Co-ordination of ANTIBIOTIC RESISTANCE AND BIOFILM FORMATION
February 24, 2017
ANTIBIOTIC RESISTANCE AND BIOFILM FORMATION IN EXTENDED SPECTRUM BETA-LACTAMASE AND NON-EXTENDED SPECTRUM BETA-LACTAMASE PRODUCING UROPATHOGENIC ESCHERICHIA COLI
Department of Goldengate international collage,Battisputali, kathmandu, Nepal and
Shree Birendra Hospital, Chhauni, Kathmandu
Uropathogenic Escherichia coli (UPEC) are the predominant causative organisms of urinary tract infections (UTI). Extended-spectrum beta-lactamases (ESBLs) confer resistance to broad spectrum beta-lactam antibiotics and they are commonly expressed by Enterobacteriaceae but the species of organisms producing these enzymes are increasing. This research provides information on UPEC, ESBL, biofilm formation and their antibiotic susceptibility pattern of E. coli from UTI patients. The study was carried out in Shree Birendra Hospital, Chhauni, Kathmandu between May to October 2013 with an objective to determine biofilm formation among ESBL producer and non producer UPEC, from UTI suspected patients. During the study, the E. coliisolated were tested for the presence of ESBL by combination disk method and biofilm detection was done by congo red agar method. Out of 1560 samples, 218 (13.98%) was found to be culture positive E.coli. Among 33% ESBL positive E. coli, 30.7% were strong and moderate biofilm producer, 15.3% weak producer and 23.61% non biofilm producer. Among uropathogens, Amikacin was found the most effective drug (87.2% sensitive), followed by Nitrofurantoin (72%), Gentamicine (61.9%) and amoxicillin was least effective (10.6% sensitive). Among 52.29% biofilm producing E. coli, maximum resistance was observed in Cephalexin where 97.8% strong producers, 90.7% moderate producer, 92.3% weak producer were resistant followed by Amoxicillin, 95.6%, 88.4%, 92.3% and 86.5% strong producer, moderate producer, weak producer were resistant. Amikacin was found to be leastresistanc among biofilm producers.17.8%, 25.6%, 15.4% and 4.8% strong producer, moderate producer, weak producer and non biofilm producer respectively were resistance to Amikacin. The ability of biofilm formation was found higher among ESBL producing than non ESBL producing strains of E. There was high tresistance rate to antimicrobial agentsamong ESBL and biofilm producingE.colisolates.
Keywords: Escherichia coli, Extended spectrum beta lactamase, Biofilm
Most urinary tract infections arise from one type of bacteria, with Escherichia coli, the primary etiologic agent among both outpatients and inpatients, accounting for 80 to 85% cases (Nicolle, 2008; Bhatta et al., 2012; Sahm et al., 2001). Antibiotic therapy which is the mainstay of treatment is dependent on a number of factors such as the predominant pathogens in the patient’s age group, antibacterial sensitivity patterns in the practice area, the clinical status of the patient, the opportunity for close follow-up, and of course, cost of treatment (Ibadin et al., 2004). The extensive and inappropriate use of antimicrobial agents has invariably resulted in the development of antibiotic resistance which, in recent years, has become a major problem worldwide (Goldstein et al., 2000). In community and hospital settings, the etiology of UTIs and the antimicrobial susceptibility of UTI causing bacteria have been changing over the years (New, 1996). Over the last decade, the treatment of choice for urinary tract infections (UTIs) has changed from cotrimoxazole to quinolone owing to the rate of resistance to cotrimoxazole and its high level of therapeutic failure (Yilmaz et al., 2005). Resistant to newer and more potent antimicrobials are no exceptions, making the therapeutic options very limited to certain antimicrobial agents like carbapenem, colistin and fosfomycin (Yilmaz et al., 2005). Extended-spectrum beta-lactamases (ESBLs) are enzymes that can hydrolyze oxyimino-beta-lactams (e.g., Cefotaxime, Ceftazidime and Ceftriaxone) and the monobactam (Aztreonam) resulting in resistance to these drugs (Patricia, 2011). ESBLs have been found most commonly in uropathogens, like E. coli and K. pneumonia (Bush and Jacoby, 2010).
Biofilm formation is a strategy employed by several microorganisms for survival in adverse conditions. The bacterial biofilm is defined as a structured community of bacterial cells enclosed in produced polymeric matrix and adherent to an inert or living surface (Costeron et al., 1999). Uropathogenic strains of E. coli account for 70-95% of the UTIs. Bacteria that invade the bladder cells and grow into structural colonies known as biofilms may be responsible for many recurrent UTIs (Ward et al., 2003). The bacteria enclosed within the biofilm are extremely resistant to treatment. This may be because the drug concentrations obtained may be insufficient in certain areas of the biofilm. The bacteria located at the base of the biofilm are metabolically inactive and are thus resistant to certain antibiotics and possess active antibiotic degradation mechanisms that contribute to avoid the accumulation of an effective drug concentration (Costern et al., 1999).
Methodology This study was conducted prospectively at Shree Birendra Hospital, Kathmandu from May to October 2013. Total of 1560 CC-MSU urine samples from UTI patients were considered for the study. Urine samples were processed using semi quantitative method. All samples were cultured in CLED agar and incubated at 37oc for 24 hours and Escherichia coli were confirmed using various biochemical tests.
Antibiotic susceptibility testing
The antimicrobial susceptibility testing of the isolates towards various antimicrobial discs was done by modified Kirby-Bauer disk diffusion method as recommended by Clinical and Laboratory Standards Institute (CLSI) using Muller Hinton Agar (MHA).
Extended Spectrum beta-lactamase (ESBL) detection test
The indicator antibiotics Ceftazidime (30μg) and Cefotaxime (30μg) were used for the screening of ESBL test. It was done by modified Kirby-Bauer disc diffusion method and zone of diameter read using CLSI guideline. An inhibition zone of ≤ 22mm for Ceftazidine and ≤ 27mm for Cefotaxime was suspected of ESBL production i.e. ESBL screening test positive. These organisms were then subjected for confirmatory ESBL detection test.
The isolates suspected as ESBL producing strains in the screening test were subjected to the phenotypic confirmatory test by combined disk diffusion method on Muller Hinton Agar. Ceftazidime (30 μg) and Ceftazidime-clavulanic acid (30 μg/ 10μg) disks were placed 20 mm apart. After incubating overnight at 37°C, ≥ 5 mm increase in the zone diameter for either antimicrobial agent which was tested in combination with clavulanic acid versus its zone when tested alone and interpreted as positive for ESBL production.
Biofilm production testing by Congo Red Agar (CRA) method
Biofilm detection test was done for both ESBL positive and negative strains of E. coli by Congo Red Agar method. This is the qualitative method to detect biofilm. CRA medium was prepared with Brain Heart Infusion (BHI) broth (37 g/L), supplemented with 5 percent sucrose (50 g/L), agar (10 g/L) and Congo Red indicator (8 g/L) (Freeman et al.,1989; Reid, 1999; Hassan et al., 2011).
First Brain Heart Infusion Agar (BHIA) with sucrose was prepared as a concentrated aqueous solution and autoclaved at 1210C for 15 minutes.Then Congo Red indicator was added to the autoclaved BHIA with sucrose at 55ºC, mixed well, poured in petri plates and left sometime to solidify.Isolates of E. coli strain were then streaked on the surface of the CRA medium and incubated aerobically at 370C for 24-48 hours.Black colored colonies with dry crystalline consistency was interpreted as positive biofilm producing strains and red colored colonies interpreted as negative for biofilm production.
On the basis of the intensity of color change of Congo Red Agar medium after inoculation of the organisms which is directly proportional to the amount of biofilm produced by the organisms, the organisms were classified in the four biofilm categories: strong producer (+++), producer (++), weak producer (+) and non producer (-) and interpreted as such (Poovendran et al., 2013; Christensen et al., 1982)
Antibiotics group Antibiotics used Total no. of E. coli isolates Suscteptibility Pattern
No. (%) No.%
Fluroquinolone Ciprofloxacin 218 132(60.6) 86(39.4)
Ofloxacin 218 118(54.1) 100(45.9)
Norfloxacin 218 134(60.5) 84(38.5)
Aminoglycoside Gentamicin 218 83(38.1) 135(61.9)
Amikacin 218 28(12.8) 190(87.2)
Sulphonamides Cotrimoxazole 218 118(54.1) 100(45.9)
Beta lactams Amoxicillin 218 195(89.4) 23(10.6)
Cephalexin 218 184(84.4) 34(15.6)
Cefixime 218 165(75.7) 53(24.3)
Ceftazidime 218 127(58.3) 91(41.7)
Cefotaxime 218 122(56) 96(44)
Ceftriaxone 218 104(47.7) 114(52.3)
Cefepime 218 107(49.1) 111(50.9)
Nitrofurans Nitrofurantoin 218t 61(28) 157(72)
Out of 1560 MSU samples 297, (19%) samples showed monomicrobial significant growth (i.e. ≥ 105 CFU/ml), 1263 (81%) samples showed no growth and 33 (2.1%) samples showed polymicrobial insignificant growth (i.e. more than two types of isolates).
Antibiotic susceptibility pattern of E.coli
Among the 218 E. coli isolates, Amikacin was found to most effective (87.2%) followed by Nitrofurantin (72%), Gentamicin (61.9%). Ceftriaxone (52.3%) and Cefepime (50.9%). Similarly Amoxicillin was found to be least sensitive (10.6%). The results are shown in Table above.
Antibiotic susceptibility pattern of ESBL producing and non producing uropathogenic E. coli
The antibiotic susceptibility pattern in ESBL producer and non producer E. coli was found to be as follows: the high percentage of sensitivity to Amikacin (77.8 vs 91.8%) followed by Nitrofurantoin (65.3% Vs 75.3%) were observed among ESBL producers and non producer. Similarly, maximum percentage of resistance to cephalosporin group i.e. Cephalexin and Ceftazidime among ESBL producer and non producer as shown in table
Antibiotics group Antibiotics used ESBL Producer (N=72) ESBL Non producer (N=146)
No (%) Resistance
No (%) Sensitive
No (%) Resistance
Fluroquinolones Ciprofloxacin 12 (16.7) 60 (83.3) 74 (50.7) 72 (49.3)
Ofloxacin 12 (16.7) 60 (83.3) 88 (60.3) 58(39.7)
Norfloxacin 11(15.3) 61 (84.7) 73 (50) 73 (50)
Aminoglycoside Gentamicin 27 (37.5) 45 (62.5) 108 (74) 38 (26)
Amikacin 56 (77.8) 16 (22.2) 134 (91.1) 12 (8.2)
Sulphonamides Cotrimoxazole 14 (19.4) 58 (80.6) 86 (58.9) 60 (41.1)
Beta lactams Amoxicillin 1 (1.4) 71 (98.6) 22 (15.1) 124 (84.9)
Cephalexin 0 (0.0) 72 (100) 34 (23.3) 112 (99)
Cefixime 0 (0.0) 72 (100) 53 (36.3) 93 (63.7)
Ceftazidime 0 (0t.0) 72 (100) 91 (62.3) 55(37.7)
Cefotaxime 0 (0.0) 72 (100) 96 (65.8) 50 (34.2)
Ceftriaxone 6 (8.3) 66 (91.7) 108 (74) 38 (26)
Cefepime 8 (11.1) 64 (88.9) 103(70.5) 43 (29.5)
Nitrofurans Nitrofurantoin 47 (65.3) 25 (34.7) 110 (75.3) 36 (24.7)
Association of ESBL production and biofilm formation among E. coli isolates
Out of 72 ESBL producing uropathogenic strains of E. coli, 22 (30.56%) were strong and moderate biofilm producer, 11 (15.27%) were weak producer and 17 (23.01%) showed no biofilm formation. Whereas among 167 non ESBL producing E. coli, 23 (15.75%) were strong biofilm producer, 21 (14.38%) were moderate biofilm producer and 15 (10.27%) were weak biofilm producer. Similarly, 87 (59.8%) were both ESBL and biofilm negative. The least number (15.27%) of ESBL positive were weak biofilm producer and 30.56% of ESBL positive were strong and moderate biofilm producer respectively. The correlation between ESBL production and biofilm formation by E. coli isolates was found statistically significant (p<0.05).
ESBL Biofilm production Total Correlation
Strong producer Moderate producer Weak producer Non producer
No % No % No % No %
Producer 22 30.56 22 30.56 11 15.27 17 23.61 72
Non producer 23 15.75 21 14.38 15 10.27 87 59.58 146
Total 45 20.64 43 19.72 26 11.9 104 47.7 218
Antibiotic Resistance pattern of Escherichia coli among Biofilm producer and non producer
In this study, the antibiotic resistance pattern of biofilm producing E. coli was found significantly higher than that of non biofilm producing E. coli. Among strong biofilm producer 75.55% (34/45), moderate producer 76.7% (33/43),and 73.1% (19/26) among weak producer and only 44.2% (46/104) among non biofilm producer were found to be Ciprofloxacin resistant whereas 60.6% (132/28) was found to be Ciprofloxacin resistant in total E. coli isolates. The correlation between biofilm production and antibiotic resistance pattern was found statistically significant (p<0.05) in most of the antibiotics (Ciprofloxacin, Ofloxacin, Norfloxacin, Amikaci, Gentamicin, Cotrimoxazole, Cephalexin, Cefixime, Ceftazidime, Cefotaxime, Ceftriaxone and Cefepime) and nonsignificant in case of, Amoxicillin and Nitrofurantoin.
Antibiotics used Resistance pattern of Biofilm producer and non producer
Strong producer N=45 Moderate producer N=43 Weak producer N=26 Non producer N=104
No (%) No (%) No (%) No (%) No (%)
Ciprofloxacin 32 (76.2) 32 (76.2) 17 (70.8) 45 (45.0) 126 (60.6)
Ofloxacin 30 (71.4) 30 (71.4) 14 (58.3) 39 (39.0) 113 (54.3)
Norfloxacin 33 (78.6) 32 (76,2) 16 (66.7) 45(45.5) 126(60.6)
Gentamicin 24 (57.1) 26 (61.9) 6 (25.0) 26 (26.0) 82 (39.4)
Amikacin 7 (16.7) 11 (26.2) 3 (12.5) 5 (5.0) 26 (12.5)
Cotrimoxazole 27 (64.3) 29 (69,0) 17 (70.8) 42 (42.0) 115(55.3)
Amoxicillin 40 (95.2) 37 (88.1) 22 (91.7) 87 (87.0) 186 (89.4)
Cephalexin 41 (97.6) 38 (90.5) 22 (91.7) 74 (74.0) 175 (84.1)
Cefixime 37 (88.1) 41 (97.6) 20 (83.3) 59 (59.0) 157 (75.5)
Ceftazidime 31 (73.8) 36 (85.7) 15 (62.7) 40 (40.0) 122 (58.7)
Cefotaxime 34 (81.1) 34 (81.0) 14 (58.3) 35 (35.0) 117 (56.2)
Ceftriaxone 29 (69.0) 28 (66.7) 15 (62.5) 28 (28.0) 100 (48.1)
Cefepime 31 (73.8) 27(64.3) 12 (50.0) 33 (33.0) 103 (49.5)
Nitrofurantoin 12 (28.6) 11 (26.2) 6 (26.0) 28 (28.0) 57 (27.4)
Out of 1560 mid stream urine samples processed, 1263 (81%) urine samples showed no significant growth and 297 (19%) were culture positive. A similar study conducted by Shrestha et al. (2007) showed the growth pattern (22.7%) and 29% positivity done by Asati and Sadawarte (2013) also support this study. In the study done by Kolawole et al. (2009) and Patel et al. (2012) showed growth positivity 60% and 46.48% respectively. The low growth positive rate observed in this study might be due to the samples from patients under treatment or for routine check up only.
In this study, the overall resistance of E. coli to antimicrobial agents was high. E. coli was found to (87.2%) sensitive to Amikacin, followed by Nitrofurantoin (72%), Gentamicin (61.9%) and least effective to Amoxicillin (89.4%) resistant followed by Cephalexin (84.4%), Cefixime (75.7%). Comparison of antibiotic sensitivity of E. coli of present study with study done by Girishbabu et al. (2013); Raza et al. (2011) showed that E coli was having good sensitivity to, Amikacin, Nitrofurantoin and Gentamicin in all of these studies. In the present study, Nitrofurantoin was found to be the second most effective drug against uropathogens and it has no role in the treatment of other infections, it can be administered orally and is highly concentrated in urine; it may therefore be the most appropriate agent for empirical use in uncomplicated UTI (Sood and Gupta, 2012).
Out of 218 uropathogenic Escherichia coli, 72 (33.2%) were found to be ESBL positive and rest of them, 146 (66.97%) were ESBL negative. In the present study, Amikacin was found to be 77.8% and 91.8% sensitive among both ESBL producer and non producer E. coli followed by Nitrofurantoin (65.3% Vs 75.3%), Gentamicin (37.5% Vs 74%), Ciprofloxacin (16.7% Vs 50.7%), Ofloxacin (16.7% Vs 60.3%), Norfloxacin (15.3% Vs 50%), Cotrimoxazol (19.4% Vs 58.9%) . 98.6% ESBL producing E. coli showed resistance to Amoxicillin whereas 84.9% non ESBL producer were resistant. Similarly, high degree of resistance by ESBL producing E. coli than non ESBL producing E. coli to Cephalosporin group of antibiotics used under study, viz. Cephalexin (100.0% Vs 99%), Ceftazidime (100.0% Vs 37.7%), Cefotaxime (100% Vs 34.2%), cefixime (100% Vs 63.7%) and Ceftriaxone (91.7% Vs 26%) and cefepime (88.9% Vs 29.5%). The findings of this study agrees with the various studies done by Wani et al. (2009); Kalaskar and Venkataramana (2012); Chatterjee et al. (2012); Vaidya (2011).
ESBLs are enzymes that mediate resistance to extended-spectrum (third generation) cephalosporins (e.g., Ceftazidime, Cefotaxime, and Ceftriaxone) and monobactams but do not affect cephamycins (e.g., Cefoxitin and Cefotetan) or Carbapenems (e.g., Meropenem or Imipenem). In addition, ESBL-producing organisms frequently show cross-resistance to many other classes of antibiotics; including aminoglycosides and fluoroquinolones, thus treatment of these infections is often a therapeutic challenge (Black et al., 2005). Extended spectrum beta-lactamase production was observed in both third generation cephalosporin susceptible and resistant groups which were in agreement with Aggarwal et al. (2009). Bacterial drug resistance may be attained through intrinsic or acquired mechanisms such as by mutations in genes targeted by the antibiotic and the transfer of resistance determinants borne on plasmids, bacteriophages, transposons, and other mobile genetic material. In general, this exchange is accomplished through the processes of transduction (via bacteriophages), conjugation and transformation. As a result of these changes, microorganisms produce various factors such as ESBL and biofilm to inactivate the used antibiotics (Alekshun and Levy, 2007; Davies and Davies, 2010).
In the present study the ability of biofilm formation by Escherichia coli isolates was determined by Congo Red Agar method. Among 218 E. coli isolates, 114 (52.29%) were biofilm producer. Among these 45 (20.6%) were found to be strong biofilm producer, 43 (19.7%) moderate producer, 26 (11.9%) weak producer and 104 (47.7%) were found to be non biofilm producer. This finding agrees with the finding of different authors from different parts of the world. Mathur et al. (2006) investigated 152 isolates for biofilm production and found 14.4% isolates strong biofilm producers, 39.4% moderate producers and 46% non biofilm producers. According to Hanna et al. (2003) biofilm formation protects bacteria from hydrodynamic flow conditions, for example in the urinary tract and against phagocytosis and host defense mechanisms, as well as antibiotics. Costerton et al. (1999) have reported that more than 50% of all bacterial infections involve biofilm formation. Similarly Nair et al. (2013); Anandkumar et al. (2012); Rijavec et al. (2008) have reported 67%, 46% and 56% positive for biofilm production respectively.
Among biofilm producing E. coli, higher antibiotic resistance was observed in strong and moderate biofilm producer. Maximum resistance of antibiotic by biofilm producing strain was observed in Cephalexin where 97.8% strong producers, 90.7% moderate producer, 92.2% weak producer and 74.0% non biofilm producer were resistant to Cephalexin followed by Amoxicillin 95.6%, 88.4%, 92.3% and 86.5% strong producer, moderate producer, weak producer and non biofilm producer. Amikacin was found to be most sensitive among biofilm producers. 17.8%, 25.6%, 15.4% and 4.8% strong producer, moderate producer, weak producer and non biofilm producer respectively were resistance to Amikacin.
In this study, the antibiotic resistance pattern of biofilm producing E. coli was found significantly higher than that of non biofilm producing E. coli. The association between biofilm production and antibiotic resistance was found to be statistically significant except in case of Amikacin, Amoxicillin and Nitrofurantoin. Microorganisms growing in a biofilm are intrinsically more resistant to antimicrobial agents than planktonic cells. High antimicrobial concentrations are required to inactivate organisms growing in a biofilm, as antibiotic resistance can increase 1,000 fold (Thien-fah and George, 2001; Stewart and Costerton, 2001). This statement supports the higher antibiotic resistance among the biofilm producing E. coli in this study.
In the present study, 72 (33%) isolates of E. coli producing ESBL and 146 (67%) non- ESBL producing isolates were compared for their ability to form biofilm. Significant difference was observed among ESBL positive and ESBL negative isolates with regard to biofilm formation ability. The isolates were classified according to Poovendran et al. (2013) and Christensen et al. (1982) to three groups as strong producer 44 (21.2%) (+++), tmoderate producer 42 (20.2%) (++) and weak producer 25 (12%) (+) on the basis of intensity of color change of the Congo Red Agar medium which is directly proportional to the amount of biofilm produced.
Out of 72 ESBL producing uropathogenic E. coli isolates, 55 (76.38%) showed biofilm formation such as, 22/72 (30.56%) were strong and moderate biofilm producer, 11/72 (15.27%) were weak biofilm producer and 17/72 (23.61%) showed no biofilm formation. Out of 146 non ESBL producing E. coli, 59 (40.4%) showed biofilm producer, of which 23/146 (15.8%) were strong biofilm producer, 21/146 (14.38%) were moderate producer, 15 (10.27%) were weak biofilm producer and 87 (59.6%) showed no biofilm formation. When comparing the potential of ESBL and Non ESBL producer among uropathogenic E. coli with regard to the biofilm formation, it was found that 30.7% Vs 15.8% ESBL and Non ESBL producing uropathogenic E. coli exhibited strong biofilm formations, 30.7% Vs 14.4% exhibited moderate biofilm formations and 15.3 Vs 10.3 exhibited weak biofilm formation. Similarly, 87 (59.6.6%) were both ESBL and biofilm negative. This shows that the ESBL producer have greater ability to form biofilm among UPEC their by increase the antibiotic resistance (Poovendran et al., 2013; Anandkumar et al., 2012).
Among cephalosporin antibiotics, Ceftriaxone was found to be most effective i.e. 52.3% sensitive followed by cefepime (50.9%). Higher resistance rate than this study was observed in the study done by Gautam et al. (2013). The findings of study done by Asti and Sadawarte, 2013 also agrees with the present study. Ciprofloxacin, Ofloxacin, Norfloxacin and Cotrimoxazol were found to be 60.6%, 54.1%, 61.5% 54.1% resistant to E. coli respectively. Lower degree of resistance rates to Nitrofurantoin (3.6%), Norfloxacin (9.4%), Gentamicin (20.4%) and Ciprofloxacin (28.4%) were documented by Kibret and Abera, (2011) from northest Ethiopia in contrast of this study but Amoxicillin was found to be equally resistance in both studies. Comparison of antibiotic sensitivity of E. coli of present study with studiy done by Girishbabu et al. (2013) shows that E. coli was having good sensitivity to Amikacin, Nitrofurantoin and Gentamicin in both studies. In present study, E. coli is less sensitive to Norfloxacin, Cefotaxime and Cotrimoxazole in comparison to antibiotic sensitivity in above study.
The main findings of this study are that there was significant difference in prevalence of UTI in male and female patients (P<0.05). The study findings showed that E. coli was the most predominant uropathogens isolated. The ability of biofilm formation was found higher among ESBL producing than non ESBL producing strains of E. coli. The correlation of biofilm formation with ESBL production by E. coli was found statistically significant (P<0.05). The status of significant bacteriuria was high in the patients of age group 21-30 years. The study showed that Amikacin was found the most effective drug against E. coli. Among ESBL and biofilm producing E. coli isolates there was high resistance rate to almost all the antimicrobial agents except a few, and alarmingly high resistance rate was observed against beta-lactam antibiotics.
1. Routine surveillance and monitoring studies should be conducted to know about different virulence facters like ESBL and Biofilm which are responsible for antibiotic resistance.
2. Amikacin, Gentamycin and Nitrofurantoin can be used for the treatment UTIs caused by ESBL and Biofilm producing organisms.
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