Screening of Urinary Tract Infection causing bacteria and their ...

Journal of Academia and Industrial Research (JAIR)Volume 2, Issue 1 June 2013 57RESEARCH ARTICLEISSN: 2278-5213Screening of Urinary Tract Infection causing bacteria and theirAntibiotic sensitivity pattern in Namakkal district, TNR. Sudha 1* , M. Rajasekara Pandian 2 and B. Senthilkumar 31 Dept. of Zoology, Manonmaniam Sundaranar University, Tirunelveli; 2 Dept. of Zoology, Arignar Anna Government Arts College,Namakkal-637001, TN, India; 3 Dept. of Medical Microbiology, Haramaya University, P.O. Box 235, Harar, EthiopiaSudha28021984@gmail.com * ; +91 9003326058______________________________________________________________________________________________AbstractUrine samples (n=100) collected from patients in and around Namakkal hospitals were screened for UrinaryTract Infection (UTI) causing bacteria and its antibiotic sensitivity pattern. UTI causing bacterial strains wereidentified as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiellapneumoniae. Antibiotic sensitivity and resistance test was carried out by disc diffusion method. The studyrevealed that UTI is most common in women and bacterial isolates were also found to be resistant againstcommercially available antibiotics. Escherichia coli strains showed 100% resistance to cefpodoxime andnovobiocin and 28% to cefamandole recording the least resistance rate. Multiple resistances toβ-lactams have been registered with high frequency among studied strains. The phenotypic tests forβ-lactamase production indicated 100% of the tested E. coli isolates were positive.Keywords: Urinary tract infection, antibiotic sensitivity pattern, Escherichia coli, cefpodoxime, β-lactamase.IntroductionUrinary Tract Infection (UTI) is a bacterial infection of anypart of the urinary tract–the kidneys, ureters, bladder andurethra. Usually a UTI manifestation includes pain, fever,and discomfort, but is easily treated–unless it spreads tothe kidneys. UTI are important because they cause acutemorbidity and may result in long term medical problems,including secondary hypertension and reduced renalfunction. It allows identification, treatment and evaluationof the children who are at risk for kidney damage. Urinarytract is normally sterile, when bacteria moves fromrectum or vagina to urethra and multiplying within urinarytract cause infection called UTI. Normally 10 5microorganisms/mL of urine from midstream collectionindicate an UTI (Modarres and Oskoii, 1997).Commonly UTI caused by gram negative bacilli E. coliwithout catheters (Stamm, 2005). Pseudomonas,Staphylococcus and Klebsiella also cause UTI but incase of hospital acquired, which are mainly due tocatheters. Commonly UTI is caused by endogenousmicroorganisms from the patient’s own bowel. ThoughE. coli and Proteus spp. are commonly and easily treatedbecause of their, sensitivity to most antibiotics however,UTIs are increasingly caused by more resistant gramnegative spp. such as Klebsiella and Pseudomonas(Huang et al., 2004). In uncomplicated UTIs, E. coli is theleading organism, whereas in complicated UTIs, gramnegative and gram-positive and often muti-resistantorganisms (Florian et al., 2006). A complicated UTI isdangerous and one needs severe treatment (CharlesBryan, 2011). UTIs may be acute and chronic.At first, patients experienced with severe and low backpain that may associate with fever due to the associatedbacteraemia and latter, these problems lastingthroughout the life. Though E. coli is common, up to10-20% of sexually active women, UTIs are micrococcalinfections (Vorland and Bowen, 2001). This infectionreaches the bladder by the ascending route with themain symptoms as urinary frequency and dysuria.Klebsiella pneumoniae is also found in bowel but in lownumber than E. coli. Although K. pneumonia is commonin nosocomial infections and cause community-acquiredUTI with bacteremia.Women are more susceptible to UTI because a woman’surethra is short, allowing quick access of bacteria to thebladder and also a woman’s urethral opening is nearsources of bacteria from the anus and vagina. Theincidence increases with age and sexual activity. Drugscan only modify the inherent functions of the concernedtissues or the cells like stimulating or depressing cellularactivity, replacing deficient substances, causing irritationor killing/weakening the invading foreign organisms.Otherwise do not create new functions. Antibacterialdrugs to restrains the synthesis of bacterial cell wallinclude β-lactams, namely penicillins, cephalosporins,carbapenems and monobactams and the glycopeptides,including vanomycin and teicoplanin (Neu, 1992;McManus, 1997). The agents of β-lactams inhibitbacterial cell wall synthesis by interfering with theenzymes required for the peptidoglycan layer synthesis.©Youth Education and Research Trust (YERT) jairjp.com Sudha et al., 2013

Journal of Academia and Industrial Research (JAIR)Volume 2, Issue 1 June 2013 58Table 1. Preliminary tests and biochemical characterization of bacterial isolates.S. aureusPositivecocciNon-motilePositiveNegativeA+A+A+A+A+NegativePositiveNegativeNegativePositivePositive-K. pneumoniaeNegativeNon-motilePositiveNegativeA+G+A+G+A+G+A+G+A+G+NegativeNegativePositivePositivePositivePositiveA/A,G+P. aeruginosaNegativerodMotilePositivePositiveA+NegativeNegativeNegativeA+G+NegativeNegativeNegativePositiveNegativePositiveAk/AE. coliNegativeshort rodMotilePositiveNegativeA+G+NegativeA+G+NegativeNegativePositivePositiveNegativeNegativeNegativePositiveA/A, G+IsolatesGramstainingMotilityCatalaseOxidaseGlucoseSucroseLactoseMannitolMaltoseIndoleMRVPCitrateUreaseNitrateTSIMR-Methyl red; VP-Vogues Proskauer; TSI – Triple sugar iron; A+- Acid production; G+ Gas production; Ak- Alkaline.Extended-spectrum β-lactamase (ESBL) producingE. coli are increasingly by acquisition of resistancecharacter from resistance organism to susceptible onethrough genetic transformation. Because of the extensiveuse of antimicrobial substances the outcome out ofmulti-resistance strains and urinary tract infections areincreasing day-by-day. Based on particularcharacteristics of the patients, UTIs are common inneurological pathology (Banciu, 2005). According toKeen (2012) in bacteria, virulence factors are oftenencoded on mobile genetic elements, such asbacteriophages and can easily spread through horizontalgene transfer. Against these backdrops, it was aimed toscreen UTI causing bacteria and its antibiotic sensitivitypattern and virulence factors among patients in andaround Namakkal hospitals.Materials and methodsSample collection: A total of 100 urine samples werecollected in each 30 mL sterile plastic bottle from thepatients located in and around Namakkal hospitals.The samples were properly labeled indicating the source,date, time of collection, sex and age of patients.The urine samples were transported in cooler boxes toLaboratory for bacteriological investigations within 4-6 hof collection.Isolation of bacteria: Culture plates of Nutrient Agar (NA)were used for the isolation of bacterial strains from urinesamples. The collected urine samples were streakeddirectly on the labeled agar plates and incubated at 37Cfor 24 h. After incubation, cultures were examined forsignificant growth. Subcultures were then made intoplates of NA and incubated for another 24 h. Cultureplates of NA, Eosin Methylene Blue Agar, MacConkeyAgar and Mannitol Salt Agar (Hi media, Bombay, India)were used.Identification of bacteria: The primary identification of thebacterial isolates was made based on colonialappearance and pigmentation. Standard biochemicaltests were performed to identify the bacterial strains.Characterization and identification of the isolates wasdone using the methods of Cowan and Steel (1985),Fawole and Oso (1988) and Cheesbrough (2004).Antibiotic susceptibility test: All isolates were subjectedinto antibiotic susceptibility test according to Nair et al.(2005). The susceptibility of isolates of E. coli toantimicrobial agents was examined by agar disc diffusionmethod. The zone of inhibition and resistance wasmeasured, recorded and interpreted according to therecommendation of the disc manufacture.©Youth Education and Research Trust (YERT) jairjp.com Sudha et al., 2013

Journal of Academia and Industrial Research (JAIR)Volume 2, Issue 1 June 2013 60Table 4. Antibiotic resistance of E. coli isolates.Strain GE CF A E Co NA T CZX CPD CE K B Va Nv Ak Am NF TM Nr Cef%ResistanceE-1 R R R R R R R R R R I I I R R R I S S S 65E-14 R R R R R R R R R I I I R R R R R S S S 70E-16 R R R R I R R R R R R I R R R R R S R R 85E-17 R R R R R R R R R R R R R R R R R S S S 85E-18 R R R R I R R R R R R I R R R R R S R R 85E-21 R I R R I R I R R R R R R R R R R S S S 70E-27 I R R R I I R I R I R R R R R R I S S S 55E-30 R R R R R R R R R I I I R R R R R S S S 70E-40 R R R I I I R R R R R R R R S R R S S S 65E-41 R R I I I R R R R R R R R R S R R R S R 75E-42 R I R R I R I R R R R R R R R R R S S S 70E-48 S I R R R I R R R I R R R R S R R R R R 75E-58 S I R I I I R R R R R R R R R R R R S R 70E-59 R I R R I R I R R R R R R R R R R S S S 70E-60 I S S R S I I R R I R R R R S R I R S S 45E-61 I S I R R R R R R I R R R R S S R R S S 60E-65 S I R R R S R R R R I R R R R S S S S R 60E-72 S R R R I S R I R R R R R R R S R S S R 65E-80 S R R R R I R R R R R R R R S R R R R S 80E-85 R I R R R S R R R R R R R R R R R S R S 80E-86 I I R I R S R I R R I R R R S S R R R S 55E-87 R R R R R R R R R R R R R R R S R R R S 90E-97 S I I I I R R R R I I R R R I S R R R S 50E-98 R I R R I R I R R R R R R R R R R S S S 70E-99 S I R R S S R R R I R R R R R S I R R S 60R = Resistance; S = Sensitive; I = Intermediate.In the present study, the most resistance rates of E. colidetected from urine culture were found to 100%resistance to Cefpodoxime (CPD) and Novobiocin (Nv)and 28% to Cefamandole. Kenneth Todar (2012)reported strains that are resistant to all available clinicallyuseful antibiotics except vancomycin. Present studyshowed 100% of the isolates were beta lactamaseproducers (Table 5). In case of cell surfacehydrophobicity analysis, the highest percentage wasobtained in female (88.54%) in 37 years of age andlowest (75.23%) in 27 years of age. In male, highestpercentage (86.95%) was observed in 53 years of ageand lowest (75.29%) in 35 years.Highest incidences (positive) for slime production wererecorded in female (60.87%) followed by male (39.13%).In female, out of 6 types of age groups, the highestprevalence was recorded in the age group between21-30 years (28.57%) and lowest in the age groupbetween 41-50 years (7.14%) and in male, highestprevalence was recorded in age groups of 11-20, 21-30,31-40 and above 50 (22.22%) and lowest in 41-50 years(11.11%) and no production in 0-10 years (Table 6).β-lactamase production indicated 100% of the testedE. coli isolates are positive and cell surfacehydrophobicity test revealed 75-88% of cells in surface ofxylene.©Youth Education and Research Trust (YERT) jairjp.com Sudha et al., 2013

Journal of Academia and Industrial Research (JAIR)Volume 2, Issue 1 June 2013 61Table 5. Virulence factors of E.coli isolates.Strain SexAgeCell surfaceβ –lactamase(years)hydrophobicityE-1 F 10 Positive 83.49E-14 F 27 Positive 75.23E-16 F 21 Positive 80.76E-17 F 15 Positive 77.31E-18 M 45 Positive 86.40E-21 M 22 Positive 82.65E-27 F 31 Positive 87.5E-30 F 22 Positive 81.18E-40 M 35 Positive 75.29E-41 F 34 Positive 79.31E-42 M 17 Positive 81.05E-48 M 59 Positive 85.71E-58 F 30 Positive 86.66E-59 F 27 Positive 81.48E-60 M 19 Positive 84.14E-61 M 37 Positive 85.56E-65 F 10 Positive 88.42E-72 M 53 Positive 86.95E-80 F 20 Positive 84.21E-85 M 25 Positive 82.29E-86 M 31 Positive 82.72E-87 F 66 Positive 76.85E-97 F 49 Positive 81.65E-98 F 52 Positive 80.35E-99 F 37 Positive 88.54Thus, the highest incidence of virulence factors, recordedby the E. coli strains became more resistance to severalantibiotics because they help the organism to overcomehost defenses and colonize or invade the urinary tract.Virulence factors are very often responsible for causingdisease in the host because they are often responsiblefor converting non-pathogenic bacteria into dangerouspathogens.ConclusionPrevalence rate of E. coli was found to be the mostcommonest organism for UTI in all age groups andshowed resistance to commonly used antibioticsespecially, 100% resistant to cefpodoxime andnovobiocin. The test isolates of E. coli tested positive inβ-lactamase, slime production and higher percentage incell surface hydrophobicity showed high antibioticresistance. But the prevalence of antimicrobial resistancecan vary according to geographical and regional location.Hence, finally we recommend the selection of antibioticshould be based on knowledge of local prevalence andwe suggest to mitigate the problem of antibioticresistance is development of new antimicrobial drugs forUTIs.References1. Alon, U., Davidai, G., Berant, M. and Merzbach, D. 1987.Five-years survey of changing patterns of susceptibility ofbacterial uropathogens to trimethoprim sulfamethoxazoleand other antimicrobial agents. Antimicrob. AgentsChemother. 31: 126-128.2. Banciu, N. 2005. Preventia infectiei si higiena in mediulspitalicesc. Editura Viata Medicala Romaneasca, Bucuresti.3. Barret, S.P., Savage, M.A., Rebec, M.P., Guyot, A.,Andrews, N. and Shrimpton, S.B. 1999. Antibiotic sensitivityof bacteria associated with community-acquired urinarytract infection in Britain. J. Antimicrob. Chemother. 44: 359-365.4. Charles Bryan. 2011. Infectious disease, chapter seven,Urinary tract infections from Infectious Disease Section ofMicrobiology and Immunology On-line. University of SouthCarolina.5. Cheesbrough, M. 2004. Morphology and characterization ofE. coli and S. aureus. District laboratory practice in tropicalcountries part II. Cambridge University. pp.157-179.6. Cowan, S.T. and Steel, K.J. 1985. Manual for theidentification of medical bacteria. 4 th edn. CambridgeUniversity Press. London. p.217.7. Czaja, C.A. and Hooton, T.M. 2006. Update acuteuncomplicated urinary tract infection in women. Postgrad.Med. 119: 39-45.8. Fawole, M.O. and Oso, B.A. 1988. Laboratory manual formicrobiology, 1 st edn. Spectrum Book Ltd., Ibadan. pp.22-45.9. Florian, M.E., Wagenlehner, K.G. and Naber, S. 2006.Treatment of bacterial urinary tract infections: Presence andfuture. Euro. Assoc. Urol. 49: 235-244.10. Foxman, B., Barlow, R., D’Arcy, H., Gillespie, B. and Sobel,J.D. 2000. Self reported incidence of urinary tract infectionand associated costs. Ann. Epidemiol. 10: 509-15.11. Freeman, D.J., Falkiner, F.R. and Keane, C.T. 1989. Newmethod for detecting slime production by coagulasenegative Staphylococci. J. Clin. Pathol. 42: 872-874.12. Gruneberg, R.N. 1994. Changes in urinary pathogens andtheir antibiotic sensitivities, 1971-1992. J. Antimicrob.Chemother. 33(A): 1-8.13. Gupta, K., Scholes, D. and Stamm, W.E. 1999. Increasingprevalence of antimicrobial resistance among uropathogenscausing acute uncomplicated cystitis in women. JAMA. 281:736-738.SexMaleFemaleSlimeproducersTable 6. Slime activity of E. coli isolates according to sex and age groups.Age group (years)0-10 11-20 21-30 31-40 41-50 Above 50Negative - - - 1 - -Weak - - - 1 - -Moderate - 1 2 1 - 2Strong - 1 - - 1 -Negative - - 1 - - -Weak 1 1 - - - -Moderate 1 1 - 3 1 -Strong - - 4 - - 2TotalNo. ofpositives914% ofoccurrence92©Youth Education and Research Trust (YERT) jairjp.com Sudha et al., 2013

Journal of Academia and Industrial Research (JAIR)Volume 2, Issue 1 June 2013 6214. Hooton, T.M. and Stamm, W.E. 1997. Diagnosis andtreatment of uncomplicated urinary tract infection. Infect.Dis. Clin. North Am. 11: 551-581.15. Huang, W.C., Wann, S.R. and Lin, S.L. 2004. Catheterassociated urinary tract infections in intensive care unitscan be reduced by prompting physicians to removeunnecessary catheters. Infect. Control Hosp. Epidemiol. 25:974-978.16. Kasper, D.L., Fauci, A.S. and Longo, D.L. 2005. Harrison’sPrinciple of International Medicine. 16 th edn. New York, NY:McGraw Hill.17. Keen, E.C. 2012. Paradigms of pathogenesis: Targeting themobile genetic elements of disease. Frontiers Cell. Infect.Microbiol. 2: 161.18. Kenneth Todar. 2012. Todar’s online textbook ofbacteriology: Bacterial resistance to antibiotics, University ofWisconsin.19. Kunin, C.M. 1997. Urinary tract infections: detection,prevention, and management, 5 th edn. Williams & Wilkins,Baltimore, Md.20. Lateef, A., Oloke, J.K. and Gueguim-kana, E.B. 2004.Antimicrobial resistance of bacterial strains isolated fromorange juice products. Afr. J. Biotechnol. 3(6): 334-338.21. Lautenbach, E., Strom, B.L. and Bilker, W.B. 2001.Epidemiological investigation of fluoroquinolone resistancein infections due to extended-spectrum beta-lactamaseproducingEscherichia coli and Klebsiella pneumoniae. Clin.Infect. Dis. 33: 1288-1294.22. Maartens, G. and Oliver, S.P. 1994. Antibiotic resistance incommunity-acquired urinary tract infection. S. Afr. Med. J.84: 600-602.23. MacGowan, A.P., Brown, N.M., Holt, H.A., Lovering, A.M.,McCulloch, S.Y. and Reeves, D.S. 1993. An eight-yearsurvey of the antimicrobial susceptibility patterns of 85,971bacteria isolated from patients in a district general hospitaland the local community. J. Antimicrob. Chemother. 31:543-557.24. McManus, M.C. 1997. Mechanisms of bacterial resistanceto antimicrobial agents. Am. J. Health Syst. Pharm. 54:1420-1433.25. Modarres, S. and Oskoii, N.N. 1997. Bacteriologic agents ofurinary tract infection in children in the Islamic Republic ofIran. Eastern Med. Health J. 3(2): 290-295.26. Nair, R.T., Kalariya. and Sumitra Chanda, T. 2005.Antibacterial activity of some selected Indian medicinalflora. Durk. J. Bid. 29: 41-47.27. Neu, H.C. 1992. The crisis in antibiotic resistance. Sci. 257:1064-1073.28. Renuart, A.J., Goldfarb, D.M., Mokomane, M., Tawanana,E.O. and Narasimhamurthy, M. 2013. Microbiology ofurinary tract infections in Gaborone, Botswana. PLoS One.8(3): e57776.29. Rosenberg, M., Gutniek, D. and Rosenberg, E. 1980.Adherence of bacteria to hydrocarbons: A simple methodfor measuring cell surface hydrophobicity. FEMS Microbiol.Lett. 9: 29-33.30. Rupinder Kaur, Geeta Walia and Manika Mehta. 2012.Prevalence of Urinary tract infections in children and theirsensitivity to various antibiotics. J. Acad. Indus. Res. 1(4):161-163.31. Stamm, W. 2005. Urinary tract infections and pylonephritis,in Harrison’s Principles of Internal Medicine (16 th edn).McGraw-Hill New York.32. Vorland and Bowen, J.M. 2001. Pharmacokinetics ofsulfluramid and its metabolite desethylsulfluramid afterintravenous and intraruminal administration of sulfluramid tosheep. Pesticide Sci. 55: 719-725.33. Winstanley, T.G., Limb, D.I., Eggington, R. and Hancock,F.1997. A 10-year survey of the antimicrobial susceptibilityof urinary tract isolates in the UK: The Microbe Baseproject. J. Antimicrob. Chemother. 40: 591-594.©Youth Education and Research Trust (YERT) jairjp.com Sudha et al., 2013