ANTIBIOTICS RESISTANCE PROFILE OF ESCHERICHIA COLI ISOLATED FROM APPARENTLY HEALTHY DOMESTIC LIVESTOCK IN SOUTH-WEST NIGERIA (OSUN STATE, NIGERIA)
This study was conducted to determine the antibiotic resistance profile of Escherichia coli isolated from apparently healthy domestic livestock viz, cow, goats, and chicken from Akure, Ondo State Nigeria, E. coli was isolated using Eosin methylene Blue Agar (EMB) and identified by conventional microbiology technique. The isolate were tested against 14 antibiotics using the disc diffusion method. A total of 42 different antibiotics resistance profile were observed with each isolate showing resistance to at least four or more drugs tested. Generally the E. coli isolates showed resistance rates of 93.8% to ampicillin, 16.% to chloramphenicol, 57.5% to cloxacillin, 75.5% to Erythromycin, 20% to Gentamicin, 60.5% to penicillin, 19.5% to streptomycin, 25.8% to Ceftazidine, 45.8% to Cefuroxine,22.2% to cefixine 30.6% to Loxacin, 65.9% to augmentin, 26% nitrofurantoin, 29.3% to ciprofloxacin, 70.3% to tetracycline. This study showed that averages number of resistance phenotypes per isolate was significantly higher for goat and cow compared with poultry. A significant public health concern observed in this study is that multi drug resistance commena E. coli strains may constitute a potential reservoir of resistance genes that could be transferred to pathogenic bacteria
TABLE OF CONTENTS
Title page ii
Table of contents vi
Introduction and Literature review 1
History of Escherichia coli 3
Escherichia coli in the gastrointestinal tract 3
Pathogenesis of Escherichia coli 3
Genome plasticity 5
Phylogeny of Escherichia coli strains 6
Therapeutic use of nonpathogenic E. coli 6
Role of normal micobiota 6
Roles in disease 6
Role in biotechnology 7
Model organism 7
Characteristics of Escherichia coli 8
History of antibiotics 8
Alexander fleming and the discovery of penicillin 9
Antibiotic resistance profile of Escherichia coli 10
Materials and Methods 12
Sample collection 12
Media preparation 12
Cultivation of Escherichia coli 13
Characterization and identification of E. coli 13
Indole test 13
Methyl red test 14
Citrate test 14
Fermentation of sugars 14
Antibiotics susceptibility test 14
Table 3.1 15
Table 3.2 16
Results analysis 17
INTRODUCTION AND LITERATURE REVIEW
Antibiotics are naturals substances secreted by bacterial and funji to kill other bacteria that are competing for limited nutrients (Bud, 2007). The term antibiotic was first used in 1942 by Selman Waksman and his collaborators in journal articles to describe substance produced by a microorganism that is cribe substances produced by a microorganism in high dilution. Many antibacterial components are relatively small molecules with a molecular weight of less than 2000 atomic mass units (Dorlands, 2010).
With advances in medicinal chemistry, most of today antibacterial chemically are semi synthetic modifications of various nautral compound (Nussbaum, 2006). These include, for example, the beta-lactam antibacterial, which include the penicillins (produced by fungi in the genus penicillum), the cephalosporins and the carbapenems. In accordance with thus many antibacterial compound are classified on the basis of chemical biosynthetic origin into natural, semisynthetic, and synthetic.
Another classification system is based on biological activity, in this classification, antibacterial are divided into two broad group according to their biological effect micro-organism bactericidal agent that kill bacteria and bacteriostatic agents that slow down or stall bacteria growth (Nussbaum, 2006).
Pencillin, the first natural antibiotics discovered by Alexander fleming in 1928.
Escherichia coli is the head of the bacterial family, entero bacteriaceae, the enteric bacteria, which are facultatively anaerobic aram-negative rods that live in the intestinal tracts of animal in health and disease. The entero bacteriaceae are among the most important bacteria medically. A number of genera (e.g. salmonella, shigella, yersinia). Several others are normal colonists of the human gastro intestinal tract (e.g. Escherichia, Enterobacter, Klebsiella) but the bacteria as well, may occasionally be associated with diseases of humans. (Kubitschek, 1990).
Physiologically, Escherichia coli is versatile and well-adapted to its characteristic habitats. It can grow in media with glucose as the sole organic constituent wild-type Escherichia coli has no growth factor requirements, and metabiologically it can transform glucose into all of the macromolecular components that make up the cell. The bacterium can grow in the presence or absence of oxygen (O2) under anaerobic conditions it will grow by means of fermentation, producing characteristic “mixed acids and gas” as end product. However, it can grow by means of anaerobic respiration, since it is able to utilize No3, No2 or fumarate as final election acceptors for respiratory election transport processes. In part, E. coli to its intestinal (anaerobic) and its extraintestinal (aerobic or anaerobic habitats) (Kubitschek, 1990).
Escherichia coli can respond to environmental signals such as chimerical, pH, temperature, osmolarity etc. in a number of very remarkable ways considering its is a unicellular organism. For example, it can sense the presence or absence of chemicals and gases in its environment and swim towards or away from them. Or it can stop swimming and grow fimbriae that will specifically attach it to a cell or surface receptor. In response to change in temperature and osmolarity, it can vary the pore diameter of its outer membrane porins to accommodate larger molecules (nutrients) or to exclude inhibitory substances. With its complex mechanisms for regulation of metabolism the bacterium can survey the chemical contents in its environment in advance of synthesizing any enzymes that metabolized these compounds. It does not wastefully produce enzymes for degradation of carbon sources unless they are available, and it does not produce enzymes for synthesis of metabolites if they are available as nutrients in the environment (Ishii et al., 2009).
Escherichia coli is a consistent inhabitant of the human intestinal tract, and it is the predominate facultative organism in the human gastro intestinal tract, however, it makes up a very small proportion of the total bacterial content. The anaerobic bacteriodles species in the bowel out number E. coli by at least 20:1. However the regular presence of E. coli. In the human intestine and faces has led to tracking the bacterium in nature as an indicator offaecal pollution and water contamination. As such, it is taken to mean that, wherever E. coli is found there may be faecal contamination by intestinal parasites of human (Fotadar et al. 2005).
SCIENTIFIC CLASSIFICATION OF ESCHERICHIA COLI
Species Escherichia coli
Binomial name Escherichia coli
HISTORY OF ESCHERICHIA COLI
Escherichia coli was first described by Theodor Escherich in 1885, as bacterium coli commune, which he isolated from the faces of new borns. It was later renamed Escherichia coli, and for many years the bacterium was simply considered to be a commensal organisms of the large intenstine. It was not until 1935 that a strain of Escherichia coli was shown to be the cause of an outbreak of diarrhea among infants. (Bread et al., 2000).
The gastrointestinal tract of most warm-blooded animals is colonized by Escherichia coli within hours or a few days after birth. The bacterium is ingested in foods or water or obtained directly from other individuals handling the infant (Hudaults et al., 2001).
The human bowel is usually colonized within 40 hours of birth. Escherichia can adhere to the mucus overlying the large intestine once established, an Escherichia coli strain may persist for months or years. Resident strains shift over a long period (weeks to month), and more rapidly after enteric infection or antimicrobial chemotherapy that perturbs the normal flora. The basis of these shifts and the ecology of Escherichia coli in the intestine of humans are poorly understood despite the vast amount of information on almost every other aspect of the organism existence. The entire DNA base sequence Escherichia coli genome has been known since 1997. (Hudaults et al., 2001).
Escherichia Coli in the Gastrointestinal Tract
The commensal E. coli strains that inhibit the large intestine of all humans and warm-blooded animals comprise no more than 1% of the total bacterial biomass. The E. coli flora is apparently in constant flux one study on the distribution of different E. coli strains colonizing the large intestine of women during a one year period (in a hospital setting) showed that 52.1% yielded one serotype, 34.9% yielded two, 4.4% yielded three, and 0.6% yielded four. The most likely source of new serotype of E-coli is acquisition by the oral route. (Hudauits et al., 2001).
Pathogenesis of Escherichia coli
Over 700 antigenic types (serotype) of E. coli are recognized based on O, H and K antigens. At one time stereotyping was important in distinguishing the small number of strains that actually cause disease. Thus, the serotype 0157:H7 (0 refers o somatic antigen; H refers to flagelar antigen) is usually responsible for causing Hus (hemolytic uremic syndrome). Nowadays, particularly for diarrheagenic strains (those that cause diarrhea) pathogenic E. coli are classified based on their unique virulence factors and can only be identified by these traits. Hence analysis for pathogenic E. coli usually requires that the isolates first be identified as E. coli before testing for virulence markers (Brussow et al., 2004). Pathogenic strains of E. coli are responsible for three types of infections in humans. Urinary tract infection (UTI), neonatal meningitis, and intestinal diseases (gastroenteristis). The diseases caused (or not caused) by a particular strain of E-coli depend on distribution and expression of an array of virulence determinants, including adhensins, invasins, toxins and abilities to withstand host defenses. (Brussow et al., 2004).
Escherichia coli encompasses an enormous population of bacteria that exhibit a very high degree of both genetic and phenotypic diversity. Genome sequencing of a large number of isolates of E. coli and related bacteria shows that a taxonomic reclassification would be desirable. However, this has not been done, largely due to its medical importance (Krieg et al., 1984) and E. coli remains one of the most diverse bacterial species: only 20% of the genome is common to all strains. (Lukjancenko et al., 2010).
A strain is a sub-group within the species that has unique characteristic 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, 2002). For example, knowing which E. coli strains are present in a water sample allow researchers to make assumptions about whether the contamination originated from a human, another manual or a bird.
A common subdivision system of E. coli, but not based on evolutionary relatedness, is by serotype, which is based on major surface antigens (O antigen: part of lipopolysacchar ride layer: H: Flagellin; K antigen: capsule), e.g. 0157:H7 (Orskov, 1977).
The first complete DNA sequence of an E. coli genome (Laboratory strain K-12 derivative mG 1655) was published in 1997. It was found to be a circular DNA molecule 4.6 million base pair in length, containing 4288 annotated protein-coding genes (organized into 2584 operons) seven ribosomal RNA (rRNA operons and 86 transfer RNA (tRNA) genes. The coding density was found to be very high, with a mean distance between genes of only 118 base pairs. The genome was observed to contain a significant number of transposable genetic elements, repeat elements, (ryptic prophages and bacteriophage remnants) (Blattner et al 1997).
Today over 60 complete genomic sequences of Escherichia and shigella species are available. Comparison of these sequences shows a remarkable amount of diversity; only about 20% of each genone represents sequences that are present in every one of the isolates while approximately 80% of each genome can vary among isolates (Lukjancenko et al., 2010). Each individual genome contains between 4,000 and 5,500 genes, but the total number of different gene among all of the sequenced E. coli strains (the pan-genome exceed 16,000. This very large variety of component, genes has been interpreted to mean that two-third of the E. coli pan-genome originated in other species and arrived through the process of horizontal gene transfer. (Zhanjbayeva et al., 2011).
Like all life forms new strain of E. coli evolve through the natural biological processes of mutation, gene duplication and horizontal gene transfer in particular 18% of the genome of the laboratory strain MG1655 was horizontal acquired since the divergence from salmonella. (Lawrence et al., 1998). In microbiology all strains of E. coli derive from E. coli K-12 or E. coli B strain some strain develop traits that can be harmful to a host animal (Nataro et al., 1998)
E. coli is the type species of genus (Escherichia and in turn Escherichia is the type genus of the family Enterobacter + “1” (Sic) + “aceae”, but from “enterobacterium” + “aceae” (enterobacteriaium being not a genus, but an alternative trrural name to enteric bacterium). (George et al, 2005). The original strain described by Escherichia is believed to be lost, consequently a new type strain (neotyped was chosen as a representative; the neotype) strain is ATCC 11775, also known as NCTC 9001 which is pathogenic to chickens has an 01: K1: H7 serotype. However, in most studies either 0157:H7 or K-12MG1656 or K-12 W3110 are used as a representative E. coli (Migula, 1895).
Phylogeny of Escherichia coli Strains
Escherichia coli is a 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 that is the inferred evolutionary history as shown below where the species is divided into six groups (Sims et al., 2011).
The link between phylogenic assistance and pathology is small e.g. the 0157:H7 serotype strains, which form an exclusive group. Group E are all enterohaerogic strains (EHEC), but not all EHEC strains are closely related. In fact four different species of shigella are nested among E-coli strains while Escherichia alberti and Escherichia fergusoni are outside of this group. All commonly used research strains of E-coli belong to group A and are derived mainly from (lifton’s K – 12 strain and to a lesser) degree from d’iterelle’s Bacillus coli strain (B strain) (07).
Therapeutic use of Nonpathogenic E. coli
Nonpathogenic E. coli strain Nissle 1917 also know as mutaflor is used as probiotic agent in medicine, mainly for the treatment of various gastro enterological diseases, including inflammatory bowel disease (Kamada, 2005).
Role as Normal Microbiota
E. coli normally colonizes an infant’s gastro intestinal tract with 40 hours 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 facilitative anaerobe of the human gastrointestinal tract (Todar K, 2007). (Facultative anaerobes are organisms that can grow in either the presence or absence of oxygen). As long as these bacteria do not acquire elements encoding for virulence factors, they remain benign commensals. (Kamada, 2005).
Roles in Disease
Virulent strains of E. coli can cause gastroenteritis, urinary tract infections and neonatal meningitis. In rarer cases, virulent strains are also responsible for hemolytic-uremic syndrome, peritonitis mastitis, septicemia and Gram-negative pneumonia (Todar, 2007).
UPEC (uropathogenic E. coli) is one of the main causes of urinary tract infection. It is part of the normal flora in the gut and can be introduced many ways. In particular for females, the direction of wiping after defecation can lead to fecal contamination of the urogenital oritices. Anal sex can also introduce these bacteria into the male urethra, and in switching from ancel to vaginal intercourse the male can also introduce UPEC to the female urogenital system.
Role in Biotechnology
E. coli play an important role in modern biological engineering and industrial microbiology (Research 2004). The work of stanely Norman cohen and Herbert Boyer in E. Coli using plasmids and restriction enzymes to create recombinant DNA become a foundation of biotechnology (Lee 1996).
E. coli is a very versatile host for the production of heterologous proteins (Russo 2003) and various protein expression systems have been developed which allow the production of recombinant proteins in E. coli one of the first useful applications of recombinant DNA technology was the manipulation of E. coli to produce human insulin (Cornelis, 2000)
Many proteins previously thought difficult or impossible to be expressed in E. coli in olded form have also been successfully expressed in E. coli (Tot 1994).
Modified E. coli cells have been used in vaccine development, bioremediation and production of immobilized enzymes. (Ruso, 2003).
E. coli is frequently used as a model organism in microbiology studies cultivated strains (e.g. E. coli K 12) are well adapted to the laboratory environment, and unlike wild type strains, have lost their ability to thrive in the intestine (Fredrick, 1997). Many lab strains lose their ability to form biofilms. These features protect wild type strains from antibodies and other chemical attracts, but require a large expenditure of energy and material resources.
In 1946, Joshua Lederberg and Edward Tatum first described the phenomenon known as bacterial conjugation using E. coli as a model bacterium (Prigent 1998 and it remains the primary model to study conjugation. E. coli was an integral part of the experiments to understand phage genetics (Prigent 1998).
By evaluating the possible combination of nanotechnologies with landscape ecology complex habitat landscapes can be generated with details at the nanoscale. On such synthetic eco-systems, evolutionary experiments with E. coli have been performed to study the spatial biophysics of adaptation in an Inland biogeography on chip (Fredrick 1997).
Characteristics of Escherichia coli
Escherichia coli commonly abbreviated as E. coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some serotype can cause serious food poisoning in human. (Escherichia coli et al 2012). The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2 (Bentley and Meganathan, 2001).
E-coli is about 2 micro meter long and 0.5μm in diameter with a cell volume of 0.6 – 0.7(μm)3 (Kubitschek HE 1990). It can live on a wide variety of substrates. E. coli uses mixed acid fermentation in anaerobic conditions, producing lactate, succinate, ethanol, acetate and carbon dioxide. (Madigan et al 2006).
Optimal growth of E. coli occurs at 370c (98.6F) but some laboratory strains can multiply at temperatures of up to 490C (120.20F) Fotadar et al 2005).
Growth can be driven by aerobic or anaerobic respiration, using a large variety of redox pairs, including the oxidation of pyruvic acid, formic acid, hydrogen and amino acids, and the reduction of substances such as oxygen, nitrate, fumarate, dimethyl sulfoxide and trimethylamine N-oxide (Ingedew 1984).
E. coli and elated bacteria posses the ability to transfer DNA via bacterial conjugation, transduction or transformation which allow genetic material to spread horizontally through an existing population. This process led to the spread of the gene encoding shigatoxin from shigella to E. coli 0157:H7, carried by a bacteriophage (Brussow H. 2004).
HISTORY OF ANTIBIOTICS
The term antibiosis meaning against life was introduced by the French bacteriologist Vuillemin as a descriptive name of the phenomenon exhibited by these early antibacterial drugs. Antibiosis was first described in 1877 in bacteria when Louis Pasteur and Robert Koch observed that an airborne bacillus could inhibit the growth of Bacillus anthraxis. These drugs were later renamed antibiotics by Selman Waksman, an American microbiologist in 1942. (Dorland, 2010).
The search for antibiotics began in the late 1800s, with the growing acceptance of the germ theory of disease, a theory which linked bacteria and other microbes to the causation of a variety of ailments. As a result, scientists began to devote time to searching for drugs that would kill these disease causing bacteria. The goal of such research was to find so called magic bullet’s that would destroy microbes without toxicity to the person taking the drug (Grigorgan et al 2006).
In 1877, Louis Pasteur showed that the bacterial disease anthrax, which can cause respiratory failure, could be rendered harmless in animals with the injection of soil bacteria. In 1887, Rudolf Emmerich showed that the intestinal infection cholera was prevented in animals that had been previously infected with the streptococcus bacterium and then injected with the cholera bacillus. While these scientist showed that bacteria could treat disease, it was until a year later, in 1888, that the German Scientist E-de Fredenreich isolated an actual product from a bacterium that had antibacterial properties. Freudenreich found that the blue pigment released in culture by the bacterium bacillus pyocyanase arrested the growth of other bacteria in the cell culture.
Alexander Fleming and the Discovery of Penicillin
In the early 1920s, the British scientist Alexander reported that a product in human tears could lyse bacterial cells. Fleming findings, which he called lysozyme, was the first example of an antibacterial agent found in humans. Like pyocyanse. Lysozyme would also prove to be a dead end in the search of an efficacious antibiotic cells.
While flaming generally receives credit for discovering penicillin, in fact technically, fleming rediscovered the substance. Through follow-up work, fleming showed experimentally that the mold produced a small substance that diffused through the agar of the plate to lyse the bacteria. He named this substances penicillin after the penicillium mold that had produced it. (Mcnulty et al., 2010).
It was not until about ten years after penicillin’s rediscovery in 1939, that Howard florey. Ernst chain and Norman hearley picked up the project. Fleming were able to overcome the technical difficulties that had plagued him, in the process spectacularly showing penicillin’s efficacy in the clinical setting cross-continent cooperation in the early 1940s a resulted in the increased scale of penicillin production.
It is not surprising that initially penicillin was used almost exclusively to teat soldiers injured during the war. That would change, though, with one fateful disaster.
Perhaps penicillin’s most important clinical trial occurred after a fore at a Boston club, which resulted in numerous burn victims being sent to Boston-area hospitals. At that time, it was common for severe burn victims to die of bacterial infections, such as those from staphylococcus. The success that physicians had in treating severely burned victims that night was largely attributed to the effects of penicillin. (Goossens et al, 2006).
In 1932, the German Gerhard Domage turned his attention away from natural antibiotics and towards synthetic ones. Domagk who investigated the effects of different channel dynes for their effects on bacterial infections, found that the protosil cured diseases caused by the streptococcus bacteria when injected into infected animals.
Around the time that florey and coworkers picked up the work on penicillin, the antibiotic gramicidin was isolated from a soli-inhabiting microbe. Gramicidin, then first natural antibiotic extracted from soil bacteria was able to arrest the growth of staphylococcus but proved highly toxic.
In 1943, Selman Waksman and his group isolated another antibacterial agent from a soil bacterium, streptomyces griseus. Waksman’s antibiotic, streptomycin, proved effective against several common infections the microbe causing tuberculosis, which had to that point resisted numerous methods of treatment. Streptomycin, through, carried with it highly toxic side effects and a fast rate of mutation, making it not a viable clinical option. (H. Gooszens and D. Guillemot, 2006).
Antibiotic Resistance Profile of Escherichia coli
Antibiotics are natural substances secreted by bacteria and fungi to kill other bacteria that are competing for limited nutrient. The introduction of antibiotics after World War I resulted in aromatic decrease of numbers of death due to bacteria infections (R. Bud, 2007). As early as 1945, fleming warned that the misuse of penicillin could lead to selection of resistant forms of bacteria. Infact, fleming had already experimentally derived such strains by varying the dosage and conditions upon which the added antibiotic to bacteria. Fleming posted that resistance to penicillin could be conferred in two ways either through the strengthening of the bacterial cell wall which the drug destroyed or through the selection of bacteria expressing mutant proteins capable of degrading penicillin.
Antibiotic usage is possibly the most important factor that promotes the emergence, selection and dissemination of antibiotic – resistant microorganism, in both veterinary and human medicine (Daniels et al, 2009). This acquired resistance occurs not only in pathogenic bacteria but also in the endogenous flora of exposed individual. It is known that genes responsible for antibiotic resistance are present in microorganism, providing them with self protection to the antibiotic compounds they produce ass defence mechanism against other microorganisms. Similarities among the genes and resistance mechanisms found in the antibiotic producers and in human pathogenic bacteria are the pools of origin of antibiotic resistance genes (Courvalin, 2008).
The antibiotic selection pressure for bacteria drug resistance in the animal is high and invariably their faecal flora contains a relatively high proportion of resistant bacteria (Whitworth et al., 2008). Colonization of the intestinal tract with resistant Escherichia coli from chicken has been shown in human volunteers and there is historical evidence that animals are a reservoir for E. coli found in humans in various countries has been reported (Frang et al., 2008).
According to Kurenbach et al, 2003 transfer of antibiotic resistance genes from Gram positive Gram negative bacteria invitro is very rare event. In vitro transfer of a naturally occurring Gram positive plasmid PIPSOI in E. faecals to E. coli has been described. Widespread reliance on antimicrobial in food animal production has resulted in a considerable rise of antimicrobial resistant strains of bacteria, complicating the treatment of infectious diseases in livestock, companion animals and humans. The selective pressure from the use of antimicrobial agents at sub therapeutic level in dairy cattle could result in the selection of those strains that contain genres for antimicrobial resistance (Call et al 2008).
Molecular tools have been used to correlate animal associated pathogens with similar pathogens affecting humans and to clearly demonstrate transferable resistance genes carried by plasmids common to both animals and humans (Pitout et al., 2009).
In the developed world, the extensive use of antibiotics in agriculture, especially for prophylactic and growth promoting purposes has generated much debate as to whether thus practice contributes significantly to increased frequencies and dissemination of resistance genes into other ecosystems. In developing countries like Nigeria, antibiotics are used only when necessary, especially if the animals fall sick, and only the sick ones are treated in such cases. According to John and Fishman (1997) will provide information on resistance trends including emerging antibiotic resistance which are essential for clinical practice. This work was therefore, undertaken to investigate the antibiotic resistance profile of E. coli isolates from apparently healthy domestic livestock viz: cow, goat and chicken..