Antibiotic Resistant Bacteria In Milk - Research Paper Example

1 Name of the College or university Topic: ANTIBIOTIC RESISTANCE BACTERIA IN MILK Student name: Student enrolment number: “TASK: A PROJECT SUBMITTED IN PARTIAL FULFILLMENT OF THE AWARD OF DEGREE IN BIOCHEMISTRY …) 1 Declaration I ……………………………………………………………., student enrolment number…………………………………….declare that this research on antibiotic resistance in bacteria in milk is my own work and has never been published before in any medical or clinical journal. Dated………………………………………………..student signature……………. Supervisors name: ………………………………………………………. Supervisor’s signature: ………………………………………………………. Date: ………………………………………………………. 2 Acknowledgements 3 Dedication 4 Abstract Five dairy milk specimens were examined for antibiotic resistance. The five milk specimens used were whole milk, semi milk, semi-skimmed milk, organic whole milk, and organic semi-milk. Each of these dairy milk specimens were exposed to six different antibiotic that were chloromphenicol, erythromycin, kanamycin, tetracycline, ampicilin and penicillin. Each of the sample was diluted from 10-1 to 10-6 in peptone water and Petri dish were marked in terms of name of agar, type of milk, dilution factor used, date and name. Bacteria were resistant to three antibiotics that were ampicillin, penicillin and vancomycin. Sensitivity tests were carried out on the antibiotics the other antibiotics; chloromophenicol, erythromycin, tetracycline and kanamycin that were incubated for three days at 37 degree Celsius. Presence of bacteria was carried out in the antibiotics that bacteria had developed resistance to using the following methods: gram stain method, catalase, oxidative and electrophoresis and determined by using dichotomous key and serological properties. 5 Table of contents 1 Declaration 2 2 Acknowledgements 3 3 Dedication 4 4 Abstract 5 5 Table of contents 6 6 List of figures 7 7 List of tables 8 8 Background 9 8.1 Statement of the problem 11 8.2 Aims and objectives 12 8.3 Hypothesis to be tested 12 8.4 Significance of the study 13 8.5 Assumptions made in the study 13 9 Introduction 14 10 Literature review 16 11 Methodology 18 11.1 Materials used 18 11.2 Chemicals used 19 11.3 Antibiotics used 19 The chemical structures of the antibiotics used 20 11.4 Specimen samples used 24 11.5 Indicators used 24 11.6 Procedures used in the study 25 11.7 Nutrient enrichment culture 25 11.8 Testing Serological properties 26 11.9 Testing presence of bacteria 26 11.10 Figure 1: The dichotomous key developed to identify the bacteria species that were present depending on biochemical tests 45 11.11 Analysis of the results 46 11.12 Conclusion 54 11.13 Future work 55 11.14 References 55 6 List of figures a. Figure 1: The dichotomous key developed to identify the bacteria species that were present depending on biochemical tests 7 List of tables a. Table 1: observations made following pour plate technique on the milk agar b. Table 2: observations made following pour plate technique on the milk agar (MA) and milk extract agar (MEA) c. Table three: observations made from use of milk agar on spread plate technique d. Table four: observations made on using spread plate technique on milk agar and milk extract agar. e. Table five: Observations made on spoiled milk samples using the spread plate technique f. Table six: repeated procedure five to validate Observations made on spoiled milk samples using the spread plate technique g. Table seven: Glucose enrichment: Observations made on spoiled milk samples using the spread plate technique h. Table eight: The procedures in step 1 were repeated and the following data obtained i. Table nine: The observations that were made on antibiotic sensitivity 8 Background Human suffer from many bacterial diseases that do not respond to the antibiotics that used to manage them. Many bacteria have been isolated from fecal matter of human beings like salmonellae spp. For instance salmonella typhimurium, clostridium perfringens, Listeria monocytogenes, Escherichia coli, and camphylobacter sp. that are very resistant to antibiotics like Amphicillin, Penicillin, Vancomycin, Chloromophenicol, Erythromycin, and Kanamycin. Similar bacteria have been successfully isolated from cattle and other livestock23 and all have shown antibiotic resistance24 to Amphicillin, Penicillin, Vancomycin, Chloromophenicol, Erythromycin, and Kanamycin. This means these bacteria are transmitted from the livestock to human through the food chain 23,24 and especially through the human consumption of milk that is contaminated with the antibiotic resistant bacteria27. This questions how the antibiotic resistant bacteria could have entered into the livestock25. These antibiotic resistant bacteria enter the livestock through two main pathways. The animal could feed on plants that were genetically engineered. First, genetically engineered crops are characterized by presence of antibiotic resistance marker genes 14that enable the genetic engineers to know if their genetic engineering process was a success. It is presence of these antibiotic resistant marker genes that triggered the bacteria to undergo mutations 14to manage their effect. In so doing, strains of bacteria are produced as a result of recombination. Secondly, feeding of healthy livestock with antibiotics in order to increase their growth rate15, a factor to ensure they sold the animals fast if they were meant for the beef industry, is a suspect to the evolution of bacteria towards drugs that treated diseases that were associated with them in the past. These antibiotics had a role of destroying bacteria16 that were present in the alimentary canal of the cattle that absorbed some of the food that the animal digested. The intended result was to enable the cattle to absorb all the food it had eaten. The main link between the cattle and human is on the milk15 that human beings consume on daily basis. It is the most likely suspect as the one that could contain the antibiotic resistant bacteria17, 32. The other areas that are worth considering are the processes through which the milk is processed, and to determine if there are bacteria that might not be present in the milk but enter the milk product as a result of the processing. 8.1 Statement of the problem Normally, milk contains bacteria that can curdle milk by acting on the lactose, glucose, casein and other proteins that are present in the milk. Production of lactic acid by anaerobic bacteria denatures milk proteins. Many bacteria that are resistant to antibiotics are passed through the food chain and this research focuses attention on whether there are bacteria that are present in the milk that may be tolerant to commonly used antibiotics like Amphicillin, Penicillin, Chloromophenicol, Erythromycin, and Kanamycin. 8.2 Aims and objectives 1. To investigate if dairy milk contains antibiotic resistant bacteria 2. To investigate the antibiotics that the bacteria present in milk is able to nullify 3. To investigate the impact of increased concentration of antibiotic towards bacterial resistance. 4. To determine the type of cell wall that is present in the bacteria that are observed in the milk by using gram stain method 5. To determine if the bacteria present in the milk is metabolically active. 8.3 Hypothesis to be tested 1. Dairy milk contains bacteria that are responsible for curdling of milk 2. The bacteria that are present in the milk may be resistant to antibiotics if the animal they come from has antibacterial that are resistant to antibiotics 3. Increasing concentration of an antibiotic should correspond with a higher rate of destruction of bacteria. 4. Bacteria that are present in the milk are not metabolically active initially because the milk is derived from a healthy animal whose immune system is functional. 8.4 Significance of the study The study would enable determination of the bacteria that are present in the milk and whether they are resistant to antibiotics. From the study , it will be possible to determine areas that the bacteria might have entered the milk and recommendations be made on how efficient the bacteria can be minimized so that they are not passed to human beings where they are likely to have adverse medical complications. 8.5 Assumptions made in the study 1. The milk samples were not exposed to air or moist conditions and were kept in sterile conditions to minimize external contaminations that could have affected the results. 2. The streak, pour plates and all physical materials that were used were sterile and no contamination on them could have distorted the results obtained. 3. The reagents used in the research like tetra methyl p- phenylene diamine, dimethyl p- phenylene diamine and hydrogen peroxide were in their pure forms to ensure their intended chemical behavior was not compromised 4. The milk was processed in bacteria free conditions. 9 Introduction Milk is a substance that is secreted by the mammals and contains many nutrients like carbohydrates, lipids, proteins, vitamins and mineral salts. milk contains micro-organisms present in it like bacteria. Other bacteria can be found in the milk as a result of feeding the cattle with genetically modified food or feeding the cattle with antibiotics. some of the antibiotics that are commonly used are Ampicilin, penicillin, chloramphenicol, streptomycin, sulphonamides, tetracycline , erythromycin, and kanamycin. The last two decades have witnessed feeding of cattle with antibiotics 15, 16,. Antibiotics have an effect of increasing the growth rate of cattle by up to 5-8% . These antibiotics are used as growth promoters 30by getting rid of gut bacteria that interfere with digestion. Following widespread use of antibiotics, many bacteria have evolved a mechanism of that enables them to grow in many antibiotics35. Bacteria manage to grow in antibiotics by producing relevant enzymes27 that cancel the effect of the antibiotic28. For instance, bacteria are able to produce penicillase that nullifies the effect of antibiotic penicillin. This has made bacteria to develop resistance against antibiotics35. The bacteria that are commonly transferred from cattle to human include Escherichia coli, salmonella typhimurium and salmonella aureas34. Antibiotic resistance of bacteria ranges from a single antibiotic resistance20 implying it can effectively the effect of one antibiotic to multiple resistance30 where the bacteria is resistant to more than one antibiotic. For example, salmonellae that are isolated from infected patients cannot be handles by antibiotics like Amphicillin, Chloromophenicol, Streptomycin, Sulphonamides and Tetracycline33 (ACSSuT). This antibiotic multi resistant bacterium, salmonella typhimurium, has a phase of type DT 104. This strain is abundant in cattle15 and is passed on to the people through the food chain16. In human beings especially, the multi resistant DT 104 strain has been successfully isolated from about 200 in 1989 to 5000 in 199623. When some antibiotic drugs were banned from use in cattle, there has been a reported decline in their quantities by a 48% margin in 1998 to 209024. 10 Literature review Penicillin kills bacteria by preventing biosynthesis of peptidoglycan14, a constituent of cell wall that makes the bacteria to lyse21 or burst as a result of entry of water through osmosis. Other antibiotics, isolated from bacteria and yeast like erythromycin, streptomycin and chloromophenicol2. Some antibiotics inhibit protein biosynthesis30 like streptomycin while other antibiotics inhibit DNA replication1. Antibiotics fall into two broad groups; broad spectrum antibiotics like tetracycline which are effective against a wide variety of bacteria and narrow spectrum bacteria like penicillin that counter a few bacteria. Sulphonamides2 are non-antibiotics similar to para-amino benzoic acid, a heterocyclic organic compound that is an essential metabolite in the reproduction of the bacteria. Sulphonamides, as competitive inhibitiors18 compete with p-amino benzoic acid for the active sites of the enzymes. Sulphonamides don’t kill the bacteria but stop it from reproducing. A bacterium counters these effects by undergoing genetic recombination3 in its plasmids. Plasmids are smaller circular pieces of DNA that are separate from the rest of bacteria’s DNA. Plasmids replicate independently of the bacteria DNA that is in automous state or may also be integrated into the bacteria’s DNA thus integrated state. Plasmids carry genes for resistance4 against effect of antibiotics. Plasmids also contain a DNA bit called F-factor4 where F denotes fertility. The F-factor carries genes for producing sex pili17 and for transfer of DNA from donor bacterium to the recipient bacterium. The F-factor inserts into the rest of the bacterium’s DNA and causes some of the DNA genes to pass into the recipient cell. Once in the recipient cell, some of the donor’s genes change places with corresponding genes of the recipient bacterium. This recombination results into a bacterium that has a new set of genes22 that are more adapted to tolerate harsh conditions that the donor was unable to survive in. the recombination of genes is a strengthening aspect of the bacteria to resist the effect of antibiotic3. Some of the genes lead into formation of enzymes that can hydrolyze any relevant antibiotic22. Other recombinant strains are able to ingest the antibiotic and use it as food. This contributes to bacteria’s resistance to antibiotics22 and questions the effectiveness of chemotherapeutic agents18 in disease management as every time a new antibiotic is used, new resistant strains emerge. 11 Methodology 11.1 Materials used a. Sterile Petri dishes b. Inoculating loop c. Micropipettes d. Microscope e. Platinum wire needle f. Nichrome wire needle g. Pour plates h. Spread plates i. Streak plates j. Darham tube k. Vials l. Velveteen disc 11.2 Chemicals used a. Hydrogen peroxide b. Water c. Dimethyl p-phenylene diamine d. Tetramethyl p-phenylene diamine e. Gram stain f. Glucose 11.3 Antibiotics used a. Amphicillin b. Penicillin c. Chloromophenicol d. Erythromycin e. Kanamycin The chemical structures of the antibiotics used Figure 2: the chemical structure of chloramphenicol Figure 3: the chemical structure of streptomycin Figure 4: the chemical structure of D-(−)-α-Aminobenzylpenicillin sodium salt Figure 5: chemical structure of Chlortetracycline Figure six: the chemical structure of 4-Amino-N-(2-pyrimidinyl) benzenesulfonamide Figure seven: the chemical structure of kanamycin Figure 8: the chemical structure of ampicillin Figure 9: the chemical structure of erythromycin Figure 10: the chemical structure of Penicillin V potassium salt 11.4 Specimen samples used a. Whole milk b. Semi milk c. Semi skimmed milk d. Organic whole milk e. Organic semi milk 11.5 Indicators used a. Bromothymol blue b. Phenophthalein c. Red and blue litmus paper 11.6 Procedures used in the study 11.6.1 Isolation of pure cultures Isolation of culture 1,2,3 was done by securing the progeny of a single cell or a group of identical cells. Various techniques were employed: single cell isolation2 by means of mechanical micromanipulator3, selective enrichment to inhibit all other undesired micro-organisms like yeasts3 that might have been present and plating 1, 4. 11.6.2 Pour plates Poured plated 3,4 were prepared by diluting the bacterial mixture in the milk samples serially in tubes of melted and cooled agar medium which were then poured into sterile Petri dishes and allowed to solidify. Dilutions were made with the inoculating loop or by use of a pipette. The poured plates contained subsurface that was within the agar as well as surface colonies. Subsurface colonies were smaller than surface bacterial colonies of the same species. The gram negative bacteria formed lenses shaped subsurface colonies29. On the other hand their surface colonies were smooth, circular and convex in cross section. 11.7 Nutrient enrichment culture This was prepared by adding glucose. The lactose fermenting bacteria were selectively enriched in a medium containing glucose. 11.8 Testing Serological properties 11.8.1 The agglutination test 11.8.1.1 Procedure An antigen was isolated from the species suspected to be salmonellae by using the dichotomous key (fig 1) and a saline suspension of the isolated bacteria that had been prepared by mixing it with 0.85 percent of sodium chloride and a corresponding antisera were incubated at 37 degrees Celsius for three hours. 11.8.1.2 Observation made The bacteria clumped together in compact granules mass. 11.9 Testing presence of bacteria The antibiotics that were used were Ampilicin, Penicilin, Kanamycin, Chloromophenicol, Erythromycin and Tetracycline. 11.9.1 Nutrient agar, peptone water and milk agar preparation 28g of nutrient agar powder was mixed with 1 liter of distilled water and heated for about 8 minutes until all the solution dissolved. It was then poured onto 100 ml bottles and then autoclaved at 121 degrees Celsius.) peptone water preparation : 1 g of peptone water powder was added into one litre of distilled water and then mixed well and then poured into 9ml beakers and then autoclaved preparation of the milk agar: 22 g of milk agar powder was mixed mixed with one litre of distilled water and then autoclaved. 11.9.1.1 Milk agar Use on the pour plate technique The nutrient agar and peptone water were first prepared4. For every sample specimen, of pasteurised whole milk, semi milk, skimmed semi milk, whole organic milk and semi organic milk, which were bought from the supermarket, were all diluted serially from 10^-1 to 10^-6. The petri dish were then marked according to type of milk sample, name of the agar, dilution factor that was present, date prepared and name, for the purpose of identification. Each of the samples was subjected to the pour plate technique and left in the incubator for seventy two hours at thirty seven degrees Celsius. 11.9.1.2 Observations made from use of the milk agar on pour plate technique Few bacteria were detected in few samples. The table below summarizes the results observed. 11.9.1.3 Table 1: observations made following pour plate technique on the milk agar Dilution factor Pasteurized Whole milk Semi milk Semi skimmed milk Organic whole milk Organic semi milk 10-1 7 102 12 10-2 4 12 10-3 3 2 10-4 10-5 5 10-6 7 11.9.2 Milk agar and milk extract agar Use on the pour plate technique 11.9.2.1 Procedure on milk agar and milk extract agar using pour plate technique The milk agar and milk extract agar and peptone water were first prepared. For every sample specimen, of pasteurized whole milk, semi milk, skimmed semi milk, whole organic milk and semi organic milk, which were bought from the supermarket, were all diluted serially from 10^-1 to 10^-6. The petri dish were then marked according to type of milk sample, name of the agar, dilution factor that was present, date prepared and name, for the purpose of identification. Each of the samples was subjected to the pour plate technique and left in the incubator for seventy two hours at thirty seven degrees Celsius. 11.9.2.2 Observations made on the use of milk agar and milk extract agar The bacteria were able to be detected in a few samples and the table below summarizes the results obtained. 11.9.2.3 Table 2: observations made following pour plate technique on the milk agar (MA) and milk extract agar (MEA) Dilution factor Whole milk Semi milk Semi skimmed milk Organic whole milk Organic semi milk Milk Agar Milk extract Agar MA MEA MA MEA MA MEA MA MEA 10-1 1 2 8 1 24 3 10-2 1 5 10-3 10-4 2 10-5 10-6 1 11.9.3 Use of spread plate technique 11.9.4 Milk agar use on spread plate technique 11.9.4.1 Procedure on spread plate technique on milk agar The milk agar and peptone water were first prepared. For every sample specimen, of pasteurized whole milk, semi milk, skimmed semi milk, whole organic milk and semi organic milk, which were bought from the supermarket, were all diluted serially from 10^-1 to 10^-6. The petri dish were then marked according to type of milk sample, name of the agar, dilution factor that was present, date prepared and name, for the purpose of identification. Each of the samples was subjected to the spread plate technique and left in the incubator for seventy two hours at thirty seven degrees Celsius. 11.9.5 Observations made on the milk agar on spread plate technique The data obtained was similar to the data obtained from the pour plate technique. The table below illustrates the results. 11.9.5.1 Table three: observations made from use of milk agar on spread plate technique Dilution factor Pasteurized Whole milk Semi milk Semi skimmed milk Organic whole milk Organic semi milk 10-1 7 102 12 10-2 4 12 10-3 3 2 10-4 10-5 5 10-6 7 The process was repeated for both milk agar and milk extract agar using spread plate technique. 11.9.6 Milk agar and milk extract agar using spread plate technique 11.9.6.1 Procedure on spread plate technique on milk agar and milk extract agar The milk agar5 and peptone water were first prepared. For every sample specimen, of pasteurized whole milk, semi milk, skimmed semi milk, whole organic milk and semi organic milk, which were bought from the supermarket, were all diluted serially from 10^-1 to 10^-6. The petri dish were then marked according to type of milk sample, name of the agar, dilution factor that was present, date prepared and name, for the purpose of identification. Each of the samples was subjected to the spread plate technique and left in the incubator for seventy two hours at thirty seven degrees Celsius. 11.9.6.2 Observations made on use of milk agar and milk extract agar on spread plate technique. There were very few bacteria present and the data was similar to the data on milk agar and milk agar extract using the pour plate technique. 11.9.6.3 Table four: observations made on using spread plate technique on milk agar and milk extract agar. Dilution factor Whole milk Semi milk Semi skimmed milk Organic whole milk Organic semi milk Milk Agar Milk extract Agar MA MEA MA MEA MA MEA MA MEA 10-1 1 2 8 1 24 3 10-2 1 5 10-3 10-4 2 10-5 10-6 1 The results obtained were satisfactory that the milk had no bacteria but after a while the milk got spoiled and that meant that there were bacteria that were present in the milk. Spread plate technique was then repeated for all the spoiled milk samples. 11.9.7 Spread plate technique on all milk samples after they expired 11.9.7.1 Procedure used on the expired milk using spread plate technique The nutrient agar5, milk agar, milk extract agar and peptone water were first prepared. For every spoiled sample specimen, of pasteurized whole milk, semi milk, skimmed semi milk, whole organic milk and semi organic milk, which were bought from the supermarket, were all diluted serially from 10^-1 to 10^-6. The petri dish were then marked according to type of milk sample, name of the agar, dilution factor that was present, date prepared and name, for the purpose of identification. Each of the samples was subjected to the spread plate technique and left in the incubator for seventy two hours at thirty seven degrees Celsius. 11.9.7.2 Observations made on spoiled milk samples using the spread plate technique Many bacteria were observed to have been present. The whole milk sample also showed presence of yeasts which is a likelihood of contamination. 11.9.7.3 Table five: Observations made on spoiled milk samples using the spread plate technique Dilution factor Whole milk Semi milk Semi skimmed milk Organic whole milk Organic semi milk AGAR NA MA MEA NA MA MEA NA MA MEA NA MA MEA NA MA MEA 10-1 652 103 52 446 86 28 218 69 28 748 129 68 547 93 52 10-2 334 82 23 198 37 17 102 42 13 356 62 42 279 37 25 10-3 158 47 86 19 5 49 21 4 146 28 15 124 19 12 10-4 72 25 37 7 22 7 72 15 73 10-5 28 11 18 9 35 5 29 10-6 7 0 8 0 11 1 13 Milk extract agar seemed to have had very few bacteria in all the samples. The procedure was repeated again and the results were as follows. 11.9.7.4 Table six: repeated procedure five to validate Observations made on spoiled milk samples using the spread plate technique Dilution factor Whole milk Semi milk Semi skimmed milk Organic whole milk Organic semi milk AGAR NA MA MEA NA MA MEA NA MA MEA NA MA MEA NA MA MEA 10-1 679 112 15 478 75 32 221 89 28 746 132 42 549 107 58 10-2 349 76 247 23 11 95 56 389 82 283 83 14 10-3 155 51 92 5 47 32 157 29 138 57 10-4 68 24 39 2 38 11 83 18 63 34 10-5 35 19 28 21 1 35 5 32 12 10-6 14 2 14 6 9 17 The milk extract agar didn’t respond well so I decided to enrich it with glucose. 11.9.7.5 Table seven: Glucose enrichment: Observations made on spoiled milk samples using the spread plate technique There was no observable change. The data below summarizes the similar results that were obtained in table six Dilution factor Whole milk Semi milk Semi skimmed milk Organic whole milk Organic semi milk AGAR NA MA MEA NA MA MEA NA MA MEA NA MA MEA NA MA MEA 10-1 679 112 15 478 75 32 221 89 28 746 132 42 549 107 58 10-2 349 76 247 23 11 95 56 389 82 283 83 14 10-3 155 51 92 5 47 32 157 29 138 57 10-4 68 24 39 2 38 11 83 18 63 34 10-5 35 19 28 21 1 35 5 32 12 10-6 14 2 14 6 9 17 11.9.8 Procedure In 100ml of the nutrient agar, 0.1g of an antibiotic was added and a 50ml sample of a specimen of milk added. Serial dilution6 of all samples was done respectively. The agar containing antibiotic pour in a dish was left to settle. 0.1ml of the sample was the measured and spread on top of the plate and left in the incubator for 7 hours at 37oC 11.9.8.1 Observations made There was no growth of bacteria in the following antibiotics: Chloromophenicol, Erythromycin, Tetracycline, and Kanamycin. There was growth in the following antibiotics: Ampilicin, and Penicilin. 11.9.9 Step 2: the concentration of antibiotic were increased by a hundred fold from 0.1g to 1g in order to compare the effect of concentration of antibiotic to bacterial resistance. 11.9.9.1 Procedure: In 100ml of the nutrient agar, one gramme of an antibiotic was added and a 50ml sample of a specimen of milk added. Serial dilution of all samples was done respectively. The agar containing antibiotic pour in a dish was left to settle. 0.1ml of the sample was the measured and spread on top of the plate and left in the incubator for 7 hours at 37oC 11.9.9.2 Observations made following increase of antibiotic concentration to one gramme. There was no growth of bacteria in the following antibiotics: Chloromophenicol, Erythromycin, Tetracycline, and Kanamycin. There was growth in the following antibiotics: Ampilicin, and Penicilin. After six days, severe growth occurred in all samples of antibiotic concentrations of 0.1g 11.9.9.3 Table eight: The procedures in step 1 were repeated and the following data obtained Dilution factor Whole milk Semi milk Semi skimmed milk Organic whole milk Organic semi milk AGAR NA MA MEA NA MA MEA NA MA MEA NA MA MEA NA MA MEA 10-1 652 103 52 446 86 28 218 69 28 748 129 68 547 93 52 10-2 334 82 23 198 37 17 102 42 13 356 62 42 279 37 25 10-3 158 47 86 19 5 49 21 4 146 28 15 124 19 12 10-4 72 25 37 7 22 7 72 15 73 10-5 28 11 18 9 35 5 29 10-6 7 0 8 0 11 1 13 The nutrient agar responded and gave better results and bacterial resistance test was done using it. The antibiotics used to test the resistance of the bacteria present were: Ampilicin, penicilin, kanamycin, chloromophenicol, erythromycin, and tetracycline. 11.9.9.4 Procedure 100ml of nutrient agar was prepared and serial dilations were made ranging from 10-1 to 10-6 and 0.01g of every antibiotic, an equivalent of ten milligrams was added to the nutrient agar. The agar containing antibiotic was then poured into Petri dish and left to settle then 0.1 ml of a sample spread on top of the plate. It was then left in the incubator for 72 hours at 37oC. 11.9.9.5 Observations made No growth with chloromophenicol, erythromycin, tetracycline and kanamycin but there were growth with ampilicin and penicillin 11.9.9.6 The procedure was repeated 100ml of nutrient agar was prepared and serial dilations were made ranging from 10-1 to 10-6 and 1g of every antibiotic, an equivalent of ten milligrams was added to the nutrient agar. The agar containing antibiotic was then poured into Petri dish and left to settle then 0.1 ml of a sample spread on top of the plate. It was then left in the incubator for 72 hours at 37oC. 11.9.9.7 Observation made After three days, there was no change. After six days, there was severe growth as before at 0.01g concentrations of the antibiotic showing milk contains penicillin, and ampiliciln resistant bacteria. 11.9.10 Sensitivity test of chloromophenicol, erythromycin, tetracycline and kanamycin was then carried out. 11.9.10.1 Procedure to determine sensitivity of antibiotics Pour agar in a Petri dish and let it to settle Obtain 0.1ml of the sample and pour on top of the agar, spread and dry. Then chloromophenicol, erythromycin, tetracycline and kanamycin disc was placed in the middle of the disc and then left in the incubator for thee days at 37 degrees Celsius. After three days a clear ring formed around the antibiotic disc and the results obtained were: 11.9.10.2 Table nine: The observations that were made on antibiotic sensitivity Antibiotics Whole milk Semi milk Semi skimmed milk Organic whole milk Organic semi milk chloromophenicol 4.9 3.8 3.2 4.6 3.7 erythromycin 4.2 3.9 3.4 4.1 3.6 kanamycin 4.3 3.4 3.0 3.9 3.6 tetracycline 4.6 3.7 2.9 3.4 3.2 11.9.11 To test the resistant bacteria in the milk This process involved the following processes 11.9.11.1 Gram stain method This was done to determine the nature of the cell wall present in the isolated bacterium 11.9.11.2 Procedure on gram stain method Smear preparations Emulsify cells very small drop of water over an area of approx 1cm squared. air dry completely and heat fix. Staining: apply crystal violet for 1 min and gently rinse off with distilled water and then flood with gram iodine for 1 min then wash with water, destain with alcohol (ethanol-20%) for about 30 seconds then wash with water and counter-strain with safarin for 10 seconds. wash with water, blot dry and then observe and record cell morphology using microscopy. 11.9.11.3 Observations made on gram stain method Some samples turned purple Other samples had mixture of purple and red stains of gram stain 11.9.12 Biochemical tests 11.9.12.1 Procedure 1: diffusion method Filter papers were impregnated6 with the test substance such as carbohydrate. A nutrient agar5 that contained appropriate indicator was inoculated by streaking with the bacterium and then the discs were placed upon the agar surface. 11.9.12.2 Observation made on diffusion method The carbohydrate diffused from the paper and a zone displaying the acid color of the indicator surrounded it 11.9.12.3 Procedure 2: using hydrogen peroxide 2-3 drops of 3 per cent of hydrogen peroxide 3,5,6 was added into the every sample or growth on a slant culture or by transferring a needle point of growth to a drop of peroxide on a slide or a Petri dish cover. 11.9.12.4 Observations made on use of hydrogen peroxide There is evolution of a colorless gas. 11.9.12.5 Procedure 3: oxidase method 3-4 drops of dimethyl p- phenylene diamine 3,4,5,6 or 3-4 drops of tetramethyl p- phenylene diamine, were added to the colony of bacteria, . The reagent that was freshly prepared as a 1% solution in water or naphthnol was added drop wise to the agar medium that has a colony of bacteria. 11.9.12.6 The Observation that was made oxidase method There is a change of color to purple black 11.9.13 Procedure 4: Electrophoresis method A mixture of amino acids was applied on a strip of absorbent paper 4, 5, 6 placed between two electrodes and a potential difference applied. 11.9.13.1 Observation made on electrophoresis The individual amino acids moved towards the electrodes. Some move towards the anode while others moved towards the cathode. 11.9.14 Testing if the bacteria present was mutant by replica plating 11.9.14.1 In table 1: there were no bacteria that were observed Mutations in bacteria7 are detected by a technique called replica plating21 which allows genetic characteristics of a bacterium to be investigated without destroying the bacteria7. 11.9.14.2 Procedure on replica plating to determine mutant behavior The procedure involved transferring the bacteria7 colonies growing on semi solid agar medium from one medium to another by using a sterile velveteen disk pressed on the plate. The first plate contained all factors that are necessary for the growth of bacterium whereas the second medium didn’t have all the required factors for the growth of the bacteria. It was not sterile though19. 11.9.14.3 Observation made on replica plating to determine presence of mutant bacteria The bacterium colony that was present in the first medium failed to grow in the second medium17. 11.9.15 The sizes of the bacteria that were identified 11.9.15.1 Table nine: the size of Escherichia Coli and salmonella aureas Bacteria identified Diameter of the bacteria in micrometres Actual size of a living bacteria depending on if it is reproducing or not Escherichia Coli 0.4-0.7 1.0-3.0 Salmonella aureas 0.8-1.0 3.0-10.0 11.10 Figure 1: The dichotomous key developed to identify the bacteria species that were present depending on biochemical tests - - 11.11 Analysis of the results Concentrations of the antibiotic 3,4 didn’t have any positive effect towards destruction of the bacteria. Once the bacteria develop sustainable resistance against any antibiotic for instance penicillin, it is not a matter of how concentrated it is. The bacteria could counter by producing penicillase, an enzyme that hydrolyses penicillin. Other bacteria evolve to start feeding on the antibiotic. For non-antibiotic drugs9 like sulphonamides that are competitive inhibitors, for the p-amino benzoic acid for the active site of the enzyme the bacterium undergoes a mutation that makes sulphonamindes non competitive inhibitors. This makes the bacteria to reproduce even in the presence of sulphonamides. The bacterium is able to undergo a specific recombination 3,9 to have genes that are able to counter the effects of streptomycin whose effect is to inhibit protein synthesis of the bacteria. This ensures that the bacterium is able to biosynthesis proteins even in the presence of streptomycin. Other antibiotics inhibit DNA replication10 of the bacteria and are handled the same way by the bacterial plasmid recombination via the positive interference of the F-factor. 11.11.1 Carbohydrate dissimilation The metabolic processes1 by which various products are formed in the durbam tube are anaerobic because free oxygen is exhausted within the inverted vial. Acids especially lactic acid and carbon dioxide is produced. The lactic acid1 is responsible for curdling the milk because the process is accompanied by denaturation of proteins. This confirmed presence of salmonellae typhimurium (fig. 1) 11.11.2 Proteolysis of milk Milk contains lactose, casein and other proteins and mineral salts1. Digestion of casein and other proteins produces dirty brownish color and the milk becomes watery. The appearance begins at the top particularly with the aerobic bacterial effect and gradually progressed down23. The medium became alkaline due to ammonia gas that was liberated during proteolysis or peptonization. De-colorization 1, 3,4,5,6 or reduction of the indicator is a characteristic behavior of bacterium in the litmus milk. Litmus shares chemical and physical properties with other dyes and has ability to behave as oxidation-reduction indicator as well as acid-base indicator meaning it accepts hydrogen ions from appropriate enzymes system and is converted into a colorless form called Leuko-dye. 4,11 Bacterial multiplication and metabolism is responsible for decolorizing litmus fast. This show bacterium is present in the milk and is not at all affected by the antibiotic. 11.11.3 In the catalase technique Hydrogen peroxide1 was reduced into water and oxygen gas that was evolved. This shows that the enzyme catalase was present and is produced by bacteria. This shows that bacteria were present. 11.11.4 Oxidation process Indopheno oxidase 1,3,6,9 reacted with tetra methyl p- phenylene diamine and the medium turned into purple black confirming presence of bacteria. 11.11.5 Evidence of proteolysis through electrophoresis The bacteria hydrolyzed the proteins into amino acids 1,8,11. Ammonia gas that evolved had been formed as a result of de-amination. The amino acids and other proteins moved to different electrodes26 depending and their rate depended on the shape, charge and size of the molecule. This proves that protein had undergone proteolysis. 11.11.6 The sizes of the bacteria Bacteria dimension 1,8,9 were difficult to determine because bacteria are prone to considerable shrinkage during preparation of the fixed and stained smears. Stained dried cells of Escherichia Coli appeared less than a third of the expected size of a living Escherichia Coli. The sizes Salmonella aureas appeared to make twenty percent of the actual expected when it is living. The size of the salmonella typhimurium was not taken because already it had been satisfactorily identified. 11.11.7 Mutant bacteria testing by replica plating The bacterium colony that grew well in the first medium failed to grow in the second medium because they were mutant 6,7,11. The mutant colonies were already present in the medium but could not be detected even in the initial medium. This draws a conclusive line that these mutants were not caused by the new environment but were selected by it. This is the basis of natural selection 25method that enables bacteria to undergo gene recombination that leads into development of antibacterial resistant bacteria. 11.11.8 Failed detection of the presence of the bacteria in the initial studies The first attempts to identify the bacteria failed to produce any results because the bacteria were not actively metabolic. The reasons behind this observation were: First, 1,4,5 when a bacterium is inoculated into a fresh liquid medium, some time elapses before it multiples. This period is called the lag phase and is when the spores of the bacteria germinate, nutrients are taken up, new enzymes are formed to metabolize the fresh medium. The populations were so low following serial dilutions 27,28. It was not possible to detect the presence of the bacteria. Second, the preservatives that were present in the bacteria were still potential threat to the growth of the bacterium. Third, the dilution process decreased the amount of bacteria per unit volume such that a sample could have failed to have any measurable bacteria but with time the few that were present multiplied as illustrated by the later results. Fourth, the bacteria were able to multiply after they adapted to the conditions of the milk media and were in the lag phase when they were adequately observed. Fifth, the dry plates also affected bacteria because the diffusion gradient of nutrients and of the waste products caused heterogenecity in the population of the bacteria. The proportional increase with time is the same through out the logarithmic phase since the doubling time is constant. The number produced in a given time will depend on number of bacteria that are present consequently, clean uncontaminated milk would take long before it gets spoiled and this is depended on the contamination level of its immediate environment 11.11.9 Serological properties Salmonellae spp. Produces antigenic substances9 that are responsible for stimulation of formation of antibodies. The suspected bacteria belonged to salmonella species 30 that produced ‘O’ antigen, the antisera was able to react specifically with the ‘O’ antigen and this confirmed that the bacterium was salmonellae. From the biochemical tests especially the catalase that led to formation of lactic acid was a confirmative towards the species of genera salmonella that was identified as salmonella typhimurium (fig 1) The bacteria that were identified in the milk were salmonella typhimurium, salmonella aureas, and Escherichia coli. How different bacteria developed resistance to antibiotics Chloramphenicol Inhibits translation on the 50S ribosomal subunit at the peptidyltransferase step (elongation inhibition) hence inhibiting Inhibits protein biosynthesis by binding to the large subunit of the bacterial ribosome and by blocking the peptidyl transfer; . Bacteriostatic. Mode of Resistance is through Acetylation by chloramphenicol acetyltransferase (cat gene). chloramphenicol-resistance is a useful genetic marker for the selection of eukaryotic cells; Resistance is usually mediated by a chloramphenicol-acetyl-transferase. it has different systemic names like D(−)-threo-2,2-Dichloro-N-[β-hydroxy-α-(hydroxymethyl)-β-(4-nitrophenyl)ethyl]acetamide, or D-(−)-threo-2-Dichloroacetamido-1-(4-nitrophenyl)-1,3-propanediol or D-threo-2,2-Dichloro-N-[β-hydroxy-α-(hydroxymethyl)-4-nitrophenethyl]acetamide. its molecular formular is Cl2CHCONHCH(CH2OH)CH(OH)C6H4NO2 and its Molecular Weight :323.13 Streptomycin sulfate salt has a molecular formular of C21H39N7O12 · 1.5H2O4S and has a molecular weight of 728.69. streptomycin Inhibits prokaryote protein synthesis by Binding to S12 protein of 30S ribosomal subunit, thus preventing the transition from initiation complex to chain-elongating ribosome, causing miscoding or inhibiting initiation. the Mode of Resistance is through Mutation in rpsL (gene for S12 ribosomal protein) prevents binding of streptomycin to ribosome. its Antimicrobial spectrum is evident in both Gram-negative and Gram-positive bacteria. D-(−)-α-Aminobenzylpenicillin sodium salt has a molecular formular of C16H18N3NaO4S and a molecular weight of 371.39 and is applied as Ampicillin sodium salt D-(−)-α-Aminobenzylpenicillin sodium salt is a Semi-synthetic penicillin is active against Gram-positive and Gram-negative bacteria. It’s a β-lactam antibiotic with an amino group side chain attached to the penicillin structure. It inhibits bacterial cell-wall synthesis (peptidoglycan cross-linking) by inactivating transpeptidases on the inner surface of the bacterial cell membrane. It is Bactericidal only to growing Escherichia coli. Bacteria become resistant to it by Cleaving β-lactam ring of ampicillin by β-lactamase. Its Antimicrobial spectrum is on both Gram-negative and Gram-positive bacteria. Tetracycline is also known as 7-Chlorotetracycline hydrochloride and has a molecular formular of C22H23ClN2O8 · HCl and a molecular weight of 515.34. it usually finds Application for Cell-permeable fluorescent probe for Ca2+ for measuring calcium flux in endoplasmic reticulum and other intracellular stores in situ. Chlortetracycline is an antibiotic produced by some strains of Streptomyces aureofaciens and Inhibits protein synthesis (elongation) by preventing binding of aminoacyl-tRNA to the 30S subunit. Antimicrobial spectrum is mainly on Gram-positive and Less active against Gram-negative bacterial than tetracycline and its Mode of Resistance is through Loss of cell wall permeability The sulphonamide used was 4-Amino-N-(2-pyrimidinyl) benzenesulfonamide that is also known as N1-(Pyrimidin-2-yl)sulfanilamide and has a molecular formula of C10H10N4O2S and Molecular Formula of 250.28. the Sulfonamide antibiotic blocks the synthesis of dihydrofolic acid by inhibiting the enzyme dihydropteroate synthase thus Inhibiting folic acid synthesis in prokaryotes.its Anti-microbial Spectrum is on both Gram positive, Gram negative, Chlamydia .Mode of bacterial Resistance occurs through Alteration of dihydropteroate synthase or alternative pathway for folic acid synthesis kanamycin B sulfate salt has a Molecular Formula of C18H37N5O10 · xH2SO4 and a Molecular Weight of 483.51 (free base basis). Kanamycin B is an aminoglycoside antibiotic that is known as 2-amino,2-deoxy-analog of kanamycin A. it binds to 70S ribosomal subunit thus inhibiting translocation and elicits miscoding. Antimicrobial spectrum is on both Gram-negative and Gram-positive bacteria, and mycoplasm Ampicillin is anhydrous and is known as D-(−)-α-Aminobenzylpenicillin. Its Molecular Formula is C16H19N3O4S and a Molecular Weight of 349.40 and inhibits the cell wall. Penicillin V potassium salt is also known as Phenoxymethylpenicillinic acid potassium salt and has a molecular formular of C16H17N2O5SKand a Molecular Weight of 388.48. It inhibits formation of cell wall. Erythromycin has a Molecular Formula of C43H75NO16 and a Molecular Weight 862.05 and is commonly referred to as Erythromycin ethyl succinate Bacteria resistance differs from one antibiotic to another and depends on the mode of action of the antibiotic. 11.12 Conclusion From the results, there exist bacteria that are present in the milk and are resistant to antibiotics. These bacteria have developed resistance to the following antibiotic: Ampilicin, penicilin, kanamycin, chloromophenicol, erythromycin and tetracycline. It was found that increasing concentration of the antibiotic doesn’t affect the resistance of the bacteria towards the antibiotic. The bacteria that are present in the milk are metabolically active because they digest starch and proteins that are present in the milk. From the results, it was concluded that initially, these bacteria are available in minute quantities and as they multiply their effect is magnified and lead into curdling of the milk. Lack of conclusive presence of bacteria in the milk specimens following serial dilution was due to loss of pathogenicity. Bacteria easily lose their pathogenecity when they are cultivated for long. The bacteria that were confirmed present in the milk were salmonella typhimurium, salmonella aureas and Escherichia coli. 11.12.1 Policy implications There should be a ban on routine administration of small doses of antibiotics to livestock in order to improve their weight gain. This will have an effect of minimizing chances of presence of antibiotic resistant bacteria in milk which when consumed by human could lead to similar scenarios. There should be withdrawal of genetically engineered crops that are fed to animals. These genetically resistant crops have antibiotic resistant marker genes. 11.13 Future work The antibiotic should be crystallized and its structure determined by using proton nuclear magnetic resonance and carbon 14 nuclear magnetic resonance in order to be certain that the antibiotic is in its pure form. After it has been confirmed that the bacteria is resistant to the antibiotic, further isolation of the decomposed products of the antibiotic should undergo the same proton and carbon NMR to determine their structures and be able to truly confirm if the compounds isolated are part of the antibiotic or are antibodies produced by the bacteria. The milk also showed presence of yeasts. These should also be studied on their behavior with the antibiotic. Many antibiotics are extracted from yeasts and bacteria. The studies should be aimed at determining if the yeasts that were identified in the presence of the bacteria are resistant to antibiotic and what gene is responsible for that. 11.14 References 1. Leone,C.A., (Ed), 1964, Taxonomic Biochemistry And Serology, New York, The Ronald Press Co. 2. Marmur, J., Falkow, S. And Mandel, M., 1963: New Approaches To Bacterial Taxonomy, Ann.Rev.Microbial, 17:329-372 3. Society Of American Bacteriologists, 1967: Manual Of Microbilogoical Methods. New York, Mcgraw-Hill Book Co., Inc. 4. Buchanan, R. E. And Gibbgons, N.E., (Eds.) 1974 :Bergey’s Manual Of Determinative Bacteriology, 8th Ed. Baltimore . The Williams and Wilkins Company Press. 5. Gibbs, B.M. And Skimmer, F.A., (1966) Identification Methods Of Microbiologists, Part A : New York Academic Press. 6. Gibbs, B. M. And Shapton, D.A. (1968), Identification Methods Of Microbiologists; Part B; New York Academic Press 7. Ainsworth, G. E. C. And Senath, Pham, (Eds), 1962, Microbial Classification. 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