INTRODUCTION
Urinary Tract Infection (UTI) is one of the most common infectious diseases
in humans (Mohammadi et al., 2010) that occur
in both community and hospital environments. It presents a spectrum of clinical
entities upon severity ranging from asymptomatic infection to acute pyelonephritis
with sepsis (Fish, 2009).
The etiology of most of the uncomplicated bacterial UTI is by the pathogens
such as E. coli, K. pneumoniae, P. mirabilis, E. faecalis,
S. saprophyticus and those of complicated UTI by E. coli, K. pneumoniae,
E. faecalis, P. aeruginosa and species of Enterobacter,
Staphylococcus, Serratia and Acinetobacter. Among bacterial UTIs,
E. coli is the most prevalent cause (Manikandan et
al., 2011; Sibi et al., 2011) accounting
for greater than 80% of the infections and appears to be a true community pathogen
(Pitout and Laupland, 2008).
UTIs are often treated with different broad-spectrum antibiotics (Akram
et al., 2007) like penicillin and cephalosporins. However the emerging
resistance among the uropathogenic strains, effective therapy with the above
mentioned antibiotics has been hindered.
This resistance towards β-lactam antibiotics is mainly mediated by the
production of a diverse set of enzymes, β- lactamases which are capable
of hydrolyzing the β-lactam ring (Bradford, 2001).
The increased use of second and third generation cephalosporins to treat infections
caused by such resistant strains has led to the development and selection of
multiple resistant organisms (Pitout and Laupland, 2008).
The production of a set of enzymes called the Extended Spectrum Beta Lactamases
(ESBLS) may result in the development of multidrug resistance (Babypadmini
and Appalaraju, 2004).
There are diverse types of ESBLs which include TEM, the first plasmid-mediated
β lactamase in gram negative bacteria named after the patient Temoniera
in Greece. SHV is another plasmid-mediated β-lactamase named for sulphydryl
variable and CTX that preferentially hydrolyze cefotaxime. OXA is another β-lactamase
belonging to molecular class D and functional group 2 day possess hydrolytic
activity against oxacillin and cloxacillin. Other β-lactamases like PER
that efficiently hydrolyzes penicillins and cephalosporins, VEB, a β-lactamase
isolated from a patient from Vietnam, GES for Guiana extended spectrum (Lee
et al., 2007) and IBC β-lactamases. The IBC-1 enzyme is a novel
integron-associated class A extended-spectrum β-lactamase that hydrolyses
all β-lactams except the cephamycins and the carbapenems (Bradford,
2001; Thomson, 2010). These infections are associated
with increased morbidity and mortality (Aggarwal et al.,
2009; Mansouri et al., 2011).
In India, the prevalence of ESBLs have been reported since 1990s (Revathi
and Singh, 1997; Karim et al., 2001; Mathai
et al., 2002; Gupta, 2007; Aggarwal
et al., 2009; Narayanaswamy and Mallika, 2011)
and very few reports of them existing in South India have been published (Babypadmini
and Appalaraju, 2004; Menon et al., 2006;
,Kingsley and Verghese 2008; Mano
and Vasanthi, 2008; Kamatchi et al., 2009;
Narayanaswamy and Mallika, 2011). Reports on the etiology
and resistance pattern of community acquired UTIs in the rapidly developing
Chennai Suburban are scarce. The objective of this study was to analyze the
antibiotic susceptibility pattern and to find out the prevalence of ESBLs among
uropathogenic E. coli and Klebsiella species isolated from
patients with community-acquired UTIs from a tertiary care hospital in the southern
suburban of Chennai.
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.
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