Microbiological analysis of retail food such as Chicken, Ham and Egg, Cheese sample were collected from various supermarkets and fresh meat shops was conducted using standard conventional methods to isolate, characterise and identify Salmonella species using API 20E phenotypic system. Six pathogenic isolates were identified and characterised including Salmonella species. The antibiotic properties of the bacteria were then revealed using disc diffusion method. All the isolates were found to be multi-drug resistant. The study helped in understanding the extent to which resistance is spread to various bacteria and in the foods in which they are most significant. The study is useful for designing better drugs that are more sophisticated and precise in treatments by eliminating the antibiotics that are found resistant and including those that showed better susceptibility. Such a selection of antibiotic is essential to cure infections caused by the resistant bacteria thereby allowing them to revert to their wild form by minimum exposure to the antibiotic to which the bacteria were resistant.
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The microbiological safety of retail food is to given an increasing importance when concerned to the public health. The extensive use of antibiotics in animal foods has caused the emergence of antibiotic resistant bacteria in humans after consumption of such foods especially of animal origin. Contaminated undercooked or raw chicken and red meat are the main agents leading to the transmittance of such pathogens. The prolonged use of antibiotics in poultry for accelerated growth, prophylaxis and for treatment has resulted in multiple resistances in various pathogenic bacteria. (Mackie & McCartney 1996)
The aim of the study focus on isolation of pathogenic bacteria resistant to antibiotics from various retail foods of which the main focus was given to Salmonella species, one of the main causative agents for food poisoning. Various food samples such as chicken, ham and egg sandwich and cheese samples were used from various retail markets to isolate the pathogenic organism. The study was conducted to find out drug resistant bacteria in retail foods even after their proclaimed hygienic standards
1.1Characteristics of Salmonella
Salmonella is one of the main causative agents for food poisoning in humans. The bacteria are gram negative, motile, non-sporing facultative anaerobic and comes under Enterobacteriaceae family. They live well in the gut of warm and cold blooded animals and can be grown in simple media. The bacteria are released into the environment through faecal contamination of water and food. They can live upto many weeks in water and years in soil if the temperature, pH and humidity are favourable. If suitable germicides are not used they can survive from sewage treatments that can contaminate coastal rivers and cause infection on consuming sea foods. They can be well distinguished by their biochemical properties and antigenic nature. They cause two main types of diseases in humans namely enteric fever or typhoid where the bacteria invade blood stream and acute gastric enteritis where bacterial infection onsets as a result of food poisoning. The bacteria multiply after intake of the contaminated food within 12-24 hours and the infection start with the onset of symptoms such as diarrhoea, stomach cramps, occasional vomiting and fever which lasts for 2-5 days that in most cases ceases spontaneously. However, it can rarely cause very severe illness and may even lead to death especially in infants, elderly or severely immune compromised persons. (Jay J 1996)
The genus Salmonella contains many serological variants 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)
1.2 Spread of Salmonella
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 ideal sources such as swine, dairy products, poultrymeat , poultry products, etc.( Suresh et al 2006).The main source of Salmonella infection by food is through chicken. Various studies have pointed out that half of the fresh and frozen chicken contains bacteria. Infection in dairy farms can lead to milk contamination if the milk or its products were directly used without proper pasteurization. Unhygienic food handling practices at home as well as in public food delivering services such as restaurants, parties can lead to a massive salmonella outbreak. (Caddow P 1989)
1.3 Nomenclature of Salmonella
Always on Time
Marked to Standard
Salmonella was classified earlier based on DNA and host specificity of the bacteria isolated. According to the clinical considerations, Salmonella typhi, Salmonella cholerae-suis, Salmonella abortus-ovis, etc were given to them. But the host specificity as the name suggested in some bacteria was not followed. Hence the newly identified strains were named according to the geographical area such as S. london, S. panama, S. stanleyville. Later on it was found out that all Salmonella serovars were formed from a single DNA hybridization group i.e., a single species composed of seven subspecies. Finally to avoid confusion with the familiar names of serovars, the species name Salmonella enterica was proposed and the subspecies were named as enterica I ,salamae II ,arizonae IIIa ,diarizonae IIIb ,houtenae IV , bongori V ,indica VI. But the Latin nomenclature may become unfamiliar in medical practices and the common vernacular terminology was preferred. (Jay J 1996)
The genus Salmonella was classified into more than 2500 serotypes with many combinations of antigens in Kauffmann-White classification. The serotypes were classified depending on the somatic O antigen and flagellar H antigen based on agglutination tests using specific antisera. Surface antigen or envelope antigen is another main antigen found in Salmonella of which Vi antigen is well known to mask serological reactions with specific antisera. Other antigens are M antigen, fimbrial antigens and R antigens. Salmonella antigens were also found in bacteria of other genera like Escherichia, Shigella, Citrobacter and Proteus. (Jay J 1996)
1.5 Incidence of Salmonella Infection
Food born infections by Salmonella serovars are causing great health concerns worldwide. Despite of the improved living styles, potentially serious but preventable disease continues to occur generally. In UK a very big increase in gastrointestinal infections was reported and the actual cases should be considered much higher as most of the food-borne illness go unreported or go without any treatment. Gastrointestinal infection by Salmonella and Campylobacter has increased while that by the enteropathogenic Escherichia coli has declined especially in children. (Caddow P 1989)
The incidence of salmonellosis in humans continues to increase and is mainly acquired from poultry, egg and egg products. According to the Communicable Diseases Report in 1986, bacterial food poisoning in UK continues to increase mainly due to the increased number of people having food in hotels and restaurants along with the trend of take-away food. Inadequate defrosting before cooking is the most common cause of salmonella food-poisoning at home. Hospital salmonella outbreaks in England and Wales from 1968 to 1977 was reviewed by the Public Health Service Salmonella Subcommittee in 1980 and 30% of the source of outbreak were reported through food. (Caddow P 1989)
1.6 Antibiotic Resistance
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 after which penicillin was introduced. 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 was 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 worldwide. (Aarestrup 1999)
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Antibiotic resistance genes from various environment were compared and sequenced and were found completely identical or with a very slight difference in the genes. 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. 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. 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. 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 hygienity inorder to reduce the food borne pathogens and to minimize their chance for developing resistance (Van et al 2007)
CHAPTER 2. MATERIALS AND METHODS
The isolation procedure was carried out three times using different samples. Chicken leg, ham and egg sandwich and semi-soft cheese sample were used for the first time. Fresh chicken and ham samples were used for the second time. Two different chicken samples from different fresh meat shops, raw egg and semi-soft cheese samples were used for the third time.
2.2 Reagents and Media
For the homogenization of sample suspension, 1% buffered peptone water was prepared and autoclaved at 1210C for 15 minutes. For enrichment of the sample suspension, Rappaport Vassiliadis(RV) broth and Selenite Cysteine(SE) broth were prepared . RV broth was autoclaved at 1150C for 15 minutes while SE broth was sterilized in boiling water bath for 15 minutes. Salmonella-Shigella (SS) agar and Bismuth Sulphite agar (BSA) were prepared for selective isolation of bacteria as the main pathogenic bacteria of interest was Salmonella. Both SS agar and BSA were boiled and directly poured without autoclaving.
Nutrient agar was used for cultural characterisation of the bacteria and gram staining reagents were used for microscopical characterisation of bacteria. For biochemical characterisation of the bacteria API 20E kit was used. Catalase and oxidase reagents were also used for biochemical characterisation of the bacteria.
For antibiotic sensitivity tests, Iso-sensitest broth and Muller-Hinton agar was prepared and autoclaved at 1210C for 15 minutes. Antibiotic susceptibility tests were performed using Mastring-S diagnostic disc M14, a ring device that contains 8 antibiotic discs impregnated in it. The antibiotics in M14 Mastring-S were Ampicillin 10 Âµg (AP), Cephalothin 5 Âµg (KF), Gentamycin 10 Âµg (GM), Colistin Sulphate 25 Âµg (CO),Streptomycin 10 Âµg (S), Sulphatriad 200 Âµg (ST),Tetracycline 25 Âµg (T) and Cotrimoxazole 25 Âµg (TS).Along with the M14 Mastring-S , individual antibiotic discs namely Meropenem 10 Âµg (MEM 10), Piperacillin 75 Âµg (PRL 75), Ciprofloxacin 5 Âµg (CIP 5) and Cefoxitin 30 Âµg (FOX 30).
2.3 Isolation of Bacteria
The food sample was homogenized in a stomacher bag in a concentration of 10-1 using buffered peptone water and serially diluted upto 10-3.
2.3.2Enrichment and Selective Isolation
As the main pathogenic bacterium of interest was Salmonella, enrichment and selective media for salmonella were used for the isolation. Hence the serially diluted samples were enriched in Selenite Cysteine broth and Rappapport Vassiliadis broth. Direct plating was also performed from the serially diluted samples in Salmonella-Shigella agar and Bismuth Sulphite agar for upto 24 hours at 370C.The suspension from the selective culture broth were also subcultured into the selective agar media the following day for overnight at 370C.All the bacteria isolated were observed for colony characteristics in nutrient agar ,viability count and biochemical tests such as gram staining , catalase test and oxidase test.
2.4 Characterization of bacteria using API 20E
The bacterial isolates from nutrient agar plates were inoculated into API 20E strips
and incubated at 370C for 18-24 hours. The numerical profiles obtained were identified using apiweb identification software.
2.5 Antibiotic Sensitivity tests
The bacterial isolates that had a very good identification after API test were selected for detection of antibiotic sensitivity. The bacterial isolates were first inoculated in isosensitest broth for 18-24 hours at 370C.Then the bacteria were spread-plated into Muller-Hinton agar (MHA) using Kirby-Bauer disc diffusion technique. M14 Mastring-S containing eight antibiotic discs was then kept in the inoculated MHA plates along with individual antibiotic discs namely 10 Âµg of Meropenem (MEM 10), 75 Âµg of Piperacillin (PRL 75), 5 Âµg of Ciprofloxacin (CIP 5) and 30 Âµg of Cefoxitin (FOX 30). M 14 Mastring-S was used since they were typically used for antibiotic susceptibility testing for gram negative bacteria. However the susceptibility of the isolates towards other Î² lactams was also studied along with a fluoroquinolone antibiotic, ciprofloxacin as well as with a carbapenem antibiotic, meropenem to understand the resistance level of the isolates towards different classes of antibiotics. The plates were then observed after overnight incubation for any zone of inhibition. The zone diameter was measured and the isolates were classified into susceptible, intermediate or resistant according to the zone diameter standards.
CHAPTER 3. RESULTS
3.1 First Isolation Of Bacteria
3.1.1 Viability Count
The number of pathogenic bacteria in retail food showed was calculated because the pathogenicity of the bacteria isolated is directly dependent on its number in the retail food. The number that was considered significant were listed in the table 3.1
Table 3.1 Viability Count from first isolation
2.7 x 103
HAM AND EGG
16 x 103
*Too many to Count
3.1.2 Cultural Characteristics
Cheese samples produced small to large, semi-translucent irregular colonies in SS agar while no growth was obtained in BSA. The colony morphology were unexpected as they were not similar to that of salmonella colonies
In ham and egg sample, small to large pink, semi-translucent colonies were obtained in SS agar while small light green opaque round colonies were obtained in BSA. The colony morphology resembled typical salmonella colonies that were expected.
Chicken samples produced medium to large white colonies with black centre in SS agar while in BSA, small to medium sized opaque black round moist colonies were formed. The colony morphology was expected as they resembled typical salmonella colonies.
3.1.3 Biochemical Characteristics
All the isolates on gram staining were found to be gram negative rods. Catalase test was positive for all isolates and oxidase test was found positive in most of the isolates except the BSA isolate from Ham and Egg sample and the BSA isolate from Chicken sample. The positive oxidase test revealed that the bacteria in the isolates were unexpected. However to find out whether they were pathogenic or not, the study was further continued with all the isolates.
Table 3.2 API Results from first isolation
2 0 1 4 0 4 6
2 0 4 1 0 4 6
2 0 0 0 0 0 4
5 1 0 7 3 2 3
6 1 7 7 1 6 4
3 1 7 7 1 7 3
*CHE 1-Cheese sample isolates from SS,CHE 2-Cheese sample isolates from BSA,HE 1- Ham and Egg sample isolates from SS,HE 2-Ham and Egg sample isolates from BSA,CHI 1-Chicken sample isolates from SS,CHI 2-Chicken sample isolates from BSA
3.2 Second Isolation of Bacteria
No growth was obtained in Ham samples on both SS and BSA plates and also in Chicken BSA plates. Growth was obtained only in Chicken SS plates. This was unexpected and can be due to sampling only from supermarkets. In the first isolation, the samples were from both fresh meat shop and from supermarkets.
3.2.1 Viability Count
The viability count of dilution 10-1 in Chicken SS plates were only found significant.
Table 3.3 Viability Count from second isolation
4.8 x 103
The chicken isolates in SS agar were large, round, moist, semi-translucent pink colonies were obtained. The morphological characteristics resembled typical Salmonella colonies
The bacteria from Chicken isolates in SS agar were gram negative rods and were catalase and oxidase positive. Hence even after the typical morphological similarity the oxidase reaction indicated that the bacteria obtained were unexpected.
Table 3.4 API Results from second isolation
2 2 4 1 0 4 6
Possibility of fluorescens/putida
3.3 Third Isolation of Bacteria
3.3.1 Viability Count
A good viability count was observed only in isolates plated in SS agar. The isolates in BSA were below the significant level. Although three types of isolates were obtained from first chicken sample, only two of them were observed as significant. However both types of colonies from the second chicken sample showed significant viability count
Table 3.5 Viability Count from third isolation
70 x 103 *
47 x 103 *
35 X 103
*From first type colony
**From second type colony
There were many bacterial isolates from the third isolation which may be due to the use of more samples from fresh meat shop.From the first chicken sample, three types of colonies were obtained in SS agar. One type of colony was small to large sized cream colour round translucent colonies while the second type of colony was small to medium pink colour round semi-translucent colonies and the third type of colony was small translucent round cream colonies with black centre. In BSA, pin-point opaque irregular black colonies were obtained. Except the first colony, all the colonies including that in BSA were typical Salmonella colonies. However all the isolates were further studied.
From the second chicken sample, two types of colonies were obtained in SS agar. The first type of colonies were small to large cream round translucent colonies while the second type of colonies were small to medium pink, round semi-translucent colonies. No growth was obtained in BSA plates. No growth was obtained in the raw egg samples in both SS and BSA. In cheese sample, pin-point, white round colonies was obtained in SS agar while no growth was obtained in BSA. Cheese isolate from SS agar failed to grow on further subculturing and was not further proceeded. Although all the chicken isolates were studied further, only the second type of colonies was typical Salmonella colonies.
Other than direct plating in selective media, all the isolates were also plated in selective agar after enrichment in SE broth and RV broth. Most of the isolates reproduced the same colonies as in direct plating while some fail to produce any growth. The only colony which was found distinct from the direct plate isolates and was typical Salmonella colony was that from the second chicken sample enriched in RV broth and plated in SS agar. The colonies were small to medium round translucent shiny colonies with black centre. The viability count of that colony was 20 x 102 cfu/g. Hence the isolate was also proceeded further.
3.3.3 Biochemical Characteristics
All the isolates obtained were found to be gram negative rods on gram staining. Catalase test was positive for all isolated while oxidase test was negative for most of the isolates except the first colony isolated from first chicken SS sample and that from the second chicken sample
Table 3.6 API Results from third isolation
Low Discrimination- 59.3%
Aeromonas hydrophila caviae/sobria 1
Very good Identification-
Escherichia coli 1
Very good identification-99.9%
Very good identification-91.5%
Aeromonas hydrophila/caviae/sobria 1/sobria 2
Very good Identification- 99.5%
Escherichia coli 1
Very good Identification-99.7%
Salmonella cholerasuis ssp arizonae
* CH1.1SS -First type of colony (small to large sized cream colour round translucent colonies) from chicken sample 1 in SS, CH1.2SS- Second type of colony (small to medium pink colour round semi-translucent colonies) from chicken sample 1 in SS, CH1.3SS- Third type of colony(small translucent round cream colonies with black centre)from chicken sample 1 in SS, CH1 BSA- Colony from chicken sample 1 in BSA, CH2.1SS- First type of colony(small to large cream round translucent colonies) from chicken sample 2 in SS, CH2.2SS- Second type of colony(small to medium pink, round semi-translucent colonies) from chicken sample 2 in SS, CH2RV- Colony from chicken sample 2 in SS after enrichment in RV broth.
3.4 Antibiotic Sensitivity Results
Six bacteria isolates with a good identification profile in API tests were selected for antibiotic sensitivity tests from all the three times of isolation procedure. Two samples which were identified as Pseudomonas aeruginosa from cheese sample and Serratia liquifaciens from ham and egg sample were selected from the first bacterial isolation. Four samples were selected from the third bacterial isolation. They were identified as Proteus mirabilis and Enterobacter cloacae from the first chicken sample and Escherichia coli 1 and Salmonella cholerasuis ssp arizonae from the second chicken sample.
A B C
D E F
Figure 1: Antibiotic Sensitivity Test in all the six isolates
A- Salmonella cholerasuis ssp arizonae, B- Escherichia coli 1,C- Enterobacter cloacae, D- Proteus mirabilis, E- Serratia liquifaciens, F- Pseudomonas aeruginosa
Table 3.7 Antibiotic Sensitivity results-Zone diameter
ANTIBIOTICS-ZONE DIAMETER (in mm)
The results revealed that all the samples were resistant towards ampicillin and cephalothin. Except for Pseudomonas aeruginosa and Serratia liquifaciens all the isolates were found resistant towards colistin sulphate. P.aeruginosa and S. liquifaciens were found to be intermediate. Except in Proteus mirabilis all the isolates were sensitive towards gentamycin. P.mirabilis was resistant towards gentamycin. Except for P .aeruginosa and S .liquifaciens that were sensitive, all the isolates were found resistant to streptomycin. All the isolates were sensitive towards sulphatriad except Escherichia coli 1 that was found resistant and Salmonella cholerasuis that showed intermediate sensitivity. Pseudomonas aeruginosa and Serratia liquifaciens were sensitive towards tetracycline while resistance was observed in P.mirabilis and Salmonella cholerasuis. E.coli 1 and Enterobacter cloacae were found intermediate. All the isolates were sensitive towards cotrimoxazole and ciprofloxacin. P.mirabilis and E.coli 1 showed resistance towards meropenem while Enterobacter cloacae and Salmonella cholerasuis showed intermediate sensitivity. However P .aeruginosa and S .liquifaciens were susceptible towards meropenem. P.aeruginosa showed resistance towards piperacillin while Enterobacter cloacae was intermediate and all other isolates were sensitive towards it. P.mirabilis and E.coli 1 were intermediate towards cefoxitin while all other isolates were resistant.
CHAPTER 4 .DISCUSSION
The main purpose of this study was to isolate and characterise Salmonella species from retail food that show antibiotic resistance for analysing the hygienic standards of retail food and enable this study for producing better drugs to which such bacteria were susceptible. However even after careful use of selective media and enrichment broth, other bacteria grew splendidly in the selective media along with the bacteria of interest, Salmonella. Since all of them were pathogenic, the study was proceeded further with all the six isolates altogether from Chicken samples, ham and egg sample and from the cheese sample that showed a high percentage of identification in API results. Hence the antibiotic resistance of the six isolates namely Pseudomonas aeruginosa, Serratia liquifaciens , Escherichia coli 1 ,Proteus mirabilis, Enterobacter cloacae and Salmonella cholerasuis ssp arizonae were studied.
4.1 Choice of method
The media used for Salmonella isolation failed to be selective towards the bacteria of choice suggesting a need for better selective media to eliminate unwanted bacteria. However the media was successful in producing Salmonella isolates that indicated that the media cannot be ignored during Salmonella isolation. A modification in the selective agents can give a better selectivity. A study conducted by comparing four different methodology for isolating Salmonella revealed a better selectivity in multiple plating using a new so called selective media, BxLH agar which was more selective in eliminating other bacteria while the growth of Salmonella was triggered. (Hoszowski and Truszcznski 1995).Hence it can be expected that for further studies requiring Salmonella isolation this modified method can be adapted for obtaining more isolates. For rapid isolation of Salmonella with great specificity, PCR compatible enrichment procedure can be used that enable DNA amplification without any DNA extraction procedures (Lofstrom et al 2004)
4.2 Bacterial Isolates and their viability
The isolation of bacteria was repeated three times from different areas for the maximum isolation of the bacteria of interest Salmonella. Hence the first isolation was concentrated on sources from both supermarkets as well as from fresh meat shops while the second isolation was concentrated only on supermarkets. However the third isolation procedure was carried out mainly from fresh meat shop. The isolates from supermarkets failed to grow in the third isolation while that from fresh meat shop produced even three types of colonies from one sample. However there were more pathogenic bacteria than the expected Salmonella from all the three steps of isolation. From the first isolation, Pseudomonas aeruginosa was isolated from cheese samples with a significant viability count of 2.7 x 103 cfu/g and Serratia liquifaciens from ham and egg sample with a significant viability count of 5.1 x 103 cfu/g. Previous studies on infected urine samples isolated eighty two Pseudomonas aeruginosa isolates that were tested for antibiotic sensitivity (Grey et al 1977). Multidrug resistant pathogenic strains of Serratia were isolated from fresh juice and fish samples for characterising their virulence factors. (Singh et al 1997) Although one isolate was obtained from the second isolation step with a viability count of 4.8 x 103 cfu/g from chicken sample, the API profile obtained was doubtful which suggested the possibility of Pseudomonas species which was already isolated from the first isolation. Hence the isolate was not studied further. From the third isolation, more bacteria were isolated from chicken samples from two different fresh meat shops. From the first meat shop, Proteus mirabilis with an insignificant viability count, E.coli 1 with a significant viability count of 45 x102 cfu/g and E.cloaceae with too high viability count were isolated. From the second meat shop, Salmonella cholerasuis with an insignificant viability count and E.coli 1 with a significant viability count of 32 x 102 cfu/g were isolated. Since E.coli 1 was isolated from both the shops, the isolate from the second meat shop was only studied further. In previous studies, three hundred Salmonella species were isolated from diseased animals and were tested for antibiotic susceptibility (Zhao et al 2007)
The three isolation steps of bacteria revealed many facts that are useful in detecting the presence of bacteria in food samples. It was identified that the chicken collected from supermarkets were less likely to be contaminated from pathogenic bacteria compared to that in fresh meat shop .This was evidently shown when chicken was collected for the second time from supermarket and only one isolate was obtained compared to that collected mainly from fresh meat shop for the third time with many isolates. From all the three steps of isolation, more bacteria were isolated from the third isolation from the fresh meat shop. Hence through this study, it was understood that the hygienic standards in fresh meat shops were poorer compared to retail supermarkets. The possible cause of bacterial contamination can be from infection in food animals due to unhygienic rearing of animals or may be due to unhygienic handling including transporting, processing and packing by infected individuals.
4.3 Antibiotic resistance and its mechanisms
The study revealed that almost all bacterial isolates showed antibiotic resistance towards Î² lactam antibiotics namely ampicillin, cephalothin, cefoxitin which may be due to excessive use of these antibiotics in animal feeds. Previous research on Serratia strains isolated from fresh juice and fish samples detected the presence in ampicillin resistant S.liquifaciens (Singh et al 1997). Resistance towards meropenem was found only in P.mirabilis and E.coli. Resistance towards Î² lactam antibiotics can be due to the production of Î² lactamases, decreased membrane permeability or due to mutation in penicillin binding proteins in the resistant bacteria. However resistance towards meropenem should be considered serious as the drug was considered as the broadest spectrum antibiotic in carbapenems according to the MYSTIC (Meropenem Yearly Susceptibility Test Information Collection) program since 1997 (Jones et al 2008).For development of resistance to this carbapenem , two mutations namely upregulated efflux and loss of OprD gene are required (Jones et al 2002).Hence the resistance in P.mirabilis and E.coli can be due to the spread of the resistant genes that lead to multi-drug resistance. However the viability count of only E.coli was obtained significant with a count of 32 x102 cfu/g. Enterobacter cloacae in the current study showed resistance towards Î² lactams and polymyxins. A study performed with clinical isolates of Enterobacter strains were obtained that produced extended spectrum Î² lactamases (Chen et al 2008).All these results showed the impact of excessive use of human antibiotics in animal feeds
All the isolates showed a good susceptibility towards fluoroquinolone drug, ciprofloxacin. A similar study was conducted in salmonella serovars in Heidelberg and the bacteria showed susceptibility towards ciprofloxacin (Zhao et al 2008).However studies were also conducted where bacteria showed resistance towards ciprofloxacin. According to surveillance for antibiotic resistance from clinical isolates in China, 42.9% of the E.coli isolates were resistant towards ciprofloxacin (Wang H et al 2005). Quinolones acts on bacteria by interfering with their DNA replication. Susceptibility of bacteria to ciprofloxacin should be studied with great concern since it is the drug of choice for treating gastrointestinal and invasive salmonella infections (Rotimi et al 2008).
Most of the isolates were resistant to colistin sulphate, a polymyxin type of antibiotic widely used in animal feeds as a growth promoter. Hence the resistance is mainly due to the excessive use of the antibiotic in animals. The drug acts by blocking the bacterial cell membrane permeability and are very effective broad spectrum antibiotic in multi-drug resistance. Hence the resistance of most isolates to an antibiotic that is very rare in developing resistance is to be noted which may be developed due to acquired resistance. The mechanism of resistance can be due to overexpression of outer membrane protein in low Mg2+ environment. Similar resistance was observed in Germany during a study of resistance in P.aeruginosa in Cystic Fibrosis patients were resistance to polymyxin B was observed. (Li et al 2005)
In the current study P. aeruginosa isolates were resistant to ampicillin , cephalothin and cefoxitin. Moreover resistance was also found in piperacillin while the isolate was sensitive to tetracycline and cotrimoxazole. Similar studies on genomically diversified isolates of P. aeruginosa from food animals, retail meat products, and food processing environments also showed resistance towards Î²-lactams (ampicillin, amoxicillin-clavulanic acid, cefoxitin, ceftiofur, and cephalothin), chloramphenicol, tetracycline, kanamycin, nalidixic acid, and sulfamethoxazole-trimethoprim (Kim et al 2007). This indicated that the isolated bacteria were only resistant to Î²-lactam antibiotics when compared to previous studies where resistance was also found in other type of antibiotics like tetracycline and cotrimoxazole. Resistance towards Î²-lactam antibiotics showed the increased use of these antibiotics in animal feeds. The viability count of P. aeruginosa isolate obtained from cheese sample was significant with a count if 2.7 x 103 cfu/g. A study on eighty two P.aeruginosa isolates from infected urine samples for Cotrimoxazole and Suphatriad susceptibility showed resistance in all isolates (Grey et al 1977).
Except one isolate most of them were susceptible towards gentamycin. Resistance in Proteus mirabilis can be due to the spread of resistant pathogens of human origin to animals that can cause infections. It can also be due to unhygienic handling of retail food which can lead to the direct transfer of bacteria from infected individuals to the food products. Most of the bacteria were resistant to Streptomycin except S.liquifaciens and P.aeruginosa which were sensitive. Aminoglycoside resistance usually occur either by production of enzyme by bacteria that can inhibit the drug action and due to membrane impermeability of bacteria to drugs.
Proteus mirabilis showed resistance towards ampicillin, cephalothin, colistin sulphate, gentamycin, streptomycin, tetracycline and meropenem. Similar studies were conducted in P.mirabilis from clinical isolates in which multiple antibiotic resistances towards gentamicin, fluoroquinolones and cotrimoxazole was found (Luzzaro et al 2001).The main antibiotics to which resistance was widespread in P.mirabilis was towards Î² lactams due to the production of Î² lactamases which can be the possible reason of multidrug resistance in the bacteria.
Most of the isolates were sensitive towards piperacillin, a penicillin drug, which may be due to its absence as an animal feed for prophylaxis. However resistance was observed in Escherichia coli which may be due to mutation in genes or by acquiring the resistant gene from other bacteria. E.coli 1 showed resistance towards ampicillin, cephalothin, colistin sulphate, streptomycin, sulphatriad, meropenem and piperacillin. The resistance towards more than one class of antibiotic indicated the presence of multidrug resistant gene transferred to the bacterial clones. The significant viability count of 32 x 102 cfu/g in E.coli 1 points out the possibility of an outbreak by the ingestion of undercooked meat. A study on antibiotic usage in food animals revealed resistance to ampicillin, streptomycin, sulphonamides, and tetracyclines (Aarestrup and Wegener 1999).Multidrug resistance in E.coli indicated the presence resistance gene in the isolate.
P.mirabilis and S.cholerasuis were found resistant to tetracycline while S.liquifaciens and P.aeruginosa were susceptible. Resistance to tetracyclines are mainly due to active pumping out of the drug from the cells and the resistance is transferred through plasmids or transposons through which new genes are acquired. Another mechanism of resistance can be due to modification of ribosomes (Magalhaes et al 1998).The antibiotic is widely used in food animals both in prophylaxis as well as in treatment that predisposes for the antibiotic resistance. Similarly a study conducted for analysing the gene patterns of E.coli in humans and porcines, tetracycline resistant genes were obtained in both human and porcine isolates. (Schwaiger et al 2010)
Salmonella cholerasuis ssp arizonae was found resistant towards ampicillin, cephalothin, colistin sulphate, streptomycin, tetracyclin and cefoxitin. Multi-drug resistance was also found in the clinical Salmonella isolates in hospitals in Taiwan, resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline and was highly prevalent in Salmonella enterica serotype typhimurium (Lauderdale et al 2006). Sixty-one percent of meat and 18% of shellfish samples were contaminated with Salmonella spp in the retail raw food sample from Vietnam and were found resistant towards tetracycline, ampicillin/amoxicillin, nalidixic acid, sulfafurazole, and streptomycin. (Van et al 2007).When compared to previous studies the Salmonella isolates also showed resistance towards Î² lactams, aminoglycosides and tetracyclines. However the viability count of the bacteria was only 20 x 102 cfu/g. But this number cannot be ignored since the number of bacteria needed for causing a salmonella infection is still a matter for debate. Many of the outbreaks occurred below 102 organisms and some had even started with a number less than 100 bacteria. (Greenwood et al 1992)
From the studies on the antibiotic resistance patterns in all the six isolates, Î² lactam resistance was found widely mainly due to the excessive use of antibiotics in animal husbandry. Multidrug resistance was observed in all isolates. It has to be noted that six animal feed additive antibiotics namely avoparcin, bacitracin, ardacin, spiramycin, tylosin and virginiamycin were banned in animals according to the Europeon Union precautionary measures. As avoparcin was a class of glycopeptides antibiotic, cross resistance was observed in vancomycin, the ultimate antibiotic for curing infections (Pugh 2002).The overuse of antibiotics as feed additives that are mainly used in humans raise the problem of cross resistance. The study points out a need for banning more antibiotics used in humans as feed additives to control the outbreak of multidrug resistant bacteria in humans. However infection by all the six isolates can be suppressed by the use of ciprofloxacin or cotrimoxazole as a drug of choice for these infections due to their susceptibility to these antibiotics.
From studying the six isolates from various retail foods, multidrug resistant bacteria were isolated indicating the poor hygienic standards in retail food. The study can be useful in developing better antibiotics namely of Î² lactam class with a better level of susceptibility. The use of antibiotics in animal feeds both for prophylaxis and treatment had increased their resistance levels. It is recommended that the antibiotics used in humans should never be used in animals that can be considered as a main cause of widespread resistance. The hygienic standards of fresh meat shops should be considered with caution. Any food products especially of animal origin should be thoroughly cooked before consumption as all the pathogenic bacteria are destroyed at that temperature. Finally the excessive and unnecessary use of the same antibiotic should be avoided which can be a predisposing factor for development of antibiotic resistance in humans.
The study can be further continued by isolation of the multidrug resistant gene by PCR. Synergistic action of different antibiotic combination on the six isolates can also be studied. These studies can reveal more specific data useful for better development of drugs.