ANTIBIOTIC SUSCEPTIBILITY PATTERN OF E. COLI AND SALMONELLA ISOLATED FROM PORK INTESTINE IN UMUAHIA, ABIA STATE

ABSTRACT

The susceptibility patterns of E. coli and salmonella species isolated from pork intestine in Umuahia, Abia state, were used for this research work. A total of 49 samples were collected, 47 isolates were positive for E. coli while 29 isolates were positive for Salmonella species. The Kirby Bauer disc diffusion method was used for the antibiotic susceptibility test. A multi disc containing eight different antibiotic, Ceftazidime (CAZ), Cefuroxime (CRX), Gentamicin (GEN), Cefixime (CXM), Ofloxacin (OFL), Augmentin (AUG),Nitrofuratoin (NIT) and Ciprofloxacin (CPR) were used for this study. E.coli had the highest susceptibility (93.6%)) to Ofloxacin followed by Gentamycin (87.2%). All the E. coli isolates were resistant to Cefixime and Augmentin. For Salmonella species, the highest susceptibility was recorded against Gentamycin (68.9%) while the least susceptibility was recoreded against Nitrofuratoin (0%). The highest resistance was recorded against Ceftazidime and Cefixime with 44.8% each. All the Salmonella isolates were resistant to more than one antibiotic confirming their multi drug resistance. This result has public health significance with Salmonella and E. coli infections being zoonotic in nature

TABLE OF CONTENTS

Title page                                                                                                                                i

Certification                                                                                                                            ii

Dedication                                                                                                                              iii

Acknowledgement                                                                                                                  iv

Table of contents                                                                                                                    v

List of tables                                                                                                                           viii      

Abstract                                                                                                                                  ix

CHAPTER ONE

1.1       Introduction                                                                                                                1

1.2       Aims and Objectives                                                                                                   2

1.3       literature Review                                                                                                         2

1.4       History of E. coli                                                                                                        3

1.5       Diversity                                                                                                                     5

1.6       Serotype                                                                                                                      5                          

1.7       Genome plasticity                                                                                                       5

1.8       Neotype strain                                                                                                             6

1.9       Phylogeny                                                                                                                   6

1.10     Roles as Normal Microbiota                                                                                       6

1.11     Role in Disease                                                                                                           7

1.12     Uses of Non Pathogenic E. coli                                                                                  7

1.13     Salmonella                                                                                                                  7

1.13.1  History of Salmonella.                                                                                                8

1.13.2  Salmonella Nomenclature                                                                                           8

1.13.3  Salmonella as Disease Causing Agent                                                                        9

1.13.4  Durability                                                                                                                    11

1.13.5  Source of Infection                                                                                                     11                 

CHAPTER TWO

2.1       Collection of Sample                                                                                                  13

2.2       Isolation and Identification of Isolates                                                                      13

2.3       Isolation and Confirmation of Salmonella                                                                 13

2.4       Procedure for Media Preparation                                                                                14

2.5       Biochemical Test and Procedures                                                                               14

2.6       Antibiotic Susceptibility Test                                                                                     16

CHAPTER THREE                                                                                                                              

Results                                                                                                                                    18

CHAPTER FOUR:

4.0       Discussion                                                                                                                   22 

4.1       Conclusion                                                                                                                  23

4.2       Suggestion for Further Studies                                                                                   23

LIST OF TABLES

Table                                                              Title                                                                       Page

1.               Identification and Characterization of the Isolates                                                        19

2.               The Percentage Occurrence of the Sensitivity and Resistance of E. coli                       20

           Isolates                                                                                                                       

3.               The Percentage Occurrence of the Sensitivity and Resistance Pattern of                     21

           Salmonella spp.                

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1       Introduction

Pigs are a major reservoir of bacterial zoonotic pathogens of which E. coli and Salmonella are the major occurring bacteria (Mc Dowell et al., 2007). Except for self consumption and scientific research, pigs are slaughtered at slaughter houses which are sometimes contaminated with the fecal material. E. coli and Salmonella belong to a group of prioritized, extended list of food and water-borne zoonoses (Cardoen et al., 2009) making it necessary for food safety authorities to focus on them as most relevant hazards in the food chain.

Food-borne diseases are an important cause of morbidity and mortality worldwide.  The contamination of meat with pathogens constitutes a major public health concern (Cohen et al., 2007). In Nigeria, processing procedures and monitoring of critical points in the meat production are not fully developed. Abattoir has become a source of infection and pollution attracting domestic and wild carnivores and rodents due to adequate slaughtering and disposal facilities (Adeyemo, 2002). Apart from incorrect processing procedures, marketing practice of meat consumed by most populace is a major area of concern (Okoli et al., 2006a).

However, previous studies have documented contamination of raw meat by bacterial pathogen (Uche and Agbo, 1985). Epidemiological reports suggest that of diarrheal illness which account for 36% of mortality cases in Nigeria (FAO/WHO, 2002).

Food contamination with antibiotic resistant bacteria can also be a major threat to public health. This is because the antibiotic resistance determinant can be transferred to other pathogenic bacteria, potentially compromising the treatment of severe bacterial infections. The susceptibility pattern and the prevalence of antimicrobial resistance among food borne pathogens have increased during recent decades (Van et al., 2007). This increase is attributed to the selection pressure created by using antimicrobial in food producing animals in addition to unregulated use of antibiotics by humans in developing countries (Vanden Bogard et al., 2000).

1.2       Aims and Objectives

The aim and objective of this study is:

·                     To investigate the prevalence of E. coli and Salmonella isolated from pork intestines in Umuahia, Abia State.

·                     To determine the antibiotic susceptibility pattern of the E. coli and Salmonella isolates.

1.3       Literature Review

E. coli is a gram-negative, facultative anaerobic, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms).The cells are about 2.0 microns (µm) long and 0.5 µm in diameter with a cell volume of 0.6-0.7µm (Kubitschek, 1990). It can live on wide variety of substrates. E. coli uses mixed acid fermentation in anaerobic conditions. Since many pathways in mixed-acid fermentation produce hydrogen gas, these pathways require the levels of hydrogen to be low, as in the case when E. coli lives together with hydrogen consuming organisms such as methanogens or sulphate reducing bacteria (Madigan and Martinko, 2006). The optimal growth of E. coli occurs at 37⁰C (98.6⁰F) but some laboratory strains can multiply at temperatures up to 49⁰C (120.2⁰F) (Fotador et al., 2005). Strains that possess flagella are motile. Their flagella have a peritrichous arrangement (Darnton et al., 2007).

 Most E. coli strains are harmless but some serotypes can cause serious food poisoning in humans and are occasionally responsible for product recall due to food contamination and hazard. The harmless strains are part of the normal flora of the gut, and can benefit their host by producing Vitamin K2 (Bentley et al.,, 1982), and by preventing the establishment of pathogen bacteria within the intestine (Hudault et al., 2001).

E.coli and related bacteria constitute about 0.1% of gut flora (Eckburg et al., 2003), and fecal oral transmission is the major route through which pathogenic strains, which are strains that have acquired certain genetic materials the body for a limited amount of time, which makes them ideal indicator organisms to test environmental samples for fecal contaminations (Feng et al., 2002). There is however a growing body of research that has examined environmentally persistent E. coli which can survive for extended period outside the host (Ishii and Sadowsky, 2008).

The bacterium can also be grown easily and inexpensively in a laboratory setting, and has been intensively investigated for over 60 years. E. coli is the most widely studied prokaryotic model organisms and an important species in the fields of biotechnology and microbiology where it has served as the host organism for the majority of work with recombinant DNA.

1.4       History of E. coli

The genera Escherichia and Salmonella diverged around 102 million years ago, which coincides with the divergence of their host: the former being found in mammals and latter in birds and reptiles (Battlistuzi et al., 2004). This was followed by a split of the Escherichian ancestor into five species (E. alberti, E. coli, E. Fergusonii, E. hermannii and E. vulneria).

In 1885, a German Pediatrician called Theodor Escherich, discovered this organism in the faeces of healthy individuals and called it “Bacterium coli commune” due to the fact it is found in the colon and early classification of prokaryotes placed these genera based on their shape and motility (Haeckel et al., 1867), during this period, Erbst Haeckel’s classification of bacteria in the kingdom monera was in place. Bacterium coli was the type species of the now invalid genus bacterium when it was revealed that the former type species “Bacterium triloculare” was missing following the reclassification as Bacillus coli by Migula in 1895 and later reclassified in the newly created genus Escherichia named after its original discoverer (Castellani and Chalmers, 1919). The genus belongs in a group of bacteria informally known as “coliforms” and is a member of the Enterobacteriaceae family (enterics) of the gamma proteobacteria (George and Garrity, 2005).

Certain strains of E. coli are a major cause of food borne illness (diarrheal illness) such as enterotoxigenic E. coli (ETEC), enteropothogenic E. coli (EPEC), entero hemorrhagic E. coli (EHEC) also called shiga toxin producing E. coli or (STEC), enteroaggregative E. coli (EAEC or EAggEc), however E. coli (EHEC) bacteria can lead to hemolyticurenic syndrome (HUS), a medical emergency that requires urgent treatment (Rohde et al., 2011).

E. coli and related bacteria posses the ability to transfer DNA via bacteria conjugation, transduction or transformation, which allows genetic material to spread horizontally through an existing population. This process of the gene encoding shiga toxin from shigella to E. coli 0157.H7 carried by a bacteriophage.

1.5       Diversity

E. coli encompasses and enormous population of bacteria that exhibit a very high degree of both genetic phenotypic diversity, and remains one of the most diverse bacterial species which only 20% of the genome is common to all strains (Lukjencenko et al., 2010). A strain is a sub-group within the species that has unique characteristics that distinguish it from other strains. These differences are often detectable only at the molecular level; however, they may result in changes to the physiology or lifecycle of the bacterium. For example, a strain may gain pathogenic capacity, the ability to use a unique carbon source, the ability to take upon a particular ecological niche or the ability to resist antimicrobial agents. Different strains of E. coli are often host specific, making it possible to determine the source of fecal contamination in environmental samples (Feng et al., 2002). Such as knowing which E. coli strains are present in a water sample allows researchers to make assumptions about whether the contamination originates from a human, another mammal or a bird.

1.6       Serotype

The most common subdivision system of E.coli that, is not based on the evolutionary relatedness, is by serotype, which is based on major surface antigens such as E.coli 0157:H7. (Orskor et al., 1977). It is however common to cite only the serogroup i.e the O-antigen. At present, about 190 serogroups are known (Stenutz et al., 2006) the common laboratory strain has a mutation that prevents the formation of an o-antigen and is thus non-typeable.

1.7       Genome Plasticity

New strains of E.coli evolve through the natural biological process of mutation, gene duplication and horizontal gene transfer, in particular 18% of the genome of the laboratory stain MG 1655 was horizontally acquired since the divergence from salmonella (Lawrence and Ochman, 1998). However, in microbiology all strains of E.coli derive from E.coli k-12 (B strains), some strains develop traits that can be harmful to host animal. These virulent strains typically cause a significant diarrhea that is unpleasant in healthy adults and is often lethal to children in the developing world (Nataro and Kaper, 1998). More virulent strains, such as 0157:H7 cause serious illness or death in the elderly, the very young or the immune compromised (Hudault et al., 2001).

1.8       Neotype Strain

E. coli is the type species of the genus (Escherichia) and in turn Escherichia is the type genus of the family Enterobacteriaceae and should be noted that the family name does not stem from the genus Enterobacter, but from enterobacterium (though enterbacterium being not a genus, but an alternative trivial name to enteric bacterium) (George et al., 2005).

1.9       Phylogeny

E. coli is species. A large number of strains belonging to this species have been isolated and characterized. In addition to serotype, they can be classified according to their phylogeny i.e the inferred evolutionary history where the species is divided into six group (Brzuszkiewicz et al., 2011), namely; Group B2, Group D, Group E, Group B1, Group A and Group B strain derivatives.

1.10     Roles as Normal Microbiota

E. coli normally colonizes an infant’s gastrointestinal tract within 40hrs of birth, arriving with food or water or with the individuals handling the child. In the bowel, it adheres to the mucus of the large intestine. It is the primary facultative anaerobe of the human gastro intestinal tract (Todar, 2007). Facultative anaerobes are organisms that can grow in either the presence or absence of oxygen. As long as these bacteria do not acquire genetic elements encoding for virulence factors, they remain commensals (Evans et al., 2007).

1.11     Role in Disease

Virulent strains of E. coli can cause gastroenteritis, urinary tract infections, and neonatal meningitis. In rare cases, virulent strains are also responsible for hemolytic-uremic syndrome, peritonitis, mastitis, and septicemia and gram negative pneumonia (Todar, 2007). UPEC (uropathogenic E.coli) is one of the main causes of urinary tract infections. It is part of the normal flora in the gut and can be introduced in many ways. In particular for females, the direction of wiping after defecation (wiping back to front) can lead to fecal contamination of the urogenital orifices. Anal sex can also introduce this bacterium into the male urethra and in switching from anal to vaginal intercourse the male can also introduce UPEC to the female urogenital system.

1.12     Uses of Nonpathogenic E. coli

Nonpathogenic E. coli strain also known as Mutaflor is used as a probiotic agent in medicine, mainly for the treatment of various gastroenterological diseases including inflammatory bowel disease (Grozdanov et al., 2004).

1.13     Salmonella

Salmonella is a genus of rod-shaped gram-negative, non-spore-forming, predominantly motile enterobacteria with diameters of about 0.7 to 1.5μm, lengths from 2 to 5μm, and flagella that grade in all directions (i.e peritrichous). They are chemoorganotrophs obtaining their energy from oxidation and reductions using organic source, and are facultative anaerobes. Most species produce hydrogen sulphide, (Clark and Barret, 1987) which can readily be detected by growing them on media containing ferrous sulphate, such as Triple Sugar Iron (TSI) or Salmonella Shigella Agar  (SSA). Most isolates exist in two phases a motile phase 1 and a non -motile phase II. Cultures that are non motile upon primary culture maybe switched to the motile phase using a cragie tube.

Salmonella is closely related to the Escherichia genus and are found worldwide in cold and warm blooded animal (including humans) in the environment. They cause illness such as typhoid fever, paratyphoid fever, and foodborne illness (Salmonellosis) (Ryan and Ray, 2004)

1.13.1  History of Salmonella

The genus Salmonella was named after Daniel Elmer Salmon, an American veterinary pathologist. While Theobald Smith was the actual discoverer of the type bacterium (Salmonella enteric var. choleraesuis) in 1885, Dr. Salmon was the administrator of the United States disease control Agency research program, and thus the organism was named after him by Smith (Food safety A to Z reference Guide, 2009). Smith and Salmon had been searching for the cause of common hog cholera and proposed this organism as the causal agent. Later research, however now show this organism (now known as Salmonella enterica) rarely causes enteric symptoms in pigs, and was thus not the agent they were seeking which was eventually shown to be a virus. However, related bacteria in the genus Salmonella were shown to cause other important infectious diseases. The genus Salmonella was finally and formally adopted in 1900 by J. Lignieres for the many species of Salmonella, after Smith first type strain Salmonella cholera.

1.13.2  Salmonella Nomenclature

Initially, each Salmonella species was named according to clinical considerations (Kanffmann, 1941). Example; Salmonella typhimurium (Mouse typhoid fever), S. choleraesuis (hog cholera). After it was recognized that host specificity did not exist for many species, new strains (Serovar or Serological Variants) received species names according to the location at which the new strain was isolated later, molecular findings led to the hypothesis that Salmonella consisted of only one species (Le Minor and Popoff, 1987), S. enterica, and the serovar were classified into six groups, (Evins et al., 1989), two of which are medically relevant. But as this now formalized nomenclature (Tindal et al., 2005) is not in harmony with the traditional usage familiar to specialist in microbiology and infectologists, the traditional nomenclature is common. Currently, there are three recognized species. S. enteric, S bongori and S. subterranean, with six main subspecies: enteric (1), Salamae (II), Arizonae (IIIa), Diarrizonae (IIIb), Houtenae (IV), and Indica (VI). Historically serotype V was bongori, which is now considered its own species (Janda and Abbott, 2006). The serovar (i.e Serotype) is a classification of Salmonella into subspecies based on antigens that the organism presents. It is based on the Kauffman white classification scheme that differentiates serological varieties from each other (Porwollik, 2011). Serotypes are usually put into subspecies groups after the genus and species, with the serovars capitalized but not italicized: an example is Salmonella enteric Serovar Typhimurium Newer methods for Salmonella typing and subtyping include genome-based methods such as pulsed field gel electrophoresis (PFGE), multiple Loci VNTR Analysis (MLVA), Multilocus sequence typing (MLST) and (multiplex) PCR-based methods (Achtman et al., 2012).

1.13.3  Salmonella as Disease Causing Agent

Salmonella infections are zoonotic and can be transferred between humans and non human animals. Many infections are due to ingestion of contaminated food (Jantsch et al., 2011). In speaking of other Salmonella serotypes, Salmonella enteritis, typhoid and paratyphoid. S. enteritis can cause serious illness because of its special virulence factors while Salmonella typhi is adapted to human and does not occur in other animals. Salmonella species are facultative intracellular pathogens that enter cells via macropinosomes (Kerr et al., 2010). Enteritis Salmonellosis or food poisoning Salmonella consist of potentially every other serotype (over a thousand) of the Salmonella bacteria, most of which have never been found in humans. These are encountered in various Salmonella species; most having never been linked to a specific host, but can also infect humans. It is therefore a zoonotic disease. The organism enters through the digestive track and must be ingested in large numbers of before it can cause diseases in healthy adults. Gastric acidity is responsible for the destruction of the majority of ingested bacteria. Salmonellosis is a disease caused by raw or undercooked food. Infection usually occurs when a person ingests foods that contains a high concentration of the bacteria, similar to a culture medium, the symptoms are usually mild, normally, no sepsis occurs, but it can occur exceptionally as a complication in elderly or weakened patients (Example, those with Hodgkin’s disease).

However, infant and young children are much more susceptible to infection easily achieved by ingesting a small number of bacteria. In infants, contamination through inhalation of bacteria, laden dust is possible. After a short incubation period of a few hours to one day, the bacteria multiply in the intestinal lumen, causing an intestinal inflammation with diarrhea that is often mucopurulent and bloody. In infants, dehydration can cause a state of severe toxicosis. Extra intestinal localizations are possible, especially Salmonella meningitis in children, osteitis etc.

Salmonella enteritis (example enterica subspecies enteritidis) can cause diarrhea, which usually does not require antibiotic treatment. However, in people at risk such as infants, small children, the elderly, Salmonella infections can become very serious, leading to complications. If these are not treated, HIV patients and those with suppressed immunity can become seriously ill. Children with sickle cell anemia who are infected with Salmonella may develop osteomyelitis.

Most people with Salmonellosis develop diarrhea, fever, vomiting, and abdominal cramps 12 to 72 hrs after infection (Brown and Brown 1978). In most cases, the illness lasts four to seven days, and most people recover without treatment. In some cases through, the diarrhea maybe so severe, the patient becomes dangerously dehydrated and most be taken to a hospital. At the hospital, the patient may receive intravenous fluids to treat the dehydration and may be given medication to provide symptomatic relief such as fever reduction. In severe cases, the Salmonella infection may spread from the intensives to the blood stream, and then to other body sites and can cause death, unless the person is treated promptly with antibiotics. The elderly, infants and those with impaired immune systems are more likely to develop severe illness.

1.13.4  Durability

Salmonella bacteria can survive for weeks outside a living body, and they are not destroyed by freezing (Sorrells et al., 1970). Ultraviolet radiation and heat accelerate their demise; they perish after being heated 55⁰C for 90mins, or 60⁰C for 12 mins (Beuchat and Heaton, 1975). To protect against Salmonella infection heating food for at least then minutes at 75⁰C is recommended, so the centre of the food reaches this temperature.

1.13.5              Source of Infection

Injected food, often gaining an unusual look or small, then is introduced into the stream of commerce; (Mermin et al., 1997)

-              Poor kitchen hygiene, especially problematic in institutional kitchens and restaurants because this can lead to a significant outbreak.

-              Excretion from either sick or infected but apparently clinically healthy people and animals (especially endangered are caregivers and animals).

-              Polluted surface water and standing water (such as in shower hoses or unused water dispensers).

-              Unhygienically thawed fowl (the meltwater contains many bacteria)

-              An association with reptiles (pet tortoises, snakes, iguanas and frogs) but primarily aquatic turtles is well described.

Salmonella can survive for some time without a host, thus, they are frequently found in polluted water, contamination from the excrement of carrier animal being particularly important.

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