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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
Current Trends in Biotechnology and PharmacyVol. 5 (1) 1054-1059 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1056plates were then inverted and incubated at 35 ±2°C for 20–24 h. After the specified incubationperiod, the zone of inhibition around each of thediscs was measured to the nearest wholemillimetre. The zone margin was defined as thatarea showing no obvious, visible growth detectedby the unaided eye. Interpretation of these zonesizes was based on Table 2 of the M100-S9NCCLS document (NCCLS, 1999).Results and DiscussionThe concept of soil as a location ofantibiotic resistance determinants, particularlythose harboured in antibiotic producers as selfprotection mechanisms has been acknowledgedfor decades. Around 57 soil samples collectedfrom different habitats (urban and agricultural)within Hyderabad, India were processed and about15,000 morphologically diverse collection ofbacteria colonies were isolated from these soilsamples and about 264 target organisms (at leastminimum one target bacterial colony type waspicked up from each soil sample) were selectedfor further phenotypic characterization based onGram staining and antibiotic susceptibility testing.The phenotypic diversity of the 264 target isolatesbased on gram staining is shown in Table 1. Itwas observed that of the total isolates, 57 belongedto the genus Bacillus, 37 were Pseudomonads,30 belonged to Enterobacteriaceae and 126represented to the group of Staphylococci. Acloser look at the distribution of the recoveredisolates indicated that only 14 actinomycetes wererecovered from these soil samples.These 264 strains were subsequentlyscreened against 15 antibiotics, including naturalproducts (such as penicillin, vancomycin anderythromycin), and completely synthetic molecules(such as ciprofloxacin and gatifloxacin). Theantibiotics encompassed all major bacterial targetsand included drugs that have been on the marketfor decades as well as several antibiotics that haveTable 1. Phenotypic density of the 264 targetbacteria isolated from the 57 soil samplesDiversity of isolates No of isolatesActinomycetes 14Staphylococci 126Bacillus 57Pseudomonads 37Enterobacteriaceae 30Table 2. Antibiotic resistance spectrum depictingmaximum number of isolates having multi-drugresistance of the soil isolatesNumber of antibiotics % of isolatesresistant to10 1.89only recently been clinically approved (such asgatifloxacin). This study provides an analysis ofthe antibiotic resistance potential of soilmicroorganisms. The frequency of resistance ishigh as observed in the study to antibiotics thathave for decades served as gold-standard fortreatment, as well as those only recently approvedfor human use. No class of antibiotic was sparedwith respect to bacterial target or natural orsynthetic origin. A broad survey of all the 264isolates, without exception suggested that everystrain in the library was found to be resistant toat least one antibiotic. The multi-drug resistancefor more than 3 antibiotics was observed in 219out of the 264 isolates (83%), with five strainsbeing resistant to more than 10 antibiotics of the15 antibiotics tested (Table 2). A comparativeInvestigations on Microbial Resistance among Bacteria Dwelling in Indian Soils
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