MATERIALS AND METHODS

A total of 131 urine samples were collected from out patients clinically suspected to have UTI from local tertiary care hospital near Chennai between December 2009 and November 2010. The clinical and demographic details regarding the identification, age, sex of the patients were recorded. After processing the samples for significant bacteriuria, the isolates were identified by performing the routine bacteriological identification tests (Koneman et al., 1997) and their colony morphology on HiCrome UTI agar (Himedia Laboratories, India). Among the isolated strains (126), only the E. coli (58) and Klebsiella species (39) isolates identified were included in the study as they were dominant.

Antibiogram assay: A panel of thirteen antimicrobial agents comprising of β lactam and non-β lactam antibiotics (Himedia laboratories, India) were tested by the disc diffusion method against the isolates of E. coli and Klebsiella species as recommended in the CLSI guidelines (CLSI, 2005). E. coli (ATCC 25922) and K. pneumoniae (ATCC 700603) were used as reference strains throughout the study.

The antibiotics included in the study were piperacillin/tazobactam (100 10 μg^-1), imipenem (10 μg), amikacin (30 μg), gentamycin (10 μg), co-trimoxazole (1.25/23.75 μg), cefuroxime (30 μg), ceftriaxone (30 μg), nitrofurantoin (300 μg), ciprofloxacin (5 μg), ceftazidime (30 μg), ampicillin (10 μg), cefotaxime (30 μg) and tobramycin (10 μg). Any isolate was considered as multidrug resistant (MDR), if it showed resistance to ≥3 antimicrobial agents (Aggarwal et al., 2009).

Detection of ESBL production: The methods used in this study involved the testing of the isolates for ESBL production against oxyimino β-lactam antibiotics following the recommendations of Clinical and Laboratory Standards Institute (CLSI) formerly NCCLS (CLSI, 2005).

Double Disc Synergy Test (DDST): In the DDST, synergy was determined between a disc of amoxyclav (20 μg amoxycillin and 10 μg clavulanic acid) and a 30 μg disc of each 3rd generation cephalosporin (3GC) (ceftazidime 30 μg mL^-1, cefotaxime 30 μg mL^-1) placed at a distance of 30 mm apart on Mueller-Hinton agar swabbed with the resistant isolates and incubated at 37°C for 18 to 24 h. The organisms were considered to produce ESBL, if the zone size around the 3GC disc extended towards the amoxyclav disc.

Cephalosporin clavulanate combination discs: The phenotype confirmatory test for ESBL production was performed with the use of ceftazidime (30 μg), cefotaxime (30 μg) with and without clavulanic acid against the isolates. Only the isolates that showed synergy in the DDST procedure were included for the test. The discs were placed on pre inoculated Mueller-Hinton agar and incubated at 37°C for 18 to 24 h. A difference of ≥5 mm between the zone diameters of either of the cephalosporin disks and their respective cephalosporin/clavulanate disk is taken to be phenotypic confirmation of ESBL production.

Micro broth dilution method: The Minimum Inhibitory Concentration (MIC) values for ceftazidime and cefotaxime with and without clavulanic acid were determined with Mueller-Hinton broth by micro broth dilution test under sterile conditions in micro titer plates. The results were interpreted as per the CLSI guidelines. Phenotypic confirmation is considered as a ≥3-two-fold-serial-dilution decrease in MIC of either cephalosporin in the presence of clavulanic acid compared to its MIC when tested alone.

Agar supplemented with clavulanate test: Antibiotic discs containing ceftazidime (30 μg), cefotaxime (30 μg) were placed on to the Mueller Hinton agar supplemented with clavulanic acid (4 μg mL^-1) and also on regular clavulanate-free Mueller-Hinton agar plates and were incubated for 18 to 24 h at 37°C. A difference in the β-lactam zone width of ≥10 mm on the two media is considered positive for ESBL production (Paterson and Bonomo, 2005; Al-Jasser, 2006).

Disc replacement method: Two amoxyclav discs were placed on Mueller-Hinton agar inoculated with the test organisms. After one hour incubation at room temperature, the amoxyclav discs were replaced with ceftazidime and cefotaxime discs on the same spot along with control discs of ceftazidime and cefotaxime placed at least 30 mm from the replaced discs and incubated at 37°C for 18 to 24 h. A positive test is indicated by a zone increase of ≥5 mm for the discs which have replaced the amoxyclav compared to the control discs (Paterson and Bonomo, 2005; Al-Jasser, 2006).

RESULTS

Out of 131 urine samples obtained, 126 samples were considered positive for UTI. The uropathogens isolated from the UTI positive samples included E. coli (58), Klebsiella species (39), Proteus mirabilis (13), Pseudomonas aeruginosa (9), Staphylococcus species (6) and Citrobacter species (1). Only E. coli and Klebsiella species were included in this study to detect ESBL production as they were the dominant strains among the other uropathogens.

The antibiogram pattern of the isolates was analyzed. Determination of the multidrug resistance showed that 59% of E. coli and 67% of Klebsiella species were MDR. All the E. coli and Klebsiella isolates showed 100% susceptibility to imipenem. Among the ESBL positive isolates, only 37% of E. coli and 36% of Klebsiella species were susceptible to amikacin and 26% of E. coli and 29% of Klebsiella species showed susceptibility to gentamycin. The non-ESBL producing isolates showed more than 50% susceptibility to other non-β lactams antibiotics with amikacin being the most effective drug (Fig. 1). Intermediate susceptibility to other antibiotics was also detected in the isolates of E. coli and Klebsiella species.

In the DDST procedure for ESBL detection, 27 E. coli (47%) isolates and 14 Klebsiella species (36%) showed a synergy and a clear zone of extension towards amoxyclav. Interestingly, three E. coli isolates (UTEC18, UTEC31 and UTEC55) showed susceptibility to ceftazidime in the routine antimicrobial susceptibility testing but revealed a synergy indicative of ESBL production.

All the 27 E. coli and 14 Klebsiella species including the three isolates which showed susceptibility to ceftazidime were confirmed to be ESBL producers by the cephalosporin/clavulanate combination method.

The Minimum Inhibitory Concentration (MIC) values of ceftazidime and cefotaxime of the ESBL positive E. coli and Klebsiella species displayed a ≥3 log 2 (two fold) dilution reduction in the presence of clavulanic acid in the broth micro dilution method. The MIC values of ESBL positive E. coli and Klebsiella species showed a high level resistance of ≥2 μg mL^-1 to ceftazidime two fold dilution reduction of ≥1 μg mL^-1 to ceftazidime/clavulanate combination. The MIC values of the ESBL positive isolates towards cefotaxime alone were ≥64 μg mL^-1 and for cefotaxime/clavulanate combination was ≥1 μg mL^-1 (Table 1, 2).

Fig. 1:	% Susceptibility of the isolates to non-β lactam antibiotics

Table 1:	Mic values of E. coli by microbroth dilution method (≥3 log 2 (two fold) decrease in values is significant)

Table 2:	Mic values of klebsiella isolates by microbroth dilution method (≥ log 2 (two fold) decrease in values is significant)

In the agar supplemented with clavulanate test all the ESBL producing E. coli and Klebsiella species displayed a zone difference of >10 mm when tested alone and in the presence of clavulanic acid (4 μg mL^-1). In the disc replacement method, a zone difference >5 mm was observed with all the ESBL positive isolates.

DISCUSSION

UTIs occur frequently in both community and hospital environments and are the most common bacterial infections in human beings. Extremes of age, female gender, sexual activity, contraception, pregnancy, instrumentation, UT obstruction, neurologic dysfunction, previous antimicrobial use and other such factors act as predisposing factors for UTI development.

Extended spectrum cephalosporins in addition to amino glycosides are important therapeutic agents in medicine and are often used for the therapeutic management of UTIs in India. The development of drug resistance could be attributed to the indiscriminate use of the antibiotics (Akram et al., 2007) and also to the availability of some of the drugs over the counter.

It has become very essential to treat and control the infections caused by the ESBL producing pathogens and hence the need for the development of novel antibiotics and the discovery of new antibacterial agents is very urgent. A report by the Infectious Diseases Society of America (IDSA) has mentioned about doripenem’s activity to be similar to meropenem and was recently approved by Food and Drugs Administration (FDA) as a therapeutic agent for complicated urinary tract infections (Boucher et al., 2009).

The study revealed E. coli and Klebsiella species to be the dominant organisms among other uropathogens. This result coincides with the previous report on the predominance of these pathogens (Shareef and Yagoub, 2006; Selvakumar and Jasmine, 2007; Karou et al., 2009; Zinnat et al., 2011). The prevalence of ESBL producers among the 131 clinical isolates was found to be 47% with E. coli and 36% with Klebsiella species. Previous reports of ESBL occurrence among uropathogenic E. coli and Klebsiella species in India were known to be 40 and 54.54%, respectively (Aggarwal et al., 2009; Patel et al., 2010). The ESBL positive isolates of E. coli and Klebsiella species were found to be multidrug resistant. This finding is correlated with that of Bhowmick and Rashid (2004). In addition to that a higher level resistance to non-β-lactam antibiotics in addition to β-lactams in the ESBL producers than non-producers was also demonstrated. This is in accordance with the previous study in South India (Selvakumar and Jasmine, 2007). Amikacin was found to be the most effective antibiotic next to imipenem against the ESBL producers. These findings are in accordance with the previous results (Babypadmini and Appalaraju, 2004). Although, susceptibility of ESBL producers to amikacin was reported as 86% in the previous study (Babypadmini and Appalaraju, 2004) only around 36% of the ESBL positive isolates showed susceptibility to amikacin. All strains of ESBL positive E. coli and Klebsiella species showed 100% susceptibility to imipenem and these results had correlated with the findings of earlier studies (Kingsley and Verghese, 2008).

The DDST procedure was found to be a reliable and efficient method for the detection of ESBL in the isolates. Three isolates which showed susceptibility to ceftazidime in the conventional antibiogram testing were confirmed to be ESBL producers. This inhibitor based detection method is cost effective and can be easily performed in all laboratories.

The MIC values of ceftazidime and cefotaxime in all the ESBL positive E. coli and Klebsiella species displayed a ≥3 log 2 (two fold) dilution reduction in the presence of clavulanic acid by broth micro dilution method. In this study, the ESBL producing isolates exhibited high level resistance to ceftazidime (up to 64 μg mL^-1) and to cefotaxime (up to 128 μg mL^-1). Previous report also demonstrated high level resistance to the above mentioned drugs (Babypadmini and Appalaraju, 2004; Kingsley and Verghese, 2008). The method was found to be reliable for ESBL detection and also to determine the appropriate drug concentration for therapy.

The agar supplemented with clavulanate test method confirmed the ESBL production in the clinical isolates but was found to be laborious. It also suffered from a drawback in preparing the clavulanate containing agar freshly before each use (Al-Jasser, 2006) and hence could not be used in a microbiology laboratory on a routine basis. However, it can be used in conjunction with DDST or cephalosporin/clavulanate combination test for ESBL detection.

The disc replacement method was a useful test in the confirmation of ESBL production in the isolates. It was found to be easy to perform and could be used in conjunction with the DDST for routine ESBL surveillance. The findings in this study have clearly indicated the high prevalence of ESBL producing organisms in the community acquired UTI among the patients in Chennai suburban.

Prevalence of ESBL producing organisms in South India have been reported in recent years (Menon et al., 2006; Kingsley and Verghese, 2008; Kamatchi et al., 2009; Narayanaswamy and Mallika, 2011) but the information on the antibiotic resistance pattern in the community acquired UTIs in the suburbs of Chennai is scarce.

Over the past few years Chennai has seen a spurt of development, both industrial as well as residential, in certain suburban localities. The pressure of an almost unstoppable increase in population in these areas has increased the risk of communicable diseases due to factors like improper sanitation and lack of hygiene.

To our knowledge this is the first report on the presence of ESBL producing uropathogenic E. coli and Klebsiella species in the southern suburban of Chennai and may be of significance because of the potential risk of transfer of ESBL producing pathogens to others in a population.

CONCLUSION

Microbial resistance to antibiotics has gained major concern because of difficulty in treating infections caused by drug resistant strains. All the ESBL producing isolates and some non-producers included in this study were found to be multidrug resistant, few being resistant to all the antibiotics tested with the exception of imipenem. Among the other antibiotics, amikacin was found to be effective. Because ESBL production in the organisms not only confers resistance to the β-lactams but also confers co-resistance to other classes of antibiotics there is a limited option in selection of antibiotics for therapy. Most of the laboratories in the suburbs do not perform regular ESBL screening procedures. Hence it can be concluded that routine and prompt ESBL screening along with conventional antimicrobial testing should be followed in all microbiology laboratories to aid the physician in the therapeutic management of infections caused by multidrug resistant strains. Also, measures to prevent and control the spread of ESBLs are the much needed approach. An antibiotic holiday could be declared by the physicians for the first few days until accurate diagnosis is made and the patients should complete the entire course of the recommended treatment. Other measures to control the spread of drug resistance include public awareness programmes involving prudent and judiciary use of antibiotics which in turn reduce the selective pressure on organisms and surveillance of infections, prompt medical compliance must be taken. Antibiotic stewardship program to enhance the clinical outcome and to control the emergence of resistance should be followed to prevent this global problem.

ACKNOWLEDGMENT

We thank the Department of Microbiology, Vels University and Chairman, Dr. Ishari K. Ganesh for providing us the opportunities for carrying out this work. We also thank Mr. J. Vijayarangan for his kind help in preparing the graph. One of the author (RB) also thanks the Vice-Chancellor and the Registrar, Periyar University for their support.