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Title: “MOLECULAR CHARACTERIZATION AND DRUG RESISTANCE OF ENTEROPATHOGENIC BACTERIA ISOLATED FROM READY TO EAT FOOD IN LUDHIANA”

Author: Naresh Kumar

Cuisine

ABSTRACT

The present study was commenced to investigate the microbiological quality of street food sold by different street vendors at Ludhiana Punjab in different area of Ludhiana. One hundred and fifty Food sample were collected from street vendors at, Chandigarh Road, Samrala Chowk, Clock tower, and Jalandhar bypass Microbial quality was assessed by MPN and samples were inoculated into various selective media such as Eosin Methylene Blue (EMB) agar, Salmonella Shigella (SS) agar, Maconky agar, blood agar. Escherichia coli, Staphylococcus aureus, Salmonella species and Bacillus cereus. Escherichia coli and Staphylococcus aureus were recovered in a significant proportion of the food,. Antibiotic sensitivity test showed that the isolates were sensitive to two antibiotics such as ciprofloxacin and gentamicin. E. coli were found resistant to ampicillin, chloramphenicol and cephalexin and Staphylococcus spp. exhibited resistant profile to ampicillin, cephalexin and vancomycin. Data of this study indicated that street food rice sold in Ludhiana is multidrug resistant food, which might cause public health hazards if these antibiotic resistant transfer to human.

Key words: Street food, multidrug resistant, microbiological assessment.

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Chapter I

INTRODUCTION

Street foods are defined as ready to eat foods and beverages prepared and or sold by vendors and hawkers especially in street and other similar public places in India. The consumption of these roadside foods is potentially increases the risk of food borne illness. Especially in developing countries it is difficult to safeguard the food from cross contamination and where unemployment is high, salaries are low, work opportunities and social programs are limited, and where urbanization is taking place. Street food vendors benefited from a positive cash flow, often avoid taxation, and can determine their own working hours. In selling snacks, complete meals, and refreshments at relatively low prices, they provide an essential service to workers, shoppers, travelers, and people on low incomes. People who depend on such food are often more interested in its convenience than in questions of its safety, quality and hygiene. The hygienic aspects of vending processes are a major source of concern for health department and running water may not be readily available. Also toilets and adequate washing facilities are rarely available. The washing of hands, utensils, and dishes is often done in buckets or bowls and insects and rodents may be attracted to sites where there is no organized sewage disposal. Finally food is not adequately protected from flies and refrigeration is usually unavailable. That is why low-income countries face the highest burden of gastrointestinal problem and other food-borne disease associated with the consumption of contaminated food.

The street food industry plays a very important role in meeting food requirements of travelers and urban dwellers, as it feeds millions of people daily with foods that are affordable and easily accessible. Several pathogenic microorganisms have been associated with. Ready to eat foods (street food) are processed and readily available for purchase and consumption. The demand for ready-to-eat convenient rising due to changing lifestyle.

Foodborne illnesses of microbial origin are a major health problem associated with street foods. In addition, resistance of foodborne microorganisms in multi-drug made the food safety situation more vulnerable in public health. Approximately, lakhs people are suffering from foodborne illness each year in Ludhiana Diarrheal diseases are the most common food poisoning cases in Ludhiana and in some cases death also noticed street foods are mostly prepared and processed manually and sold to the public at various lorry terminals, by the roadside or by travelling vendors. It was also observed that most of the vendors are migrated from Utter Pradesh and Bihar they are not qualified and do not have knowledge of hygiene while handling and preparing the food

 As street food business requires low investment, most of the vendors were found to own the business.

It is obvious no microbial analysis of street food so far performed in Ludhiana. The objectives of research work were therefore focused on the following points:

1. To investigate the microbial quality of foods sold in streets and predisposing factors to their contamination.

2. This may be an attempt to make aware common people regarding microbial contamination of street vended food and health hygienic.

3. To estimate the prevalence of Multi Drug Resistant microorganisms from the isolates

Done  up here  

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CHAPTER II

REVIEW OF LITERATURE

The review of literature is performed with a view to obtaining information relating to the present research work under taken. The contributions made by numerous research workers are compiled and the publications are listed in the bibliography. Recent works on microbiological research of street food are illustrated considering consumer's protection and public health hazards. Moreover recent works on risk assessment and monitoring of foods and their environment with particular reference to safety maintenance and wholesomeness are pinpointed.

Prevalence of microorganism in street food

Odu NN and Akano UM  (2012) The study was carried out to analyze the microbial quality of shawarma purchased in Port Harcourt city. Twelve (12) Shawarma samples were randomly sampled from (3) local eateries and four (4) home prepared samples were analyzed for the presence of microorganism using appropriate selective media. Inoculations were done using the spread plate technique. The total viable count (TVC) of bacterial population in all shawarma samples were in the range of 2.0x103 to  1.8x106cfu/g. Generally, the vegetables recorded the highest (1.8x106) number of bacterial growth especially in Elelenwo. Also, when all elements where combined, Elelenwo still had the highest total viable bacterial count (1.1x106). The total coliform count ranged from 1.9x103 to 9.4x105, with Elelenwo having the highest count. The range for staphylococci count was 1.9x103 to 5.3x103, with Choba recording the highest. The home made sample which was used as a control had significantly smaller total viable count of aerobic bacteria. Some fungal species was isolated from the dough and vegetables and this includes; Rhizopus stolonifer and Aspergillus niger and the total fungal count ranged from 2.0x104 to 8.1x104, with Government Residential Area

(GRA) having the highest count. The TVC for aerobic mesophillic bacteria for all 3 locations and the home made samples were 1.1x106cfu/g, 8.0x105cfu/g, 9.0x105cfu/g and 4.2x103cfu/g respectively, with Elelenwo and GRA having the highest TVC, while the total viable bacterial count for both Choba and home-made samples were the lowest (8.0x105 and 4.2x103). It showed that bacterial isolates were most predominant (84.6%) compared to the fungi isolates (15.4%). The frequency of occurrences of the eight genera of pathogenic bacteria isolated from all shawarma samples showed that Proteus spp. (22.7%) was the most predominant. This was followed by Escherichia

coli (13.6%), Bacillus spp. (13.6%) and Staphylococcus aureus (13.6%). Enterobacter aerogens (9.1%), Klebsiella spp. (9.1%), Serratia marcescens (9.1%), and Micrococcus spp. (9.1%) were least predominant. The study showed that the home made samples, which were prepared in the right sanitary condition, showed that contamination may be

as a result of poor manufacturing practices employed by the food vendors. This is of public health concern as these organisms are known causes of food-borne diseases and food intoxications.

UNICEF, 2005).

 (2010) This study was conducted to examine the level of bacterial contamination in some selected cooked food in Ogbomosho, Nigeria and to determine the antibiotic susceptibility profile of the bacterial contaminant. A total of nine (9) organisms were isolated, the isolates were subjected to various biochemical tests and the isolates were identified as Bacillus licheniformis, Aeromonas hydrophila, Enterobacter aerogenes, Bacillus cereus, Proteus mirabilis, Pseudomonas putida, Proteus vulgaris, Pseudomonas cholororaphi and Proteus morganii. Survival vival of isolates at different temperature ranges of 50-80oC was determined and it was discovered that as the temperature increased the growth of the isolates decreased. Survival of isolates at different pH ranges was determined using Spectrophotometer at wavelength of 560 nm as the pH changed to basicity from acidity growth of isolates increased. Effect of different concentration of Sodium Chloride (NaCl) on the growth of isolates shows that the rate of growth of isolates decreased as the concentration of NaCl increased. Finally, antibiotic susceptibility test was conducted and the result indicated 53.85% resistance while 46.15% are sensitive to the antibiotics.

María Consuelo Colombia In another study undertaken in Alberta during 1996 and 1999, 209 strains of Salmonella, obtained from food animals were isolated and 17 antimicrobial drugs were tested and ,11.8% of strains were positive for resistance. These strains were commonly resistant to tetracycline (35.4%), streptomycin(32.5%), sulfamethoxazole (28.7%), ticarcillin (27.3%) and ampicillin (26.8%)[9]. Salmonella enterica serovar Heidelberg frequently causes foodborne illness in humans. The authors compared the prevalence of Salmonella serotype Heidelberg in a sampling of 20,295 meats, including chicken breast, ground turkey, ground beef and pork ribs, collected between 2002 and 2006 a total of 298 Salmonella serovar Heidelberg isolates were recovered, representing 21.6% of all Salmonella serovars from retail meats. One hundred seventy-eight (59.7%) were from ground turkey, 110 (36.9%) were from chicken breast, and 10 (3.4%) were from pork chops; none was found in ground beef. One hundred ninety-eight isolates (66.4%) were resistant to at least one compound, and 49 (16.4%) were resistant to at least five compounds. Six strains (2.0%), all ground turkey, were resistant to at least nine antimicrobial agents. The greatest resistance in isolates from poultry was to tetracycline (39.9%), followed by streptomycin (37.8%), sulfamethoxazole (27.7%), gentamicin (25.7%), kanamycin (21.5%), ampicillin (19.8%), amoxicillin-clavulanate (10.4%) and ceftiofur (9.0%). These data indicate that Salmonella serovar Heidelberg is a common serovar in retail poultry meat and includes widespread clones of multidrug-resistant strains

Geert Huys,1* Klaas D'Haene,

In the present study, a collection of 187 Enterococcus food isolates mainly originating from European cheeses were studied for the phenotypic and genotypic assessment of tetracycline (TC) resistance. A total of 45 isolates (24%) encompassing the species Enterococcus faecalis (n = 33), E. durans (n = 7), E. faecium (n = 3), E.

casseliflavus (n = 1), and E. gallinarum (n = 1) displayed phenotypic resistance to TC with MIC ranges of 16 to 256 µg/ml. Eight of these strains exhibited multi resistance to TC, erythromycin, and chloramphenicol. By PCR detection, TC resistance could be linked to the presence of the tet(M) (n = 43), tet(L) (n =16), and tet (S) (n = 1) genes. In 15 isolates, including all of those for which the MIC was 256 µg/ml, both tet (M) and tet(L) were found. Furthermore, all tet(M)-containing enterococci also harbored a member of the Tn916-Tn1545 conjugative transposon family, of which 12 erythromycin-resistant isolates also contained the erm(B) gene.Filter mating experiments revealed that 10 E. faecalis isolates, 3 E. durans isolates, and 1 E. faecium isolate could transfer either tet(M), tet(L), or both of these genes to E. faecalis recipient strain JH2-2. In most cases in which only tet(M) was transferred, no detectable plasmids were acquired by JH2-2 but instead all trans conjugants contained a member of the Tn916-Tn1545 family. Sequencing analysis of PCR amplicons and evolutionary modeling showed that a subset of the transferable tet(M) genes belonged to four sequence homology groups (SHGs) showing an internal homology of >99.6%. Two of these SHGs contained tet(M) mosaic structures previously found in Tn916 elements and on Lactobacillus and Neisseria plasmids, respectively, whereas the other two SHGs probably represent new phylogenetic lineages of this gene.

needs for understanding the hygiene knowledge and practices of food vendors to ensure hygienic preparation of street foods.

Mugampoza et al. (2013) referred occurrence of Escherichia coli and Salmonella spp. in street-vended food in Nakawa Division, Uganda. In particular, food samples including fish stew, meat stew, fried eggs, salads, and unbottled drinking water were taken for microbial analysis from the study area. In the results, E. coli levels were found to be 100% and 60% in Nakawa and Naguru Parishes respectively. Microbial enumeration of Salmonella spp. was negative for all samples tested. Vendors who did not meet adequate personal hygiene standards, adjusted odds ratio, AdjOR: 0.96, CI: 0.85-0.99 and those who stored food at inappropriate temperatures, AdjOR: 0.88, CI: 0.47-0.97 were more likely to have their food exposed to pathogen spreading vectors. The presented results indicate that street foods vended in Nakawa and Naguru Parishes are a source of contamination as elucidated by presence of E. coli.

Tabashsum et al. (2013) determined prevalence of foodborne pathogens and spoilage microorganisms and their drug resistant status in different street foods of Dhaka city. For this assessment, 39 street foods samples of 13 kinds were collected from Motijheel area, the busiest part of the Dhaka city area. These samples were analyzed for foodborne pathogens including, Salmonella spp., Escherichia coli O157, O111, O26 and other E. coli, other coliforms, Cronobacter sakazakii, Yersinia spp., Listeria spp., Staphylococcus spp., and spoilage microorganisms including Enterococcus spp., Pseudomonas spp., Bacillus spp., and lactic acid fermenting bacteria (LAB). The average natural aerobic bacterial population varied from 3.0 ± 0.04 log CFU/g to 8.8 ± 0.02 log CFU/g and the average coliform count varied from 2.0 ± 0.01 log CFU/g to 7.5 ± 0.02 log CFU/g. In addition, Salmonella spp. and Escherichia coli (O157, O111, O26) were identified in 2 street food samples, other E. coli were found in 5 samples,

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coliform bacteria was found in 28 samples and Enterococcus spp. in 10 samples, out of 39 food sample analyzed. Moreover, Listeria spp. were detected in 15 samples, Yersinia spp. in 10 samples, Enterobacter sakazakii in 8 samples, and Staphylococcus spp. in all 39 samples. Among the spoilage organisms, Bacillus spp. were identified in 12 food samples, Pseudomonas spp. in 15 food samples and lactic acid fermenting bacteria (LAB) in 24 samples, out of the 39 samples tested.

Ates et al. (2011) studied microbiological analysis of stuffed mussels sold in the streets. Thirty samples (600 stuffed mussels in total) were collected periodically and microbiological analyses were performed by standard procedures for

Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella sp., Clostridium sp. In terms of Salmonella sp., approximately 50% of samples were not suitable for consumption. Besides, in accordance with Turkish Food Codex Microbiological Criteria Announcement in terms of E. coli 30%, in terms of B. cereus 80%, in terms of S. aureus 76.6%, in terms of Clostridium perfringens 13.3% of these samples were not suitable for consumption.

Chukwuemeka et al. (2011) evaluated the bacteriological quality of food and water consumed in Nsukka, Enugu state, Nigeria, using three bacteria enumeration methods. Data obtained are assumed to reflect the level of personal and environmental hygiene in the study population. Ten types of foods—beans, yam, abacha, okpa, moimoi, pear, cassava foofoo, rice, agidi, and garri—and 10 water samples were evaluated for bacteriological quality, precisely determining the level of coliform contamination, using the most probable number (MPN), lactose fermentation count (LFC), and Escherichia coli count (ECC) methods. Bacterial counts differed significantly (p<0.05) among the various food samples. Escherichia coli and Klebsiella pneumoniae were the two major coliforms identified among 98 coliform isolates obtained from the various food samples, of which 78 (79.6%) were assumed to be of human origin on account of their ability to grow at 44 °C. The level of coliform contamination in the food samples from vendors and restaurants (geometric mean count 7.64-

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9.21; MPN ≥50) were above the accepted 104 colony -forming unit/g or MPN ≤10 limits.

Tambekar et al. (2011) determined bacteriological quality of street vended food panipuri. Forty water sample of panipuri were aseptically collected from eleven locations of Amravati City. Analysis of the food samples revealed that 93% of panipuri water samples had high loads of bacterial pathogens such as

Escherichia  coli  (41%),  Staphylococcus  aureus  (31%),  Klebsiella  sp.  (20%),

Pseudomonas sp. (5%) and yeast (3%). It is suggested that regular monitoring of the quality of street foods must be practiced to avoid any food-borne infection in future.

Ayeh et al. (2009) studied prevalence of intestinal parasitic infections among food vendors in Accra, Ghana. Random sampling was used to select 204 food vendors from 7 metropolitan areas in Accra. The parasitological profiles of stool samples from the vendors sampled were developed using direct smear, formalin-ethyl acetate sedimentation method, modified ZiehlNeelsen, and trichrome staining techniques. The overall prevalence of parasitic infection was 21.6%, with helminthic (15.2%) predominating over protozoan (6.4%) infections. Seven different parasites were identified: Ascaris lumbricoides (5.0%),

Strongyloides stercoralis (4.4%), Enterobius vermicularis (4.1%), Cryptosporidium parvum (2.5%), Giardia lamblia (2.0%), Ancylostoma duodenale (2.0%), and

Entamoeba histolytica (2.0%). The study indicated high levels of gastrointestinal parasitic infection among food vendors in the metropolis.

Tunung et al. (2007) determined incidence and characterization of Salmonella species in street food and clinical samples. The isolates were characterized by their antibiotic resistance, plasmid profiles and randomly amplified polymorphic DNA (RAPD) sequences. A total of 24 salmonellae, belonging to seven different serotypes, were isolated from 129 different street-vended foods and drinks and 12 clinical samples (rectal swabs). The encountered serotypes from street foods were Salmonella Biafra (n=8), Salmonella Braenderup (n=3) and Salmonella Weltevreden (n=1), and from clinical samples were Salmonella Typhi

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(n=8), Salmonella Typhimurium (n=2), Salmonella Paratyphi A (n=1) and Salmonella Paratyphi B (n=1). The results showed no similarities in the types of Salmonella serotypes from street food and clinical samples examined. The Salmonella strains were resistant to one or more of the 14 tested antibiotics. Seventeen isolates harbored plasmids, with plasmid sizes ranging from 3.0 to 38.5 MDa

2.3 Isolation of bacteria from street food

Madueke et al. (2014) studied microbiological analysis of street foods. Fried yam, fried potato, fried plantain, akara, fish and suya were analyzed for their microbial load. The samples were analyzed for bacteria and fungi using standard procedures. Analysis of the food samples revealed mean total bacterial count ranging from 5.0 x 104 cfu/g (akara) to 2.08 x 107 cfu/g (fish). Mean coliform count ranged from 5.0 x 104 cfu/g (yam) to1.0 x 107 cfu/g (suya), and fungal count ranged from 1.5 x 105 cfu/g (yam) to 6.0 x 105 cfu/g (fish). The organisms encountered included: Bacillus cereus, Staphylococcus aureus, Streptococcus sp., Enterobacter sp, Escherichia coli, Shigella dysenteriae, Klebsiella, Pseudomonas, Micrococcus, Flavobacterium, Mucor, Penicillium sp., Aspergillus niger, Aspergillus flavus, Fusarium sp. and Rhizopus stolonifer. The coliform counts were high (≥ 105) in most of the samples.

Sina et al (2011) studied characterization of Staphylococcus aureus isolated from street foods, toxin profile and prevalence of antibiotic resistance. S. aureus was isolated on growth agar media and confirmed by gram staining, catalase activity, and coagulation of citrated rabbit plasma. The leucotoxins and epidermolysins were identificated by radial immune precipitation. Enterotoxins and TSST-1 were assessed by agglutination method. The antimicrobial susceptibility was tested by diffusion method on Mueller Hinton agar. About 56.25% of food dishes analyzed were contaminated. They produce Panton and Valentine Leucocidin (13.33%) and various enterotoxins such as, the enterotoxins A (56.29%), B (37.77%), D (13.33%), the TSST (15.92%), the leucotoxin LukE/LukD (69.25%), and the Epidermolysin A (14.07%). About 15.18% of the S. aureus colonies were resistant to methicillin. This study reveals

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a wide range of S. aureus food contamination producing various disease mediated toxins.

Das et al. (2010) assessed microbiological quality of chaats sold in Bangalore. Analysis of the food samples revealed high loads of bacterial pathogens such as

Streptococcus faecalis, Escherichia coli, Staphylococcus aureus, Bacillus sp., Klebsiella sp. and Pseudomonas sp. Total viable counts of bacteria in all the samples varied between 0.4-3.0x104 cfu g-1, faecal coliforms between 0.03-0.14x104 cfu g-1 and faecal streptococci between 0.2-11x104 cfu g-1. Aciduric yeasts like Saccharomyces and filamentous fungi like Mucor and Rhizopus were also encountered. Salmonella and Vibrio cholerae were not detected in any of the samples. The presence of faecal streptococci and coliforms indicated faecal contamination of the processing water as well as the prevailing unhygienic conditions related to the location of the food stalls.

Furlaneto et al. (2010) stated hygienic-sanitary condition, microbiological quality and antimicrobial susceptibility of isolated strains from sandwiches sold in street markets. Sanitary conditions were evaluated in 47 street-vending places, and 20 samples were randomly collected for analyzing the contamination with Aerobic mesophilic bacteria, Staphylococcus aureus, total coliforms and coliforms at 45 degrees C, and Salmonella. The sanitary conditions were inadequate in 79.2% of the evaluated items. All sandwiches (100%) were contaminated with mesophilic bacteria, coliforms at 35 degrees C and fecal coliform, and 45% with E. coli, S. aureus was found in 55% of samples, and in 25% these values were higher than those allowed by legislation. No Salmonella sp. was isolated from any of the tested samples.

Modarressi et al. (2010) isolated Salmonella enterica from chicken, beef and street foods in Malaysia. Eighty-eight non-repeat Salmonella isolates from 300 food samples were isolated and characterized using conventional culture, biochemical and serological methods and confirmation of Salmonella was determined by polymerase chain reaction (PCR) using specific primers. Among the 88 Salmonella isolates, 11 serovars [Corvallis (n=33), Typhimurium(n=18),

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Hadar(n=7), Enteritidis (n=5), Weltevreden (n=5), Agona(n=5), Newport (n=4),

Albany (n=3), Istanbul (n=2), Emek(n=1) and Wandsworth (n=1)] were identified.

Cerna et al. (2009) isolated Mycobacterium mucogenicum from street-vended chili sauces. Fifty-one street-vended chili sauces were collected in different areas of Mexico City during the spring of 2007. NTM were recovered from 6% (3 of 51) of samples, and in all cases the identified species was Mycobacterium mucogenicum. This mycobacterium has been associated with human illness; therefore, street-vended chili sauces are a potential source of NTM infection.

Mensah et al. (2002) investigated the microbial quality of foods sold on streets of Accra and factors predisposing to their contamination. Standard methods were used for the enumeration, isolation, and identification of bacteria. Most vendors were educated and exhibited good hygiene behaviour. Diarrhoea was defined as the passage of ³3 stools per day) by 110 vendors (94.0%), but none associated diarrhoea with bloody stools; only 21 (17.9%) associated diarrhoea with germs. The surroundings of the vending sites were clean, but four sites (3.4%) were classified as very dirty. Examinations were made of 511 menu items, classified as breakfast/snack foods, main dishes, soups and sauces, and cold dishes. Mesophilic bacteria were detected in 356 foods (69.7%): 28 contained Bacillus cereus (5.5%), 163 contained Staphylococcus aureus (31.9%) and 172 contained Enterobacteriaceae (33.7%). The microbial quality of most of the foods was within the acceptable limits but samples of salads, macaroni, fufu, omotuo and red pepper had unacceptable levels of contamination. Shigella sonnei and enteroaggregative Escherichia coli were isolated from macaroni, rice, and tomato stew, and Salmonella arizonae from light soup. Street foods can be sources of enteropathogens.

Kaneko et al. (1999) investigated raw vegetables cut for salad, cooked salad, cooked rice, boiled noodles, bean curd, and cooked Japanese foods were purchased in 27 retail shops in Tokyo. Intact vegetables before being processed and ready-to-eat fresh salad products were obtained from two food factories located in the suburbs of Tokyo. Two hundred thirty-eight retail samples, 137

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samples of intact vegetables, and 159 samples of fresh products were examined for aerobic plate count (APC), coliforms, Escherichia coli, Listeria spp.,

Staphylococcus aureus, and Bacillus cereus. The APC of retail foods were 2.1 to 5.7 log CFU/g, and the range for the coliforms was 0.1 to 2.3 log CFU/g. The APC and coliform values showed that the raw vegetables cut for salad were the most heavily contaminated among the six kinds of ready-to-eat foods examined. Although L. monocytogenes was not detected, two samples of raw vegetables and five kinds of cooked foods yielded Listeria spp. and S. aureus was detected in one sample of Japanese cooked food. The APC of the intact vegetables were 2.9 to 7.3 log CFU/g upon arrival and 2.2 to 7.2 log CFU/g after 3 days storage at 10 degrees C. The APC of the fresh products were 3.4 to 7.6 log CFU/g upon arrival and 4.7 to 8.7 log CFU/g after 3 days storage at 10 degrees C. The isolation rates for coliforms were 6.1 to 50% for intact vegetables and 50 to 66.7% for fresh products. E. coli was detected only in the fresh products. B. cereus was isolated from 20.1% (17 of 81) of the intact vegetables and 9.2% (8 of 87) of the fresh products.

2.4 Identification of bacteria from street food

2.4.1 Cultural characterization

Konuku et al. (2012) stated that Staphylococcus aureus on mannitol salt agar showed colonies that fermented mannitol and appeared golden yellow.

Hossain et al. (2002) reported that the Salmonella spp. produced small round and smooth colonies on nutrient agar and opaque, translucent and colorless colonies on Salmonella-Shigella (SS) agar. The organisms produced colorless, pale, transparent colonies on MacConkey agar and small, round, low convex, translucent, pale and red color colony on Brilliant green agar (BGA) against pinkish background which was initially green in color.

Sharada et al. (1999) reported that E. coli colonies on Nutrient agar (NA) were smooth, moist, low and convex. The colony on Eosin Methylene Blue (EMB) agar plate appeared as dark with characteristics metallic sheen.

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2.4.2 Morphological characterization by staining techniques

Mahmud (2009) found the Bacillus spp. as Gram positive rods with spores arranged in pairs and also in long chain.

Samad (2005) stated that Salmonella are facultatively anaerobic, Gram negative bacilli and usually enter the body via the gastrointestinal tract where they can persist for long period of time.

Thomas et al. (2005) reported E. coli as an enteric bacillus which has belonged to the family Enterobacteriaceae. It is Gram-negative, rod shaped, motile, capsulated, non-acid fast, non-spore former and facultative anaerobes.

2.4.3 Biochemical characterization

Konuku et al. (2012) isolated Staphylococcus aureus from grapes where the organism showed negative results for indole, methyl red, Voges-Proskauer reaction, hydrogen sulphide, gelatin liquefaction, and oxidase test and showed positive results with citrate, lactose, sucrose, glucose, lipid hydrolysis, catalase, and starch hydrolysis test .

Hossain (2002) found that Salmonella ferment dextrose, maltose and manitol with production of acid and gas but no fermentation was observed in lactose and sucrose.

Thomas (1998) performed some biochemical tests with E. coli and concluded that most E. coli ferment lactose; reduce nitrates and methyl-red positive. Approximately 10% of the species were late lactose fermenters; some of them were non-lactose fermenters.

2.4.4 Molecular characterization

Darahi et al. (2008) determined DNA finger printing of ten E. coli O157:H7 strains based on random amplified polymorphic DNA (RAPD) technique. Ten E. coli O157:H7 strains isolated from children with either hemorrhagic colitis (HC) or hemolytic uremic syndrome (HUS). Their DNA was extracted and

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further amplified by RAPD-PCR using 53 decamer primers. In addition, genetic distance and cluster analysis were estimated. RAPD-PCR analysis proved to be of great value in designing a variety of molecular based epidemiological studies that focuses on the identification and characterization of E. coli O157:H7.

Gibotti et al. (2004) studied the E. coli ipa genes by RFLP-PCR assays for enteroinvasive E. coli (EIEC) serotypes.

Guan et al. (2002) identified E. coli by the amplification of 16S rRNA gene by PCR.

2.5 Antibiogram of bacteria isolated from food samples

Kibret and Tadesse (2013) assessed the prevalence and antibiogram of bacteria from street vended white lupin in Bahir Dar Town. A total of 40 samples were processed for detection of indicator bacteria and pathogens. The total coliform counts were 954.2±385 and 756.2±447.3 at the surface and the core of white lupin, respectively. On the other hand, the fecal coliform counts were 880.9±396.6 and 662.1±461.9 at surface and the core, respectively. There was a statistically significant difference in total colifoms and fecal coliform counts between the surface and core of white lupin (p <0.05). Escherichia coli 29 (72.5%), Salmonella spp. 23 (57.5%) and Shigella spp. 8 (20%) were the pathogens isolated. Most bacterial isolates were resistant to tetracycline, cotriamoxazole and erythromycin whereas many of them were sensitive to chloramphenicol, nalidixic acid, gentamicin and ciprofloxacin. The overall multiple antimicrobial resistances rate was 75%.This study revealed contamination of white lupin and a potential health to consumers, and the bacteria isolated showed high rates of multiple drug resistance.

Lin and Modarressi (2011) analyzed antimicrobial resistance of Salmonella isolates from 300 meat products (raw beef, chicken meat and street foods). A total of 88 non-duplicate Salmonella from 66 (22.0%) retail meat and 22 (7.5%) street food samples were recovered and 11 serovars were identified. Among the 88 Salmonella isolates, the highest resistance was to tetracycline (73.8%),

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followed by sulfonamide (63.6%), streptomycin (57.9%), nalidixic acid (44.3%), trimethoprim-sulfamethoxazole (19.3%), ampicillin (17.0%), chloramphenicol (10.2%), cephalotin (8.0%), kanamycin (6.8%), ciprofloxacin (2.2%) gentamycin (2.2%), cefoxitin (2.2%), amoxicillin-clavulanate (1.0%) and amikacin (1.0%). Sixty-seven percent of the isolates (59/88) were multidrug resistant (MDR). Four integron-positive isolates could transfer resistance phenotypes to the recipient strain, E. coli J53 via conjugation. These data revealed that the Salmonella isolates recovered from the retail meats and cooked street foods were resistant to multiple antimicrobials, which can be transmitted to humans through food products.

Singh et al. (2011) tested 100 samples of street-purchased burgers. A total of 225 isolates were recovered from 100 samples. It was observed that burger sample have the prevalence of Escherichia coli (42.22%), Salmonella(31.11%) and Staphylococcus sp. (22.66%), Escherichia coli sp. showed the highest percentage. The isolated bacteria showed the sensitivity against some of the antibiotics. The Amoxycillin, Ciprofloxacin, Gentamicin, Ofloxacin and Tetracycline were very effective against the Salmonella, Staphylococcus aureus showed high resistance to Chloramphenicol in the present study.

Tagoe et al. (2011) conducted antibiotic sensitivity of bacteria isolated from sachet water popularly known as Pure water. Bacteria counts ranges between 2.8x103-5.9x105 cfu mL-1 in all Sachet water brands with different bacterial isolates which included E. coli, Coagulase negative Staphylococcus, S. aureus, E. faecalis, K. aerogenes, M. catarrhalis, B. cereus, L. monocytogenes and Enterobacter sp. etc. The degree of resistance of isolates to the antibiotics ranges from 50.0-87.5%, with multiple drug resistance to 4-7 antibiotics. The isolates showed 100% resistance to Ampicillin, Flucloxacillin and Penicillin, while none of them was resistant to Gentamycin. The resistance to other antibiotics ranged from 93.3% for Erythromycin and Cefuroxime, 60% for Co-trimoxazole and 20% for Tetracycline. The results indicate the presence of antibiotic resistant bacteria in sachet water consumed in the Metropolis with its attendant potential health.

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2.6 Safety and public health significance of street food

Khairuzzaman et al. (2014) studied food safety challenges towards safe, healthy and nutritious street Foods in Bangladesh. In this study socio demographic characteristics, common hazards, and occupational hazards of street food vendors, microbial risk associated with street food, food safety interventions and control measures, regulatory aspects and legal requirements, financial constraints, and attitudes is described.

Mirriam et al. (2012) conducted a study on foodborne pathogens recovered from ready-to-eat foods from roadside cafeterias and retail outlets in Alice. Microbiological analysis was conducted on 252 samples which included vegetables, potatoes, rice, pies, beef and chicken stew. The isolates were identified using biochemical tests and the API 20E, API 20NE and API Listeria kits. High levels of total aerobic count were observed in vegetables, 6.8 ± 0.07 followed by rice, 6.7 ± 1.7 while pies had the lowest count (2.58 ± 0.24). Organisms isolated included: Listeria spp. (22%), Enterobacter spp. (18%),

Aeromonas hydrophila (12%), Klebsiell aoxytoca (8%), Proteus mirabilis (6.3%), Staphylococcus aureus (3.2%) and Pseudomonas luteola (2.4%). There was a statistically significant difference (p < 0.05) in the prevalence of foodborne pathogens from hygienic and unhygienic cafeterias. The results indicated that most of the ready-to-eat food samples examined in this study did not meet bacteriological quality standards, therefore posing potential risks to consumers.

Amoah et al. (2011) studied low-cost options for reducing consumer health risks from farm to fork where crops are irrigated with polluted water. The research focused on three entry points: farm-based measures, market-based measures, and street-restaurant-based measures to determine effective methods for the reduction of pathogens, using faecal coliforms and helminths as indicators. The focus was set on street food restaurants. At farm level, the studies showed that sedimentation ponds and filtration techniques like sand filters could reduce the number of helminth eggs to acceptable levels in both dry and wet seasons. Among various irrigation methods, low-head bucket drip

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kits achieved high removal levels of up to 6 log units of faecal coliforms on vegetables. The tested market-based interventions had generally less impact on the contamination carried over from the farm, like through the support of microbial die-off but they remain important to prevent new or additional contamination.

Muyanja et al. (2011) studied risk factors, practices and knowledge of street food vendors with respect to food safety and hygiene. A total of 225 street food vendors were investigated. Street vendors (87.6%) were women and with low education level. Vendors had access to tap water within 5 min walk. Non-disposable plates/cups were the commonly used for vending food. Use of soap and cold water for washing utensils was common practice. Wash water recycled several times and only changed when very cloudy and soapy. Toilet facilities were dominated by pour/flash toilet and pit latrine. Masaka (64.3%) and Jinja (38.9%) vendors disposed off the garbage at the vending sites whereas in Kampala (92.8%) used gunny bags. Cooked food was handled at ground level and exposed to flies. Masaka vendors (68.6%) had no hygiene regulations governing the street food vending business whereas Kampala (75.9%) and Jinja (65.3%) indicated hygiene regulations were enforced onsite management by local government.

Rane (2011) stated that street food vending has become an important public health issue and a great concern to everybody. This is due to widespread food borne diseases, due to the mushrooming of wayside food vendors who lack an adequate understanding of the basic food safety issues. Major sources contributing to microbial contamination are the place of preparation, utensils for cooking and serving, raw materials, time and temperature abuse of cooked foods and the personal hygiene of vendors. Various studies have identified the sources of food safety issues involved in street foods to be microorganism belonging to the genus Bacillus, Staphylococcus, Clostridium, Vibrio, Campylobacter, Listeria, Salmonella.

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Silva et al. (2011) referred informal street food trade systems constitute a high risk for public health. In this work, sanitary conditions of street food vending areas, as well as social and economic aspects of salesmen established in the Campus of Porangabucu of the Federal University of Ceara - Brazil had been investigated. Data were collected from 24 points of sales in the period between August and October 2009. It was found that most of the recommendations of the RDC n. 218 are not observed, yielding the following results: the group is made up mostly of men (58.3%) showing incomplete basic education; 66.6% of the salesmen wore ornaments during the handling of food; the lack of trash disposal was evidenced in 62.5% of the places; it was observed the presence of dirt on the utensils used in 37.5% of sales outlets and 42% of vendors did not conserve food in appropriate temperature and containers.

Berbicz et al. (2010) stated that the street food commerce might cause health risks to the population. Microbiology analyzes of 42 food samples were collected in the marketing points in Maringa (PR) city. The microbiological analyses found contamination by Coliforms at 45 degrees C in 14.3% of the samples and by Staphylococcus positive coagulase in 2.4% of the samples (RDC 12/2001). No contamination by Salmonella spp. and B. cereus was found in any of the samples. In the first checklist, a 33.4% non-conformity was detected. The Good Practices training program was enforced in 12 hours. After the second checklist accomplished in a sample of 10 street food points of sale, and there could be observed that some aspects such as hygiene have improved.

Abdalla et al. (2009) studied the evaluation of food safety knowledge and practices of street food vendors in Atbara city between March and April, 2008. The questionnaires respondents were 28% male and 72% were female, 48% of them had primary school education while 42% were illiterates. The most prevalent isolated bacteria from cooked meals, bottled drink and fresh juice were; Escherichia coli, Staphylococcus aureus and Bacillus sp. The viable bacterial counts were 4.6 CFU/ml, 3.7 CFU/ml and 4.1 CFU/ml for cooked meals, bottled drink and juice, respectively.

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Kisla et al. (2008) collected fifty samples (750 stuffed mussels in total) periodically and microbiological analyses were performed by standard procedures for aerobic plate count, coliforms, fecal coliforms, Enterobacteriaceae, Staphylococcus aureus, Bacillus cereus and Vibrio spp. Aerobic plate counts above 5 log CFU/g were obtained in 16 and 72% of stuffing mixture samples at high and low ambient temperatures, respectively, and average aerobic plate counts of outer surface samples at high and low ambient temperatures were 3.21 and 4.34 log CFU/ml, respectively. The prevalence and the count levels of coliforms, fecal coliforms, Enterobacteriaceae, and Vibrio spp. (except for the prevalence of Vibrio spp. in stuffing mixture samples) in the samples at high ambient temperatures were considerably higher compared with those at low ambient temperatures (P<0.05). High frequencies of pathogens S. aureus and B. cereus were found in stuffing mixture samples at high ambient temperatures, with averages of 2.84 and 2.94 log CFU/g, respectively (P<0.05). The result of this investigation indicates that stuffed mussels as a street food may constitute a potential health hazard.

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