roger et al. (2001).

It was observed that freshly obtained cassava waste water was acidic. This is

not the usual pH for growth of most microorganisms and hence would mitigate the

survival and growth of most microorganisms. However, experiments showed that

various microorganisms would tolerate this pH; thus the latter would begin the

breakdown of fresh cassava waste water and alter the pH to favour succession by

others as shown in Tables 7, 8 and 9. These other microorganisms raise the pH to the

level where Salmonella and Shigella would thrive. A physiological study of the

survival of soil microorganisms at pH levels within the range found in cassava waste

water showed that microorganisms such as Citrobacter diversus, E.coli and Bacillus

spp. appeared after 24 hr of incubation while Candida pelliculosa appeared after 48

hr. It was not quite surprising to find these bacterial strains in the cassava processing

site because they have been implicated in cassava fermentation as was mentioned

earlier. Since these microorganisms survived most in the cyanide-enriched medium,

they could have started the fermentation process.

When the experiment was repeated by adjusting the pH of the medium to 3.0,

there were observable changes in the pH during the period of incubation. The soil

sample from Ibagwa 1 had an increase to a pH of 4.80 within 24 hours and yielded

Bacillus spp., Arthrobacter spp., Candida pelliculosa, and Candida tropicalis. After

48 hours incubation, the pH rose to 7.10 and the medium yielded E. coli,

Staphylococcus spp., and Candida guillermondii; and in 72 hours the pH increased

further to 8.00 and the microorganisms present were Corynebacterium spp.,

Trichosporon cutaneum and Torulopsis glabrata. The soil sample from Ibagwa site 2

also had increase of pH to 5.20 after 24 hour incubation, yielding Bacillus spp., E.coli,

Salmonella spp., Candida pelliculosa and Candida tropicalis. The pH increased to

7.60 in 48 hours and yielded Candida pelliculosa only while after 72hr; it increased

further to 8.40 and yielded Micrococcus spp. only. This notable and steady increase

in the pH of the media containing the soil samples yielded different groups of

microorganisms. Therefore, those microorganisms initially isolated could have begun

the fermentation process and with the breakdown, the pH increased to favour growth

of other microorganisms. Interestingly, the consortium of microorganisms isolated

from the soil samples have been implicated in cassava fermentation in other studies

58

(Oguntoyinbo, 2007 & Coulin et al., 2006) and have also been involved in cyanide

degradation (Baxter and Cumming,2006).

Microbial succession in the fermentation process was investigated. Those

microorganisms which started the breakdown gave way to others (Table 10). This also

shows that microorganisms survived at different pH levels. During the cassava

fermentation, the pH was observed to decline from 6.84 to 3.70 by the 7th day of

fermentation; this remained constant until the 20th day when this pH increased to 4.00

and was constant until the 28th day of fermentation (Figure 3). This probably

explained why traditional fermentation of cassava is said to take four to six days in

order to effect sufficient detoxification of the roots (Kimaryo et al, 2000;

Oguntoyinbo, 2007). This makes the fermented food safe for human consumption

due to a reduction of pH, linamarase activity, total cyanide levels and increase in acid

levels (Caplice and Fitzgerald, 1999). Moreover, the longer the fermentation period,

the less the residual cyanide content of the final fermented products (garri)

(Odoemelam, 2005).

The microbial succession study for cassava fermentation showed a

predominance of the yeasts from the first day of the fermentation (pH 6.84) to the

fifth day (pH 4.10). This could be attributed to the yeasts’ low metabolism which

allows them to grow at even low pH imposed by the lactic acid bacteria

(Oguntoyinbo, 2006). The coexistence and positive interactions involving yeasts and

lactic acid bacteria in fermented cassava have also been documented by Mante et al.,

(2003). Candida krusei and Candida tropicalis have been reported to help in the

modification of cassava texture during fermentation, they have been found to exhibit

linamarase activity and are capable of breaking down the cyanogenic glycosides

present in cassava (Amoa-Awua et al., 1997). The yeasts presumably were involved

in breaking down the starch into simpler sugars utilized by the bacteria. There was

absence of growth in the different plating media used from the 7th day to the 19th day

of fermentation which had a pH of 3.70 (Table 10). This can be attributed to the low

pH in which most microorganisms cannot survive. The presence of Bacillus spp. and

Corynebacterium spp. was observed at pH 4.00 and these have been considered to

play important roles in the fermentation process (Oyewole and Odunfa, 1988).

Experimentally, the survival and amplification of Salmonella typhi and

Shigella dysenteriae in the cassava waste water in the absence of other microbial

interactions was investigated. The cyanide content of two known varieties of cassava

59

was determined and the result showed NR8082 to be higher than TMS 92/0326 (Table

11). The fermentation process for the two known varieties of cassava was carried out

for five days. The results showed decrease to acidic pH for both varieties of cassava

(Figure 4). The results presented in the Figures 6 to 9 showed these microorganisms

grew to a peak at about 60hr and then declined in population. The observed growth in

these studies could be due to the presence of trace nutrients which helped them to

survive but when this was depleted, their growth declined. Salmonella typhi in TMS

92/0326 (Figure 8) had a continuous increase in growth up to the 120th hr of

incubation. The same test organism was observed to decline in growth in NR8082,

while the other test organism, Shigella dysenteriae showed decline in growth in both

NR8082 and TMS 92/0326 waste water. Apparently, the NR8082 variety of cassava

with the higher cyanide content could not support the amplification of the test

organisms for a longer period of time. It can be said that the presence of cyanide in

waste water was an inhibitory factor to the amplification of the pathogens. Likewise,

the absence of other contaminants present in the environment play a major role in

inhibiting the amplification of these test organisms in the purified waste water.

Although the acid nature of the cassava waste water will probably mitigate the growth

and survival of most microorganisms. The presence of other microorganisms can raise

the pH and also utilize the cyanide, thereby encouraging the survival of the pathogens.

These other microorganisms presumably originated from the soil, water, and other

external sources like the activities of man and animals in the surroundings. Therefore,

the ultimate survival of Salmonella and Shigella depends on the presence of other

organisms which will break the cyanide and raise the pH.

The main objective of this work was to evaluate cassava waste water as a

possible medium for multiplication and a vehicle for dissemination of pathogens

particularly Salmonella and Shigella which are known to spread through contaminated

water, vegetables, etc. From the results shown in Table 1, and Figure 2, though,

Shigella is said to remain viable for a limited time outside the human body

(Niyogi,2005), other studies have revealed its ability to be viable when inoculated on

some materials (Islam et al., 2001). Therefore, its presence in the environment is of

public health significance. The presence of other gram-negative bacteria in these

processing sites should not be overlooked. Some of them like the Citrobacter spp.,

Proteus spp., E. coli, Klebsiella spp., Enterobacter spp., Serratia spp., Yersinia spp.,

and Pseudomonas spp. have been reported to be pathogenic to man; most being agents

60

of diarrheal diseases, urinary tract infections and nosocomial pathogens (Murray et

al., 1998). Where hygiene is poorly maintained, contamination of food and drinking

water will be very common.

It is apparent however, that a variety of environmental factors maybe

implicated in the survival of Salmonella and Shigella pathogens in the environment.

The cassava waste water is usually not treated before being discharged into the

environment, so when left to lie in the environment, other contaminants come in to

render it suitable for microbial proliferation. Consequently, animal droppings and

human wastes are usually washed off by surface water and the likelihood of this

mixing with the waste water is high. The putrid nature of the waste water attracts

houseflies to it. Levine and Levine (1991) have shown that Salmonella typhimurium

and Shigella can multiply in the gut of the housefly and can be excreted for weeks or

longer. For Salmonella spp., animal products have been reported as the vehicle of

transmission (Lewis, 1998, Murray et al.,1998) as well as infected animals especially

cats, swine and dogs (W.H.O.,2005). Occasionally, contaminated food and water may

be the vehicle of transmission (A.P.H.A., 1992). This means that sources of water

supply in the community if not properly taken care of can be contaminated.

The presence of Salmonella and Shigella pathogens in the processing sites

pose a serious health hazard to man. Infections caused by these pathogens have been

reported to be the major cause of morbidity and mortality (Vargas et al., 2004).

Moreover, their possession of multi-drug resistant traits also complicates the

treatment procedures (Brooks et al., 2003; Iwalokun et al., 2001 & Wang et al.,

2005). Every likely means of their transmission should be discouraged. Cassava waste

water should therefore, be considered as a means of further spread of these pathogens

and adequate attention should be given to its discharge in the environment.

Therefore, cassava waste water left in the environment to ferment is a potential

medium for Salmonella and Shigella and such potential medium should be handled as

human waste. People involved in cassava processing should use septic tanks to get rid

of the offensive odor as well as the microorganisms involved. This environmental

perspective must be taken into consideration in formulating measures to control

diarrheal diseases in Nigeria and other parts of the world.

61

CONCLUSION

From the result of this work, it is concluded that:

• Some cassava processing sites were contaminated with Salmonella and

Shigella showing that the organisms survive and multiply in the waste water.

• The presence of other pathogenic microorganisms in these sites makes them

likely sources of spread of infections.

• When cassava waste water disposal sites are not properly treated and

inspected, vectors such as flies can breed and spread the pathogens.

• Foods such as vegetables should not be cultivated near such sites to avoid their

contamination with these pathogens.

• The fermentation of cassava up to five days should be encouraged since this

helps in making the fermented food (garri) much safer for consumption.

• Cassava processing sites should not be located near living quarters.

62

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APPENDICES

Appendix 1. pH changes of the two varieties of Cassava with time.

Fermentation p H Readings

Duration (hr) NR8082 TMS92/0326

0 4.43 6.65

24 4.33 3.95

48 4.47 3.96

72 4.32 3.86

96 4.57 3.83

120 3.98 3.36

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Appendix 2. Absorbance Readings (_nm) at different Time intervals. Sample Time intervals (hr) 0 24 48 72 96 120

NRST 0.048 0.108 0.140 0.139 0.122 0.098 NRSHD 0.058 0.148 0.140 0.163 0.169 0.124 TST 0.040 0.098 0.111 0.135 0.124 0.172 TSHD 0.060 0.160 0.238 0.173 0.147 0.133

KEY: NRST = Salmonella typhi in NR8082 Cassava waste water. NRSHD = Shigella dysenteriae in NR8082 Cassava waste water. TST = Salmonella typhi in TMS 92/0326 Cassava waste water. TSHD = Shigella dysenteriae in TMS 92/0326 Cassava waste water.