STUDY OF PRESENCE OF MULTI-DRUG RESISTANT ORGANISMS FROM A PHARMACEUTICAL EFFLUENT. RESEARCH ARTICLE

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1 NTENATONAL JOUNAL OF PHAMACEUTC & DUG ANALY VOL.4 UE 2, 2016; ; N: EEACH ATCLE TUDY OF PEENCE OF MULTDUG ETANT OGANM FOM A PHAMACEUTCAL EFFLUENT. Naik A*, Dpak. Dpartmnt of Biotchnology, Jai Hind Collg, Churchgat, Mumbai. Dat civd: 4 th Fbruary 2016; Dat Accptd: 19 th Fbruary 2016 Dat publishd: 23 rd Fbruary naik.amruta95@gmail.com Abstract: On of th wondr discovris of th 20 th cntury is Antibiotics. But th ral wondr and a grat caus of concrn is th ris of antibiotic rsistanc in hospitals, communitis and th nvironmnt. Thr ar many mchanisms by which microorganisms can dvlop rsistanc. This study aims at finding th rol of microorganisms isolatd from th fflunt of a pharmacutical industry in sprading drug rsistanc. Th diffrnt microorganisms isolatd from th fflunt wr E.coli, Psudomonas spp, Entrobactr spp, and.aurus. Thir antibiotic snsitivity pattrn was studid. Th rsults from th study showd th prsnc of multidrug rsistant organisms in th fflunt sampl. About % isolats wr rsistant to Ampicillin, Cftriaxon, and Gntamicin, 33.33% to Ciprofloxacin whil 100% showd rsistanc to Cftazidim and Erythromycin Ky words: Antibiotics, pharmacutical fflunt, multidrug rsistant organisms. ntroduction: Th problm of antibiotic rsistanc is univrsal. Multidrug rsistanc has dvlopd in almost all bactrial pathogns. A varity of nosocomial infctions hav bn rportd du to ths highly rsistant bactria. Uncontrolld and xcssiv us of antibiotics by humans and animals, rsults in an incras in antibiotic rsistanc and caus th sprad of rsistanc gns in nvironmntal sampls 1. Thrfor antibiotic rsistanc is not only found in pathognic bactria but also in nvironmntal organisms inhabiting trrstrial and aquatic habitats. Highr numbrs of rsistant bactria occur in pollutd habitats compard with unpollutd habitats, indicating that humans hav contributd substantially to th incrasd proportion of rsistant bactria occurring in th nvironmnt 2. ndian pharmacutical companis supply 20% of th world s gnric drugs 3. Th fflunt coming out of ths industris nds to b tratd wll bfor it is dischargd in th natural watr bodis. Th rsistanc gns may b transfrrd from nvironmntal bactria to human pathogns which is th main risk for public halth. Th input of rsistant bactria into th nvironmnt sms to b an important sourc of rsistanc in th nvironmnt 4. Antibiotics xrt a slction in favour of rsistant bactria. Th rsistant bactria adapt to nvironmntal conditions and srv as vctors for sprad of antibiotic rsistanc 5. Th fflunt coming out of a pharmacutical industry contains antibiotic and antibiotic rsistant bactria which can transfr drug rsistanc horizontally to pathogns. To th bst of our knowldg not many studis hav bn undrtakn to study this phnomna. This study attmpts to gnrat information rgarding th prsnc of microorganisms in th tratd fflunt of a pharmacutical industry locatd in th outskirts of Mumbai and th drug rsistanc pattrn of ths isolats. Matrials and mthods : ampl collction: Th tratd fflunt was collctd from a pharmacutical company locatd in th outskirts of Mumbai. Th sampl was collctd in stril conical flask and stord at 4 0 C till us. solation and charactrization: Bactria wr isolatd and purifid by strak plat mthod on standard slctiv and diffrntial mdia. Th plats wr incubatd at 37 0 C for 24 hrs. [Fig.1] solatd culturs wr idntifid by biochmical tsts. staining was carrid out. Catalas tst, Citrat utilization tst, ndol production tst, Mthyl dvogs Proskur (MVP) tst, Uras tst and frmntation of carbohydrats lik glucos, lactos and sucros wr carrid out for th idntification. [Fig. 2] 85

2 Naik A t al, nt J. Pharm. Drug. Anal, Vol: 4, ssu: 2, 2016; 8589 Fig. 1: solation on Mac ac conky s agar plat Antimicrobial uscptibility Tst (AT): AT for all th isolats was prformd as pr Ki Kirby Baur disc diffusion mthod 6 using th antibiotics otics mntiond blow. Th mdia usd was Mullr llr Hinton agar pr prpard as pr standard procdur. cdur. Th dnsity of th cu cultur was adjustd to 0.5 McFarland s stand dard. With th hlp of stril cotton swabs, cultur was inoculatd on th mdium so as to obtain a lawn cultur. ant obusing stril forcps th discs of various antibiotics taind from Hi Mdia wr placd on th plat. Th plats wr incubatd ovrnight night at 370C. Th zon sizs wr intrprtd as pr th standard chart and th organisms wr classifid as snsitiv, intrmdiat and rsistant to th various antibiotics. cs. Discs containing co Ampicillin /ulbactum (10/10 µg), Cftriaxon (30µg), Ciprofloxacin (5µg), Cftazidim (30µg), Erythromycin Eryt (15µg), Gntamicin (10µg), Gntamicin (30µg), Norfloxacin (10µg) wr usd to tst organisms organ and discs containing Amoxycillin (30µg), Chloramphnicol (30µg), Cphotaxim (30µg), Cfpim (30µg), Ciprofloxacin Cipro (30µg), Cftizoxim(30µg), Erythromycin Erythro (15µg), PnicillinG (10 units)) wr usd to tst positiv organorga isms. All th isolats wr r maintaind at rfrigration rfrigr tmpratur tur on Nutrint Agar slants. Working stocks wr maintaind taind and usd for furthr studis. stu Fig. 2: Biochmical tsts for idntification of o organisms. ndol tst Catalas tst Mthy rrd tst Uras tst Vogus proskaur tst Citrat utilization Carbohydrat frmntation f 86

3 Naik A t al, nt J. Pharm. Drug. Anal, Vol: 4, ssu: 2, 2016; 8589 Fig.3: Antibiotic snsitivity tst sult and Discussion: A total of four typs of bactria wr isolatd from th fflunt sampl collctd. Out of ths four typs, on was positiv and thr wr. Using th idntif idntification tchniqus thy wr confirmd as Entrobactr spp. (T1), Eschrichia coli (TLF), Psudomonas spp. (TLNF) and taphylococcus aurus (T4). Tabl 1 shows th rsult of bioch biochmical tsts. Tst cultur T1 TLF TLNF T4 s natur positiv Morphology hort rod Coccobacilli od Carbohydrat Organism idntindol Mthyl VogsCitrat fid Uras frmntation rd proskaur utilization production Glc Lac uc Entrobactr spp. AG AG A Cocci AG AG AG Eschrichia coli Psudomonas spp. Catalas taphylococcus Aurus Lgnd : : positiv ; : ; A : acid id production ; G : gas production. Glc : glucos ; Lac : lactos ; uc : sucros. s Tabl 2 shows th AT rsults for th various antibiotics usd. Tst cultur T1 TLF TLNF ANTBOTC TETED Ampicillin A 10/10 Cftriaxon Ci 30 GAM NEGATVE OGANM Ciproflox Ciprofloxa Cftazidim Erythromycin cin CF 5 CAZ 30 E 15 GAM POTVE OGANM Amoxicil Chloromphnic Cp Cphotaxim Cfpim Ciprofloxalin ol CPM 30 cin Am 30 C 30 CE 30 Cf 30 T4 Lgnd: rsistant ; snsitiv ; intrmd intrmdiat. Gntamicin G 10 Gntamicin G 30 Norfloxaci n Nx 10 Cftizoxim Ck 30 Erythromycin E 15 PnicillinG P 10 87

4 Naik A t al, nt J. Pharm. Drug. Anal, Vol: 4, ssu: 2, 2016; s Numbr of drugs to which organism is 0 Entrobactr spp. Psudomonas spp. A graph showing organisms against numbr of drugs to which thy ar rsistant % 20 0 A (10) CF (5) E (15) G(30) A graph rprsnting th numbr of organisms (in %) rsistant against antibiotics usd (with concntrations). AT was carrid out for all th isolats. Entrobactr spp. was rsistant to Ampicillin (10µg), Cftriaxon (30µg), Ciprofloxacin (5µg), Cftazidim (30µg), Erythromycin (15µg), Gntamicin (10µg), Gntamicin(30µ µg). Eschrichia coli was rsistant to Cftazidim (30µg) and Erythromycin (15µg). Psudomonas spp. was rsistant to Ampicillin (10µg), Cftriaxon (30µg), Cftazidim (30µg), Erythro and mycin (15µg), Gntamicin (10µg), Gntamicin(30µg) taphylococcus aurus was not rsistant to any of th anti to Am biotics usd. About % isolats wr rsistant picillin, Cftriaxon, and Gntamicin. Cftazidim and Erythromycin showd 100% rsistanc % isolats wr rsistant to Ciprofloxacin. Whil Norfloxacin showd 0% rsistanc. [Fig.3] Th rsults from th prsnt study showd th prsnc of multidrug rsistant organisms lik Entrobactr spp., Eschrichia coli, and Psudomonas spp. in th tratd fflunt sampl of th pharmacutical company. Th company usd activatd sludg mthod for th tratmnt of th wast which was thn dischargd in th watr body. An t al 2011 showd that othr study carrid out by Ashour group of gnus taphylococcus contributs to 38.4% of total sampls whil gnus Micrococci to 22.4%, Bacillus spp. 35% and Klbsilla spp 1% 7,8.All th gram strains wr rsistant to Cftazidim and Erythromycin. n a study conductd by Latf t al (2003) th fflunt of a pharmacutical company was xamind microbiologi includd taphylococcus cally. Th organisms ncountrd aurus, Eschrichia coli, Protus vulgaris, rratia marcscns and Psudomonas aruginosa. Th rsistancs of th 25 bacfflunt to th frquntly trial strains isolatd from th usd antibiotics wr considrd. About 80% of th iso 76% to Citrofurantoin, lats wr rsistant to Amoxycillin, 64% to Cotrimoxazol and Augmntin, 60% wr rsistant to Nalidixic acid, 52% wr rsistant to Ttracyclin and Ofloxacin, whil rsistanc of 12% was obtaind for Gn tamicin 9. sults of thir study ar in agrmnt with th prsnt study to som xtnt. 88

5 Naik A t al, nt J. Pharm. Drug. Anal, Vol: 4, ssu: 2, 2016; 8589 Conclusion: ndia has shown to b a brding ground for around 50 drug rsistant microbs du to xtnsiv usag and impropr disposal of pharmacutical drugs into th nvironmnt 10. Constant prsnc of antibiotics in its nvironmnt has ld to th gradual adaptation of bactria to tolrat highr doss of antibiotics. Th antibiotic dos not tchnically caus th rsistanc, but allows it to happn by crating a situation whr an alrady xisting variant can flourish. This study was conductd at a vry primary lvl to find out if thr ar drug rsistant organisms prsnt in th wast gnratd in th pharmacutical company. From this study it can b concludd that multidrug rsistant microbs ar prsnt in th wast which can transmit th rsistanc to pathogns. inc not much data is availabl from this kind of study th nd of th hour is to find various ways by which sprad of drug rsistanc by this mchanism can b chckd. Taking into considration its ffcts, stringnt rgulation is rquird from industris and rgulatory agncis in ndia. Monitoring th amount of drugs prsnt in th wast and propr disposal is ssntial. frncs: 1. vrsn A, Koihn J, Frankin A. High prvalnc of vanconmycin rsistant Entrococci in wdish wastwatr. Appl. Environmntal Microbiology. 2002; 68: Pathak P, Gaw A, Battacharj JW. Distribution and antibiotic rsistanc among arobic htrotrophic bactria from rivrs in rlation to pollution. J Environmntal cinc Halth A.1993; 28: is rsponsibl for antibiotic rsistanc/22jan Dhundi, B Pradhan, K Mishra (2010). Multipl drug rsistanc in bactrial isolats from liquid wasts gnratd in cntral hospitals of Npal. Kathmandu. Uni Md J. 8 (29): Krus H.ndirct transfr of antibiotic rsistanc gns to man. Actc Vt cand. 1999; 92: Baur AW, Kirby MM, hrris JC, Truck M., (1966). Antibiotic suscptibility tsting by a standardizd singl disk mthod. Am J Clin Pathol. 45: ahaab Farooq, fra Ghori and Faiqa Abdur ub.dntification and multipl drug rsistanc of bactria, isolatd from pharmacutical industrial fflunt (slamabad, Pakistan). NT J CU C : E: Ashour M, Mansy M, Eissa M E (2011). Microbiological Environmntal Monitoring in Pharmacutical Facility. Egypt Acad Journal Bio ci. 3(1): Latf A (2003). Th Microbiology of a Pharmacutical Efflunt and ts Public Halth mplications. World J Microb Bio. 20: Gal J, Narayan A (2012). Drug dfying grms from ndia spd postantibiotic ra. Bloombrg Markts magazin. 89