full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (1) 1054-1059 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1055counteract resistance. For example, Vancomycinwas discovered in the 1950’s and did not showany resistance until the mid 1980’s. After whichcertain nosocomial pathogens developedresistance and by early 1990’s molecular studiesdemonstrated that this mechanism prevailed inthe soil microorganisms proving that an earlyintervention would have prevented this emergence.A recent study by Knapp et al. (5) expressedconcern that increased antibiotic resistance insoils could have broad consequences to publichealth through potential exposure through waterand food supplies. Their results suggested thatthere may be a progressively increasing chanceof encountering organisms in soils collected fromin and around Netherlands exhibiting resistanceto antimicrobial therapy. They concluded thatfurther studies need be performed globally so thatthe scope and possible ramifications of their resultscould be better understood. Considering thesefacts, we performed these studies with Indian soilsto prove this hypothesis and generate baselinedata under Indian context on antibiotic resistancein bacteria living in soils collected from differenthabitats.Materials and MethodsRaw materials : Different antibiotics likeamoxicillin, ampicillin, ciprofloxacin, erythromycin,penicillin G, tetracycline, vancomycin, amikacin,chloramphenicol, gentamicin, gatifloxacin,kanamycin, neomycin, streptomycin,cycloheximide and rifamycin, and different mediasuch as Muller Hinton Agar were procured fromHiMedia Laboratories Pvt. Ltd., Mumbai, India.Collection of soil samples and isolation ofbacteria : Soil samples of about one gram werecollected with sterile metal spatulas from a depthof 1–5 cm from different habitats in and aroundHyderabad, India, like residential garden soil, farmsoil, public park soil, agricultural field soils,composting manure soils and soils near factoriesand lakes in sterile collection bottles. The entirecontents of each tube were suspended in 50 mlsterile water and vortexed on a shaker. Dilutionsof 1:10 and 1:100 were prepared from thesuspensions. Amounts of 1 ml from the originalsuspension and from each of the dilutions werepipetted into sterile Petri dishes containing 15 mlof Muller Hinton agar. Fungal growth wasprevented by the addition of 30 µg/ml ofcycloheximide to the medium. The Petri disheswere incubated at 35°C for 18–24 h in anincubator until the colonies were fully formed.Pure colonies were obtained after repeated subculturingand the pure colonies were maintainedon nutrient agar slants. Preliminarycharacterization of the isolates was based oncolony morphology and Gram staining.Antibiotic susceptibility testing : Target isolateswere tested for susceptibility to specifiedantibiotics using a standard agar disc diffusionmethod. Isolated colonies were initially culturedin nutrient broth at 35 ± 2°C for 24 hours and thecell density was adjusted to McFarland 0·5turbidity standard, resulting in a suspensioncontaining 1–2 × 10 8 cfu ml -1 . Within 15 min afteradjusting the turbidity, a sterile swab was dippedinto the adjusted suspension for inoculation andthen uniformly streaked over the entire surfaceof a Mueller–Hinton agar (MHA) plate and thenthe drug-impregnated discs were placed on thesurface of these inoculated MHA plates. Testingfor antibiotic susceptibility was performedfollowing the testing and quality assurancepractices outlined in the M2-A6 NCCLSDocument (NCCLS, 1997a). The panel includedamoxicillin (Amx), ampicillin (Amp), ciprofloxacin(Cip), erythromycin (Ery), penicillin G (Pen),tetracycline (Tet), vancomycin (Van), amikacin(Amk), chloramphenicol (Chl), gentamicin (Gen),gatifloxacin (Gat), kanamycin (Kan), neomycin(Neo), streptomycin (Str) and rifamycin (Rif). TheGanesh Kumar et al