Research Journal of Recent Sciences ______ ______________________________ ______ ____ ___ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 124 - 129 (201 3 ) Res. J. Recent . Sci. International Science Congress Association 124 Isolation and Characterization of Multi drug Resistant Super Pathogens from soil S amples Collected from Hospitals Prasad Chandan 1 , Mishra R . P 2 , Ali Asif 3 , Gangwar V.S. 4 and Chand Shweta 5 1 Dept . of Chemistry, D.A.V College, Kanpur, INDIA 2 R and D Division , MRD Life Sciences, Lucknow, INDIA 3 Integral University, Lucknow, INDIA 4 Dept of Chemistry, V.S.S.D College, Kanpur, INDIA 5 Dept of Chemistry, Christ Church College, Kanpur, INDIA Available online at: www.isca.in Received 1 4 th September 2012, revised 19 th January 201 3 , accepted 02 nd February 201 3 Abstract Soil samples from two different city hospitals were collected , pre - treated along with several antibiotics for primary screening of numerous microbes and were culture d after serial dilution over sterile nutrient agar plates. A total of three isolated were identified and purified from the samples, further screened for individual antibiotics at their respective varying concentrati ons and all the three isolates were found to be strong resistant against antibiotics selected in the study. Morphological, biochemical and physiological properties were analysed for all the isolates. Keywords: MDR pathogens, hospital samples, Acinetobacter baumannii , drug resistance. Introduc tion Antimicrobial resistance is not new, but the number of resistant organisms, the geographic locations affected by drug resistance, and the breadth of resistance in single organisms are unprecedented and mounting. Diseases and disease agents that were o nce thought to be controlled by antibiotics are returning in new leagues resistant to these therapies 1,2 . Drug - resistant strains initially appeared in hospitals, where most antibiotics were being used. Sulfonamide - resistant Streptoccoccus pyogenes emerged in military hospitals in the 1930s. Penicillin resistant Staphylococcus aureus confronted London civilian hospitals very soon after the introduction of penicillin in the 1940. Similarly, Mycobacterium tuberculosis with resistance to streptomycin emerged in the community soon after the discovery of this antibiotic 3, 4 . Resistance to multiple drugs was first detected among enteric bacteria - namely, Escherichia coli , Shigella and Salmonella — in the late 1950s to early 1960s 5 . Such strains posed severe clinical p roblems and cost lives, particularly in developing countries 6 . Nevertheless, the resistance problem was perceived by some, most notably those in the industrialized world, as a curiosity of little health concern confined to gastrointestinal organisms in dis tant countries 7 - 9 . This attitude changed in the 1970s when Haemophilus influenza and Neisseria gonorrhoeae , organisms that cause respiratory and genitourinary disease, respectively, emerged with resistance to ampicillin and, in the case of Haemophilus , wit h resistance to chloramphenicol and tetracycline as well. Fuelled by increasing antimicrobial use, the frequency of resistance escalated in many different bacteria, especially in developing countries where antimicrobials were readily available without pres cription. Poor sanitation conditions aided spread and small healthcare budgets prevented access to new effective but more expensive antibiotics. Individuals may succumb to MDR infections because all available drugs have failed, especially in the developing world. Notable global examples include hospital and community MDR strains of Mycobacterium tuberculosis , Enterococcus faecium , Enterobacter cloacae , Klebsiella pneumoniae , S. aureus , Acinetobacter baumanii and Pseudomonas aeruginosa (World Health Organiza tion website). In developing countries, MDR enteric disease agents such as Salmonella Enteritidis , Shigella flexneri and Vibrio cholera threaten and circumvent public health measures 10, 11 . Overall, in the United States and the United Kingdom, 40 – 60% of no socomial S. aureus strains are methicillin - resistant (MRSA) and usually MDR. More deaths are associated with MRSA than with methicillin - sensitive strains. A steadily increasing, small proportion of MRSA also now shows low - level resistance to Vancomycin (th e Drug of choice), leading to treatment failure. For the increase cause of such infection in hospitals, at least two mechanisms have been documented 12 . First, antimicrobial - resistant flora may be endemic within the institution and may be transferred to the patient within the hospital setting. Second, a small population of antimicrobial - resistant bacteria that are a part of patient's endogenous flora at the time of hospitalization may emerge under the selective pressure of antibiotics and become the dominant flora. Material and Methods The soil sample was taken from hospital wastages dumping site. Soil sample was chosen because of higher probability of finding bacterial stains of localized zone mainly obtained from dump hospital wastages which may include me dicines, edibles, Re search Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 124 - 129 (201 3 ) Res. J. Recent. Sci. International Science Congress Association 125 patient’s dressings etc , so there might be probability of finding large amount of pathogenic bacteria. The soil sample was taken from Vivekanand Polyclinic and Ram Manohar Lohia hospital, Lucknow, Uttar Pradesh. The samples were pre - incu bated in two flasks - one containing 10.0% antibiotic supplemented with nutrient broth media and other containing only 10.0% antibiotic. Both flasks were incubated for one week at 37ºC. After one week prepared inoculums were serially diluted and spread on NA plates and incubated overnight. For further characterisation Glucose, maltose dextrose and mannitol fermentation tests were performed on their respective broth followed by glucose oxidase, nitrate reductase, urease and MRVP tests. To check their growth potential all the isolates were selected for antibiotic sensitivity tests. Antibiotic sensitivity test was performed by gel diffusion method by taking different concentrations of antibiotics. Antibiotics used were ofloxacin, ciprofloxacin, cefitriaxine, ma hacef, roxithromycin, norflox, amoxicillin, ampicillin, odoxil, amoxyclav, uricomycin, tetracycline, zone of inhibition was calculated for different concentrations 13 . Growth optimization tests were performed to check for the growth enhancement and growth inhibitory materials (other than antibiotics). In test tubes containing distilled water 1% of yeast, beef extract, maltose, lemon juice, blood, peptone, blood, starch and grape juice were taken and optical density was measured after a week. Then pH opti mization tests was performed at 0.2% beef extract with the pH of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 in separate test tubes. Microbial growth kinetics analysis was performed for all three isolates in nutrient broth media by measuring the optical density . The effects of metals are both stimulatory and inhibitory on microorganisms. This test was performed to check the growth of microbes. 0.1% Cu +2 , Pb +2 , Mg +2 , Ca +2 and Fe +2 ions in each test tubes was added in 0.2% beef extract and optical density was m easured. The isolates were also grown on different growth elicitors for studying their growth optimization study conditions. 0.2% Beef extract media in separate test tubes Growth elicitors included 0.1% Maltose, Starch, Dextrose, Sucrose and Glycine. Res ults and Discussion 3 bacterial isolates were obtained. The 1 st and 3 rd isolate showed latexy mocoid growth while 2 nd one showed growth with greenish pigmentation The 1 st and 2 nd isolates were identified as Gram negative cocci while third isolate as Gram negative bacilli, all showing positive results for catalase test indicating their aerobic nature On the basis of following biochemical tests the 1 and 2 isolate were characterised as Gram negative aerobic cocci coming under Nisseria species whereas cultur e 3 was characterized as Gram negative aerobic curved rod coming under Alkaligene Sp. Category . Table - 1 Table showing comparative colony morphology of the bacterial isolates from different samples Colony Morphology 1 st isolate 2 nd isolate 3 rd isolate Ma rgin Entire Entire Entire Surface Texture Rough Smooth Smooth Elevation Elevated Elevated Elevated Optical Texture Slightly Convex Slightly Convex Slightly Convex Pigmentation Dim Light Greenish Dim Light CULTURE 1 CULTURE 2 CULTURE 3 Figure - 1 Photographs showing pure cultures of the isolates on NA media after incubating at 37 0 C for 48 h Re search Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 124 - 129 (201 3 ) Res. J. Recent. Sci. International Science Congress Association 126 Table - 2 Showing results of different biochemical test on culture 1, 2 and 3 Tests Culture 1 Culture 2 Culture 3 Glucose fermentation + + + Glucose oxidase + _ _ Dextrose fermentation + + + Maltose fermentation + _ + Mannitol fermentation + _ _ Citrate + + + Nitrate reduction + _ + Urease + + + MR _ _ _ VP + + + Figure - 2 Figure showing zone of inhibition of various antibioti c s for first isolate Figure - 3 Figure showing zone of inhibition of various antibiotics for second isolate Figure - 4 Figure showing zone of inhibition of various antibiotics for third isolate Microbial growth kinetics analysis: After measurement of o ptical density of all the three isolates it was observed that culture 1 gave the best growth. Table showing analysis of growth pattern of all the three microbes in a given time interval . Media optimisation of Isolates: All the three isolates showed optimu m growth in beef and yeast extract media no growth was observed in case of lemon juice. Table showing growth of all the three cultures on various components for growth optimization Re search Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 124 - 129 (201 3 ) Res. J. Recent. Sci. International Science Congress Association 127 Table - 3 Table showing zones of inhibition for all the three isolates aga inst different antibiotics Antibiotics Culture 1 Culture 2 Culture 3 Ofloxacin 2.40 2.30 0.00 Tetracycline 0.00 0.00 0.00 Mahacef 1.50 1.00 1.10 Ciproflox 2.10 2.80 1.60 Amicacin 0.00 0.00 0.00 Amoxyclav 0.00 0.00 0.00 Amoxycilin 0.00 0.00 0.00 Rox ithromycin 0.00 0.00 0.00 Cefatriaxone 0.00 0.00 0.00 Zenflox 0.00 0.00 0.00 Odoxil 0.00 0.00 0.00 Metrogyl 0.00 0.00 0.00 Table - 4 Table showing growth pattern of isolates in different media components S.n Media (1%) Mrsra 1102 mrsra 1101 mrsra 1103 1 Starch + 0 0 2 Yeast extract +++++ ++++ ++++ 3 Beef extract ++++ ++++ ++++ 4 Tryptone +++ ++ ++ 5 Maltose ++++ + + 6 Lemon juice 0 0 0 7 Grape juice 0 + + 8 Blood ++ N.A N.A Table - 5 Table showing growth pattern of isolates in different media co mponents Time(hr) 1 st isolate 2 nd isolate 3 rd isolate 1. 0.001 0.000 0.000 2. 0.101 0.005 0.004 3. 0.216 0.045 0.039 4. 0.315 0.086 0.094 5. 0.384 0.159 0.176 6. 0.409 0.239 0.310 7. 0.415 0.341 0.350 8. 0.429 0.349 0.361 9. 0.431 0.351 0.391 10. 0.435 0.350 0.398 11. 0.430 0.349 0.390 Effect of Metal ions on Growth: In presence of different metal ions, maximum growth occurred in iron and no growth occurred in copper but maximum pigmentation occurred in calcium ions Discussions: Characteriza tion of all isolates gave surprising results showing the emergence of Nisseria and Alkaligenes spp. as MDR pathogens which are generally not. The cause of increasing resistance among the bacteria might be due to development of MDR efflux pump against that drug due to its prolonged exposure at contaminated hospital dumping sites, due to mixing of both MDR and non MDR strains of pathogens at hospital waste disposal site resulting in genetic recombination of plasmids between two bacteria thriving at same place one of which might be MDR or induction of multi drug resistance by proteins secreted by MDR bacteria 14 - 17 . Antibiotic such as Ciprofloxacin, Ofloxacin belonging to Quinilone family are considered to be best medicines in case of MDR infections and have bro ad spectrum effects 18, 19 . As per the working mechanism it was observed that drug directly dealing with DNA replication i.e DNA Gyrase inhibition by ciprofloxacin are more potent and effective and are less proned to development of resistance by bacteria un less there is development of MDR efflux pump to Re search Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 124 - 129 (201 3 ) Res. J. Recent. Sci. International Science Congress Association 128 that drug unlike inhibition of protein synthesis as done by tetracycline or cell membrane destruption by penicillin family of drugs 20, 21 . Protein source supplement like beef extract, yeast extract are the be st source required for bacterial growth and proliferation, even they can support growth without any other additional nutrient supplement required. Bacteria usually produce its secondary metabolite in stress conditions in order to survive 22 . Carbohydrate, a mino acids, multivitamin capsules etc and metal ions acts as growth elicitors in case of many bacterial strains. The place of emergence of new and pathogenic strains of MDR bacteria can be hospital itself if not taken care of hospital dumped wastages. Tab le - 6 Effect of metal ions on growth of 3 rd isolate Metal ions (0.1%) Growth Pigment Cu +2 _ + Pb +2 + _ Mg +2 ++ ++ Ca +2 +++ +++ Fe +2 ++++ _ Zn +2 + _ Table - 7 growth pattern of the third isolate under different elicitors Elicitors Growth Maltose ++ S tarch ++++ Dextrose ++ Sucrose ++ Glycine _ Conclusion Hospitals including patient ward, operation theatre and ICU are a potent platform for spreading of pathogens among individuals either healthy or unhealthy persons also there is a maximum exposure of pathogens against various drugs. The bacterial isolates purified from those hospital samples showed more resistance against drugs in comparison to microbes isolated from normal soil samples. 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