Screening of Urinary Tract Infection causing bacteria and their Antibiotic sensitivity pattern in Namakkal district, TN

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1 Volume 2, Issue 1 June RESEARCH ARTICLE ISSN: Screening of Urinary Tract Infection causing bacteria and their Antibiotic sensitivity pattern in Namakkal district, TN R. Sudha 1*, M. Rajasekara Pandian 2 and B. Senthilkumar 3 1 Dept. of Zoology, Manonmaniam Sundaranar University, Tirunelveli; 2 Dept. of Zoology, Arignar Anna Government Arts College, Namakkal , TN, India; 3 Dept. of Medical Microbiology, Haramaya University, P.O. Box 235, Harar, Ethiopia Sudha @gmail.com * ; Abstract Urine samples (n=100) collected from patients in and around Namakkal hospitals were screened for Urinary Tract Infection (UTI) causing bacteria and its antibiotic sensitivity pattern. UTI causing bacterial strains were identified as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae. Antibiotic sensitivity and resistance test was carried out by disc diffusion method. The study revealed that UTI is most common in women and bacterial isolates were also found to be resistant against commercially available antibiotics. Escherichia coli strains showed 100% resistance to cefpodoxime and novobiocin and 28% to cefamandole recording the least resistance rate. Multiple resistances to β-lactams have been registered with high frequency among studied strains. The phenotypic tests for β-lactamase production indicated 100% of the tested E. coli isolates were positive. Keywords: Urinary tract infection, antibiotic sensitivity pattern, Escherichia coli, cefpodoxime, β-lactamase. Introduction Urinary Tract Infection (UTI) is a bacterial infection of any part of the urinary tract the kidneys, ureters, bladder and urethra. Usually a UTI manifestation includes pain, fever, and discomfort, but is easily treated unless it spreads to the kidneys. UTI are important because they cause acute morbidity and may result in long term medical problems, including secondary hypertension and reduced renal function. It allows identification, treatment and evaluation of the children who are at risk for kidney damage. Urinary tract is normally sterile, when bacteria moves from rectum or vagina to urethra and multiplying within urinary tract cause infection called UTI. Normally 10 5 microorganisms/ml of urine from midstream collection indicate an UTI (Modarres and Oskoii, 1997). Commonly UTI caused by gram negative bacilli E. coli without catheters (Stamm, 2005). Pseudomonas, Staphylococcus and Klebsiella also cause UTI but in case of hospital acquired, which are mainly due to catheters. Commonly UTI is caused by endogenous microorganisms from the patient s own bowel. Though E. coli and Proteus spp. are commonly and easily treated because of their, sensitivity to most antibiotics however, UTIs are increasingly caused by more resistant gram negative spp. such as Klebsiella and Pseudomonas (Huang et al., 2004). In uncomplicated UTIs, E. coli is the leading organism, whereas in complicated UTIs, gram negative and gram-positive and often muti-resistant organisms (Florian et al., 2006). A complicated UTI is dangerous and one needs severe treatment (Charles Bryan, 2011). UTIs may be acute and chronic. At first, patients experienced with severe and low back pain that may associate with fever due to the associated bacteraemia and latter, these problems lasting throughout the life. Though E. coli is common, up to 10-20% of sexually active women, UTIs are micrococcal infections (Vorland and Bowen, 2001). This infection reaches the bladder by the ascending route with the main symptoms as urinary frequency and dysuria. Klebsiella pneumoniae is also found in bowel but in low number than E. coli. Although K. pneumonia is common in nosocomial infections and cause community-acquired UTI with bacteremia. Women are more susceptible to UTI because a woman s urethra is short, allowing quick access of bacteria to the bladder and also a woman s urethral opening is near sources of bacteria from the anus and vagina. The incidence increases with age and sexual activity. Drugs can only modify the inherent functions of the concerned tissues or the cells like stimulating or depressing cellular activity, replacing deficient substances, causing irritation or killing/weakening the invading foreign organisms. Otherwise do not create new functions. Antibacterial drugs to restrains the synthesis of bacterial cell wall include β-lactams, namely penicillins, cephalosporins, carbapenems and monobactams and the glycopeptides, including vanomycin and teicoplanin (Neu, 1992; McManus, 1997). The agents of β-lactams inhibit bacterial cell wall synthesis by interfering with the enzymes required for the peptidoglycan layer synthesis.

2 Volume 2, Issue 1 June Table 1. Preliminary tests and biochemical characterization of bacterial isolates. S. aureus cocci Non-motile - K. pneumoniae Non-motile A/A,G+ P. aeruginosa rod Motile Ak/A E. coli short rod Motile A/A, G+ Isolates Gram staining Motility Catalase Oxidase Glucose Sucrose Lactose Mannitol Maltose Indole MR VP Citrate Urease Nitrate TSI MR-Methyl red; VP-Vogues Proskauer; TSI Triple sugar iron; - Acid production; G+ Gas production; Ak- Alkaline. Extended-spectrum β-lactamase (ESBL) producing E. coli are increasingly by acquisition of resistance character from resistance organism to susceptible one through genetic transformation. Because of the extensive use of antimicrobial substances the outcome out of multi-resistance strains and urinary tract infections are increasing day-by-day. Based on particular characteristics of the patients, UTIs are common in neurological pathology (Banciu, 2005). According to Keen (2012) in bacteria, virulence factors are often encoded on mobile genetic elements, such as bacteriophages and can easily spread through horizontal gene transfer. Against these backdrops, it was aimed to screen UTI causing bacteria and its antibiotic sensitivity pattern and virulence factors among patients in and around Namakkal hospitals. Materials and methods Sample collection: A total of 100 urine samples were collected in each 30 ml sterile plastic bottle from the patients 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 to Laboratory for bacteriological investigations within 4-6 h of collection. Isolation of bacteria: Culture plates of Nutrient Agar (NA) were used for the isolation of bacterial strains from urine samples. The collected urine samples were streaked directly on the labeled agar plates and incubated at 37 C for 24 h. After incubation, cultures were examined for significant growth. Subcultures were then made into plates of NA and incubated for another 24 h. Culture plates of NA, Eosin Methylene Blue Agar, MacConkey Agar and Mannitol Salt Agar (Hi media, Bombay, India) were used. Identification of bacteria: The primary identification of the bacterial isolates was made based on colonial appearance and pigmentation. Standard biochemical tests were performed to identify the bacterial strains. Characterization and identification of the isolates was done using the methods of Cowan and Steel (1985), Fawole and Oso (1988) and Cheesbrough (2004). Antibiotic susceptibility test: All isolates were subjected into antibiotic susceptibility test according to Nair et al. (2005). The susceptibility of isolates of E. coli to antimicrobial agents was examined by agar disc diffusion method. The zone of inhibition and resistance was measured, recorded and interpreted according to the recommendation of the disc manufacture.

3 Volume 2, Issue 1 June Table 2. Cultural characteristics of the bacterial isolates. Isolates No. of Eosin methylene Nutrient agar MacConkey Agar isolates blue agar E. coli 25 Whitish colonies Pink colour colonies Green-metallic sheen colonies - P. aeruginosa 14 Greenish colonies Colorless colonies - - Brown, K. pneumoniae 7 - Pink color colonies dark-centered, - mucoid colonies S. aureus 11 Whitish colonies - - Mannitol salt agar Yellow color colonies Table 3. Prevalence of E.coli among patients in and around Namakkal hospitals. Sex Age group (years) % of Above 50 occurrence Male 0/1 2/5 2/6 3/7 1/5 2/9 10/33 (30.30%) Female 2/4 2/10 5/23 3/11 1/4 2/15 15/67 (22.39%) Total % of occurrence 25 Tests for virulence factors: The prevalent bacterial strain was used for testing virulence factors by various methods. Slime activity of test isolates were confirmed by Congo red plate method according to Freeman et al. (1989). Bright orange or red colonies were considered as positive. Different intensities in the dye uptake were expressed as + (weak), ++ (moderate) and +++ (strong), whereas non-pigmented colonies were considered as non-slime producers. Microbial surface hydrophobicity was assessed with xylene according to Rosenberg et al. (1980). The degree of hydrophobicity was calculated as [1-(a1/a0)] 100(%). Here a1 is OD of microbial suspension added with xylene (OD of the aqueous phase) and a0 is OD of microbial cells in sterile phosphate-buffered saline (ph 7.1). Beta lactamase production was assayed using the method of Lateef et al. (2004). The presence of clear colorless zones around the bacterial growth is an indication of β-lactamase production. Results and discussion Totally 57% of isolates were obtained by morphological and biochemical tests from urine samples collected from patients. Standard biochemical tests were performed to identify the bacterial strains from urine samples. Characterization and identification of the isolates was done using the methods of Cowan and Steel (1985), Fawole and Oso (1988) and Cheesbrough (2004). The dominant isolates were identified as Escherichia coli (43.86%) followed by Pseudomonas aeruginosa (24.56%), Staphylococcus aureus (19.3%) and Klebsiella pneumoniae (12.28%). Table 1 and 2 shows the biochemical results and cultural characteristics of the isolated bacterial isolates. Rupinder Kaur et al. (2012) reported that E. coli (71.7%) was most common in UTI followed by K. pneumonia (15.3%), S. aureus (4.3%) and P. aeruginosa (4.3%) in her study. Escherichia coli is the predominant pathogen in UTI and there is a high chance of recurrent infection by E. coli within first 6 months (Foxman et al., 2000). Several studies revealed that UTI is associated with E. coli in 80% of cases (Kasper et al., 2005); 90% of all UTIs (Kunin, 1997); E. coli are often occur in both community and hospital acquired UTI (Gruneberg, 1994; MacGowan et al., 1993; Barret et al., 1999). Urinary tract infection is more likely to occur in women and 50-60% of adult women experience UTI during their lifetime (Foxman et al., 2000; Czaja and Hooton, 2006). In the present study, we noted a similar trend that the sex wise distribution showed inclination towards women (Table 3). The UTI prevalence is highly dependent on age and gender. The higher incidence of UTI was recorded in female (60%). In female out of 6 types of age groups, the highest prevalence was recorded in the age group of years (33.33%) and followed by (20%) and lowest in (6.67%) age groups. In case of male, the highest prevalence was recorded in years (30%) followed by 11-20, and above 50 (20%) (Table 3). The extensive use of antibiotics in the treatment of bacterial infections led to development of multi-resistance strains. Ampicillin/amoxicillin and co-trimoxazole continued to lose their sensitivity so it is better to avoid it in empiric treatment of UTI (Winstanley et al., 1997; Renuart et al., 2013). Some studies revealed that the prevalence of resistance among community of E. coli strains ranged from 5-60% for these drugs (Alon et al., 1987; Gruneberg, 1994; Maartens and Oliver, 1994; Hooton and Stamm, 1997; Gupta et al., 1999). In the present study, the resistance rates of E. coli detected from urine culture was found to be 100% against Cefpodoxime (CPD) and Novobiocin (Nv), 96% Vancomycin (Va), 88% Ceftizoxime (CZX) respectively (Table 4). Additionally, the most sensitivity rates were reported for 72% cefamandole (Cef) (Table 4). Escherichia coli isolates producing ESBLs are significantly more frequently found to be resistant to other antibiotics, in particular fluroquinolones (Lautenbach et al., 2001).

4 Volume 2, Issue 1 June Table 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 % Resistance E-1 R R R R R R R R R R I I I R R R I S S S 65 E-14 R R R R R R R R R I I I R R R R R S S S 70 E-16 R R R R I R R R R R R I R R R R R S R R 85 E-17 R R R R R R R R R R R R R R R R R S S S 85 E-18 R R R R I R R R R R R I R R R R R S R R 85 E-21 R I R R I R I R R R R R R R R R R S S S 70 E-27 I R R R I I R I R I R R R R R R I S S S 55 E-30 R R R R R R R R R I I I R R R R R S S S 70 E-40 R R R I I I R R R R R R R R S R R S S S 65 E-41 R R I I I R R R R R R R R R S R R R S R 75 E-42 R I R R I R I R R R R R R R R R R S S S 70 E-48 S I R R R I R R R I R R R R S R R R R R 75 E-58 S I R I I I R R R R R R R R R R R R S R 70 E-59 R I R R I R I R R R R R R R R R R S S S 70 E-60 I S S R S I I R R I R R R R S R I R S S 45 E-61 I S I R R R R R R I R R R R S S R R S S 60 E-65 S I R R R S R R R R I R R R R S S S S R 60 E-72 S R R R I S R I R R R R R R R S R S S R 65 E-80 S R R R R I R R R R R R R R S R R R R S 80 E-85 R I R R R S R R R R R R R R R R R S R S 80 E-86 I I R I R S R I R R I R R R S S R R R S 55 E-87 R R R R R R R R R R R R R R R S R R R S 90 E-97 S I I I I R R R R I I R R R I S R R R S 50 E-98 R I R R I R I R R R R R R R R R R S S S 70 E-99 S I R R S S R R R I R R R R R S I R R S 60 R = Resistance; S = Sensitive; I = Intermediate. In the present study, the most resistance rates of E. coli detected 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 clinically useful antibiotics except vancomycin. Present study showed 100% of the isolates were beta lactamase producers (Table 5). In case of cell surface hydrophobicity analysis, the highest percentage was obtained in female (88.54%) in 37 years of age and lowest (75.23%) in 27 years of age. In male, highest percentage (86.95%) was observed in 53 years of age and lowest (75.29%) in 35 years. Highest incidences (positive) for slime production were recorded in female (60.87%) followed by male (39.13%). In female, out of 6 types of age groups, the highest prevalence was recorded in the age group between years (28.57%) and lowest in the age group between years (7.14%) and in male, highest prevalence was recorded in age groups of 11-20, 21-30, and above 50 (22.22%) and lowest in years (11.11%) and no production in 0-10 years (Table 6). β-lactamase production indicated 100% of the tested E. coli isolates are positive and cell surface hydrophobicity test revealed 75-88% of cells in surface of xylene.

5 Volume 2, Issue 1 June Table 5. Virulence factors of E.coli isolates. Strain Sex Age Cell surface β lactamase (years) hydrophobicity E-1 F E-14 F E-16 F E-17 F E-18 M E-21 M E-27 F E-30 F E-40 M E-41 F E-42 M E-48 M E-58 F E-59 F E-60 M E-61 M E-65 F E-72 M E-80 F E-85 M E-86 M E-87 F E-97 F E-98 F E-99 F Thus, the highest incidence of virulence factors, recorded by the E. coli strains became more resistance to several antibiotics because they help the organism to overcome host defenses and colonize or invade the urinary tract. Virulence factors are very often responsible for causing disease in the host because they are often responsible for converting non-pathogenic bacteria into dangerous pathogens. Conclusion Prevalence rate of E. coli was found to be the most commonest organism for UTI in all age groups and showed resistance to commonly used antibiotics especially, 100% resistant to cefpodoxime and novobiocin. The test isolates of E. coli tested positive in β-lactamase, slime production and higher percentage in cell surface hydrophobicity showed high antibiotic resistance. But the prevalence of antimicrobial resistance can vary according to geographical and regional location. Hence, finally we recommend the selection of antibiotic should be based on knowledge of local prevalence and we suggest to mitigate the problem of antibiotic resistance is development of new antimicrobial drugs for UTIs. References 1. Alon, U., Davidai, G., Berant, M. and Merzbach, D Five-years survey of changing patterns of susceptibility of bacterial uropathogens to trimethoprim sulfamethoxazole and other antimicrobial agents. Antimicrob. Agents Chemother. 31: Banciu, N Preventia infectiei si higiena in mediul spitalicesc. Editura Viata Medicala Romaneasca, Bucuresti. 3. Barret, S.P., Savage, M.A., Rebec, M.P., Guyot, A., Andrews, N. and Shrimpton, S.B Antibiotic sensitivity of bacteria associated with community-acquired urinary tract infection in Britain. J. Antimicrob. Chemother. 44: Charles Bryan Infectious disease, chapter seven, Urinary tract infections from Infectious Disease Section of Microbiology and Immunology On-line. University of South Carolina. 5. Cheesbrough, M Morphology and characterization of E. coli and S. aureus. District laboratory practice in tropical countries part II. Cambridge University. pp Cowan, S.T. and Steel, K.J Manual for the identification of medical bacteria. 4 th edn. Cambridge University Press. London. p Czaja, C.A. and Hooton, T.M Update acute uncomplicated urinary tract infection in women. Postgrad. Med. 119: Fawole, M.O. and Oso, B.A Laboratory manual for microbiology, 1 st edn. Spectrum Book Ltd., Ibadan. pp Florian, M.E., Wagenlehner, K.G. and Naber, S Treatment of bacterial urinary tract infections: Presence and future. Euro. Assoc. Urol. 49: Foxman, B., Barlow, R., D Arcy, H., Gillespie, B. and Sobel, J.D Self reported incidence of urinary tract infection and associated costs. Ann. Epidemiol. 10: Freeman, D.J., Falkiner, F.R. and Keane, C.T New method for detecting slime production by coagulase negative Staphylococci. J. Clin. Pathol. 42: Gruneberg, R.N Changes in urinary pathogens and their antibiotic sensitivities, J. Antimicrob. Chemother. 33(A): Gupta, K., Scholes, D. and Stamm, W.E Increasing prevalence of antimicrobial resistance among uropathogens causing acute uncomplicated cystitis in women. JAMA. 281: Sex Male Female Slime producers Table 6. Slime activity of E. coli isolates according to sex and age groups. Age group (years) Above Weak Moderate Strong Weak Moderate Strong Total No. of positives 9 14 % of occurrence 92

6 Volume 2, Issue 1 June Hooton, T.M. and Stamm, W.E Diagnosis and treatment of uncomplicated urinary tract infection. Infect. Dis. Clin. North Am. 11: Huang, W.C., Wann, S.R. and Lin, S.L Catheter associated urinary tract infections in intensive care units can be reduced by prompting physicians to remove unnecessary catheters. Infect. Control Hosp. Epidemiol. 25: Kasper, D.L., Fauci, A.S. and Longo, D.L Harrison s Principle of International Medicine. 16 th edn. New York, NY: McGraw Hill. 17. Keen, E.C Paradigms of pathogenesis: Targeting the mobile genetic elements of disease. Frontiers Cell. Infect. Microbiol. 2: Kenneth Todar Todar s online textbook of bacteriology: Bacterial resistance to antibiotics, University of Wisconsin. 19. Kunin, C.M 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 Antimicrobial resistance of bacterial strains isolated from orange juice products. Afr. J. Biotechnol. 3(6): Lautenbach, E., Strom, B.L. and Bilker, W.B Epidemiological investigation of fluoroquinolone resistance in infections due to extended-spectrum beta-lactamaseproducing Escherichia coli and Klebsiella pneumoniae. Clin. Infect. Dis. 33: Maartens, G. and Oliver, S.P Antibiotic resistance in community-acquired urinary tract infection. S. Afr. Med. J. 84: MacGowan, A.P., Brown, N.M., Holt, H.A., Lovering, A.M., McCulloch, S.Y. and Reeves, D.S An eight-year survey of the antimicrobial susceptibility patterns of 85,971 bacteria isolated from patients in a district general hospital and the local community. J. Antimicrob. Chemother. 31: McManus, M.C Mechanisms of bacterial resistance to antimicrobial agents. Am. J. Health Syst. Pharm. 54: Modarres, S. and Oskoii, N.N Bacteriologic agents of urinary tract infection in children in the Islamic Republic of Iran. Eastern Med. Health J. 3(2): Nair, R.T., Kalariya. and Sumitra Chanda, T Antibacterial activity of some selected Indian medicinal flora. Durk. J. Bid. 29: Neu, H.C The crisis in antibiotic resistance. Sci. 257: Renuart, A.J., Goldfarb, D.M., Mokomane, M., Tawanana, E.O. and Narasimhamurthy, M Microbiology of urinary tract infections in Gaborone, Botswana. PLoS One. 8(3): e Rosenberg, M., Gutniek, D. and Rosenberg, E Adherence of bacteria to hydrocarbons: A simple method for measuring cell surface hydrophobicity. FEMS Microbiol. Lett. 9: Rupinder Kaur, Geeta Walia and Manika Mehta Prevalence of Urinary tract infections in children and their sensitivity to various antibiotics. J. Acad. Indus. Res. 1(4): Stamm, W 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 Pharmacokinetics of sulfluramid and its metabolite desethylsulfluramid after intravenous and intraruminal administration of sulfluramid to sheep. Pesticide Sci. 55: Winstanley, T.G., Limb, D.I., Eggington, R. and Hancock, F A 10-year survey of the antimicrobial susceptibility of urinary tract isolates in the UK: The Microbe Base project. J. Antimicrob. Chemother. 40: