PLASMID PROFILE OF ANTIBIOTIC RESISTANT SALOMONELLA SPP ISOLATED FROM POULTRY PRODUCT

ABSTRACT

The antibacterial resistance patterns of salmonella species isolated from poultry droppings in imo state university of owerri (imsu) the samples comprises of egg shell, soil in poultry farm feather, faces, clocal swab were analyzed microbiologically. Isolation of salmonella was carried out on salmonella Shigella agar medium and identified according to their microscope and biochemical reaction bacterial isolates were obtained, identified and confirmed as salmonella species as identified from several biochemical examinations. The isolates were subtested to antibiotics susceptibility test using disc diffusion test on Mueller  Hinton Agar. High resistance was recorded against septrin(100%) chloramphednicol (100%), streptomycin (100%), taruid (100%), auginetin (100%), sparfloxacin(66%), ciprofloxacin (66%), Amoxacillon(91%), Gentermicin(91%) and pefloxacon(91%) plasmid profile was also carried out to determine and characterized antibiotics traits in these results reveal the presence of drug resistant salmonella in commercial poultry feeds in owerrl metropolis. This poultry farm feather, faeces, can serve as a channel for transfer of resistant strains of bacteria to poultry birds as well as human and the environment

feather, faeces, can serve as a channel for transfer of resistant strains of bacteria to poultry birds as well as human and the environment

 

 

 

 

CHAPTER ONE

1.0                     INTRODUCTION/ LITERATURE REVIEW       

1.1    INTRODUCTION

Food-borne disease and food poisoning have increasily become a heath concern worldwide with species of  salmonella spp.  have for long been reported as leading cause of food borne infections (Whitwort et al., 2008). In developed and developing countries salmonella serotypes have been extensively incriminated as the most important zoonotic pathogens in several countries worldwide (Akinyemi et al., 2007). They are responsible for the significant morbidity and mortality in both humans and animals (Akinyemi et al., 2007), each year an estimated 1.3billion cases resulting to about 3million death occur worldwide due to salmonellosis alone (winkor et al., 2000). In spite of the important of poultry as the major element in the human food chain, it has been frequently labeled as one of the most important sources of food poisoning due to salmonella serovars causing the majority of food borne outbreaks worldwide (European food safety agency., 2004). Because of the importance of salmonella spp. As the cause of a food-borne disease, typing methods such as plasmid profiling have been used to trace the outbreak to the contaminated source for public health intervention (Akinyemi et al., 2007). Plasmid analysis has recently been used to investigate an outbreak of multi resistant (R-type Acssuspi) salmonella isolates from poultry products or used in solving outbreaks caused by other salmonella and hygiene as well as immunization and proper nutrition has provided major benefit in human life expectancy ( world health organization; 2002), however the increased utilization of antibioticsin both public and veterinary setting has led to the emergence of antibiotic resistance and as a conse avence poses a serious threat to public health safety (world health organization: 2002). The abuse of antibiotics around the globe for preservation of food materials treating animals with different kinds of disease or preventative purpose have selected for antibiotic resistant strains. These strains which exhibit drug resistant continue to multiply when challenged with an antibiotic (Ryder et al., 2013).

Enteric fever commonly known as typhoid fever, a systematic infection is one of the major disease caused by salmonella paratyphoid (Nataro et al: 2000). Animal faces are potential source of antibiotic resistant bacterial if released into the environment, resistant strains may contaminate water and food source which causes salmonella which can be a potential threat to human health (Pitout et al: 2009)when consumed. Poultry birds have frequently been incriminated as a means of salmonella contamination and conseavently acts as major source of pathogen in humans (Baevmler et al., 2000) when infection spread beyond the intestinal tract, appropriate anti-microbial therapy can be lifesaving (Shan and Korejo 2012). Today the industrialized countries have agreed that the development of bacterial resistance must be prevented in order to control the spread of these bacterial pathogen to human (Helmuth; 2000). According to (Abdellah et al., 2009) the extensive use of those in human and animals has led to an increased in bacterial multidrug resistant among several bacteria strains including salmonella 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 this practice contributes significantly to increased frequencies and dissemination of resistance genes into other eco-system (Carraminana et al., 2004) reported that 40% of the antibiotics produced in the united states were used in stock feeds. Bacterial isolates obtained by (Carraminana et al., 2004) from a poultry slaughter house in Spain had high percentages of resistance to many antibiotics. In developing countries like Nigeria, antibiotics are used only when necessary, especially of the animal fall, and only the sick ones treated in such cases (Nsofor and Iroegbu: 2013), there is currently a worldwide trend to reduce the use of antibiotic in animal food due to the contamination of meat product with antibiotic resides as well as the concern that some therapeutic treatments for human diseases might be jeopardize due to the appearance of resistant bacteria (Kabir, 2009). During the last decade, use of antimicrobial drugs for growth promotion and therapeutic treatment in food animals has received much attention. However even in the absence of heavy use of antibiotics, it is important to identify and monitor susceptibility profiles of bacterial isolates, particularly of commensal organisms. This according to (John and Fishman, 2002) will provide information on resistance trends including emerging antibiotic resistance which are essential for clinical practice. This singular factor has shown the need to probe further into the prevalence of antibiotic resistant organisms in poultry feed sold in Nigeria.

• STATEMENT OF PROBLEM

Salmonellosis is an infectious disease which often occurs through contaminated food, especially food products with an animal origin such as meat, chicken, egg, animal foods. In developing countries estimation of salmonellosis is difficult because there has not been sufficient surveillance, during the two past decades, the emergence of antibiotic resistant. Salmonella has become a serious problem worldwide. Wide usage of antibiotic in the diet of domestic animals has made drug resistant bacterial which could be transferred to human beings. In recent years, problem of resistant strains to multiple drugs (MOR) is increasing and most studies in Nigeria and other countries have shown high resistance of salmonella strains to several antibiotics. It is necessary to monitor the prevalence and antimicrobial resistance of food-borne pathogens for effective food safety planning and targeted interventions.

 

• AIM AND OBJECTIVE

• To isolate, identify and characterize salmonella species from poultry product.
• To determine the susceptibility pattern of salmonella species isolated from poultry product to some antibiotics.
• To suggest measures that should be applied to reduce the risk of introducing salmonella species in poultry products.
• To assess the public health implications of the poultry feeds.

1.4 LITERATURE REVIEW

1.4.1 SALMONELLA

1.4.2 TAXONOMY

Salmonella are mostly defined as a medically important gram-negative rod, non-spore forming bacterial (Murray et al., 2009), motile by flagella and most heterogeneous bacterial. These microbes belong to the Enterobacteriaceae family with more than 40 genera and hundreds of species and sub-species. These can be found worldwide in soil, water, vegetation and normal intestinal floral of many animals including humans, commonly medical important, enterobacteriaceae family including species of Salmoneua, Proteus, Escherichia coli, Kiebsiella, Shigella, Morganeua, Enterbacter, Citrobacter and Serratia (Murray et al., 2009).

1.4.3 SALMONELLA IN POULTRY.

The process of colonization of Salmonella covers humans and animals including livestock, poultry, rodents, reptiles and birds (Hirsh et al., 2004). The first case of salmonella infections was reported in 1899. Most common age for infection in poultry is under 2 weeks and rare over 4 weeks of birds. The whole flock can be affected with 100% morbidity and less than 20% mortality rate. All serotypes of salmonella have almost similar clinical signs with depression, ruffled feathers, diarrhea, swollen eyelids and sudden death (Murray et al., 2009). Whereas broad-spectrum antibiotics are effective against a broad range of bacteria, in this lesson we will look more closely at these general types of antibiotics, and we will see what makes a given antibiotic fit into each category

1. Bactericidal Antibiotics

Bactericidal antibiotics kill bacterial directly. The word “cidal” means kill, like in the words homicide or suicide. How do bactericidal antibiotics actually kill bacterial? Well, there are many different antibiotics that all have different mechanisms, and you can learn more about them in other lessons. But here are couple examples: the antibiotic polymyxin B injures the plasma membrane of bacteria, allowing their contents to leak out. Under normal circumstances, bacteria and other cells have to keep a perfect balance of ions on both sides of the plasma membrane because of osmosis. Polymyxin B disrupt this balance, and also lets other important molecules, like DNA and RNA leak out, so the bacterium is a goner (Itah and Essien, 2005).

1. Bacteriostatic antibiotics

In contrast to bactericidal antibiotics, bacteriostatic antibiotic stops bacterial from growing. This word is also easy to remember the suffix “static” means staying stable. The bacterial don’t die, but they can’t grow or replicate either. So, how do bacteriostatic antibiotics help clear up an infection, if they don’t actually kill bacteria? Well, bacterial normally divide really quickly in our bodies, and their numbers can get totally out of control. But if an antibiotic stops them from growing and dividing, the host’s immune system will be able to get rid of the bacterial ribosome, so that no new proteins can be made. This doesn’t kill the bacterial: they already have this proteins they need to survive for a while. However, they can’t replicate, because they would need to make tons of new proteins in order to make a whole new bacterial cell. Another class of bacteriostatic antibiotics is the sulfa drugs. They prevent this production of important metabolites that the bacterium needs in order to make new DNA, RNA and proteins. Now we understand the differences between bactericidal and bacteriostatic antibiotics. Let’s look at the difference between broad-spectrum and narrow-spectrum antibiotics (Chin-A-Woeng et al., 2000).

• Narrow-spectrum antibiotics

Narrow-spectrum antibiotics are only effective against narrow range of bacterial for example; penicivin G is very effective at killing gram-positive bacteria, but not very effective against gram negative. Why is that? What causes an antibiotic to have a narrow-spectrum of antibiotic antimicrobial activity? Often it has to do with the ability of the antibiotic to penetrate inside of the bacterium. Gram-positive bacteria have a relatively loose outer wall that many antibiotics can diffuse through. However, a gram-negative bacteria have a complex outer layer that prevents the passage of many layer or fat-soluble molecules. Another reason that antibiotics can have a narrow-spectrum of activity can be their targets molecules. If an antibiotics target a molecule that a bacterium doesn’t even have, of course it won’t be effective against that bacterium. For example, isoniazid specifically targets my co-bacteria, such as the bacterial that causes tuberculosis, it’s specific because it prevents the synthesis of my-colic acids, which are found in the cell walls of mycobacteria, but not most other types of bacteria, it’s good to treat patients with antibiotics that have a narrow spectrum of activity, because then the “good” bacterial that normally live inside of us won’t all get killed off along with the pathogen that caused the infection. However, when a patient comes into a clinic with an infection, it’s often not clear exactly which microbe is causing it. So in the case of severe infection, when it’s really important that an antibiotic work quickly, so that the patient can survives narrow-species (Ivanov et al., 2006).

1.4.4 ANTIBIOTIC RESISTANCE

Antibiotic resistance occurs when an antibiotic has lost its ability to effectively control or kill bacterial growth: in other words, the bacteria are “resistant” and continue to multiply in the presence of therapeutic levels of antibiotic (Poole, 2004).

1.4.5 MECHANISMS OF ANTIBIOTIC RESISTANCE

Resistance to antibiotics can be caused by four general mechanisms

• The inactivation or modification of the antibiotic.
• An alteration in the target site of the antibiotic that reduces its binding capacity.
• The modification of metabolic pathways to circumvent the antibiotic effect
• The reduced intracellular antibiotic accumulation by decreasing permeability and / or increasing active efflux of the antibiotic (Dubey and Maheshwari, 2005).

1.4.6 β- LACTAM ANTIBIOTICS

β-Lactam antibiotics (beta lactam antibiotics) are broad class of antibiotics consisting of all antibiotics agents that contain a β-lactam ring in their molecular structures. This includes Penicillin derivatives (Penans), Cephalosporin’s (Cephens), mono bactams and carbapenems.

Most β-lactam antibiotics work by inhibiting cell wall biosynthesis in the bacterial organism and are the most widely used group of antibiotics up until 2003, when measures by sales, more than half of all commercial available antibiotics in use were β-lactam compounds. Bacteria also develop resistance to β-lactam antibiotics by synthesizing β-lactam ring to overcome this resistance, β-lactam antibiotics are often given by β-lactamase inhibitors such as Elavulante acid (Helmutu, R., 2000).

1.4.7 MODE OF ACTION OF ANTIBIOTICS

Different antibiotics have different mode of action, owing to the nature of their structure and degree of affinity to certain targets sites within bacteria cells (Dubey, 2005)

1. INHIBITION OF CELL WALL SYNTHESIS: While the cells of human and animals do not have cell wall, this structure is critical for the life and survival of bacterial species, a drug that targets cell walls can therefore selectively kill or inhabit bacterial organisms, examples: penicillin, Cephalosporin’s, Bacteriacin and Vancomycin.
2. INHIBITORS OF CELL MEMBRANE FUNCTION:Cell membrane are important barriers that segregate and regulate the intra and extracellular flow of substance. A disruption or damage to this structure could result in leakage of important solutes essential for the cells, because this structure is found both in eukaryotic and prokaryotic cells, the action of this class of antibiotics are often poorly selective and can often be toxic for systemic use in the mammalian host. Most clinical usage is therefore limited to tropical applications. Examples: polymyxin B and colastin.

• INHIBITORS OF PROTEIN SYSTHESIS: Enzymes and cellular structures are primarily made of protein. Protein synthesis is an essential process necessary for the multiplication and survival of all bacterial cells. Several types of antibacterial agents target bacterial protein synthesis by binding to either the 30s or 50s sub-unit of intracellular ribosomes. This activity then results in the disruption of the normal cellular metabolism of bacterial, and conseavently leads to the death of organism or the inhibition of its growth and multiplication. Examples aminoglycosides, macrolides, Lincosamides, Streptogramins, chloramphenicol, Tetracycline’s.

1. INHIBITORS OF NUCLEIC ACID SYNTHESIS: DNA and RNA are keys to the replication of all living forms including bacteria, some antibiotics work by binding to components involved in the process of DNA or RNA synthesis, which causes interference of the normal cellular processes which will ultimately compromise bacterial multiplication and survival. For examples: quinolones, metronidazole, and rifampin.
2. INHIBITORS OF OTHER METABOLIC PROCESSES: other antibiotics act on selected cellular processes essential for the survival of the bacterial pathogens. For example, both sulfonamides and trimethoprim disrupt the folic acid pathway. Which is a necessary step for bacterial to produce precusors important for DNA synthesis. Sulfonamides target and bind to dihydropteroate synthase trimethophrim inhibit dihydrofolate reductase: both of these enzymes are essential for the production of folic acid, a vitamin synthesized by bacteria, but not humans.

 

1.4.8 SALMONELLA VIABILITY IN POULTRY PRODUCT

Estimated survival time in poultry product is more than 98 days (Juven et al. , 2005), found than viability of  S.typhimurium in poultry product, at room temperature is 71weeks and in litter, 78 weeks. Furthermore at 7⁰c the organisms may survive up to 79weeks in feed and litter. More than 80⁰c temperature is required for elimination of salmonella from feed during steam conditioning (Blankenship et al., 2006)

1.4.9 DISSEMINATION OF SALMONELLA

One of the leading causes of food borne infection in the world is still due to S. enteritidis by consuming poultry products including eggs and meats (Rabsch et al 2001: Murray et al., 2009).

Food poisoning in human beings is closely related by the use of poultry products which are contaminated with salmonella (Nashed, 2005). Internal content of eggs can be contaminated by S. enteritidis (Gast and Beard. 2009) in a study (Campbel et al., 2000), found that birds contamination to healthy birds due to salmonella present in the environment. In poultry production cycle, various factors are responsible for the introduction of salmonella including humans, rodents, feeds, broiler, house and hatchery (Patrick et al., 2004) investigated that poultry eggs without proper cooking method were a major risk factors for the outbreak is S.enteritidis illness from the year 1980’s in the united states, furthermore, the describe that control effort   prevent S.enteritidis illness from the region during the year 1985 through 1990. The national salmonella surveillance system (COC) was developing to collect data of outbreaks from all locations. They include information of city, country, states location of food preparation and consumption. The results from 1985 to 1995 showed that incident rate of S.enteritidis increased from 2.38 to 3.9 per 100000 population while with a decline of 4.9% it came down to 1.98% per 100000 in 1999. The reason for this decline in infection and outbreak could not be proved. It was through that the implementation of prevention and control measures played a major role during the 1990’s. These control measures mainly dealt with safe handling methods including proper cooking of eggs, regulations regarding refrigeration, quality assurance programs, educational messages, on from testing and trace ability.

1.5 CAPABILITY OF MODIFICATION

Salmonella can develop resistance against routine elimination practices of sanitation, chemical treatment and antibacterial drugs. Mostly the responsible factors in the introduction of organism into the poultry production chain are fed, hatching, poultry house, rodent and man. The infection in one bird can spread organisms internally or externally to all birds in the house. The poultry transport system provides the path for organism to transfer from one broiler delivery to at least the feather of the birds to another delivery. He reinvested that researchers and equipment manufacturers should emphasize on improving microbiological avality of poultry carcass. Furthermore, modification of advance techniaves like automatic evisceration of carcass, spray immersion chilling and freezing of carcass in plastic bag can be valuable methods to reduce the microbial load on carcass (Shirota et al., 2001)

1.6 FACTORS RESPONSIBLE FOR TRANSFER OF SALMONELLA CONTAMINATION

Animals can get infections when they are fed with salmonella contaminated feed. In the region were endemic infection is well controlled or absent, salmonella contaminated feed is a major source for introducing salmonella in animal food production. (European food safety authority, 2008).

Shirota et al., (2001) investigated that enteritidis strain of salmonella isolated from poultry farm belonged to the same phage type and was genetically related with the S.enteritidis obtained from feed, this study was conducted during 1993-1998 in eastern japan to observe the genetic relationship of salmonella serotypes which were contaminating eggs through poultry feed in layer farms.

1.7 MAJOR SOURCE OF SALMONELLA DISSEMINATION DURING FEED PRODUCTION

The data from the study of jones from Richardson (2004) confirmed that dust and feed ingredients can be major source of salmonella contamination during the feed milling process. They collected samples from three (3) feeds mills which were individually producing 100000-400000 tons of feeds every year. Five different locations were selected  for sampling from each mill including ingredient receiving area, mixer, pelleting mill , cooler and out loading region, a total of 886 samples were collected with a range of 68 samples from feed ingredients, 189 samples from dust and 629 samples from feed. The temperature were also recorded from each sample which was collected from the pelleting mill. The results showed significantly higher enterobacteriaceae counts in those feed samples which were also positive for salmonella as compared to the feed samples which were not contaminated with salmonella. The data illustrated that maintaining high temperature during pellet making was not uneven. However, they suggested that 85 0c temperature is required during pelleting to eliminate salmonella completely. They concluded that contamination rate was related with the management practice of mill. Furthermore, dust and feed ingredients remained the main source of contamination. Davies and Wray (2010) conducted an investigation to compare salmonella status in poultry feed mill and on farm home mixers, they collected sample from four (4) feeds mills.

1.8 HYGIENE IN POULTRY FARMS

It is important to reduce chance of infection to a minimum measure for preventing disease which include the following.

1. Keeping the chicken house clean and dry at all times.
2. Disinfect all litter materials before use.
3. Locate the chicken farm at least n100m away from other chicken farms.
4. Control rodent and mills
5. Keep away visitors from chicken farms.
6. Clean the chicken and feeders regularly.
7. Clean the chicken house thoroughly and disinfect after disposing birds.
8. Remove the dead birds.

1.9 SALMONELLOSIS

Salmonella infection (salmonellosis) is a bacterial disease of intestinal tract caused by salmonella species, a gram-negative facultative anaerobic rod shaped bacterium that belong to the family Enterobacteriaceae (Kenneth, 2005). Salmonella species are disseminated in the natural environment through humans and animals exertion. Evans and Reperts. (2004). Salmonella infection is one of the most common forms of zoonosis with infection being transmitted directly from animals to humans which are commonly in poultry farms. Salmonellosis occurs in most countries and affects all animal species. Infections mechanisms starts within 12-72 hours after infection of bacterium with inflammation of the stomach, intestine and diarrhea of febrile illness, effective salmonella vaccines are available for poultry and animals, only typhoid fever vaccines are available for humans and non-typhoidal salmonellosis (Evans and Reperts, 2004).

1.9.1 SYMPTOMS OF SALMONELLOSIS.

The bacterium reduces responses in the animal, it is infecting and this is what typically causes the symptoms father than any direct toxin produced (Evans and Reperts, 2004). Symptoms are usually gastro intestinal nausea, vomiting, abdominal cramps and bloody diarrhea with mucus, headache, fatigue and rose spots are also possible. These symptoms can be severe especially in young children and the elderly once. Symptoms last generally up to a week and can appear 12-70 hours after injecting the bacterium (Evans and Reperts, 2004). After the bacterium infection reactive Curthritis (Reitor Syndrones) can develop (Workin et al., 2011) in the sickle cell anemia osteomyelitis due to salmonella infection is much more common than in general population, through infection is the cause of osteomyelitis in sickle cell anemia patients.

1.9.2 DIAGNOSIS OF SALMONELLA INFECTIONS

Salmonella species can be detected in stool, in cases of bacteremia of invasive illness, the bacteria can also be defected in the blood, urine or rare occasions in tissues. The test consists of growing the bacteria in culture (Miller and Paaves, 2005). A feed blood or other samples is placed in nutrient broth or an agar and incubated for 2-3 days. After that, a microbiologist confirms it’s by identity by looking at biochemical reaction. Treatment with bacterial growth in culture and leads to a negative test even when salmonella causes the infection (Miller and Paaves, 2005).

1.9.3 TREATMENT OF SALMONELLA INFECTION

Salmonella gastro intestinal usually resolves in 5-7 days and most do not require treatment other than oral fluids. Persons with several diarrhea dehydration with intravenous fluids. Anyone with a suspected salmonella infection should be tested for salmonella, although most people recover from salmonella without any need for antibiotic treatment, in contrast, antibiotics are recommended for persons at risk of invasive disease including infants younger than three (3) months of age (Murray et al., 2009).

1.9.4 SALMONELLA CONTROL MEASURE IN ANIMAL FEED.

Jones (2011) reviewed practical salmonella control measure in animal feed. He suggested that control measures can be divided into three (3) broad categories

1. Attempts should be made to prevent the contamination from entering the facility.
2. A system can be developed to reduce conditions which enhance microbial growth within due processing facility.
3. Design the procedure which eliminates the pathogen.

He describes further that to assess the accurate contamination rate, it essential to collect the samples especially from the feed materials which are supposed to enter the facility. Developing a system from continuous control of dust and oil or fat accumulations is necessary task because it provides the excellent medium for the growth and spread of microbes within feed manufacturing facilities. Similarly uncontaminated feed material can be obtained by controlling rodents, wild bird’s sanitation of transport vehicles and restricting personnel access to facilities and equipment. Hazard analysis approach can be used to discover and disrupt vie location involved in multiplication of salmonella. He concluded that thermal process and pelleting may not be enough method to eliminat4e the salmonella completely from feed and recontamination may occur during cooling or transportation, the goal of controlling the salmonella can be achieved by the addition of chemical compounds including organic and formal dehyde.

Macioroski et al., (2006), discussed the role of management to control food borne salmonella species in animal feed and feed ingredients in a review study. They acknowledged that feed ingredients and environment which habor salmonella can mix contamination in feed which results in the cross contamination from feed to the animals. Anti-microbial compounds and management strategies are been developed to reduce the colonization and elimination of salmonella from gastrointestinal tracts of the animals. Feed additives tracts while use of antibiotics may prohibitive due to production cost factor. Concluding this review, they suggested that use of NACCP approach to identify critical areas and develop more efficient monitoring and sampling techniques for individual feed batches would minimize the impact of salmonella in the future.