Antibiotics are among the most frequently prescribed medications in modern medicines. The cell protection strategies in the organisms, development of resistance in previously susceptible microbes, the inevitable progression of microbes exposed to antibiotics to develop resistance, were the nesisities that ensures the need for continual cycles of discovery and development of new antibiotics. A large variety of antibiotics are available in the drug market today, several others being added regularly in combat with various pathogens that cause disease in humans as well as in animals. Our present study focused to investigate the change in efficacy of commonly used antibiotics such as amoxicillin, ampicillin, sparfloxacin, cefixime. We have collected antibiotics with before and after their expiry dates. A simple eukaryotic model organism Saccharomyces cerevisiae is used to study the comparative understanding of this microbe with these different antibiotics. In our investigation we found that response of Sacchromyces cerevisiae towards different antibiotics varied in its intricacies. Fresh forms of antibiotics have significantly inhibiting the growth of Saccharomyces cerevisiae as compared to expired forms. The observations revealed that expired forms of antibiotics loose their efficacy drastically

A S, Vickram

Devi, N Prathyusha

Kumar S., Venkat

Pathy M, Ramesh

Rajeswari, Devi

S, Siddeswari

Sasvihalli, Radha S

Sridharan, T B

V, Salma

English

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 Research in Biotechnology, 3(1): 29-35, 2012                                                          ISSN: 2229-791X      www.researchinbiotechnology.com      Regular Article Study on Efficacy of Expired and Active Forms of Various Antibiotics on Saccharomyces cerevisiae  T B Sridharan, Radha S Sasvihalli, N Prathyusha Devi, Siddeswari S, SalmaV,  Vickram A S, Devi Rajeswari, Ramesh Pathy M, Venkat Kumar S.  School of Bio Sciences and Technology, VIT University, Vellore 632 014, Tamil Nadu, India   Antibiotics are among the most frequently prescribed medications in modern medicines. The cell protection strategies in the organisms, development of resistance in previously susceptible microbes, the inevitable progression of microbes exposed to antibiotics to develop resistance, were the nesisities that ensures the need for continual cycles of discovery and development of new antibiotics. A large variety of antibiotics are available in the drug market today, several others being added regularly in combat with various pathogens that cause disease in humans as well as in animals. Our present study focused to investigate the change in efficacy of commonly used antibiotics such as amoxicillin, ampicillin, sparfloxacin, cefixime. We have collected antibiotics with before and after their expiry dates. A simple eukaryotic model organism Saccharomyces cerevisiae is used to study the comparative understanding of this microbe with these different antibiotics. In our investigation we found that response of Sacchromyces cerevisiae towards different antibiotics varied in its intricacies. Fresh forms of antibiotics have significantly inhibiting the growth of Saccharomyces cerevisiae as compared to expired forms. The observations revealed that expired forms of antibiotics loose their efficacy drastically.  Keywords: Antibiotic resistance, Drug efficacy, Microbes, Saccharomyces cerevisiae.   Antibiotics are the substances that can be produced or derived from certain fungi or bacteria and other micro organisms that can destroy or inhibit growth of other microbes. Their mechanisms of action include (1) interaction with the cell membrane, leading to leakage of cytoplasmic components; (2) interference with the synthesis of membrane components; (3) interference with nucleic acid synthesis; and (4) interference with microtubule assembly.  The origins of natural antibiotics are noted along with cell protection strategies in the producer organism and the development of resistance in previously susceptible microbes. Some bacteria are naturally resistant to certain antibiotics (inherent resistance). Clinical resistance is commonly due to the emergence of resistant organisms following antibiotic treatment (acquired resistance). This emergence, in turn is due to selection of resistant mutants of the infective species (endogenous resistance) or, usually to transfer of resistance genes from other naturally resistant species (exogenous resistance). The inevitable progression of microbes exposed to antibiotics to develop resistance ensures the need for continual cycles to discover and development of new antibiotics. So, new antibiotics have to be designed and produced almost at a regular  T.B. Sridharan et al. /Research in Biotechnology, 3(1): 29-35, 2012  30  basis to overcome this problem. Choosing the right drug at the appropriate concentration and the required time is a way to combat drug resistance. In addition to this, all the antibiotics available in the market today lose their efficacy after their expiry period. The danger of taking expired antibiotics is not only loosing their chemical integrity, but they could play a role in creating antibiotic resistance. Many antibiotics are coated with a smooth film that ensures the medication gets released at the right point in the digestive tract. If that coating is compromised, the antibiotic may be released into blood stream too soon, and cause intestinal complications such as diarrhoea. It could also irritate stomach lining. There is always a risk to benefit ratio considered while developing a drug. The risk to benefit ratio should in general be less risk and more beneficial to the patient where as in the case of the expired drugs the risk factor is increasing drastically. In our present study we investigated the changes in efficacy of some commonly used antibiotics: Amoxicillin, Ampicillin, and Sparfloxacin, Cefixime before and after their expiry period. We have used a simple eukaryotic model organism, Saccharomyces cerevisiae.  MATERIALS AND METHODS: Strain: Saccharomyces cerevisiae is used as a test organism for this study. Medium: The strain was grown in a liquid broth of YPD medium, prepared using 2g of dextrose, 2g of peptone and 1 g of yeast extract dissolved in 100 ml of double distilled water, and the pH was  adjusted to 6.8 and then sterilized. To this 2 % of gelling agent, agar is used. Drugs: Both active (non-expired) and expired drugs of: Amoxicillin, Ampicillin, Sparfloxacin, Cefixime. Method:  5 ml of the media are taken in each of seven test tubes. 1% seed culture is added to these tubes containing 5ml medium and drugs with varrious concentrations were added. The inoculated experimental cultures were grown at room temperature for 48 h. The harvested cultures were measured for its turbidity at 600 nm using UV-VIS spectrophotometer (Ultraspec-1100 of Amersham Biosciences). The optical density (OD) values were tabulated for microbial growth obtained from non-expired and expired drug of different concentrations. Similarly repeated for the other drugs also, 3 such experiments were conducted for each drug (Table 1-4).These data were represented in graphs (Fig 1-4) Cell count: Liquid culture was taken and diluted in ratio of 1:10. The liquid sample containing immobilized cells is placed on the haemocytometer chamber; it is covered with a cover glass, and leave for capillary action to completely fill the chamber with the sample. Looking at the chamber through a microscope, the number of cells in the chamber was determined by counting. The number of cells of Saccharomyces grown both in expired as well as in non-expired antibiotics containing YPD medium was given in the tables 1-4 is obtained from the average of the three experiments.  RESULTS AND DISCUSSIONS: A reduction in the microbial population was observed in all of the four active drugs and expired drug at higher concentration which was taken under study. Expired drug at lower concentration showed increase in the growth of microorganism  As observed from the OD values (figure 1,2,3&4) and viable cell count from haemocytometer (figure 5,6,7&8). The drug concentration (both active & expired) and microbial growth were inversely proportional. There was significant reduction in microbial population with the increased concentration of active & expired drug.   T.B. Sridharan et al. /Research in Biotechnology, 3(1): 29-35, 2012  31  A flaggy curve of inhibition was observed in microbial population when treated with expired drug of Amoxicillin indicating no change in microbial population. But, there was increase in population at higher concentration. A bell shaped curve was observed when treated with active Amoxcilin under low concentration of Amoxicillin the microbial population and at higher concentration the population was decreased (figure 1)  Figure 1: Graph shows the OD values of microbial growth treated with active & expired Amoxicillin. The concentration of amoxicillin taken is given in x axis and the OD values of microbial growth is taken in y axis. Similar inhibitory curve was observed when treated with expired drug of Ampicillin at very high concentration of expired drug there was decrease in population showing slightly linear line. At high concentration of 35mg both active and expired drug showed same effect (figure 2). Flagy shaped inhibitory curve was observed when treated with both active and expired Sparfloxacin, indicating no population fluctuation. As the expired drug taken under study was of 2 months expiry. So, close O.D. values were obtained between active and expired drugs (Figure 3) When treated with expired and active Cefixime similar curves were obtained at high concentration showing decrease in microbial population. At certain points there was coincidence in OD values due to expired duration was less. But yet low concentration of 0 to 10 mg range the bell shaped curve was observed with expired drug and declined line was observed with active drug (Figure 4).  Table 1: Data shows the OD values of microbial growth under various concentration of Amoxillin. 5ml of YPD medium was taken in each of  15 boiling test tubes were inoculated with 50µl of 48h grown seed culture and 50, 100, 150, 200, 250, 300, 350µl of  expired and non-expired Amoxillin stock (10 mg/ml) solution along with control (0.0µl). The inoculated cultures were allowed to grow at room temperature in a shaker at 150rpm for 48 h. Harvested cultures were measured for its growth based on its turbidity using UV-VIS spectrophotometer at 600nm. The OD values for the individual tubes were tabulated, and are the average of 3 such experiments.Counting was done using haemocytometer .The number of cells mentioned here are the averages of 3 such experiments.   Concentration of Amoxillin in mg OD of non-expired Amoxillin at 600 nm OD of expired Amoxillin at 600nm Number of cells from non- expired Amoxillin Number of cells from expired Amoxillin 0 0 0 0 0 5 0.895 0.37 73.666 138.666 10 0.786 0.608 104.66 125 15 0.727 0.563 86 102.333 20 0.611 0.563 92.333 90.666 25 0.57 0.456 86.333 109.666 30 0.455 0.351 69.333 84.333 35 0.56 0.295 46.666 111.666   T.B. Sridharan et al. /Research in Biotechnology, 3(1): 29-35, 2012  32  Table 2: Data shows the OD values of microbial growth under various concentration of Ampicillin. 5 ml of YPD medium was taken in each of 15 boiling test tubes were inoculated with 50 µl of 48hr grown seed culture and 50,100,150,200,250,300,350µl of expired and non-expired Ampicillin stock (10 mg/ml) solution along with control (0.0µl). The inoculated cultures were allowed to grow at room temperature in a shaker at 150 rpm for 48hr.Harvested cultures were measured for its growth based on its turbidity using UV-VIS spectrophotometer at 600nm.The OD values for the individual tubes were tabulated, and  are the averages of 3 such experiments. Pippetted out 10µl of harvested culture from all 14 tubes and control into an e tube and diuted them with 990µl of distilled water and cell counting was done using haemocytometer.   Concentration of Ampicillin in mg O.D of non-expiredAmpicillin at 600nm O.D of expired Ampicillin at 600nm Number of cells from non-expired Ampicillin Number of cells from expired Ampicillin 0 0 0 0 0 5 0.754 0.613 111 122.333 10 0.683 0.562 98 113.666 15 0.663 0.554 101 115.333 20 0.76 0.49 76 97.333 25 0.438 0.411 69.666 80.333 30 0.364 0.308 55 62.666 35 0.285 0.228 40.666 47.666  Table 3: Data shows the OD values of microbial growth under various concentration of Sparfloxacin. 5ml of YPD medium was taken in each of  15 boiling test tubes were inoculated with 50µl of 48hrs grown seed culture and 50,100,150,200,250,300,350µl of  expired and non-expired Sparfloxacin  stock (10mg/ml) solution along with control (0.0µl).The inoculated cultures were allowed to grow at room temperature in a shaker at 150rpm for 48hr. Harvested cultures were measured for its growth based on its turbidity using UV-VIS spectrophotometer at 600nm.The OD values for the individual tubes were tabulated below are the averages of 3 such experiments. Pippetted out 10µl of harvested culture from all 14 tubes and control into an e tube and diluted them with 990µl of distilled water and cell counting was done using haemocytometer.   Concentration of Sparfloxacin O.D of non-expired Sparfloxacin at 600nm O.D. of expired Sparfloxacin at 600nm Number of cells from non-expired Sparfloxacin Number of cells from expired Sparfloxacin 0 0 0 0 0 5 0.782 0.694 128 102 10 0.705 0.656 110 89 15 0.591 0.544 77 69 20 0.511 0.463 65 57 25 0.433 0.381 51 42 30 0.358 0.308 35 31 35 0.267 0.245 26 20  With expired Amoxcilin flagy shaped curve was shown at low concentration indicating no fluctuation in cell count. But at high concentration a wavy shaped curve was observed indicating high rate of fluctuation in viable cell counts. With low concentration of active Amoxicillin, a bell shaped curve was observed with increase and decrease in cell count and at high concentration. A declined curve of slightly bell shaped was observed showing decreased cell count. At 20 mg concentration, the cell count was same for both active & expired drug (Figure 5)   T.B. Sridharan et al. /Research in Biotechnology, 3(1): 29-35, 2012  33  Table 4:  Data shows the OD values of microbial growth under various concentration of Cefixime. 5ml of YPD medium was taken in each of 15 boiling test tubes were inoculated with 50µl of 48hr grown seed culture and 50, 100, 150, 200,250,300,350µl of  expired and non-expired Cefixime stock (10mg/ml) solution along with control (0.0µl).The inoculated cultures were allowed to grow in room temperature in a shaker at 150rpm for 48hs. Harvested cultures were measured for its growth based on its turbidity using UV-VIS spectrophotometer at 600nm.The OD values for the individual tubes were tabulated below are the averages of 3 such experiments. Pippetted out 10µl of harvested culture from all 14 tubes and control into an e tube and diluted them with 990µl of distilled water and cell counting was done using haemocytometer.   Concentration of Cefixime O.D. of non-expired Cefixime at 600nm O.D. of expired Cefixime at 600nm Number of cells from non-expired Cefixime Number of cells from expired Cefixime 0 0 0 0 0 5 0.74 0.782 120 132 10 0.723 0.827 101 145 15 0.673 0.716 83 92 20 0.627 0.69 72 86 25 0.386 0.514 46 65 30 0.326 0.368 35 41 35 0.256 0.286 20 31   Figure 2: Graph shows the OD values of microbial growth treated with active & expired ampicillin. The concentration of ampicillin taken is given in x axis and the OD values of microbial growth is taken in y axis. Similar systematic flaggy shaped inhibitory curve was observed with only change in height of their peaksat at low concentration, when treated with active & expired Ampicillin. At higher concentration of expired Ampicillin, continuous declined linear line was observed. But with active drug, it was slight flagy shaped indicating the fluctuation in cell count (Figure 6). A flagy shaped curved was observed with both active & expired drugs of sparfloxacin with only change in height of the curve (Figure 7). With expired Cfixime, on cells a constant increase in the cell count was observed at low concentration and a constant decrease in cell count was observed at high concentration. With active cefixime, at low concentration a flag shaped curve depicting a high cell count  was observed. Cell counts were similar for high concentration of both active and expired cefixime. (Figure 8)   Figure 3: Graph shows the OD values of microbial growth treated with active & expired sparfloxacin. The concentration of sparfloxacin taken is given in x axis and the OD values of microbial growth is taken in y axis.   T.B. Sridharan et al. /Research in Biotechnology, 3(1): 29-35, 2012  34   Figure 4: Graph shows the OD values of microbial growth treated with active & expired cefixime. The concentration of sparfloxacin taken is given in x axis and the OD values of microbial growth is taken in y axis.   Figure 5: Graph shows the number of cells observed from microbial growth treated with active and expired amoxicillin. The concentration of amoxicillin taken is given in x axis & the number of cells observed in microbial growth is taken in y axis.                         Figure 6: Graph shows the number of cells observed from microbial growth treated with active and expired ampicillin. The concentration of ampicillin taken is given in x axis & the number of cells observed in microbial growth is taken in y axis.   Figure 7: Graph shows the number of cells observed from microbial growth treated with active and expired Sparfloxacin. The concentration of sparfloxacin taken is given in x axis & the number of cells observed in microbial growth is taken in y axis.  Figure 8: Graph shows the number of cells observed from microbial growth treated with active and expired Cefixime. The concentration of cefixime taken is given in x axis & the number of cells observed in microbial growth is taken in y axis.  CONCLUSION: The cell protection mechanisms  of the cells are becoming more strategic and their development of resistance to antibiotics is incresing, demanding the need for the continual cycles of discovery and development of new antibiotics. Futures of these work mainly focusing to check the incresing antibiotic resistance. Every company manufacturing  T.B. Sridharan et al. /Research in Biotechnology, 3(1): 29-35, 2012  35  the antibiotics should ensure that the risk to benefit ratio is less. The  efficacy of the antibiotics after the expiry will lead to lose its chemical integrity and also play a role in the development of antibiotic resistance .So, the Government should take proper measures that the expired drugs are not in use.  REFERENCES Davis. (1994). Inactivation of antibiotics and dissemination of resistance genes. Science 264: 375-382. Fluit, A, C. and F. J. Schmitz. (2004). Resistance integrons and super integrons.clin microbial.infect. 10: 272-288 Vazquez, D. (1979). Inhibitors of protein biosynthesis, (eds) springer, Berlin. Roberts, M. C. (2003). Amoxcillin therapy: update clein. infect. Dix. 36: 475-483 Roe, M. T., Vega, E. and S. D. Pillai. (2003). Antimicrobial resistance markers of class 1 and class 2 integron-bearing from irrigation water and sediments. Emerge.infect. Dis.  9: 822-826 Neu, (1992) H. C. Neu, The crisis in antibiotic resistance, science 257 (1992), PP. 1064-1073 Hamilton Miller. J M T (2004). Antibiotic resistance from two perspectives: man and microbe. Int, J. Antimicrobial agents 23: 209-212 T. B. Sridharan et al. Determination of change in efficacy of various antibiotics before and after expiry: A study. Journal of pharmacy research 2010, 3(5), 1068-1073.    

Study on Efficacy of Expired and Active Forms of Various Antibiotics on Saccharomyces cerevisiae

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Study on Efficacy of Expired and Active Forms of Various Antibiotics on Saccharomyces cerevisiae

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