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PROJECT TOPIC AND MATERIAL ON MICROBIAL ASSESSMENT OF RAW MEAT AT ABATTOIR AND ANTIBIOTIC SUSCEPTIBILITY
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Four (4) samples of raw meat were collected from two abattoirs namely: Kwata Abattoir at Awka and Amansea Abattoir all in Awka South, Anambra State Nigeria. The isolation and characterization of bacteria and fungi in the raw meat were studied. A total of eighth (8) bacterial isolates from beef and intestine samples were obtained and characterized as Escherichia Coli, Streptococcus Spp, Salmonella Typhi and Pseudomonas aeruginosa showing on Table 3 and three (3) fungi isolates were obtained which are Aspergillus, Mucor and Chrysonilia sitophila.Infected meat, however, should be eliminated through systematic meat inspection in production, and consequently, consumers will more often encounter meat exogenously spoiled by bacteria or fungi after the death of the animal.
Food safety remains a critical issue with outbreaks of foodborne illness resulting in substantial costs to individuals, the food industry and the economy (Kaferstein et al., 1997). Despite advances in food science and technology, foodborne diseases remain one of the major public health and economic problems all over the world (WHO, 1995 and Legnani et al., 2004). The risk of foodborne illness has increased markedly over the last 20 years, with nearly a quarter of the population at higher risk for illness (CDC, 2003; 2004). For instance in the United States, 76 million people get sick, 325,000 hospitalizations, 5,000 Americans die each year from foodborne illness and 2,366,000 cases, 21,138 hospitalizations and 718 deaths in England and Wales (Mead et al.,1999 and Adak et al., 2002). There are about 5.4 million cases of foodborne disease in Australia each year (OzFoodNet, 2006). Hence, trends in foodborne illness in the developed countries indicate that the incidence of foodborne illness is increasing, and that it is likely to remain a threat to public health well into this century (Crerar et al., 1996).
There are many and varied sources of organisms causing food poisoning. Most cases of food poisoning are caused by bacteria which arise from animal, human or environmental sources (Gracey et al., 1999). Contaminated raw meat is one of the main sources of foodborne illnesses (Bhandare et al., 2007). Specific sources that contribute microbial contamination to animal carcasses and to fresh meat during slaughter and dressing include the faeces, the hide, water, air, intestinal contents, lymph nodes, processing equipment, and humans (Sofos, 2005), and can be transferred to the carcass during skin removal and evisceration (Hansson et al., 2000; Reid et al., 2002). The types of microorganisms and extent of contamination present on the final product are influenced by sanitation procedures, hygienic practices, application of food safety interventions, type and extent of product handling and processing, and the conditions of storage and distribution (Sofos, 2005).
There are four major pathogens that have frequently been associated with meat and meat products including Salmonella species, Campylobacter species, Listeria monocytogenes, and Escherichia coli O157:H7. These organisms have been linked to a number of cases of human illness (Mershal et al., 2010).
Salmonella is the most frequently reported cause of foodborne illness (Birhaneselassie and Williams, 2013). Foodborne salmonellosis often follows consumption of contaminated animal products, which usually results from infected animals used in food production or from contamination of the carcasses or edible organs (Alemayehu et al., 2002). Salmonella infection in meat animals arises from intensive rearing practices and the use of contaminated feeds (Ejeta et al., 2004). Cross-contamination of carcasses with Salmonella can also occur during slaughtering operations (Baird-Parker, 1990). Stress associated with transport of animals to abattoir augments shedding of Salmonella by carrier animals and this may contribute to the spread of the organism to other animals in the slaughter plant (Isaacson et al., 1999).
Slaughtering procedures potentially involve many risks of both direct and cross contamination of carcasses and meat surfaces. During slaughter, faecal contamination of edible organs with subsequent contamination of the carcass may occur. This can be carried through all slaughter procedures up to the processing of the raw products, which are important sources of Salmonella in the human food chain (Edwards et al., 1997). Contamination of equipment, utensils and hands of workers can spread Salmonella to uncontaminated carcasses and parts, which can occur in subsequent handling, processing, transport, storage, distribution and preparation for consumption (Ejeta et al., 2004).
Salmonellosis causes significant morbidity and mortality in both humans and animals and has a substantial global socioeconomic impact (Tassios et al., 1997). There are 16 million annual cases of typhoid fever, 1.3 billion cases of gastroenteritis and 3 million deaths worldwide due to Salmonella (Bhunia, 2008). Mortality due to Salmonella infections is mainly a health problem in developing countries, but morbidity due to acute Salmonella infections also has important socio-economic impact in industrialised nations (Hansen- Wester and Hensel, 2001). Salmonella infections in the United States account for roughly 19,336 hospitalizations, 17,000 quality adjusted life years lost and $3.3 billion in total medical expenditures and lost productivity each year (Batz et al., 2011). For human salmonellosis in the Netherlands, the costs are estimated to be between 32 and 90 million Euro per year (van Pelt and Valkenburgh, 2001).
Salmonella infections are very common and an important public health problem in many parts of the world. Studies in different countries indicated that Salmonellae are wide spread in small ruminants (Nabbut and Al-Nakhli, 1982 and Chandra et al., 2007). Research to date, as well as unpublished reports from different health institutions in Ethiopia have indicated that salmonellosis is a common problem and also showed the presence of a number of serogroups/ serotypes in humans, animals, animal food products and other foods (Nyeleti et al., 2000; Muleta and Ashenafi, 2001; Molla et al., 2003; Tibaijuka et al., 2003; Woldemariam et al., 2005, Asrat, 2008 and Akafete and Haileleul, 2011).
Moreover, an increase in the resistance of Salmonella to commonly used antimicrobials has been also noted in both public health and veterinary sectors in Ethiopia (Molla et al., 1999; Molla et al., 2003 and Asrat, 2008). The extensive use of the first line drugs has led to the development of multiple drug resistance at a level which could pose a serious problem in the near future (Getenet, 2008). Although, little study has so far been undertaken to isolate Salmonella from goat‟s meat in Ethiopia (Molla et al., 1999, Woldemariam et al., 2005; Wassie, 2004 and Akafete and Haileleul, 2011) from central part of the country and export abattoirs, there was no report regarding the status of the Salmonella from Dire Dawa municipal abattoir.
Problem Statement and Justification of the Study
In spite of the increased consumer demand on food safety standards for beef in Nigeria municipality there are still poor hygiene and sanitary practices along the food production chain which contribute to unacceptable level of microbial load in meat. This poses a health risk to consumers. Although several studies have been conducted to assess the degree of meat losses due to contamination of carcasses and offals (Mtenga et al., 2000), detection of zoonotic conditions through post mortem inspection (Komba et al., 2012) and occurrence of Thermophilic Compylobacter spp in cattle slaughtered at Morogoro municipal abattoir (Nonga et al., 2010), limited studies have been conducted to assess microbial contamination of beef along the production chain from the abattoir to retail meat outlets. In order to minimize public health risks, there is a need to assess microbial contamination of beef along the production chain and point out the main contaminated points that would require interventions through a HACCP system and education for different actors on beef enterprise.
Objective of the Study
The overall objective of the study was to determine the extent of microbial contamination and associated risk factors in beef production chain from abattoir to retail meat outlets in Morogoro municipality.
- i) To identify risk factors contribute to microbial contamination of beef from the abattoir to retail meat outlets.
- ii) To establish the main beef microbial contamination points from the abattoir to retail meat outlets.
iii) To determine the extent of microbial contamination of beef along the production chain from the abattoir to retail meat outlets.
Overview of food hygiene and food safety
Foodborne diseases remain a real and formidable problem in both developed and developing countries, causing great human suffering and significant economic losses. Up to one third of the population of developed countries may be affected by foodborne diseases each year, and the problem is likely to be even more widespread in developing countries, where food and water-borne diarrhoeal diseases kill an estimated 2.2 million people each year, most of them Children (FAO/WHO, 2006). The problem is severe in developing countries due to difficulties in securing optimal hygienic food handling practices. In developing countries, up to an estimated 70% of cases of diarrheal disease are associated with the consumption of contaminated food (Knife and Abera, 2007).
Food safety is therefore a fundamental public health concern, and achieving a safe food supply poses major challenges for national food safety officials. Changing global patterns of food production, international trade, technology, public expectations for health protection and many other factors have created an increasingly demanding environment in which food safety systems operate. An array of foodborne hazards both familiar and new, pose risks to health and obstacles to international trade in foods. These risks must be assessed and managed to meet growing and increasingly complex sets of national objectives (CAC, 2007).
Food hygiene and food safety practices
Foodborne diseases are common in developing countries including Ethiopia because of the prevailing poor food handling and sanitation practices, inadequate food safety laws, weak regulatory systems, lack of financial resources to invest in safer equipment, and lack of education for food handlers (WHO, 2004). National Hygiene and Sanitation Strategy program (MoH, 2005) reported that in Ethiopia more than 250,000 children die every year from sanitation and hygiene related diseases and about 60% of the disease burden was related to poor hygiene and sanitation in Ethiopia. Unsafe sources, contaminated raw food items, improper food storage, poor personal hygiene during food preparation, inadequate cooling and reheating of food items and a prolonged time lapse between preparing and consuming food items were mentioned as contributing factors for outbreak of foodborne diseases (Linda du and Irma, 2005).
Studies conducted in different parts of the country showed the poor sanitary conditions of catering establishments and presence of pathogenic organisms like campylobacter, Salmonella, Staphylococcus aureus, Bacillus cereus and Escherichia coli, (Bayleyegn et al., 2003; Abera et al., 2006; Knife and Abera, 2007; Tefera et al., 2009 and Mekonnen et al., 2013).
Of the foods intended for humans, those of animal origin tend to be most hazardous unless the principles of food hygiene are employed. Animal products such as meats, fish and their products are generally regarded as high-risk commodity in respect of pathogen contents, natural toxins and other possible contaminants and adulterants (Yousuf et al., 2008). Bacterial contamination of meat products is an unavoidable consequence of meat processing (Jones et al., 2008). Even if data regarding meat borne diseases in Ethiopia are extremely scarce, a few studies conducted in different parts of the country have shown the public health importance of several bacterial pathogens associated with foods of animal origin (Bayleyegn et al., 2003; Ejeta et al., 2004: Adem et al., 2008; Kumar et al., 2009 and Tefera et al., 2009). Salmonella remains a leading etiological agent in bacterial foodborne diseases (D‟Aoust, 1991).
Indicator Organisms on Meat
The safety of raw meat products can be estimated based on indicator organism including TVC, TCC and TFC counts of mesophilic (Barros et al., 2007). Their presence indicate the possibility of finding pathogenic bacteria. TVC gives a quantitative idea about the presence of microorganisms such as bacteria, yeast and mould in samples. The coliform bacteria group consists of several genera of bacteria within the family Enterobacteriaceae. Total coliforms are a group of bacteria that are widespread in nature.
All members of the total coliforms group can occur in human faeces, but some can also be present in animal manure, soil, sub-merged wood and in other places outside the human body. The usefulness of total coliforms as an indicator of faecal contamination depends on the extent to which the bacteria species found are faecal and human in origin. Faecal coliforms are good indicator of contamination from human or other animal waste products and they indicate greater risk of exposure to pathogenic organisms than total coliforms (Moore and Griffith, 2002). Control measures that reduce the number of bacterial load will reduce the risk of pathogenic bacteria on meat.
Common Microorganisms Present in Meat and Meat Products
Microorganisms of relevance with regard to meat hygiene include helminths, moulds, bacteria and viruses. Within these groups, bacteria play the most important role. Parasites are of insignificant value in meat which has passed meat inspection, or where efficient internal parasite control programmes or measure are in place. The most frequently identified bacterial pathogen associated with consumption of beef products are Salmonella spp, Compylobacter spp, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Clostridium perfringens, Yersinia enterocolitica, Bacillus cereus and Vibrio parahaemolyticus (Biswas et al., 2011). Compylobacter spp, Salmonella spp and Escherichia coli are often present in fresh meat and poultry (Zhao et al., 2001). Ali et al. (2010) reported the foodborne pathogens isolated from meat samples in retail meat shops. They included Escherichia coli O157:H7, Listeria spp, Salmonella enteritidis and Shigella species while in meat handling equipments in retail shops were Staphylococcus and Shigella spp. Soyiri et al. (2008) isolated Staphylococcus aureus, Bacillus cereus, Clostridium perfringens and Escherichia coli in beef samples from butchers. Moreover, the faecal coliforms such as Escherichia coli are generally considered as indisputable indicators of faecal contamination from warm blooded animals (Yousuf et al., 2008).
The Effects of Bacteria in Meat and Meat Products
Food animals are useful as they supply quality protein and revenues to man, but on the other hand they serve as vehicles of disease pathogens. Raw meat remains an important and probably the major source of human food borne infection with pathogenic bacteria. In spite of decades of effort to control them, it has been difficult to obtain food animals free of pathogenic bacteria (Wilfred and Fairoze, 2011). The effects that microbial contaminants cause on meat include spoilage of the meat, food poisoning and condemnation of carcasses which results into reduction of income to farmers as well as meat sellers. Consumers and meat handlers may acquire bacterial diseases such as Anthrax, Q-fever, Campylobacteriosis, Ornithosis, Botulism, Staphylococcus food poisoning, Salmonellosis, Brucellosis, Erysipelas, Streptococcosis, Tetanus, Yersiniosis, Clostridiosis, Listeriosis, Glanders, Leptospirosis and Tuberculosis due to poor handling of food animals and meat (Adeyemo, 2002).
Incidences of Microbial Load in Meat, Handling Equipments and Facilities
The microbiological profile in meat products is the key criteria for determining quality and safety of fresh produce. Ideally, meat should be considered as wholesome when pathogens of concern are absent or if present should be at low number depending on their toxin or metabolites produced (Biswas et al., 2011). Bhandare et al. (2009) reported TVC at 5.8 ± 0.17 log CFU/cm2 and 6.05 ± 0.25 log CFU/cm2 in modern Indian and traditional meat shops respectively. In abattoir, the highest TVC were observed on floor 7.19 ± 0.18 log CFU/cm2 and the lowest values in water 3.90 ± 0.07 log CFU/cm2 , while in retail meat shops the highest were observed on floor 7.45 ± 0.46 log CFU/cm2 and the lowest on the plastic bags 3.08 ± 0.24 log CFU/cm2. Barros et al. (2007) reported contamination level by mesophilic aerobe count in samples from retail establishments and slaughterhouse equipments at 4.68 log CFU/cm2, Total Coliforms at 2.55 log CFU/cm2 and that of Escherichia coli at 1.8 log CFU/cm2 respectively.
In other studies Nouichi et al. (2009) reported microbial load as indicated by TVC, TCC and TFC in bovine carcass slaughtered at El-Harrach slaughter house in Algeria at 4.48 ± 0.63, 2.92 ± 0.43 log CFU/cm2 and 2.60 ± 0.32 log CFU/cm2 respectively. Kumar et al. (2010) found a high total aerobic plate count of 75.91 % in beef produced and marketed in some parts of Tigray region with high percentage of unsatisfactory quality. Ukut et al. (2010) reported microbial load on fresh meat sold in Calabar Metropolis markets at 2.24 x 104- 5.01 x104 CFU/g and 1.05 x 103 – 3.72 x 103 CFU/g for TVC and TCC respectively.
Source of Beef Contamination
Unless the animals are infected the meat of freshly slaughtered animals are generally sterile. The presence of microorganisms on post slaughtered carcasses is due to contamination occurring immediately before, during and after slaughter. The microbial contaminations of carcasses occur mainly during processing and manipulation during skinning, evisceration, processing at abattoir and retailers establishments (Gill, 1998). The main sources of meat contamination include; animal/carcasses source, on farm factors, transport factors, abattoir and butchers facilities, parasites and wild animals, meat van, abattoir and retail meat outlet workers.
Faecal matter is a major source of contamination and can reach carcasses through direct deposition as well as by indirect contact through contaminated carcasses, equipments, workers, installations and air (Borch and Arinder, 2002). Faeces as well as soil adhering to animals are carried into abattoir on hair, hides, hooves and tail of animals. Contact between carcasses and hides allow a mixture of microorganisms to be introduced on the carcasses. These contaminating microorganisms are derived from the animal’s pre slaughter environment that may be of faecal, soil, water or feed origin (Bell, 1997). Infected body fluid such as urine, milk, blood, mucus, rumen fluid, intestinal fluid and fluid from excised abscess can be another source of carcasses contamination (Galland, 1997).
On farm factors
Body condition may affect the pathogens load. Weak animals lie down more often than healthy ones, thereby increasing the likelihood of contaminating hides. Contacts between animals at auction barns may increase the pathogen load (Galland, 1997). The exterior of the animals harbours large number and different types of microorganisms from soil, water, feed, manure as well as its natural flora (Mtenga et al., 2000).
Transportation of slaughter animals
The transport factors such as the type and cleanliness of transport facility, distance travelled and duration of journey, harshness of ride, overpopulation of animals in the conveyance and frequency of stops, may affect and contribute to pathogen load (Galland, 1997).
Abattoir and butchers facilities
The abattoir and beef retail outlet environments play important roles in contamination of meat. Site selection and availability of good quality portable water are important factors to consider when selecting site for constructing abattoir or retail meat outlets since it affects the quality of meat. Meat contamination in abattoirs and retail meat outlets result from the use of contaminated water, unhygienic practices like poor handling, use of contaminated tables to display meat intended for sale and the use of contaminated knives and other equipments in cutting operations (Fasanmi et al., 2010).
The length of time animals are held at the abattoir before slaughter can affect the pathogen load by increasing the probability of exposure and infections. Sanitation of walk ways, pen floor, railings, feed and water affect the pathogen load (Galland, 1997). Dirt, soil, body discharges and excreta from animals in holding pens or lairages are primary sources of contamination of carcasses in the later stages of the operation. This happens irrespective of whether or not the animals are fit and have passed ante mortem inspection. Adzitey et al. (2011a) reported the possible sources of contaminations arising from the cutting knives, intestinal contents, chopping boards, hides, meat handlers, containers, vehicle for transporting carcasses and the meat selling environment. It has been reported by Ali et al. (2010) that knives, wooden boards and weighing scales from retail shops are sources of bacterial contamination particularly Staphylococcus aureus and Shigella species. Akinro et al. (2009) reported that with inadequate slaughtering and disposal facilities, the abattoir becomes a source of infection and pollution, attracting domestic and wild carnivores, rodents and flies, which are vectors of diseases. Refrigerator or freezers are essential storage facilities used to prevent spoilage of meat following prolonged storage at room temperature and hence keep meat safe for long period of time.
Parasites and wild animals
With inadequate slaughtering and disposal facilities attracting flies, domestic animals, wild carnivores and rodents, abattoir/slaughter houses become among the important sources of microbial contamination (Adeyemo, 2002).
The vehicles used to transport meat from abattoir to retail meat outlets may act as sources of contamination since often lack regular cleanliness and are not well covered leading to contamination by dusts, insects and flies. Sulley, (2006) reported contamination of meat resulting from other means of transport such as motor-bikes and bicycles due to insufficient vans and trucks. On the other hand, the few transport available were not properly cleaned and thus contained high microbial loads (Sulley, 2006).
Abattoir and retail meat outlet workers
The hygienic condition of the abattoir and retail meat outlet workers has potential to contribute contamination in beef before and after processing. Adetunde et al. (2011) reported that unclean slaughter men’s hands, butcher arms, clothing and equipment used in carcass dressing process accounted for the microbial contamination and also the study of Jeffery, (2003) revealed that the worker hands and their equipments were among the main sources of meat contamination.
Overview of Salmonella
The Salmonella bacterium was first described by Theobald Smith (1859-1934) and then in
1885, two American veterinarians, Salmon and Smith isolated the bacterium causing hog cholera from infected pigs (Salmon and Smith, 1886). The name Salmonella was subsequently adopted in honor of Dr. Salmon. Over the decades following the pioneering work of Salmon and Smith, many other Salmonella were isolated from both animals and humans (Widal, 1896; Getenet, 2008). The antigenic classification or serotyping of Salmonella used today is a result of years of study of antibody interactions with bacterial surface antigens by Kauffman and White in the 1920s to 1940s (Kauffmann, 1950). According to this Kauffmann-White scheme, each Salmonella serotype is recognized by its possession of a particular lipopolysaccharide (LPS) or O antigen and a flagellar or H antigen. This led to the description of more than 2500 serotypes at present (Brenner et al., 2000; Popoff et al., 1998 and Popoff et al., 2004).
Classification and nomenclature
Historically Salmonella had been named based on the original places of isolation such as Salmonella London and Salmonella Indiana. This nomenclature system was replaced by the classification based on the susceptibility of isolates to different selected bacteriophages which is also known as phage typing. Phage typing is generally employed when the origin and characteristic of an outbreak must be determined by differentiating the isolates of the same serotype. It is very reproducible when international standard sets of typing phages are used More than 200 definitive phage types (DT) have been reported so far. For example, S. Typhimurium DT104 designates a particular phage type for Typhimurium isolates (Hanes, 2003; Andrews and Baumler, 2005 and Pui et al., 2011).
Epidemiologic classification of Salmonella is based on the host preferences. The first group includes host-restricted serotypes that infect only humans such as S. Typhi. The second group includes host-adapted serotypes which are associated with one host species but can cause disease in other hosts serotypes such as S. Pullorum in avian. The third group includes the remaining serotypes. Typically, Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Heidelberg are the three most frequent serotypes recovered from humans each year (Gray and Fedorka-Cray, 2002 and Boyen et al., 2008).
The genus consists of two species: the first is S. enterica which is divided into six subspecies; S. enterica subsp. enterica, S. enterica subsp. salamae, S. enterica subsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp. houtenae and S. enterica subsp. indica; and the second is S. bongori (formerly called S. enterica subsp. bongori) (WHO, 2003c). Salmonella enterica subspecies I is mainly isolated from warm-blooded animals and accounts for more than 99% of clinical isolates whereas remaining subspecies and S. bongori are mainly isolated from cold-blooded animals and account for less than 1% of clinical isolates. As an example, the Kauffmann species Salmonella Typhimurium is now designated as Salmonella enterica subspecies I serotype Typhimurium. Under the modern nomenclature system, the subspecies information is often omitted and culture is called S. enterica serotype Typhimurium and in subsequent appearance, it is written as S. Typhimurium. This system of nomenclature is used nowadays to bring uniformity in reporting (Andrews and Baumler, 2005 and Parry, 2006).
Kauffmann-White scheme classifies Salmonella according to three major antigenic determinants composed of flagellar H antigens, somatic O antigens and virulence (Vi) capsular K antigens. This was adopted by the International Association of Microbiologists in 1934. Agglutination by antibodies specific for the various O antigens is employed to group Salmonellae into the 6 serogroups: A, B, C1, C2, D and E. For instance, S. Paratyphi A, B, C and S. Typhi express O antigens of serogroups A, B, C1 and D, respectively. More than 99% of Salmonella strains causing human infections belong to Salmonella enterica subspecies enterica. Although not common, cross-reactivity between O antigens of Salmonella and other genera of Enterobacteriaceae do occur (Pui et al., 2011).
Therefore, further classification of serotypes is based on the antigenicity of the flagellar H antigens which are highly specific for Salmonella (Scherer and Miller, 2001). In brief, O antigens are lipopolysaccharide (LPS) of the outer bacterial membrane. They are heat stable, resistant to alcohol and dilute acids. H antigens are heat-labile proteins associated with the peritrichous flagella and can be expressed in one of two phases. The phase 1 H antigens are specific and associated with the immunological identity of the particular serovars whereas phase 2 antigens are non-specific antigens containing different antigenic subunit proteins which can be shared by many serovars. K antigens which are heat- sensitive carbohydrates are produced by Salmonella serovars that express a surface-bound polysaccharide capsular antigen (Hu and Kopecko, 2003; Yousef and Carlstrom, 2003).
General characteristics of Salmonella
Salmonella make up a large genus of gram-negative bacilli within the family Enterobacteriaceae and it constitute a genus of more than 2300 serotypes that are highly adapted for growth in both humans and animals and that cause a wide spectrum of disease. The growth of S. typhi and S. paratyphi is restricted to human hosts, in whom these organisms cause enteric (typhoid) fever. The remainder of Salmonella serotypes, referred to as non-typhoidal Salmonella, can colonize the gastrointestinal tracts of a broad range of animals, including mammals, reptiles, birds, and insects. More than 200 of these serotypes are pathogenic to humans, in whom they often cause gastroenteritis and can also be associated with localized infections and/or bacteremia (Fuaci and Jameson, 2005).
Members of the genus Salmonella are ubiquitous pathogens found in humans and livestock, wild animals, reptiles, birds, insects (Getenet, 2008) and can multiply under various environmental conditions outside the living hosts (Pui et al., 2011). Salmonellae are gram-negative, non-spore forming, facultative anaerobic bacilli, and 2 to 3 by 0.4 to 0.6 μm in size (Getenet, 2008). They do not require sodium chloride for growth, but can grow in the presence of 0.4 to 4%. Most Salmonella serotypes grow at temperature range of 5 to 47°C with optimum temperature of 35 to 37°C but some can grow at temperature as low as 2 to 4°C or as high as 54°C . They are sensitive to heat and often killed at temperature of 70°C or above. Salmonella grow in a pH range of 4 to 9 with the optimum between 6.5 and 7.5. They require high water activity (aw) between 0.99 and 0.94 (pure water aw=1.0) yet can survive at water activity less than 0.2 such as in dried foods. Complete inhibition of growth occurs at temperatures less than 7°C, pH less than 3.8 or water activity less than 0.94 (Pui et al., 2011).
Like other members of the family Enterobacteriaceae, they produce acid on glucose fermentation; reduce nitrates to nitrite, and don‟t produce cytochrome oxidase. In addition; all Salmonellae except S. gallinarum-pullorum are motile by means of peritrichous flagella, and all but S. typhi produce gas (H2S) on sugar fermentation (Fuaci and Jameson, 2005 and Getenet, 2008). Salmonella are non- capsulated except S. Typhi, S. Paratyphi C and some strain of S. Dublin (Getenet, 2008).
Geographic distribution and host range
Salmonella is one of the leading causes of bacterial foodborne disease in industrialized as well as developing countries even though the incidence seems to vary between countries (Radostits et al., 1994; D‟Aoust, 1997; Molla et al., 2003 and Chiu et al., 2004). The wide variations in the national prevalence of Salmonellosis likely arise from limited scope of studies and lack of coordinated epidemiological surveillance systems, under-reporting of cases and the presence of other diseases considered being of high priority (Radostits et al., 1994 and Molla et al., 2003).
The epidemiology of salmonellosis is complex largely because there are more than 2,500 distinct serotypes (serovars) with different reservoirs and diverse geographic incidences. Changes in food consumption, production, and distribution have led to an increasing frequency of multistate outbreaks associated with fresh produced and processed foods (Rounds et al., 2010).
According to the WHO Global Salm-Surv, during 2000-2002, S. Enteritidis was by far the most common serotype reported from humans globally. In 2002, it accounted for 65% of all isolates, followed by S. Typhimurium at (12%) and S. Newport at (4%). Among non- human isolates, S. Typhimurium was the most commonly reported serotype in all the three years, accounting for (17%) of isolates in 2002 followed by S. Heidelberg (11%) and S. Enteritidis (9%). Salmonella Enteritidis, S. Typhimurium and S. Typhi were ranked among the fifteen most common human serotypes in all regions of the world throughout the three year study period. Salmonella Agona, S. Infantis, S. Montevideo, S. Saintpaul, S. Hadar, S. Mbandaka, S. Newport, S. Thompson, S. Heidelberg and S. Virchow were also widespread. In Africa in 2002, S. Enteritidis and S. Typhimurium were each reported from approximately one fourth of isolates from humans (Galanis et al., 2006 and Swaminathan et al., 2006).
Reservoir host and source of infection
Salmonellosis is the most common foodborne disease in both developing and developed countries, although incidence rates vary according to the country (Stevens et al., 2006). The fecal wastes from infected animals and humans are important sources of bacterial contamination of the environment and the food chain (Ponce et al., 2008). Members of Salmonella enterica subspecies enterica are widely distributed in the environment and in the intestinal tracts of animals (Anjum et al., 2011). People can become infected following a failure of personal hygiene after contact with infected animals and or other infected people. Environmental contamination, especially untreated water is also important (Gracey et al., 1999). Most human infections are acquired through consumption of contaminated food of animal origin (Gracey et al., 1999 and Anjum et al., 2011).
Foods of animal origin, particularly meat, poultry, and, in some instances, unpasteurized egg products are considered to be the primary sources of human salmonellosis (Tauxe, 1991; Nielsen et al., 1995; Wray and Davies, 2000; Acha and Szyfres, 2001; White et al., 2001). It has been reported that livestock and their products can contribute to as much as 96% of the total Salmonella infection in humans (Dahal, 2007). Most of these food products, e.g. beef, mutton and poultry, become contaminated during slaughter and processing, from the gut contents of healthy excreting animals. In the same way, all food that is produced or processed in a contaminated environment may become contaminated with Salmonellae and be responsible for outbreaks or separate cases of disease as a result of faults in transport, storage, or preparation (D‟Aoust, 1997). Unlike S. typhi and S. paratyphi, whose only reservoir is humans; non-typhoidal salmonellosis is acquired from multiple animal reservoirs (Fuaci and Jameson, 2005).
A less common source of non-typhoidal Salmonella infections is exposure to pets, especially reptiles. Fecal carriage rates in reptiles can be more than 90%. It is estimated that approximately 74,000 infections with Salmonella result from exposure to reptiles and amphibians in the United States each year (AAP, 2013). Iince 1986, an increase in the popularity of non-banned reptiles, including iguanas, has been followed by increases in rates of Salmonella infections. Other pets, including African hedgehogs, snakes, birds, rodents, baby chicks, ducklings, dogs, and cats, can also serve as potential vectors (Fuaci and Jameson, 2005).
Mode of transmission
Salmonella infection appears to be one of the most common examples of an enteric disease that is transmitted from animals to humans. The transmission occurs both through food products, such as meat, dairy products, and eggs, and by direct contact between animals and humans through the fecal-oral route (Olsvik, et al., 1985).
Foodborne salmonellosis often follows consumption of contaminated animal products such as raw meat, poultry and eggs. Not washing fresh fruits and vegetables before eating them, as well as not thoroughly cleaning work surfaces used to prepare raw meat and other foods in the kitchen can also be source of Salmonella. Food can also be contaminated by food handlers who do not thoroughly wash their hands with soap after handling raw meat or after using the bathroom (WHO, 1989). Salmonella infections are primarily of foodborne origin but can also occur through contact with infected animals, humans, other feces (Rounds et al., 2010).
The main mode of transmission is from food products contaminated with animal products or waste most commonly eggs and poultry but also undercooked meat, unpasteurized dairy products, seafood, and fresh produced. S. enteritidis associated with chicken eggs is emerging as a major cause of foodborne disease. Approximately 1 in 20,000 eggs is thought to be infected with S. enteritidis. Between 1974 and 1994, there was a fivefold increase (from 5% to 25%) in the isolation of S. enteritidis from eggs in the United States; in 1998, the U.S. Department of Agriculture estimated that 80% of all salmonellosis cases were caused by infected eggs (Fuaci and Jameson, 2005).
The outcome of a Salmonella infection is determined by the status of the host and status of the bacterium. The status of the bacterium is determined by the so called virulence factors which is described as follows (Van Asten and van Dijk, 2005).
Salmonellosis in the human host is generally associated with Salmonella enterica subspecies enterica and acute infections can present in one of four ways: enteric fever, gastro-enteritis, bacteremia, and extra intestinal (EI) focal infection. As with other infectious diseases the course and outcome of the infection are dependent upon a variety of factors including inoculating dose, immune status of the host and genetic background of both host and infecting organism (Getenet, 2008). Broadly speaking the Salmonella enterica from human infections can be subdivided in to two groups: the enteric fever (typhoidal) group and non-typhoidal Salmonella (NTS), which typically cause gastroenteritis but can cause invasive disease under certain conditions (Selander et al., 1990).
All Salmonella infections begin with the ingestion of organisms in contaminated food or water (Francis et al., 1992 and Fuaci and Jameson, 2005). The infectious dose of Salmonella varies from 103 to 106 colony-forming units. This variability probably reflects the ability of Salmonellae to resist the low pH of the stomach a powerful component of host defense (Fuaci and Jameson, 2005). After leaving the stomach, Salmonella must traverse the mucosal layer overlaying the epithelium of the small intestine. After crossing the mucosal layer overlaying the intestinal epithelium, Salmonella interacts with both enterocytes and Microfolds cells (Mcells) (Francis et al., 1992). The organisms are rapidly internalized and transported into submucosal lymphoid tissue where they may enter into systemic circulation. Salmonella have also the ability to induce non phagocytic epithelial cells by a process known as bacterial mediated endocytosis. This process involves the formation of large membrane ruffles around the organism and cytoskeleton rearrangement (Francis et al., 1992). Salmonella is then internalized within bound vacuoles through which organisms‟ trancytose from the apical to the basolateral surface (Rathman et al., 1997). Once it crosses the intestinal epithelium, Salmonella serotypes that cause systemic infections entermacrophages, and migration of infected macrophages to other organs of reticulo-endothelial systems probably facilitates the dissemination of bacteria in the host (Getenet, 2008).
Gastroenteritis due to NTS may persist with fever, nausea, vomiting, abdominal pain and symptoms may continue for over a week. In contrast, the early symptoms of enteric fever are often vague, and may include a dry cough, severe headache, anorexia, fever and a tendency to constipation rather than diarrhoea (Parry et al., 2002). If enteric fever is not treated on time, serious complication like hemorrhage from ulcers can occur during the third week of illness or perforation of the peyer‟s patches (PP) can cause generalized peritonitis and septicemias; these are the commonest cause of death in typhoid fever. With the introduction of early and appropriate antibiotic therapy, the average case fatality rates for typhoid are less than 1% (Everest et al., 2001).
Salmonella infections in animals
Salmonella have a wide variety of domestic and wild animal hosts. The infection may or may not be clinically apparent. In the subclinical form, the animal may have a latent infection and harbor the pathogen in its lymph nodes, or it may be a carrier and eliminate the agent in its fecal material briefly, intermittently, or persistently. In domestic animals, there are several well-known clinical enteritis due to species-adapted serotypes, such as S. pullorum or S. abortus equi. Other clinically apparent or in apparent infections are caused by serotypes with multiple hosts (PAHO, 2001).
The principal causes of clinical salmonellosis in cattle are serotype Dublin and S. Typhimurium. Other serotypes can sometimes be isolated from sick animals. Salmonellosis in adult cattle occurs sporadically, but in calves it usually acquires epizootic proportions. The disease generally occurs when stress factors are involved. Serotype dublin, adapted to cattle, has a focal geographic distribution. In the Americas, outbreaks have been confirmed in the western United States, Venezuela, Brazil, and Argentina. It also occurs in Europe and South Africa. In adult cattle, the disease begins with high fever and the appearance of blood clots in the feces, followed by profuse diarrhea, and then a drop in body temperature to normal. Signs of abdominal pain are very pronounced. The disease may be fatal within a few days or the animal may recover, in which case it often becomes a carrier and new cases appear. Calves are more susceptible than adults, and in them the infection gives rise to true epidemic outbreaks, often with high mortality. Septicemia and death are frequent in newborns. The carrier state is less frequent among young animals and occurs primarily in adult cattle. The infection is almost always spread by the feces of a cow that is shedding the agent, but it may also originate from milk (PAHO, 2001).
Swine are host to numerous Salmonella serotypes and are the principal reservoir of S. choleraesuis. Serotypes that attack swine include S. enteritidis, S. Typhimurium, and S. dublin. S. choleraesuis is very invasive and causes septicemia; it may be isolated from the blood or from any organ. Swine are particularly susceptible and experience epidemic outbreaks between 2 and 4 months of age, but the infection also appears in mature animals, almost always as isolated cases. The most frequent symptoms are fever and diarrhea. The infection usually originates from a carrier pig or contaminated food. Infection by other serotypes may sometimes give rise to serious outbreaks of salmonellosis with high mortality. Because of the frequency with which swine are infected with different types of Salmonellae, pork products have often been a source of human infection (PAHO, 2001).
Cases of clinical salmonellosis in sheep and goats are infrequent. The most common serotype found in gastroenteritis cases is S. typhimurium, but many other serotypes have also been isolated. Serotype S. abortus ovis, which causes abortions in the last two months of pregnancy and gastroenteritis in sheep and goats, seems to be restricted to Europe and the Middle East (PAHO, 2001). Horses are also susceptible to Salmonellae, particularly S. typhimurium. Salmonella enteritis occurs in these animals, sometimes causing high mortality. Calves suffer from acute enteritis with diarrhea and fever; dehydration may be rapid. Nosocomial transmission has been seen in hospitalized horses (Bauerfeind et al., 1992).
In recent years, a high prevalence of infection caused by numerous serotypes has been confirmed in cats and dogs. These animals may be asymptomatic carriers or may suffer from gastroenteritic salmonellosis with varying degrees of severity. Dogs can contract the infection by eating the feces of other dogs, other domestic or peridomestic animals, or man. Dogs and cats can also be infected by contaminated food. In addition, dogs can transmit the disease to man. Treatment for these animals consists mainly of fluid and electrolyte replacement (PAHO, 2001).
Two serotypes, S. pullorum and S. gallinarum, are adapted to domestic fowl. They are not very pathogenic for man, although cases of salmonellosis caused by these serotypes have been described in children. Many other serotypes are frequently isolated from domestic poultry; for that reason, these animals are considered one of the principal reservoirs of Salmonellae. Pullorum disease, caused by serotype S. pullorum, and fowl typhoid, caused by S. gallinarum, produce serious economic losses on poultry farms if not adequately controlled. Both diseases are distributed worldwide and give rise to outbreaks with high morbidity and mortality. Pullorum disease appears during the first 2 weeks of life and causes high mortality. The agent is transmitted vertically as well as horizontally. Carrier birds lay infected eggs that contaminate incubators and hatcheries. Fowl typhoid occurs mainly in adult birds and is transmitted by the fecal matter of carrier fowl. On an affected poultry farm, recuperating birds and apparently healthy birds are reservoirs of infection. Salmonella un-adapted to fowl also infect them frequently. Nearly all the serotypes that attack man infect fowl as well. Some of these serotypes are isolated from healthy birds. The infection in adult birds is generally asymptomatic, but during the first few weeks of life, its clinical picture is similar to pullorum disease (loss of appetite, nervous symptoms, and blockage of the cloaca with diarrheal fecal matter). The highest mortality occurs during the first two weeks of life. Most losses occur between six and ten days after hatching (PAHO, 2001).
Rodents become infected with the serotypes prevalent in the environment in which they live. Rodents found in and around food processing plants can be an important source of human infection. Of 974 free-living wild animals examined in Panama, 3.4% were found to be infected, principally by serotype S. enteritidis and, less frequently, by S. arizonae (Arizona hinshawii) and Edwardsiella. The highest rate of infection (11.8%) was found among the 195 marsupials examined. Outbreaks of salmonellosis among wild animals held in captivity in zoos or on pelt farms are not unusual. Salmonella infection in cold-blooded animals has merited special attention. An infection rate of 37% was found in 311 reptiles examined live or necropsied at the National Zoo in Washington, D.C. The highest rate of infection was observed in snakes (55%) and the lowest in turtles (3%). The Salmonellae isolated were 24 different serotypes formerly classified under the common name of S. enteritidis, 1 strain of S. choleraesuis, and 39 of S. arizonae. No disease in their hosts was attributed to these bacteria, but they may act together with other agents to cause opportunistic infections (PAHO, 2001).
Salmonella infections in humans
Salmonella infections in humans can range from a self-limited gastroenteritis usually associated with non-typhoidal Salmonella (NTS) to typhoidal fever with complications such as a fatal intestinal perforation (OIÉ, 2000). Non-typhoidal Salmonella is one of the principal causes of food poisoning worldwide with an estimated annual incidence of 1.3 billion cases and 3 million deaths each year (Torpdahl et al., 2007). Outbreaks of salmonellosis have been reported for decades, but within the past 25 years the disease has increased in incidence in many continents. The disease appears to be most prevalent in areas of intensive animal husbandry (OIÉ, 2000).
The incubation period in people is variable but is usually between 12 and 36 hours. The typical presenting symptom is diarrhea but this may be accompanied by nausea and abdominal pain, although vomiting is not usual. There may also be a headache and fever. While the infection is normally self-limiting and does not require antibiotic treatment, occasionally, with more invasive Salmonella such as S. Virchow, bacteremia can occur. The infection is rarely fatal in people (Gracey et al., 1999).
Salmonellosis is most commonly caused by S. enterica subsp. typhimurium or S. enterica subsp. enteritidis. Secondly, S. enterica subsp. typhi and S. enterica subsp. paratyphi are the causes of typhoid fever or paratyphoid fever, respectively. Salmonella can replicate both inside the vacuoles of host cells and in the external environment. Salmonella are the second most common pathogens isolated from humans with gastroenteric disease in developed countries (Buncic, 2006).
Salmonella Typhimurium and S. enteritidis occur in the gastro intestinal tract of animals, including livestock. The disease is self-limiting, but can be severe in young, elderly or otherwise IC (immunecompromised) people. Salmonella invade epithelial cells in the ileum and proliferate in the lamina propria and profuse, watery diarrhoea results. Some isolates produce a heat-labile enterotoxin, which initiates diarrhoea. Sequelae include post- enteritis reactive arthritis and reiter‟s syndrome and systemic infection can result. Individuals can develop carrier status of up to 6 months in duration. The infectious dose varies, from only a few CFU to >105 CFU, so growth of the pathogen in foods has not been a factor in all cases of foodborne salmonellosis, but appears to have been in some.
Salmonella penetrate the intestinal epithelium, possibly proliferating in macrophages and polymorphs, pass into mesenteric lymph nodes, liver or spleen then cause septicemia. Peritonitis and subsequent death can occur. Ulceration of the ileum can occur if organisms multiply in the bile of the gall bladder and cause re-infection. Any food could be a vehicle of infection if contaminated with human faeces. Foods known to have been vehicles of typhoid fever include raw milk, shellfish and meat. However, typhoid fever is predominantly spread by water contaminated with human faeces (Buncic, 2006).
Isolation and identification of Salmonella
Salmonella can be isolated either from tissues collected aseptically at necropsy or from faces, rectal swabs, environmental samples, food products and feedstuffs. When infection of the reproductive organs, abortion occurs, it is necessary to culture fetal stomach contents, placenta and vaginal swabs and, in the case of poultry, embryonated eggs. Individual samples for bacteriological tests should be collected as aseptically as possible by following the respective standards. Moreover, precaution should be taken to avoid cross contamination of samples during transit and at the laboratory. Packages should also be kept cool and accompanied by adequate information (OIE, 2005). Non-typhoidal Salmonella gastroenteritis is diagnosed when Salmonella is cultured from stool (Fuaci and Jameson,
2005). The isolation and identification of Salmonella can be performed using techniques recommended by International Organizations for Standardization (ISO-6579, 2002), and those recommended by the Global Salmonella Surveillance (GSS) and National Health Services for Wales (NHS) (Zelalem et al., 2011).
The two EU approved methods for Salmonella detection in food and animal feedstuffs are ISO-6579:2002 and NMKL 71 (Nordic Committee on Food Analysis) (Carrique-Mas and Davies, 2008). ISO-6579:2002 is sensitive, but complex and expensive. It consists of pre- enrichment of the sample in BPW followed by selective enrichment in MKTTn and RVS. From each enrichment medium, plating onto two agar media plates (one of which is Xylose-Lysine Desoxycholate [XLD] agar) is carried out after 24 h and 48 h of incubation. Up to five colonies per plate have to be confirmed, which may potentially involve the confirmation of up to 40 presumptive colonies (ISO-6579, 2002). Conventional cultural methods for the detection of foodborne Salmonella species generally consist of five distinct and successive steps. These are pre-enrichment in nonselective media and selective enrichment in broth media, plating on differential agar, biochemical screening and serological conformation (D‟Aoust, 2001).
For confirmation, it is recommended that at least five colonies be identified in the case of epidemiological studies. If on one dish there are fewer than five typical or suspect colonies, take for confirmation all the typical or suspect colonies. Streak the selected colonies onto the surface of pre-dried nutrient agar plates in a manner which will allow well-isolated colonies to develop. Incubate the inoculated plates at 37 °C ± 1 °C for 24 h ± 3 h and pure cultures is used for biochemical and serological confirmation (ISO-6579,2002).
Confirmation can be made using biochemically using triple sugar iron agar (TSI) (Oxoid CM0277, Basingstoke, England), Christensen‟s urea agar (Oxoid CM53, Basingstoke, England), lysine iron agar (LIA) (Oxoid CM381, Basingstoke, England), Voges Proskauer (VP), methyl red (MR) (Micromaster Thane, India), and Indole tests (Becton Dickinson, USA) (Zelalem et al., 2011). Typical Salmonella cultures show alkaline (red) slants and acid (yellow) butts with gas formation (bubbles) and (in about 90 % of the cases) formation of hydrogen sulfide (blackening of the agar) When lactose-positive Salmonella is isolated, the TSI slant is yellow. Thus, preliminary confirmation of Salmonella cultures shall not be based on the results of the TSI agar test only (ISO-6579, 2002).
Serological confirmation and serotyping– Agglutination tests, ELISA, anti-globulin and compliment fixation tests have been used to detect antibody responses to Salmonella infections (Quinn et al., 1999). The detection of the presence of Salmonella O-, Vi- and H- antigens is tested by slide agglutination with the appropriate sera, from pure colonies and after auto-agglutinable strains have been eliminated. This method of relies on the antibody/antigen reaction between a test culture and commercially prepared antiserum (ISO-6579, 2002).
Antimicrobial susceptibility tests and resistance profile
There are three test methods (disk diffusion, broth dilution and agar dilution). Antimicrobial susceptibility testing methods that consistently provide reproducible and repeatable results is when followed correctly (CLSI, 2008).
Disk diffusion – Disk diffusion refers to the diffusion of an antimicrobial agent of a specified concentration from disks, tablets or strips, into the solid culture medium that has been seeded with the selected inoculum isolated in a pure culture. Disk diffusion is based on the determination of an inhibition zone proportional to the bacterial susceptibility to the antimicrobial present in the disk. The diffusion of the antimicrobial agent into the seeded culture media results in a gradient of the antimicrobial. When the concentration of the antimicrobial becomes so diluted that it can no longer inhibit the growth of the test bacterium, the zone of inhibition is demarcated. The diameter of this zone of inhibition around the antimicrobial disk is related to minimum inhibitory concentration (MIC) for that particular bacterium/antimicrobial combination; the zone of inhibition correlates inversely with the MIC of the test bacterium. Generally, the larger the zone of inhibition, the lower the concentration of antimicrobial required to inhibit the growth of the organisms. However, this depends on the concentration of antibiotic in the disk and its infusibility (OIE, 2012).
Disk diffusion is straightforward to perform, reproducible, and does not require expensive equipment. Its main advantages are: low cost, ease in modifying test antimicrobial disks when required, can be used as a screening test against large numbers of isolates, can identify a subset of isolates for further testing by other methods, such as determination of MICs. Manual measurement of zones of inhibition may be time-consuming. Automated zone-reading devices are available that can be integrated with laboratory reporting and data-handling systems. The disks should be distributed evenly so that the zones of inhibition around antimicrobial discs in the disc diffusion test do not overlap to such a degree that the zone of inhibition cannot be determined. Generally this can be accomplished if the discs are no closer than 24 mm from centre to centre, though this is dependent on disk concentration and the ability of the antimicrobial to diffuse in agar (OIE, 2012).
Broth and agar dilution methods- The aim of the broth and agar dilution methods is to determine the lowest concentration of the assayed antimicrobial that inhibits the visible growth of the bacterium being tested (MIC, usually expressed in μg/ml or mg/litre). However, the MIC does not always represent an absolute value. The „true‟ MIC is a point between the lowest test concentration that inhibits the growth of the bacterium and the next lower test concentration. Therefore, MIC determinations performed using a dilution series may be considered to have an inherent variation of one dilution. Antimicrobial ranges should encompass both the interpretive criteria (susceptible, intermediate and resistant) for a specific bacterium/antibiotic combination and appropriate quality control reference organisms. Antimicrobial susceptibility dilution methods appear to be more reproducible and quantitative than agar disk diffusion. However, antibiotics are usually tested in doubling dilutions, which can produce inexact MIC data. The selection of an AST methodology may be based on the following factors: ease of performance, flexibility, adaptability to automated or semi-automated systems, cost, reproducibility, reliability, accuracy, the organisms and the antimicrobials of interest in that particular OIE Member, availability of suitable validation data for the range of organisms to be susceptibility tested (OIE, 2012).
Salmonella species are leading causes of acute gastroenteritis in several countries and salmonellosis remains an important public health problem worldwide, particularly in the developing countries (Rotimi et al., 2008). The situation is more aggravated by the ever increasing rate of antimicrobial resistance strains (Zelalem et al., 2011). In recent years problems related to Salmonella have increased significantly, both in terms of the incidence and severity of cases of human Salmonellosis. Since the beginning of the 1990s, strains of Salmonella which are resistant to a range of antimicrobials including the first choice agents for treatment of humans have emerged and are threatening to become a serious public health problem. Drug resistant Salmonella emerge in response to antimicrobial usage in humans and in food animals so, selective pressure from the use of antimicrobials is a major driving force behind the emergence of resistance. Multi-drug resistance to critically important antimicrobials is compounding the problem (WHO, 2005). There are reports of high prevalence of resistance in Salmonella isolates from countries such as Taiwan (Lauderdale et al., 2006), India (Mandal et al., 2004, 2006), The Netherlands (Duijkeren et al., 2003), resistant isolates from France (Weill et al., 2006), Canada (Poppe et al., 2006), and Ethiopia (Molla et al., 2003).
A particular concern with S. Typhimurium DT 104 is that it has resistance to many antibiotics and often acquires resistance to others. Most strains are resistant to ampicillin, chloramphenicol, streptomycin, the sulphonamides and tetracycline. Recent resistance additions include resistance to trimethoprim and of particular concern, to the fluoroquinolones. Resistance to this latter group of antibiotics is a major worry as they are among the drugs of choice for the treatment of invasive Salmonella in humans. There is considerable debate as to what factors result in the emergence of antibiotic resistant strains of bacteria and it is alleged that antibiotic use in animals is part of the problem. Equally the use or misuse of antibiotics in humans for example also leads to the development of antibiotic resistance. The continuing development of antibiotic resistance may lead to sufficient pressure ultimately to restrict the antibiotics available to the veterinary profession for animal treatment (Gracey et al., 1999).
Antimicrobial resistant Salmonella are increasing due to the use of antimicrobial agents in food animals (Threlfall, 2002; Molla et al., 2003; Lynch et al., 2006; Molla et al., 2006; Zewdu and Cornelius, 2009) at sub-therapeutic level or prophylactic doses which may promote on-farm selection of antimicrobial resistant strains and markedly increase the human health risks associated with consumption of contaminated meat products (Molla et al., 2003; Molla et al., 2006; Zewdu and Cornelius, 2009). Cattle have been implicated as a source of human infection with antimicrobial resistant Salmonella through direct contact with livestock and through the isolation of antimicrobial resistant Salmonella from raw milk, cheddar cheese, and hamburger meat traced to dairy farms. Antimicrobial use in animal production systems has long been suspected to be a cause of the emergence and dissemination of antimicrobial resistant Salmonella (Alexander et al., 2009).
This spread of antimicrobial resistance through the food chain is regarded as a major public health issue (Threlfall, 2002 and Lynch et al., 2006). The appearance of both plasmid mediated antibiotic resistant against conventional anti- Salmonella drugs and chromosomal resistance to quinolones and fluoroquinolones has reduced therapeutic options for Salmonella septicemia in humans (Nor Elmadiena et al., 2012)
Gastroenteritis caused by Salmonella is usually a self-limiting disease (Richards et al.,
1993 and Fuaci and Jameson, 2005) and diarrhea resolves within three to seven days and fever within seventy two hours (Fuaci and Jameson, 2005). Accordingly therapy should be directed primarily to the replacement of fluid and electrolyte losses. Therefore, antimicrobials should not be used routinely to treat uncomplicated non-typhoidal Salmonella gastroenteritis or to reduce convalescent stool excretion (Richards et al., 1993). However, antimicrobial therapy should be considered for any systemic infection (Parry et al., 2002).
Antibiotic treatment usually is not recommended and in some studies has prolonged carriage of Salmonella. Neonates, the elderly, and the immunosuppressed (e.g., HIV- infected patients) with non-typhoidal Salmonella gastroenteritis are especially susceptible to dehydration and dissemination and may require hospitalization and antibiotic therapy (Fuaci and Jameson, 2005). Because of the increasing prevalence of antimicrobial resistance, empirical therapy for life threatening bacteremia or local infection suspected to be caused by non-typhoidal Salmonella should include a third generation cephalosporin and a quinolone until susceptibility patterns are known. Amoxicillin and trimethoprim- sulfamethoxazole are effective in eradication of long-term carriage. The high concentration of amoxicillin and quinolone in bile and the superior intracellular penetration of quinolone are theoretical advantages over trimethoprim-sulfamethoxazole (WHO, 2003a).
Antibiotics are coined from the Greek words “anti” meaning against and “bios” meaning life (Prescott et al., 2002). Antibiotics maybe defined as substances produced by the natural metabolic process of some microorganisms that can inhibit or destroy other organism even in minute amount with very little effect on the host (Majorie et al., 2006). To be effective, antimicrobial therapy must disrupt a necessary component of a microbe„s structure or metabolism to the extent that microbes are killed and their growth inhibited. At the same time, the drug must be safe for humans while not being overly toxic causing allergies or disrupting normal flora (Majorie et al., 2006).
Antimicrobial agents are the most important agents in the treatment of bacterial infections and thus the worldwide increase in antibiotic resistant bacteria is of major concern. Antibiotic resistance is an adaptive response in which microorganism begin to tolerate an amount of drug that would ordinarily be inhibitory (Prescott et al., 2002). Antibiotic resistance can also be a type of drug resistance in which microorganisms are able to survive exposure to an antibiotic.
Some bacteria are said to have innate resistance against antibiotics and this typically reflects variations in the structure of their all envelop. Resistance or reduced susceptibility may also be phenotypic resulting from adaptation to growth with a specific environment (Huges and Russel, 1998). The origin of antibiotic resistance genes are unclear, however, studies using clinical isolates collected before introduction of antibiotics demonstrated susceptibility, although conjugative plasmids were present. Resistance can be achieved by horizontal acquisition of resistance genes, mobilized via insertion sequences, transposons and conjugative plasmids, by recombination of foreign DNA into the chromosome or by mutation in different chromosomal loci.
The primary cause of antibiotic resistance is antibiotic use both within and outside medicine and in veterinary medicine. The greater the duration of exposure, the greater the risk of development of resistance, irrespective of the severity of the need for antibiotics.
The widespread use of antibiotics both inside and outside of medicine is playing a significant role in the emergence of resistant bacteria. Some of the causes include:
- The addition of antibiotics to the feed of livestock and this use among others leads to the development of resistant strains of bacteria.
- In some countries, antibiotics are sold over the counter without a prescription which also leads to the development of resistant strains.
- In supposedly well-regulated human medicine, the major problem of the emergence of resistant bacteria is the misuse and overuse of antibiotics by doctors as well as patients which include:
- Shortening the course of the antibiotic because of the fact that they now feel better.
- Taking antibiotic doses less than those prescribed and recommended.
- People who insist on antibiotics and physicians prescribe them as they feel they do not have time to explain why they are not necessary.
- Physicians who do not know when to prescribe antibiotics
- Physicians that is overly cautious for medical legal reasons.
- Poor hand hygiene by hospital staff has been associated with the spread of resistant organisms.
4) The household use of antibacterial in soaps and other products.
5) The unsound practices of the pharmaceutical manufacturing industry.
Mechanism of Resistance
Microbial populations develop resistance to antimicrobials through several mechanisms. The rate at which an individual gene mutates to express an antimicrobial resistance phenotype is a complex phenomenon in which environment, cell physiology, bacterial genetics and population dynamics all play roles (Neu, 1992). In addition, for full resistance to occur, mutations most develop within multiple genes because of genetic redundancy in the antimicrobial targets (Wikipedia, 2001).
Bacterial cells can acquire genetic sequence from other organism through several processes. First, there is the take up of naked DNA from their immediate surroundings by a process termed “transformation”. The frequency with which bacteria acquire DNA from the environment depends on several factors including cell wall structure and bacterial species with transfer frequencies being as low as 10-7. Bacteria cell have to be “competent” to acquire extraneous DNA by transformation (Arcangioli et al., 1999). This process of transformation is complex, and there are differences in the process among Gram positive and Gram negative bacteria. The process involves the specific recognition sequences in order for the DNA to be taken up by the bacteria (Elkins et al., 1999).
Bacteria can also exchange and acquire genetic material through “conjugation” of self replicating extra chromosomal DNA or plasmids. This requires physical contact between cells which allows the plasmids to be exchanged between donor and recipients‟ cells (Cloeckaert et al., 2000).
A third mechanism of horizontal gene transfer is the introduction of genetic material into a bacterium by a bacteriophage or “transductions”. In this method, the virus attaches and injects its own nucleic acids to the bacterial cell which in some cases facilitates the introduction of new genes into the bacterial genome (Sambrook et al., 1989).
Transposons which are genetic elements confered to selectable phenotype flanked by two insertion sequences are involved in horizontal gene transfer event between bacteria. Transposons are unique in that they have the ability of transferring themselves from one genetic locus and move to other taxis. Transposons can be transferred through all of the methods mentioned above namely conjugation, transformation and transduction (Neu, 1992).
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