Antibiotic Resistance In Salmonella Biology Essay

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Dairy products are to be collected from various supermarkets and analysed microbiologically using standard conventional methods to isolate and identify Salmonella species. Further the subtypes are to be confirmed using serological tests. The antibiotic properties of the bacteria are then to be revealed using standard conventional methods and the resistance pattern to various antibiotics will be obtained and can be plotted in a graph with the help of Microsoft excel. The study aims at understanding the extent to which resistance is spread in the society by analysing the antibiotic activity of various dairy products. The study can help us in understanding the properties of pathogenic bacteria in more detail that is useful for designing drugs that are more sophisticated and precise in treatments.

Keywords: Salmonella, Antibiotic resistance, dairy products

1. AIM

To detect the antibiotic resistance of Salmonella species isolated from dairy products

2. OBJECTIVES

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To isolate and characterize Salmonella species from dairy products which shows antibiotic resistance.

3. INTRODUCTION

The pathogen widely distributed in nature and well known for causing foodborne infections include Salmonella species. The Salmonellae are members of Enterobacteriaceae characterised as gram negative, facultative anaerobic, non-sporing bacilli that mainly cause food borne gastroenteritis. They are well studied in biochemical, structural and molecular levels .They can grow in simple media and can be distinguished by their biochemical properties and antigenic nature. (Jay J 1996)

3.1 Pathogenicity

In humans they mainly cause two types of diseases namely enteric fever or salmonellosis where the bacteria invade blood stream of the host and acute gastroenteritis in which infection is spread through food contamination. Infants, immunosuppressed patients and patients suffering from blood borne diseases are more susceptible to salmonella infections than healthy individuals.(Banwart G 1979)

In typhoid the bacteria invade the intestinal mucosa and are infiltrated in the mesenteric lymph nodes from where they multiply and are released along with the endotoxin lipopolysaccharide that results in septicemia. (Banwart G 1979)

Foodborne toxic infections caused by Salmonella species (e.g., S. typhimurium) start an onset of symptoms such as vomiting, diarrhoea, fever that takes about 12-24 hours followed by an intake of contaminated food and last for 2-5 days and cures spontaneously. However S.enteritidis infections can become more severe due to diarrhoea which can even cause death without proper antibiotics. (Banwart G 1979)

3.2 Antigenicity

There are mainly three types of antigens namely somatic o antigen or cell wall antigen, surface antigen or envelope antigen and flagellar H antigen. Vi antigen is a well known surface antigen which can mask serological reactions when treated with specific antisera. (Jay J 1996)

3.3 Habitats

Salmonella live principally in human and animal intestinal tract. S.typhi and S.paratyphi A are strict human pathogens while S.abortusovis, S.gallinarum, and S.typhisuis are ovine, avian, and porcine serovars respectively (Banwart G 1979). Salmonellosis in humans is spread through faecal contamination of water or food. The bacteria are disseminated to the environment through human or animal excreta. The bacteria can survive upto several weeks in water and several years in soil if favourable conditions of temperature, pH and humidity are available. They can survive sewage treatments if suitable germicide is not used during processing. Such water can contaminate coastal river and cause an infection on consuming sea foods. (Arvanitidou et al 1997)

3.4 Antibiotic Susceptibility

Antibiotic resistance of salmonella has increased drastically in the last decade due to frequent use of antibiotics clinically in humans and animals and due to the use of growth promoting antibiotics in animal feeds as well as its use therapeutically or prophylactically (Threlfall et al 2000). Plasmid borne resistance is more frequent in bacteria causing infections in infants e.g. (S.typhimurium, S.panama, S.wien, S.infantis) Resistance to Ampicillin, Chloramphenicol, Streptomycin, Tetracycline, Kanamycin and Sulfonamides is commonly observed (Jay J 1996)

4. LITERATURE REVIEW

The genus Salmonella contains serological variants of more than 2500 types that include Salmonella enterica serovar typhi causing typhoid and Salmonella paratyphi A and B causing paratyphoid fevers (Tennant et al 2010). A large number of non-typhoidal infectious salmonella cause gastroenteritis in normal individuals. Multiple studies conducted on infants revealed the importance of non-typhoidal Salmonella mainly Salmonella typhimurium and Salmonella enteritidis as infectious pathogens. In immunocompromised and debilitated individuals they become invasive leading to sepsis, bacteremia and focal infections like meningitis. (Tennant et al 2010)

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A study conducted on sea food for detecting salmonella was found to be much higher when PCR was used .The number was quite high in fish, shrimp and oysters compared to conventional isolation methods. The use of PCR helped in isolation of the bacteria with better specificity and lesser time when compared to usual methods that takes longer time for selective plating and biochemical tests. Many PCR primers were used to detect the bacteria but the most specific one found positive in all samples was hns (Shabarinatha et al 2007)

The spread of communicable disease in England and Wales from dairy products by Salmonella and Campylobacter in large number were studied and was found to be preventive if heat treatments were given universally (Galbraith et al 1982).Multiple-resistance against chloramphenicol, ampicillin, streptomycin, sulfamethoxazole, tetracycline, and ticarcillin was found among the salmonella species isolated from food animals and foods in Alberta (Johnson et al 2005)

Polymerase Chain reactions can be employed to identify the bacteria based on O and H serotyping. (Tennant et al 2010).Specific genes present in Salmonella can be selected using Basic Local Alignment Search Tool that helps in finding the sequence in various Salmonella serovars subspecies and compare their patterns. This enhances the knowledge regarding the epidemiological and taxonomical studies about the bacteria. (Kim et al 2006)

Food born infections by Salmonella serovars are causing great health concerns worldwide. In many developed countries ingestion of contaminated food , water or poultry products had caused non-typhoidal salmonellosis (Xia et al 2009).The bacteria can be isolated from sources such as swine, dairy products, poultrymeat , poultry products, etc.( Suresh et al 2006).Antibiotic resistance in pathogenic bacteria can make treatments ineffective which can cause a major health concern. Earlier antibiotic resistance was considered to be dependent strictly on the pathogen. Currently the focus is given to the resistance genes that are transferred between non-pathogenic bacteria in human and animal flora or between environmental and clinically important pathogens. (Sorum et al 2002)

Before the introduction of antibiotics in medicine the plasmids isolated from pathogenic bacteria showed little resistance. Then penicillin was introduced in therapeutical field. Soon penicillin resistant bacteria evolved followed by new resistant forms on introducing new antibiotics. The pharmaceutical industry tried to resolve this by introducing new antibiotics resulting in an increased emergence of antibiotics in practice worldwide. As a result numerous studies were conducted to compare the relationship between the increased use of antibiotics and resistance pattern and their close relationship was revealed. (Aarestrup 1999)

The use of antibiotics as growth factors in animals has increased the resistance in animals. This also caused the development of new antibiotic resistance in humans. Hence E. coli showed resistance to Streptothricin, an antibiotic used in animals and a clone was isolated from humans with urinary tract infections. Similar resistance were observed in humans after the use of apramycin, enrofloxacin, macrolide in animals. The change in resistance pattern after the withdrawal of the antibiotic found resistant varied according to the species. Majority of them showed a higher decrease in their resistance level while others remained the same. It was also found out that the resistance is highly dependent on the amount of antibiotic used. Although guidelines were given to select an antibiotic that is not used clinically as growth promoter, they are not often followed resulting in a wide spread resistance world wide. (Aarestrup 1999)

Antibiotic resistance genes from various environment were compared and sequenced and were found completely identical or with a very slight difference in the genes (Sorum et al 2002). Sulphonamide resistance genes isolated from gram negative bacteria of entirely different ecological niches such as Vibrio cholerae, Salmonella, Escherichia coli, Aeromonas salmonicida and from the plant pathogen Erwinia amylovora were found identical indicating a common origin (Sorum et al 2002).However to track the origin of sulphonamide resistance in food products seems quite difficult. Aminoglycoside resistance was found by chemically modifying the target site of the enzyme acting with aminoglycoside molecule. Trimethoprim resistance in most of the gram negative bacteria was encoded in the gene cassettes of integrons and was wide spread. (Sorum et al 2002)

Studies using retail raw meat and poultry samples from supermarkets revealed heavy contamination with Salmonella species indicating unhygienic handling and processing of food and its distribution without refrigeration (Van et al 2007). The contamination was highest in beef and chicken samples compared to pork and shell fish which may vary according to the region. A similar pattern was also obtained for antibiotic resistance. The bacterial isolates generally showed resistance towards Tetracycline, Ampicillin and Streptomycin reflecting their use in animal husbandry while resistance with Nalidixic acid was found occasionally (Van et al 2007). Multiple resistances towards Tetracycline, Ampicillin or Amoxylin, Sulphonamide and Naldixic acid were detected. The study illustrated the level of resistance and warning to give more attention in food processing and personnel hygiene inorder to reduce the food borne pathogens and to minimize their chance for developing resistance (Van et al 2007)

5. MATERIALS AND METHODS

5.1 Source

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Retail foods based on dairy products such as milk, milk products, cheese, etc. from various supermarkets are to be collected. (Roberts et al 1995)

5.2 Isolation of Bacteria

The food sample can be homogenised in 1% buffered peptone water in a ratio 1:9. Addition of surfactants such as tergitol can improve isolation. The test sample could then be subcultured in 10 ml of Rappaport-Vassiliadis broth by adding 0.1ml of the sample and in 10 ml of Selenite cystine broth by adding 1 ml of test sample. Subculture a loopful of each broth to two selective media namely Salmonella Shigella agar and Bismuth Sulphide agar. The former media should be incubated at 37°c for 20-24 hours and the latter incubated upto 48 hours.The agar plates are examined for typical colonies. (Roberts et al 1995)

5.3 Characterisation of bacteria

Biochemical tests using triple sugar iron agar or lysine iron agar slopes, urease test , sucrose or lactose media, indole test and citrate utilization tests are to be performed. Acid butt and alkaline slope in triple sugar agar test, alkaline reaction in lysine iron agar with hydrogen sulphide production , citrate negative, urease negative, sucrose or lactose negative are characteristic for salmonella species (Roberts et al 1995).Presumptive isolates can then be serotyped using commercial Salmonella agglutinating sera to confirm the serotypes (Greenwood et al 1992)

5.4 Antimicrobial Susceptibility Tests

Antibiotic resistance of salmonella isolated can then be performed using disc diffusion test based on the standard procedure of the Clinical and Laboratory Standards Institute. According to the zone diameter the isolates can be classified into susceptible, intermediate or resistant according to the zone diameter standards The disc concentrations used are ampicillin 10 μg, amoxicillin 10 μg, amoxicillin-clavulanic acid 30 μg, cephalothin 30 μg , chloramphenicol 30 μg, ciprofloxacin 5 μg , enrofloxacin 5 μg, tetracycline 30 μg, gentamicin 10 μg, kanamycin 30 μg , nalidixic acid 30 μg , norfloxacin 10 μg , sulfafurazole 300 μg , streptomycin 10 μg , and trimethoprim 5 μg. Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as the reference strains(Van et al 2007).The antibiotic sensitivity pattern obtained can plotted in a graph to understand the growth pattern using Microsoft excel.Minimum Inhibitory Concentration can be calculated using microdilution method based on the guidelines of the Clinical and Laboratory Standards Institute (Lauderdale et al 2006).

5.5 Time scale

The source can be selected within one week from various supermarkets. The isolation , characterisation of bacteria and confirmation using agglutinating sera can be completed within two weeks. The antimicrobial activity can be completed within one week

6. HYPOTHESIS

Salmonella species are to be isolated from dairy products using standard microbiological techniques. The bacteria identified and confirmed using serotyping with specific antisera should then be tested using various antibiotic testing procedures. The antibiotic activity of the bacteria are then analysed and checked for multiple resistance. The study will point out the possible reason for causing such types of resistance and their inter-relationship with other kinds of food. This gives pharmacists to design better drugs to eliminate the potential danger that the resistant bacteria cause to the public and suggest possible remedies to prevent such resistance in future