Pakistan Joumal of Biological Sciences 7 (12): 2081-2084, 2004 ISSN 1028-8880 © 2004 Asian Network for Seientifie Information Isolation, Identification and Antibiotic Resistance Profile of Indigenous Bacterial Isolates from Urinary Tract Infection Patients Nadia Gul, Talat Y, Mujahid and Samia Ahmad Department of Microbiology, University of Karachi, Karachi-75270, Pakistan Abstract: Sixty-five bacterial strains were isolated from urine samples of patients suffering from winary ‘act infection and identified by conventional methods. Eighty pescent of total isolated organisms were found tobe gram negative while remaining 21% were gram positive. Among gram negatives, coli and gram positive S. aureus and S. pyogene were the most prevalent organism. The percentage of gram negative isolates was 2s follows Escherichia coli (47.6) followed by Pseulomonas aeruginosa (9.2%), Klebsiella pneumoniae (7.6%), Enterobacter aerogenes (6.1%), Proteus mirabilis and Serratia marcescens (4.0% each), The percentage of gram positive isolates includes, Siophylocovcus aureus and Streptococcus pyogenes (4.6% each), Enterococcus faecalis, Staphylococcus epidermidis and Bacillus subtilis (3% each) and Staphylococcus saprophyticus (1.5%). The antibiotic resistance of identified organisms was carried out by dise-iffusion method with commercially available ce of fifteen antibicties having different mode of actions such as cell wall synthesis inhibitors, membrane permeability alternatives, protein synthesis inhibitors and DNA. synthesis inhibitors. Gram negatives showed more resisiance to these antibioties as compared to gram positive organisms, The most effective antibiotic for gram negative UTI isolates is gentamycin showing 69.2% efficacy, then sulfamethoxazole-trimethoprim (SXT-TMP) with 55% efficacy and then kanamycin having 50% efficacy. Among gram positives, chloramphenicol is most effective with 84.6% efficaey, then ofloxacin and gentamycin with 76.9% effieacy and then norfloxacin with 69.2% efficacy Key words: Urinary tract infection, antibiotics, antibacterial, resistance, sensitive INTRODUCTION Urinary Tract Infections (UTIs) are among the most common bacterial infections ranging from asymptomatic to severe sepsis”, It also contributes the most common nosocomial infection in many hospitals and accounts for approximately 35% of all hospital-acquired infections. UTT cause increased morbidity and economic cost” Bacteria are the primary organisms that cause UTI Gram negatives cause 80-85% and gram positives cause 15-20%, Among, gram negatives Escherichia coli is the most frequent pathogen but in complicated UTIL the prevalence of other antibiotic resistance organisms increases such as Klebsiella, Proteus, Serratia, Enterobacter, Pseudomonas. Among gram positives S. saprophyticus, E, faecalis, S. agalactiae, S. pyogenes, S. aureus and Bacillus subtilis are wsvally prevalent and fre resistant to a variety of different antibiotics” Enterococcus isolates cause 2.3% of UTI and best known, as antibiotic resistant opportunistic pathogens". Varieties of antibiotics are available for UTIs and the choice is depend upon many factors including severity of infection and primary or recurrent infection, Antibiotics used for treatment of UTI includes amoxycillin, cephalosporins, tetracycline, nitrofurantoin but fluoroquinolones are most commonly used For catheter induced infection antibiotics in combinations are effective against a wide variety of microorganisms includes fluoroquinolones, ampicillin’ Gentamicin, imipenem eilastatin” Development of resistant strains is a common problem in antimicrobial chemotherapy. Among ‘uropathogens the rate of resistance is high and frequency of resistance to antibiotics and drugs is directly linked! to consumption of antibiotics”. According to Tomasz” gram positive bacteria are the most common cause of nosocomial infections, Enterococci are best-known antibiotic resistant opportunistic pathogens, resistant to vancomycin’. No single antibiotic is bactericidal. for ‘Vancomycin Resistant Enterococei (VRE) and Methcilin- Resistant Staphylococcus (MRSA) and combination therapy is mandatory. Present study was based on the isolation-identification and antibiotic renatance pattem of about sixty-five indigenous bacterial isolates from UTI patient Corresponding Author: Samia Almac, Department of Microbiology, University of Karachi, Karachi-75270, Pakistan ‘Tel; 03002343201 E-mail: samiaz@genys net 2081Pak. J Biol. Si, MATERIALS AND METHODS Isolation and identification of UT isolates: Sixty-five tasine samples from UTE patients were collected from dierent pathological laboratories of cosmopolitan city of Karachi, For the wolation of UTI causing strains, loop Fall of urine sample was streaked on to ntrient agar plate and incubated at 37°C for 24 h. Next day individual colonies were selected amt identified on the bases of, morphological, cultural and biochemical characteristics” For identification of gram negative bacteria: To check morphological characteristics, gram-staining, capsule staining (Anthony's method) and motility test were performed. To check the growth patter, different media incloding MacConkey’s agar no.3, Bosine Methylene Blue agar (BioM laboratories, USA) were wsed. For biochemical characteristics, sugar fermentation (lactose, glucose, mannitol, maltose, sucrose and xylose), TSI, IMVIC (indole, MR. VP, citrate) and nitrate tests were performed" For identification of gram positive bacteria: To check morphological characteristics, gram-staining and capsule staining (Anthony's method) was performed. To check the growth pattem, different media including MacConkey’s agar no3, Nutrient agar, Brain Heart Infusion agar, Mannitol Salt agar and blood agar base (Oxoid) supplemented with 58 sheep blood were used. For biochemical characteristics, sugar fermentation, cxidase, catalase, coagulase, novabiocin, optochin, bacitracin and bile csculin sensitivity test were performed"! Maintenance of clinical isolates: Stock cultures were maintained in vials by growing the UTI isolates in 3 mL nutrient broth and next day overlaying with 3 mL, 40% elycerol™. Vials were then freazed at -70°C. Determination of antibiotic resistance profile: UTI isolates were subjected to antibiotic resistance sereening by dlise diffusion method. For this purpose, laws of UT isolates was made on nutrient agar plates with the help of wire-loop. Then commercially available antibiotic dises were placed on lawn of culture and plates were incubated at 37°C for 24h, Next day presence or absence of zone of Inhibition around the antibiotic discs was observed" Anntibioties used were penicillin Gand amoxicillin (cell wall, synthesis inhibitors), polymyxin B (membrane permeability alternatives), tetracycline, kanamycin, tobramycin, neomycin, gentamycin, chloramphenicol, ethyLhydrocupreine hydrechloride (optechin) and (12): 2051-2054, 2008 lineomyein (protein synthesis inhibitors), ciprofloxacin, norfloxacin, sulfamethexazole-trimethoprim (SXT-TMP) and ofloxacin (DNA synthesis inhibitors), RESULTS AND DISCUSSION Urinary Traet Infections (UTIs) are among the most commonly prevalent infections in clinical practice, The purpose of the present study is to ckscribe the susceptibility or resistance profile of multi-drug resistant isolates from urinary tract infection. Sixty-five isolates from different pathological laboratories of Karachi (Pakistan) were isolated and identified by conventional methods. Identification of the causative organism and its susceptibility to antimicrobials is important, so that proper drug is chosen to teat the patient in early stages of UM". Peroentage of different gram negative and gram positive isolates in urinary tract infections is depicted in ‘Table 1. The frequeney of gram negative enteric bacteria causing UT is more then gram positives. Among gram negatives, E. colt was the most predeminant organism. ‘The percentage of gram negative isolates was as follows Escherichia coli (47.6%) followed by Pseudomonas aeruginosa (9.2%,) Klebsiella pneumoniae (7.6%), Enterobacter aerogenes (6.1%), Proteus mirabilis and ‘Serratia marcescens (4.6% each). The percentage of gram positive isolates includes, Staphyfocaccus aureus anc Streptococeus pyogenes (4.6% each), Enterococcus faecalis, Staphylococcus epidermidis ond Bacillus ‘subtilis G% each) and Staphylococcus saprophsticus (1.3%), Present results are in close agreement with Ali! which reported organisms responsible for UTI include Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Staphylococcus spp. and Pseudomonas aeruginosa. Previous studies have also demonstrated that 2, coli isthe most frequent etiological agent causing community and hospital acquired UTIs?" Al the isolates were screened for drug resistance profile by dise-diffusion method with commercially available disc of penicillin G, amoxicillin (cell wall synthesis inhibitors), polymyxin B (cell membrane altemative), gentamycin, Kanamyein, neomycin, tetracycline, tobramycin, chloramphenicol, ethyl- hhydrocupreins hydrochloride Coptochin) and lincomycin (protein synthesis inhibitors), ciprofloxacin, ofloxacin, sulfamethoxazoletrimethoprim (SXT-TMP) and norfloxacin (DNA synthesis inhibitors) Table 2 indicates the resistance level against commonly used antibiotics in uuinary tract infection, Nearly all the isolates (aram positive and gram negative) were found to be resistant against most of the antbioties, whereas gram negative shows more resistance as compare to gram positive 2082Pak. J. Biol. Sei, ? (12): 2051-2054, 2004 ‘Toble 1: Pecctages of gam negative and gam postive batt sled faut inal 60 Bact Toulanber Totsloot — gumnetnesad ila sgn —_UTT soa _ gramps Gram negative Eel 51 406 « Kpmsmone Ss 16 ° arog 4 et 7 P mics 5 4 ‘ Smarcercen 45 6 Peneima 6 82 8 (Gram poative 13, Scamvur 3 46 Pn ‘Sepldrmis 2 80 15 Sgpopintin 1 is « ‘Sogou 3 45 2 2B facals 2 30 15 ‘ais 2 30 15 “Tble2 Tapenade sities ang UT les “Tota Na. af aoe (5) antec Disecode_Sentive Reviews %FMeay Peiclin Pe 2 6 30 Ameiclin A ° sss PobmmoxinB 1 o 1s Gentanyein ” a3 Kenan 36 2 Neeawen x ’ 7 2s Teroeeine Ty 2 6 30 Tetempein Tas a 2 aso Chloratinical Cy u ares} Liseaest 2 6 “30 Cipro Cp » 38 Norovacin Nor u u Dfteace Of u 4 pact op ° soo Silimetsyazce: Se s mn sa7 trinetgr “Tuble 3: Percentage ebevenes of de sabes saubot gram postive sng negaive UT oes Peete etn Antibictie Grompostive £0) Grmnetive Tsu oo $880 a8 7690 @2 uso soo a0 na 4610 26 00 500 Linens 1338 a0 Ciao, as10 480 ‘Neflevain 20 $80 Ditosace 600 as praca 200 a0 Silfmahocctetrimadaprin 3070 ss organisms, Resistance to aminoglycosides _and chloramphenicol in gram negative bacilli often mediated by B-lactamases which are unaffected by exposure of the bacterium to the potential drugs". Interestingly. resistance 10 penicillin G, amoxicillin, polymyxin B, neomycin, tetracycline, tobramycin, lincomycin. and coptochin is more frequent in our study, It has been angued that there isa direct relation between the antibiotic used and the frequeney and kinds of antibictic-resistant strains inkuuman beings!” The resistance to antimierobial agents can readily be iensferred among becteria by transmissible clements’plasmids!™. These resistant organisms can pass their resistance genes to their offspring by replication or torelated bacteria through conjugation”, Epidemiological studies have suggested that antibiotic resistance genes emerge in microbial populations within 5 years of the therapeutic introduction of an antibiotié™), Further. the antibiotic resistance genes (found in human and animal isolates) could have originated in the industrial microbes that are used for the production of antibiotics In our studies, the most effective antibiotic for gram negative UTI isolates is gentamycin showing 69.2% efficacy, then sulfamethoxazolestrimethoprim (SXT-TMP), which shows 35% efffcacy and then kanamycin showing 509% efficacy Among gram positives, chloramphenicol is most effective with 84.6% efficacy. then ofloxacin and gentamycin with 76.9% efficacy ane then norfloxacin with 69.2% efficacy (Table 3). Ciproffoxacin is highly active against UTE pathogens!and maintain excellent level of susceptibility famong common UTI pathogens, In the present study ciprofloxacin showed 46.1% efficacy against gram positives and 48.0% against gram negative bacteria. Due to frequent use susceptibility to ciproflaxacin decreases in a stepwise manner”! and resistance rates increasing among, UTL isolates. So wide apread empirical use of fluoroquinolones. should be discouraged became of potential promotion of resistance!” Gram positive bacteria are also most frequent cause of nosocomial diseases and difficult to teat because of their high frequency of drug resistance”. Due to antibiotic resistance andl limited antimicrcbial activity of antibiotics some strains of disease causing bacteria are now untreatable such as_vancomycin-resistance Enterococcus. 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