Investigation of Tetracycline Antibacterial Activities

Investigation of tetracycline Anti bacteriuml ActivitiesNOR FAEZAH BINTI ROSMIDIEscherichia coli is a Gram-negative bacterium, which discovered and named after Theodor Escherichia and belong to family Entero bacteriumceae family. E. coli is classified as same stem as other bacterial such as Bordetella pertussis, Borrelia burgdoferi, Chlamydia trachomatis, Helicobacter pylori and many more species that be well cognise in medical world. E. coli transmitting can be found all over the world and more common in tropical country. This infection especially can be transmitted through diet because the infections atomic number 18 mainly due to begrime food. Thus, this infection can be called as food-borned disease. An individual can get infected when consumed foods that are contaminated with dirt. However, not every individual that consumed contaminated food can get the infection. This is because, in that respect are certain criteria to be fulfilled. Firstly, thither must be charact er of infection, which means, E. coli must present in the food. Then, the individual is eating food contaminated with pathogen, as the main itinerary of infection is through orofecal. Lastly, the bodys immune organisation cannot get rid of the pathogen, which eventually ca apply disease. This study is conducted to isolate, characterize and survey germicide activities of E. coli in media and take out. A total of 3 sample is unruffled from the stock culture and labelled as EY1, E27 and E31. closing off is made on the eosin methylene blue (EMB) agar and identified by using one thousand staining under microscope. To check for the germicide activities, minimum inhibitory minginess (MIC), minimum bactericidal concentration (MBC) and time knock down render are done. Results reveals that the MIC value for E. coli is 128 and MBC value is 1024. duration-kill seek kink disclose that the chemical reaction of tetracycline to the bacteria in media and milk slightly different furthe r yet, able to achieve bactericidal effect of the drug.1.0 INTRODUCTION1.1 IntroductionMastitis, or inflammation of the mammary gland, is predominantly due to the effects of infection by bacterial pathogens, although mycotic or algal microbes play a role in some cases (Ronald J. Erskine, 2016b). in that respect are many factors that influence the development of mastitis including microbial, military and environmental (Contreras Rodrguez, 2011). Any opportunistic microbes that can invade tissue and causation infection can lead to mastitis. The pathogen that use this transmission mode are Staphylococcus aureus, Streptococcus agalactiae and Corynebacterium bovis. They may spread from oxen to another cattle through aerosol transmission and invade the cup of tea causing bacteremia. there is other route of infection, which is through the environment of the cattle. The pathogens that associated with environmental reservoirs are Escherichia coli and Trueperella pyogenes (Ronald J. Ers kine, 2016a) Post-milking tit dipping, dry cow therapy, well-maintained milking equipment, and culling of cows with chronic mastitis have successfully controlled contractable mastitis bacteria such as Streptococcus agalactiae and Staphylococcus aureus (Makovec Ruegg, 2003). Milk samples poised from cows on Wisconsin dairy farms clearly demonstrated that environmental bacteria are the most common causes of clinical mastitis. (Pamela L. Ruegg, 2015)Once inside the handbag, E. coli procreate rapidly, causing influx of the inflammatory cells. As neutrophils ingest and kill bacteria, endotoxin is released and along with other inflammatory mediators causes severe local inflammation. This inflammatory response is characterized by increased vascular permeability, changes in milk composition and damage to the mammary epithelial cells, which may result in the characteristic watery or serous milk secretion.The treatment for mastitis include administration of antimicrobial via the intrama mmary route or parentally (Gruet, Maincent, Berthelot, Kaltsatos, 2001). Unfortunately, despite the best possible antimicrobial treatments, failures of bacteriologic recruit are common, especially for S. aureus mastitis, and antimicrobial resistance (AMR) is considered one of the reasons for measly cure rates (Candrasekaran et al., 2014).There are other reasons, which are the focusing and iatrogenic factors, drug factors, mastitis-causing organism factors and also mammary gland factor. However, the major reason is the insufficient contact of the antimicrobial with the mastitis-causing organisms at the site of infection is a major cause of mastitis treatment failure (Kiro R. Petrovski, 2007). Some bacteria such as S. aureus can localize inside host cells past restrict the concentration of drug in the cells. E. coli can form biofilms, a condition where the microorganisms structured within an array of exopolysaccharides (EPS) and adhering to a living or inert surface with the f unction of protecting the microorganisms in stress environments (Costa, Espeschit, Pieri, Benjamin, Moreira, 2014). The stalkless cells ar scatd in biofilms tend to be more resistant to antimicrobial therapy compared with plantonic free cells, due to the slow diffusion of the antimicrobial in the upcountry layers of the biofilm (Costa et al., 2014). Also, to be effective, drugs need to be free and not inhibited by other components. Some drug may have high similarity binding towards protein. Thus, some component in the milk, for example, casein, may bind to the antibiotic and reduce the free the drugs in the udder. In the case of S. aureus infections during lactation, it has been estimated that the bacteriological cure rate is only within 25% to 50% only (Kiro R. Petrovski, 2015).1.2 bother StatementEscherichia coli is one of the major cause of mastitis development. Based on the seek done in Wisconsin dairy farm, 21% of the mastitis case result from the E. coli infection. in spi te of of all the treatment given, the therapy has not been successful. Hence, the aim of this study is to investigate the antibacterial activities of Tetracycline against E. coli from clinical mastitis in goth media and milk.1.3 enquiry QuestionsThis study is knowing to answer either tetracycline can demonstrate equivalent antimicrobial faculty against E.coli in addition medium and in milk?1.4 Research HypothesesThis study is constructed to answer the hypothesis, either tetracycline demonstrates equivalent antimicrobial efficacy against E. coli in growth medium and milk.1.5 Research ObjectivesThis study is conducted to achieve a set of following objectives -To suss out minimum inhibitory concentrations (MIC) of Enrofloxacin and against the pathogenic E. coliTo determine minimum bactericidal concentrations (MBC) of Enrofloxacin and against the pathogenic E. coliTo perform time kill assay of Enrofloxacin against E. coli2.0 LITERATURE REVIEW2.1 Definition of mastitisMastitis is sw elling of the mammary gland that frequently caused by bacteria ingoing the teat canal and moving up to the udder and there are two types of mastitis, which are contagious mastitis and also environmental mastitis (Dairy Australia, 2007). Contagious or cow-associated mastitis generally related to the bacterial infection that causing disease, which are Staphylococcus aureus and Streptococcus agalactiae (Dairy Australia, 2007). The bacteria present in the udders or on teat skin and spread either by splashes of disease-ridden milk or sprays during stripping on milkers hands or teatcup liners and by print flow of milk between teatcups (Dairy Australia, 2007). Meanwhile environmental mastitis evanesce when the provide which used to house the cattle is contaminated (Ronald J. Erskine, 2016a). It is not only occupation on the bedding of the cattle, the infected soil, manure, calving pads and water host bacteria can also causing this disease to developed (Dairy Australia, 2007).Mastitis come in two forms, which are subclinical and clinical, however, subclinical form is rare to be find (Ronald J. Erskine, 2016a).2.1Escherichia coliEscherichia coli is no longer bizarre in our communities. This Enterobacteriaceae is a Gram-negative bacteria. It cannot sporulate and can be found commonly in the intestine. This bacteria have a characteristic of glucose fermenting, acid producing, nitrate minify and oxidase negative. It also have facultative anaerobic characteristic which can grow easily at 37C. Physically, E. coli is petty in size, approximately 1-2 x 30-30 , with flagella. It has no capsule and also no mucus layer. There are several antigen structures that play an important role for the identification of this bacteria, which are K antigen, H antigens and O antigen. K antigen can be found in the capsules, a compound in the group of polysaccharide. H antigen can be found in flagella and O antigen can be found in the cells or the body as it is a substance in the group of lipopolysaccharide and polysaccharide (Wiwanitkit, 2011).Despite the fact that most of E. coli are harmless and beneficial, there are species that have the ability to cause disease, and they are divided into groups fit in to the mechanism of pathogenesis. The groups are enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC) and enteroaggregative Escherichia coli (EAEC). EHEC is a group that can cause hemorrhagic colitis and can end up with a specific crookedness called uremia haemolytic syndrome. EIEC or also known as Shiga toxin-producing E. coli (STEC) causes licentiousness. ETEC is a major cause of travelers diarrhea and diarrhea in group of infants in developing countries. E. coli toxic products from the gut causes problems to the patients body. EPEC is a major cause of diarrhea in infants and EAEC is a new pathogen (Wiwanitkit, 2011).2.2 MastitisMastitis is the inflammation of the mammary gland and udder tissue, and is a major endemic disease of dairy cattle. It commonly occurs as an immune response to bacterial invasion of the teat canal by variety of bacterial sources present on the farm, and can also occur as a result of chemical, mechanical or thermal disfigurement to the cows udder (AHDB Dairy, 2017).3.0 MATERIALS AND METHODS3.1 Samples collectionThis study is conducted in laboratory of dexterity of Veterinary Medicine in University Malaysia Kelantan. The bacteria sample, which is Escherichia coli is isolated from the stock samples of the laboratory. There are three samples, which are EY1, E27 and E31.3.2 Samples preparationThe samples are cultured on the nutrient agar for 18 to 24 hours at 37C.3.3 Isolation and identification of Escherichia coliThe bacterial colony on the nutrient agar is then cultured on the eosin methylene blue (EMB) agar and gram staining is done.3.4 Minimum Inhibitory ConcentrationMinimum inhibitory concentration (MIC) are determined using the brot h micro dilution method. A range concentrations of antimicrobial is prepared in a 96 well microplate, followed by inoculation of bacteria culture to yield approximately 5105 cfu/ml in a 250 final volume. The plate is then incubated in 37C for 18 hours. The lowest concentration of antimicrobial that inhibit bacterial growth is taken as MIC value.3.5 Minimum Bactericidal ConcentrationThe dilution representing the MIC are aliquot volume of the broth and spot onto Nutrient agar. The agar is then incubated 18 hours with 37C. No growth indicate that antibiotic is bactericidal at that concentration. If there is present growth indicative that antibiotic work as antibiotic only particularly on the dilution.3.6 Time Kill hindranceTube containing designated concentration of antimicrobial will be inoculated with 105 cfu/ml of bacteria in Nutrient agar, followed by incubation at 37C, in an incubator shaker at 200 rpm. At 0, 1, 4, 24 hour post inoculation, 50 aliquots will be taken out, serially diluted in saline and plated on Nutrient agar. Plates will be incubated for 18 hours at 37C followed by colony counting. An antimicrobial is considered bactericidal if there is 3-log decrease in cfu/ml of bacteria counts, as compared to the growth control. 4.0 RESULTS4.1 Isolation and identification of Escherichia coliColony of E. coli on EMB agar is small to medium circle in size and shape, with dark green and glossy, embossed and polish appearance.4.4 Time Kill curbSAMPLE EY1 (x103)T0T1T4T24MEDIA support cx1201351501X MIC421121one hundred ten2X MIC26422605X MIC201211015X MIC131310 take outCONTROL1231351411561X MIC46351731102X MIC4626111005X MIC32148015X MIC371340Table 1 Time kill tab Results for Sample EY1 in x103SAMPLE EY1 (x106)T0T1T4T24MEDIACONTROL5.56.06.757.51X MIC2.10.551.055.52X MIC1.32.11.305X MIC1.00.60.55015X MIC0.650.650.050milkCONTROL6.156.757.057.81X MIC2.31.758.655.52X MIC2.31.30.555.05X MIC1.60.70.4015X MIC1.850.650.20Table 2 Time kill arrest Results for Sam ple EY1 in x106Figure 3 Time-kill curve for E. coli (EY1) against Tetracycline on media and milkSAMPLE E27 (x103)T0T1T4T24MEDIACONTROL1131261411541X MIC6429491112X MIC583518105X MIC42222015X MIC301110 take outCONTROL1271391451611X MIC48361801002X MIC5852301115X MIC412218515X MIC4722205Table 3 Time kill undertake Results for Sample E27 in x103SAMPLE E27 (x106)T0T1T4T24MEDIACONTROL5.656.37.057.71X MIC3.21.452.455.552X MIC2.91.750.90.55X MIC2.11.10.1015X MIC1.50.550.050MILKCONTROL6.356.957.258.051X MIC2.41.89.05.02X MIC2.92.61.55.555X MIC2.051.10.90.2515X MIC2.351.11.00.25Table 4 Time kill Assay Results for Sample E27 in x106Figure 4 Time-kill curve for E. coli (E27) against Tetracycline on media and milkSAMPLE E31 (x103)T0T1T4T24MEDIACONTROL1151211371451X MIC5941391142X MIC573727435X MIC553923015X MIC243370MILKCONTROL1211401491651X MIC56401801082X MIC7150451165X MIC51261210215X MIC5825130Table 5 Time kill Assay Results for Sample E31 in x103SAMPLE E31 (x106)T0T1T4T24MEDIACONTROL 5.756.056.857.251X MIC2.952.051.955.72X MIC2.851.851.352.155X MIC2.751.951.15015X MIC1.21.650.350MILKCONTROL6.057.07.458.251X MIC2.82.09.05.42X MIC3.552.52.255.85X MIC2.551.30.65.115X MIC2.91.250.650Table 6 Time kill Assay Results for Sample E31 in x106 Figure 5 Time-kill curve for E. coli (E31) against Tetracycline on media and milk