ABSTRACT
The increased prevalence of Salmonella contamination in poultry has gained considerable scientific attention during the last few decades. The research work was done to determine the plasmid profile of antibiotic resistant Species isolated from poultry products. In the reaserch, 24 strains of Salmonella isolated where tested to ten antibiotics using the disc diffusion method, to determine their susceptibility. The zone of inhibition was observed and recorded in percentage. 24 strains showed 100% resistant to septrin, chloramphenicol, streptomycin, tarvid, agumentin, higher resistant was observed against amoxacilin (19%), Gentamicin (91%) and pefloxacin(91%). The isolates showed lower resistant of 66% against sparfloxacin and ciprofloxacin. While 6 isolates was sensitivity against gentamicin, amoxacilin, sparfloxacin, and ciprofloxacin. Six resistant strain of Salmonella isolates was selected at random to determine the plasmid profile. The result obtained showed that 1, 2, 6 in the DNA ladder are positive to plasmid with the molecular weight of 0.5kb – 48.5kb, while 3, 4, 5, are negative to plasmid.
CHAPTER ONE
1.0 INTRODUCTION/LITERATURE REVIEW
1.1 INTRODUCTION
The emergence of antibiotic-resistant Salmonella strains has become a major public health concern. Salmonella species are widely distributed in the environment that causes a diverse spectrum of diseases in human and animals. Non typhoidal Salmonella species are among the foremost bacterial pathogens implicated in food-borne gastroenteritis worldwide (Foley et al., 2006). The World Health Organization (WHO) has estimated that annually 1.3 billion cases of acute gastroenteritis or diarrhea due to non typhoid salmonellosis causing 3 million deaths. In India an estimated 4,00,000 children below 5 years age die each year due to diarrhoea (Sudershan et al., 2009). Salmonella is most often transmitted to humans through the food chain, with over 95% of salmonellosis cases attributable to the consumption of undercooked or mishandled beef, chicken and eggs etc., (Foley et al., 2006). A variety of antimicrobial agents have been used to treat the salmonellosis. An increasing rate of antimicrobial resistance in Salmonella has been reported in many developing and developed countries (Threlfall et al., 1993). Briggs and Fratamsco (1999) reported that the frequency of resistance is presumably due to extensive use of antimicrobial agents in human and veterinary medicine. Furthermore, resistance to combinations of several classes of antimicrobials has led to the emergence of Multi-Drug Resistant (MDR) strains that may pass from food animals to humans (White et al., 2001). The genes involved in resistance in Salmonella are often plasmid-born and therefore potentially transmissible to other pathogenic enteric microorganism with genetic factors which control antibiotic resistance. Spread of antibiotic resistance plasmids in Salmonella from chickens to human handlers (White et al., 2001) or of antibiotic-resistant microorganisms from poultry to humans in various countries (Poppe et al., 2002) has been reported.
Poultry in India started as a backyard activity and in the past three decades it has gone through a revolutionary change. Today India ranks 8th in broiler production in the world. Statistics of 2002 show that India produced 1.2 billion kg of broiler meat per annum which gave an annual per capita consumption of 1.2 kg (Asha, 2004). Chicken is considered to be the most widely produced in Namakkal (Latitude:11°.13’ 48” N; Longitude: 78° 10’ 12” E). Thus, the poultry industry is one of the major sources of economy. There are mass production centres in various parts of the country especially in Southern states, Tamil Nadu, Karnaka and Andhra Pradesh. Though the consumption has been promoted, no effective steps are taken to monitor the quality of chicken meat and no guidelines have been prescribed for the meat processing in retail markets. The present study has been taken up primarily to assess the level of Salmonella contamination in meat with special reference to S. enteritidis. The export of poultry product from this region is increasingly on the rise. The possibility for transfer of antibiotic resistance genes among humans, animals and the environment is a direct threat to public health. The incidence of antibiotic-resistant Salmonella may pose health risk to many populations in world.
AIM AND OBJECTIVES
The aim of this study is to evaluate the plasmid profile of salmonella species isolated and its objectives is as stated
- To isolates salmonella spp.
- Determination of antibiotic sensitivity on the isolated salmonella spp.
- Evaluation of the plasmid profile of salmonella spp.
1.2 LITERATURE REVIEW
1.2.1 SALMONELLA
Is a genus of rod-shaped (bacillus) Gram-negative bacteria of the Enterobacteriaceae family. The two species of Salmonella are Salmonella bongori and Salmonella enterica. Salmonella enterica is further divided into six subspecies and over 2500 serovars.
Salmonellae are found worldwide in both cold-blooded and warm-blooded animals, and in the environment. Strains of Salmonella cause illnesses such as typhoid fever, paratyphoid fever, and food poisoning (salmonellosis) (Su, and Chiu, 2007).
Salmonella species are non-spore-forming, predominantly motile enterobacteria with cell diameters between approximately 0.7 and 1.5 µm, lengths from 2 to 5 µm, and peritrichous flagella (flagella that are all around the cell body). They are chemoorganotrophs, obtaining their energy from oxidation and reduction reactions using organic sources, and are facultative anaerobes, capable of surviving with or without oxygen.
| Scientific classification of Salmonella | |
| Superkingdom: | Bacteria |
| Kingdom: | Bacteria |
| Phylum: | Proteobacteria |
| Class: | Gammaproteobacteria |
| Order: | Enterobacteriales |
| Family: | Enterobacteriaceae |
|
Genus: |
Salmonella Lignieres 1900 |
| Species | |
| S.bongori S. enterica |
|
The genus Salmonella is part of the family of Enterobacteriaceae. Its taxonomy has been revised and has the potential to confuse. The genus comprises two species, Salmonella bongori and Salmonella enterica, the latter of which is divided into six subspecies: enterica, salamae, arizonae, diarizonae, houtenae, and indica.[3][4] The taxonomic group contains more than 2500 serovars, defined on the basis of the somatic O (lipopolysaccharide) and flagellar H antigens (the Kauffman–White classification). The full name of a serovar is given as, for example, Salmonella enterica subsp. enterica serovar Typhimurium, but can be abbreviated to Salmonella Typhimurium. Further differentiation of strains to assist clinicoepidemiological investigation may be achieved by antibiogram and by supra- or subgenomic techniques such as pulsed-field gel electrophoresis, multilocus sequence typing, and, increasingly, whole genome sequencing. Historically, salmonellae have been clinically categorized as invasive (typhoidal) or noninvasive (nontyphoidal salmonellae) based on host preference and disease manifestations in humans (Porwollik, et al., 2004).
1.2.2 SALMONELLA ENTERICA
The bacteria Salmonella is commonly associated with food poisoning in countries all over the world. There are two species of Salmonella: S. enterica and S. bongori. However, the species that most people refer to when they talk about Salmonella is S. enterica. This species is divided into a subset comprising of serovars. In S. enterica alone, there are over 2,000 serovars of bacteria. Salmonella infections can originate from household pets containing the bacteria, particularly reptiles, improperly prepared meats and seafood, or the surfaces of raw eggs, fruits, or vegetables that have not been adequately disinfected. The most common cause of food poisoning is associated with the serovar S. Enteritidis. These outbreaks are attributed to contaminated poultry, chicken eggs, and products that contain eggs. Because of the high demand for poultry and poultry products, farms can become highly unsanitary. For example, chicken farms contain thousands up on thousands of chickens all living in the same area. Dead chickens are left for days before they are removed from the others. If one chicken contains the Salmonella bacteria, the organism can spread rapidly through thousands of others. When these chickens are processed and sent to stores all over the country, a mass Salmonella outbreak can occur (Porwollik, et al., 2004). When humans contract this disease, they can expel some of the Salmonella bacteria in their feces. In areas with poor sanitation, these bacteria can get into the water system where they can infect another person or animal and begin a whole new cycle of transmission.
Poultry eggs can contain Salmonella bacteria both on the surface as well as inside. After hens lay eggs, they sit on top of them to keep them warm. While sitting, fecal matter can transfer from the chicken to the surface of the egg. If the chicken is infected with Salmonella, the eggs must be properly disinfected in order to prevent disease. This is a larger problem in developing countries rather than the US or UK. The more common method of transmittance in the US is attributed to eggs infected with Salmonella on the inside. Salmonella Enteritidis can infect the ovaries of a hen and thus contaminate the egg before the shell forms (Porwollik, et al., 2004). Because the bacterium silently infects the hen’s ovaries, the hen does not show any signs of an illness. Therefore, seemingly healthy hens transmit this disease.
In the United States alone, Salmonella is responsible for 1.4 million infections, 15,000 hospitalization, and 400 deaths per year. These statistics are attributed to all serovars of Salmonella enterica not solely Enteritidis.
1.2.3 CHARACTERISTICS OF SALMONELLA ENTERICA
Salmonella enterica is one of two Salmonella species (enterica and bongori) and a member of the Enterobacteriaceae family (Su and Chiu, 2007). Salmonella enterica spp. is subdivided into 6 subspecies (enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV) and indica (VI) ). The usual habitat for subspecies enterica (I) is warm-blooded animals (Murray, et al., 2007). The usual habitat for subspecies II, IIIa, IIIb, IV and VI is cold-blooded animals and the environment (Porwollik, et al., 2004). All species of Salmonella can infect humans. Salmonella enterica subspecies enterica has 2610 different serotypes; the most well known being serotypes Typhi, Paratyphi, Enteriditis, Typhimurium and Choleraesuis (Su and Chiu, 2007). The serotypes are characterized by three surface antigens: the flagellar “H” antigen, the oligosaccharide “O” antigen and the polysaccharide “Vi” antigen (found in Typhi and Paratyphi serotypes)(Bronze and Greenfield, 2005). Salmonella enterica is a facultative anaerobe and is a gram negative, motile and non-sporing rod that is 0.7-1.5 by 2.0-5.0 µm in size (Bronze and Greenfield, 2005).
1.2.4 PATHOGENICITY/TOXICITY
Salmonella enterica can cause four different clinical manifestations: gastroenteritis, bacteremia, enteric fever, and an asymptomatic carrier state. It is more common in children under the age of 5, adults 20-30 year olds, and patients 70 years or older (Ryan and Ray, 2004).
Gastroenteritis: Gastroenteritis or “food poisoning” is usually characterized by sudden nausea, vomiting, abdominal cramps, diarrhea, headache chills and fever up to 39 ºC. The symptoms can be mild to severe and may last between 5-7 days (Ryan and Ray, 2004). The Typhimurium serotype is the most common cause of gastroenteritis and there are an estimated 1.3 billion cases and 3 million deaths annually (1.4 million cases and 600 deaths in the US alone) due to non-typhoidal Salmonella (Brock, et al., 2003). In well resourced countries with low levels of invasive complications, the mortality rate due to non-typhoidal Salmonella is lower then 1%; however, in developing countries, the mortality rate can be as high as 24% (Chimalizeni, et al., 2010).
Bacteremia: Bacteremia occurs in 3-10% of individuals infected with Salmonella enterica and certain serotypes (particularly serotype Choleraesuis) have higher mortality rates (Bronze and Greenfield, 2005). Immunosuppressed individuals and patients with comorbid medical conditions (e.g. HIV-AIDS, diabetes, mellitus, malignancy, corrhosis, chronic granulomatous disease, sickle cell disease, lymphoproliferative disease, or collagen vascular disease) have a higher risk of developing bacteremia due to a Salmonella infection (Bronze and Greenfield, 2005). Bacteremia can cause septic shock; endocarditis, especially in patients older than 50 or with heart conditions; infection of the aorta, especially in patients with pre-existing atherosclerotic disease; liver, spleen, and biliary tract infections in patients with underlying structural abnormalities; mesenteric lymphadenitis; osteomyelitis in long bones and vertebrae; urinary tract infection; pneumonia; pulmonary abscess; brain abscess; subdural and epidural empyema; meningitis; CNS infections (rarely); and death (Bronze and Greenfield, 2005).
Enteric fever: Also known as typhoid fever, this infection is caused by serotypes Typhi and Paratyphi (Ryan and Ray, 2004). Enteric fever is characterized by fever (rising within 72 hours after the onset of illness) and headache, brachycardia, faint rose-colored rash on the abdomen and chest, anorexia, abdominal pain, myalgias, malaise, diarrhea (more common in children) or constipation (more common in adults), hepatosplenomegaly, segmental ileus, meningismus, and neuropsychiatric manifestations (Bronze and Greenfield, 2005). Less common symptoms are sore throat, cough, and bloody diarrhoea. Complications include myocarditis, encephalopathy, intravascular coagulation, infections of the biliary tree and intestinal tract, urinary tract infection, and metastatic lesions in bone, joints, liver, and meninges (Ryan and Ray, 2004). The most severe complication (occurs in about 3% of patients) is haemorrhage due to perforations of the terminal ileum of proximal colon walls (Ryan and Ray, 2004). If untreated, the fever can last for weeks; however, with proper antimicrobial therapy, patients usually recover within 10-14 days (Ryan and Ray, 2004). The disease is milder in children and, if treated, has a mortality rate of less than 1%; untreated cases can have a mortality rate greater than 10 % (Bronze and Greenfield, 2005).
1.2.5 EPIDEMIOLOGY
Infections with Salmonella enterica occur worldwide; however, certain diseases are more prevalent in different regions. Non-typhoid salmonellosis is more common in industrialized countries whereas enteric fever is mostly found in developing countries (with the most cases in Asia) (Bronze and Greenfield, 2005). There are about 1.3 billion cases of non-typhoid salmonellosis worldwide each year and the WHO estimates that there are 17 million cases and over 500,000 deaths each year caused by typhoid fever (Bronze and Greenfield, 2005). There is a peak in disease in the summer and fall, and it is most common in children (Ryan and Ray, 2004). In the developing world, salmonellosis contributes to childhood diarrhoea morbidity and mortality as bacteria are responsible for about 20% of cases (Bronze and Greenfield, 2005). Epidemics of salmonellosis have been reported in institutions such as hospitals and nursing homes (Ryan and Ray, 2004).
1.2.6 HOST RANGE
For serotypes causing non-typhoidal salmonellosis, the primary hosts are domestic and wild animals such as cattle, swine, poultry, wild birds, and pets (particularly reptiles) as well as flies (Krauss H., et al., 2003). Humans are usually the final host (Krauss H., et al., 2003). For Salmonella Typhi, humans are the only known host (Ryan and Ray, 2004).
1.2.7 INFECTIOUS DOSE
The infectious dose varies with the serotype. For non-typhoidal salmonellosis, the infectious dose is approximately 103 bacilli (Ryan and Ray, 2004). For enteric fever, the infectious dose is about 105 bacilli by ingestion (Collins and Kennedy, 1983). Patients with achlorhydria, depressed cell-mediated immunity, or who are elderly may become infected with at a lower infectious dose (Ryan and Ray, 2004). The infectious dose may also be dependent on the level of acidity in the patient’s stomach (Bronze and Greenfield, 2005).
1.2.8 MODE OF TRANSMISSION
Human infection usually occurs when consuming contaminated foods and water, contact with infected feces, as well as contact with infective animals, animal feed, or humans (Davis, et al., 2004). Foods that pose a higher risk include meat, poultry, milk products, and egg products (Brock, et al., 2010). In hospitals, the bacteria have been spread by personnel in pediatric wards, either on their hands or on inadequately disinfected scopes. Flies can infect foods which can also be a risk for transmission to humans (Greeberg, 1964).
1.2.9 INCUBATION PERIOD
For non-typhoidal salmonellosis, the incubation period is variable, depends on the inoculum size, and usually ranges between 5 and 72 hours. For typhoid fever, the incubation period can be between 3 and 60 days, although most infections occur 7-14 days after contamination. The incubation period for typhoid fever is highly variable and depends on inoculum size, host susceptibility, and the bacterial strain (Murray, et al., 2007).
1.2.10 COMMUNICABILITY
Humans can spread the disease for as long as they shed the bacterium in their feces (Emerson H., 1939). Certain carriers shed the bacteria for years and 5 % of patients recovering from non-typhoidal salmonellosis can shed the bacteria for 20 weeks. Animals can have a latent or carrier state where they excrete the organism briefly, intermittently or persistently (Bronze and Greenfield, 2005).
1.2.11 RESERVOIR
For non-typhoidal salmonellosis, the reservoir hosts are domestic and wild animals such as cattle, swine, poultry, wild birds, flies and pets (particularly reptiles), as well as other humans (with the chronic carrier state). For serotype Typhi, humans with the chronic carrier state are the only reservoir for the disease (Richmond and McKinney, 1999).
1.2.12 DRUG SUSCEPTIBILITY
Susceptible to chloramphenicol, ciproflaxin, amoxicillin, co-trimoxazole, trimethprim-sulfonamid, cephalosporins and norfloxacin. Some resistance to chloramphenicol has been reported and, in 1989, 32% of strains were multi-drug resistant (Bronze and Greenfield, 2005).
1.2.13 SUSCEPTIBILITY TO DISINFECTANTS
Gram negative bacteria are susceptible to 2-5% phenol, 1% sodium hypochlorite, 4% formaldehyde, 2% glutaraldehyde, 70% ethanol, 70% propanol, 2% peracetic acid, 3-6% hydrogen peroxide, quaternary ammonium compounds and iodophors; however, Salmonella spp. is resistant to nitrites (Block, 2001).
