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Full Length Article Amit Pandey* , Neha Rajput , Mohammad Nadeem Aslam and Bharat Choudhary 1. 1R&D Division, MRD LifeSciences, Lucknow, India. 2. Alpine Institute of Management and Technology, Dehradun, India. 3. APS University, Rewa, M.P., India Five MDR cultures of bacteria were isolated from four different locations of Lucknow and Kanpur, respectively. Based upon Gram staining four isolates (A1, A2, C2 and G3) were identified as Gram negative out of which A1 was mixed culture that is cocci + rod and A2 and G3 were Gram negative rods. Culture N was identified as Gram positive mixed culture that is cocci + rod. Based upon the biochemical analysis G3 bacterial culture was identified as Pseudomonas aeruginosa (Table 1), for multi-drug resistance (MDR) test seven different antibiotics in different concentration were used. It was observed that A1 bacterial culture showed resistance to amoxycilline (100 µg/ml), cefixime (100 µg/ml), ofloxacin (50 µg/ml), roxithromycin (20 µg/ml), ampicilline (50 µg/ml) and tetracycline (50 µg/ml) while A2 culture was resistant to amoxycilline (50 µg/ml) and cefixime (1 mg/ml). C2 was resistant to amoxycilline (10 mg/ml), cefixime (1 mg/ml), roxithromycin (10 mg/ml), ampicilline (20 µg/ml) and tetracycline (20 µg/ml). G3 was resistant to amoxycillin (1 mg/ml) and cefixime (1 mg/ml). And N culture was resistant to amoxycilline (100 µg/ml), cefixime (1 mg/ml), roxithromycin (50 µg/ml), ampicilline (50 µg/ml) and tetracycline (20 µg/ml).
Keywords: Multi-Drug Resistance (MDR), Susceptibility; Antibiotics; Bacterial Isolates; Broad Spectrum; Minimum
Inhibitory Concentration (MIC).
Abbreviations : µg- Microgram; ml- Mililitre; mg - Miligram; R - Resistant; S- Sensitive; AR-Antibiotic Resistance;
hrs.-Hours; MBC -Minimum Bactericidal Concentration; MIC- Minimum Inhibitory Concentration;
MDR- Multi-Drug Resistance
bacterium to be effective against MDR bacterial cultures. The microorganisms have a defense system against various antibiotics and Antibiotic resistance is a considerable term while studying about they grow even in presence of antibiotics by getting resistant. It was diseases in human, animals and plants. Mostly the antibiotic resistant suggested that soil microbes harbour antibiotic resistant (AR) genes with bacterial cultures are found in clinical and veterinary land areas whereas a diverse gene sequencing (Jalal, et al., 2010). The antibiotic sensitivity the existence of antibiotic resistance (AR) bacteria in water has also is expressed in terms of minimal inhibitory concentration (MIC) and observed and supported to be present in higher concentrations and minimal bactericidal concentration (MBC) and it give quantitative data. diversity in hospital areas as compared to domestic areas. Antibiotics are These quantitative results are useful in predicting the tissue, blood or either synthesized industrially or also produced by microorganisms; urine levels of antibiotics that must be attained to assure inhibition or these antibiotics have microstatic or microcidal activity. Mainly these killing. In comparison to clinical or hospital area soil, the soil from antibiotics producing microbes interrupt the microbial metabolism by various aquatic places were taken for isolating the AR pathogens. The various mechanisms (Jalal et al., 2010). Several researches were main objective of this work is to isolate the multi--drug resistant (MDR) performed that dealt with advanced antimicrobial resistance in bacteria pathogens from the aquatic places of Lucknow and Kanpur, U.P against isolated from food, animal and environment (Jensen et al., 2001). The various antibiotics and to know the MIC and MBC values for various use of antibiotics is increasing continuously in different fields like antibiotics in presence of the isolated pathogens. veterinary medicine, agriculture, medicines etc. but the awareness of knowledge regarding the quantity of antibiotics present in the Materials and Methods
environment after their use is very less (Hirsch et al., 1998). The population of antibiotic resistance (AR) bacteria in the areas where The soil samples were collected from five different locations of antibiotics are used is common but existence of AR bacteria in aquatic Lucknow and Kanpur; A (Sewage water, Lucknow), C (Pond water, environment is also increasing readily (Schwartz et al., 2003). It is Lucknow), G (Ganga river, Kanpur) and N (Gomti river, Lucknow). observed that resistance in bacteria could be – (i) intrinsic resistance i.e. Samples were serially diluted and bacteria were isolated on Nutrient natural resistance of bacteria to certain antibiotics. (ii) Acquired resistance i.e. the susceptible that become resistant by adapting itself through genetic changes. (iii) Multi--drug resistance i.e. the resistance of E- Mail: amit@mrdlifesciences.com Full Length Article Isolation and Characterization of Multi-Drug Resistant Cultures, Amit Pandey et al., Biochemical tests Isolate C2 Isolate G3 Agar, two bacterial colonies were selected and designated as: A1, A2, C1, C2, G1, G2, N1 and N2, respectively. Bacterial colonies were sub Cellular morphology cultured several times up to pure culture, Catalase activity maintained for further biochemical analysis, and preserved at low temperature. After isolation, identification of the bacterial isolates Voges proskauer's test was carried out according to Bergey's Manual of Citrate utilization Determinative Bacteriology (Buchanan and Gibbonns, 1978). All the bacterial isolates were analyzed for their King's B medium test sensitivity against commonly used antibiotics (Pseudomonas by agar well diffusion method. Different antibiotics having different mechanisms of action were taken as tetracycline, amoxycilline, ampicilline, roxithromycin, ofloxacin, cefixime Table 1. Biochemical analysis of isolated MDR bacterial cultures.
and ciprofloxacin of concentration 5 µg/ml, 10 µg/ml, 20 µg/ml, 30 µg/ml, 50 µg/ml, 100 µg/ml, 1 mg/ml and 10 mg/ml. Wells were prepared on nutrient agar plate previously spreaded with 50 µl of isolated bacterial broth culture. These wells were loaded with 50µl of antibiotic solution and then plates were incubated at 37°C for overnight, then the zone of inhibition were examined. The obtained multi- characterized by microscopic and Biochemical characterized by staining (Gram staining and Endospore staining) and biochemical activity (IMVIC) as par Bergey's manual given in Aneja Table 2. MDR test for sample A1 [Note: R= resistant, S= sensitive].
(2003). King's B medium is a confirmation medium for detection and differentiation of P s e u d o m o n a s a e r u g i n o s a f r o m o t h e r Pseudomonas based on fluorescein (pyoverdin) production and pyocyanin inhibition. Peptone, potassium hydrogen phosphate, heptahydrated magnesium sulfate (MgSO .7H O) and bacteriological agar were added to distilled water (pH 7.0 – 7.2). Solution was autoclaved and poured in test tube to prepare slants. The isolates were inoculated on slants and kept for incubation at 37°C for 48 hrs.
MIC is the lowest concentration which resulted in maintenance or reduction of inoculum viability". Only antibiotic that shows inhibitory Table 3. MDR test for sample A2 [Note: R= resistant, S= sensitive]. activity toward the bacterial isolate using the Kirby-Bauer methods are tested further. The active antibiotics were serially diluted to make a range of antibiotic concentrations that encompasses the concentration used in the (Kirby- Five MDR cultures of bacteria were isolated from four different Bauer method, 1966). 20 µl of test bacterium was added to all tubes locations of Lucknow and Kanpur. Based upon Gram staining, four having 3 ml of nutrient broth which was serially diluted with antibiotic. isolates (A1, A2, C2 and G3) were identified as Gram negative out of After incubation, the MIC is identified as the least concentration of which A1 was mixed culture i.e. cocci + rod and A2 and G3 were Gram antibiotic that inhibits the growth of the test bacterium. The Minimal negative rods. Culture N was identified as Gram positive mixed culture Bactericidal Concentration (MBC) is determined by spreading the that is cocci + rod. All isolated bacterial cultures were aerobic. Based bacterial colonies on nutrient agar plate which was obtained by MIC. upon the biochemical analysis, G3 bacterial culture was identified as Pseudomonas aeruginosa (Table 1). For the multi- drug resistance Full Length Article Authors to provide, P.A. Mary Helen et al., Isolation and Characterization of Multi-Drug Resistant Cultures, Amit Pandey et al., Concentration Amoxy (MDR) test seven different antibiotics in different concentration were used. It was observed that A1 bacterial culture showed resistance to amoxicilline (100 µg/ml), cefixime (100 µg/ml), ofloxacin (50 µg/ml), roxithromycin (20 µg/ml), ampicilline (50 µg/ml) and tetracycline (50 µg/ml). A2 culture was resistant to amoxicilline (50 µg/ml) and cefixime (1 mg/ml). C2 was resistant to amoxicilline (10 mg/ml), cefixime (1 mg/ml), roxithromycin (10 mg/ml), ampicillin (20 µg/ml) and tetracycline (20 µg/ml). G3 was resistant to amoxicilline (1mg/ml) and cefixime (1 mg/ml). And N culture was Table 4. MDR test for sample C2 [Note: R= resistant, S= sensitive].
resistant to amoxicilline (100 µg/ml), cefixime (1 mg/ml), roxithromycin (50 µg/ml), ampicilline (50 µg/ml) and tetracycline (20 µg/ml). It was observed Concentration Amoxy that even after showing resistance at higher concentrations amoxicilline was sensitive at concentration 20 µg/ml against four cultures (A2, C2, G3 and N) which was considered as MIC value of amoxicilline. Roxithromycin also showed sensitivity at concentration 30 µg/ml against culture N which was considered as its MIC value.
Antibiotics are chemotherapeutic agents that have revolutionized the treatment of infectious disease – turning life-threatening diseases into more Table 5. MDR test for sample G3 [Note: R= resistant, S= sensitive].
manageable and treatable conditions. Resistance became a major challenge to the treatment of Concentration Amoxy infectious diseases shortly after the introduction of antibiotics. The objective of the experiment was to isolate multi-drug resistant bacteria which can use antibiotics as carbon source for their nutrition and growth. Bacteria gain resistance through various methods: some bacteria make an antibiotic ineffective before the drug can kill them; some strains alter the drug attack site so that the antibiotic becomes ineffective; some rapidly pump out the antibiotic–antibiotic efflux. Some bacteria have a natural resistance to antibiotics but others become resistant through genetic mutation or by acquiring resistance from another bacterium Table 6. MDR test for sample N [Note: R= resistant, S= sensitive].
(Alliance for the Prudent Use of Antibiotics, 1999). Five multi-drug resistant bacterial cultures were isolated from all sampling points. Out of which only one was identified as Pseudomonas aeruginosa. This culture causes infections of wounds, burns, eyes and ears (Lateef, 2003). The antibiotic sensitivity pattern of the five bacterial isolates is shown in Tables (2–6). All five cultures were resistant to cefixime and amoxycillin even at higher concentrations (100 µg), which was much higher compare to work done by Lateef (2003) this process was done with the help of Agar well diffusion method which was earlier done by Bauer et al. (1966). Only A1 isolate was resistant to ofloxacin.A1, C2 & N isolates were resistant to roxithromycin, ampicillin and tetracycline. None of the five isolates were resistant to Table 7. MIC value of culture A2 against Cefixime ciprofloxacin i.e. all were susceptible to ciprofloxacin. After MIC the (Initial concentration= 1mg/ml) antibiotics amoxycillin, cefixime showed resistance at higher concentration (0.25 mg/ml). After MBC it was observed that the MDR






Full Length Article Isolation and Characterization of Multi-Drug Resistant Cultures, Amit Pandey et al., Figure 1. Resistance shown by isolated MDR culture C at higher Table 8. MIC value of culture C2 against Cefixime concentration (100 µg/ml, 1 mg/ml & 10 mg/ml) of cefixime (Initial concentration= 1mg/ml) and amoxycilline Figure 2. Resistance shown by MDR isolates at lower concentrations of cefixime and roxithromycin for cultures A2 & C2 Table 9. MIC value of culture C2 against Amoxycilline (Initial concentration= 1mg/ml) Figure 3. Resistance shown by isolates N & A2 with amoxycilline Table 10. MIC value of culture A2 against Amoxycilline (Initial concentration= 1mg/ml) cultures are bacteriostatic as they show growth even at higher concentrations of antibiotics.
At the end of all the experiments it was identified that out of all bacterial cultures isolated from various aquatic places, five were multi--drug Figure 4. MBC culture plates of the isolates (Cultures showing the resistant. Isolates A2 & C2 are Gram negative rods, isolates A1 & G3 are growth in the presence of Antibiotics) A2: culture against Cefixime Gram negative mixed cultures and only one isolate N is Gram positive C2: culture against Amoxycilline mixed culture, all isolates were catalase positive. Isolate G3 was identified as Pseudomonas aeruginosa and A2 of family Enterobacteriaceae. All these five isolates were multi--drug resistant as they were resistant to various antibiotics used which were of different mechanism of action. After MIC & MBC tests it was observed that the isolated cultures were bacteriostatic in nature. I am very grateful and my heartiest thanks to Mr. Manoj Verma, Director, MRD LifeSciences (P) Limited, Lucknow, Mr. R.P. Mishra (Research Scientist), Mr. Jahir Alam Khan (Research Scientist), & Ms. Chanda Sinha (Research Scientist), MRDLS, Lucknow, for there kind support Figure 5. Confirmatory test of Pseudomonas aeruginosa in G3 isolate throughout the research work. I am also thankful to the almighty without (Pink colonies in King's B Media). Full Length Article Isolation and Characterization of Multi-Drug Resistant Cultures, Amit Pandey et al., whose consent anything is possible.
Flavobacterium/Bacteroides line of phylogenetic descent. Syst. Appl. Microbiol. 21: 374-383.
Jalal, K.C.A., Nur Fatin U.T., Mardiana M.A., Akbar John B., Kamaruzzaman Y.B., et al., 2010. Antibiotic resistance microbes in Aneja, K.R., 2003. Experiments in Microbiology, Plant Pathology and tropical mangrove sediments in east coast peninsular, Malaysia. African Biotechnology. New Age International (P) Limited: New Delhi Vol. 4. P. Journal of Microbiology Research. 8:640-645 Jensen LB., Baloda S., Boye M., Aarestrup FM. 2001. Antimicrobial Alliance for the Prudent Use of Antibiotics, 1999. Antimicrobials in the resistance among Pseudomonas spp. and the Bacillus cereus group United States. Strategies Developed by an NGO: 1-5.
isolated from Danish agricultural soil. Environ. Int. 26: 581-587 Bauer AW., Kirby WM., Sheris J.C., Turck M. 1966. Antibiotic Lateef, A., 2003. The microbiology of a pharmaceutical effluent and its susceptibility testing by a standardized single disc method. Am. J. Clin. public health implications 3: 212-218.
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Schwartz T., Kohenen W., Jansen B., Obst U., 2003. Detection of Buchanan R.E. and Gibbonns E.N. 1978. Bergey's Manual of antibiotic resistant bacteria and their resistance genes in waste water, Determinative Bacteriology. Williams and Wilken Co.: Baltimore surface water, and drinking water biofilms. FEMS Microbiol. Ecol. 43: Hirsch P., Ludwig W., Hethke C., Sittig M., Hoffmann B., Gallikowski Vincent D.S.J. and DVM., MA., 1999 Informing Public Policy on CA., 1998. Hymenobacter roseosalivarius gen. Nov. From continental Agricultural Use of Antarctic soils and sandstone: bacteria of the Cytophaga/ Citation: Amit P., Neha R., Mohammad N A., Bharat C. 2011. Isolation and characterization of multi-drug resistant cultures from aquatic areas. Adv Bio Tech 11(4):16-20

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