PHENOTYPIC DETECTION OF EXTENDED-SPECTRUM BETA LACTAMASES (ESBLS) PRODUCING ORGANISMS AMONG ENTEROBACTERIACEAE AND THEIR ANTIBIOTIC RESISTANCE PATTERN IN URINARY TRACT INFECTION (UTI) AT KUMAUN REGION, UTTARAKHAND.
Extended-spectrum [beta]-lactamase (ESBLs) enzymes are plasmid-mediated enzymes with capacity to hydrolyse and thus inactivate broad-spectrum [beta]-lactam antibiotics, which in turn confer a decreased susceptibility against commonly used antibiotic drugs, such as penicillins and extended-spectrum cephalosporins. 
A wide range of Enterobacteriaceae family members express Extended-spectrum [beta]lactamase (ESBLs) enzyme. The prevalence of extended spectrum beta-lactamase (ESBL) producing organisms among clinical isolates vary greatly worldwide and is rapidly changing over time. Urinary tract infection is a common bacterial disease, often contributes to a frequent cause of morbidity in outpatients as well as hospitalised patients.  Although, UTIs occur in both men and women, clinical studies suggest that the overall prevalence of UTI is higher in women. The introduction of antimicrobial therapy has contributed significantly to the management of UTIs. Still the major problem with routine antibiotic therapies is the rapid emergence of antimicrobial resistance in clinical settings and the community.  The resistance pattern of community acquired uropathogens has not been extensively studied in the Indian subcontinent. [4-6] Detection of ESBL producing organism from samples such as urine may be important, because this represents an epidemiologic marker of colonisation and therefore there is potential for transfer of such organisms to other patients.Pl The study was carried out to determine the prevalence of ESBL among the Enterobacteriaceae uropathogens and knowing resistance pattern of such multidrug resistant pathogens will help in the appropriate usage of antimicrobial agents and prevent further emergence of resistant strains.
MATERIALS AND METHODS
A Descriptive study of total 2438 urine samples were processed from symptomatic UTI cases attending or admitted to the Government Medical College and Hospital, Haldwani from November 2013 to September 2015. Urine samples were inoculated onto cystine lactose electrolyte-deficient agar (CLED agar) using a fixed volume loop (0.01 mL). Culture plates were aerobically incubated at 37[degrees]C for 18 to 24 hrs. and examined for growth of pathogenic microorganisms. Colony count of [less than or equal to] [10.sup.5] CFU/ mL was considered to be significant. The antibiotic discs (concentrations in [micro]g) included in the panel for Enterobacteriaceae isolates were ampicillin (10 [micro]g), gentamicin (10 [micro]g), amikacin (30 [micro]g), ciprofloxacin (5 [micro]g), levofloxacin (5 [micro]g), norfloxacin (10 [micro]g), nitrofurantoin (300 [micro]g) and cotrimoxazole (1.25/ 23.75 [micro]g). Ceftazidime (30 [micro]g) and ceftazidime/clavulanic acid (30 Hg/10 [micro]g) were included with antibiotics panel to screening as well as for detection of ESBL producing Enterobacteriaceae. E-test strip was applied for confirmation of ESBL producing Enterobacteriaceae isolates. The discs were provided by Hi-Media Laboratories Pvt. Ltd., Mumbai.
Tests for ESBL Production in Members of Family Enterobacteriaceae
Isolates of family Enterobacteriaceae that were considered to be positive for ESBL production by the phenotypic confirmatory disc diffusion test (PCDDT) were subjected to the E-test
1. CLSI Phenotypic Confirmation Test --The ceftazidime (30 [micro]g) discs alone and in combination with clavulanic acid (ceftazidime + clavulanic acid, 30/10 [micro]g disc) were applied onto a plate of Mueller-Hinton Agar (MHA), which was inoculated with the lawn culture of the test strain. The plates were incubated overnight at 37[degrees]C. An increase of [greater than or equal to] 5 mm in the zone of inhibition of the combination disc in comparison to the ceftazidime disc alone was considered to be a marker for ESBL production.
2. E-test--Minimum inhibitory concentration (MIC) was determined for the ESBL Enterobacteriaceae isolates by E-test using ESBL strips (HiMedia Laboratories Pvt. Ltd., Mumbai). The ESBL E-strip is a plastic drug-impregnated strip, strips were impregnated with cefotaxime (CTX) at one end and cefotaxime + clavulanic acid (CTX+) at another end. A lawn culture of the test organism was inoculated on Mueller-Hinton Agar (MHA). The E-test ESBL strip was placed on the centre of the plate. The plates were incubated aerobically at 37[degrees]C for 16-18 hours. The MIC was interpreted as the value at the intersection of the growth ellipse with the strip. The isolate was confirmed to be an ESBL producer when the ratio of the MIC value of cefotaxime to the MIC value of cefotaxime in combination with clavulanic acid was more than 8. The ESBL production was also confirmed when no zone was obtained for cefotaxime, but zone was observed in cefotaxime and clavulanic acid combination.
Escherichia coli ATCC 25922 were used as the negative control and Klebsiella pneumoniae ATCC 700603 was used as the positive control.
Date Entry and Analysis
Data entry will be done in MS Excel and Statistical analysis was done. Among the Enterobacteriaceae isolates obtained from sample, screening for ESBL producing Enterobacteriaceae was done and quantified in percentages and tabulated. Antibiotic resistance pattern of ESBL producing Enterobacteriaceae isolates obtained was quantified in percentage and tabulated.
A total of 695 clinical isolates were obtained from 2438 urine samples. Out of these 695 isolates 454 (65.32%) isolates were from Enterobacteriaceae family, out of which a total of 188 (41.4%) isolates were ESBLs producing Enterobacteriaceae.
Among the ESBL producing Enterobacteriaceae, Escherichia coli was found in maximum number of isolates. Whereas the higher prevalence detected in Klebsiella pneumoniae (65.71%) followed by Escherichia coli (40.56%), Klebsiella oxytoca (37.5%), Proteus species (34.28%), Citrobacter freundii (29.41%) and Providencia rettgeri (25%). A high degree of resistance to ampicillin and cotrimoxazole were found among these ESBL producing Enterobacteriaceae isolates (98.4% and 93.08% respectively). High degree of resistance was also seen with fluoroquinolones and gentamicin. The most susceptible drug against these strains was nitrofurantoin and amikacin, which showed 15.95% and 25% resistances respectively.
In the current era, extended-spectrum [beta]-lactamases (ESBLs) organisms are a major global problem in the clinical and community settings due to the increasing use of broad-spectrum antimicrobial agents. The total prevalence of ESBL producing Enterobacteriaceae was found to be 41.4% in our study, which was approximately equal to Tillekeratne et al  and Metri et al,  which reported the ESBL prevalence of 40.2% and 39.1% respectively in urine specimen among Enterobacteriaceae. Hanan A et al  and Shashwati et al  also reported 51% and 50% prevalence respectively. Mohanty et al  reports the higher prevalence of 68.78% for ESBL producing Enterobacteriaceae organisms. This study showed the degree of ESBL prevalence is high among Klebsiella pneumoniae (65.71%) followed by E. coli (40.56%), Proteus spp. (34.28%), Citrobacter freundii (29.41%) and Providencia spp. (25%). Afridi F et alM study showed highest frequency for ESBL production in Klebsiella species (84.61%) followed by Escherichia coli (68.55%), Enterobacter species (36.84%) and Proteus mirabilis (28.57%). Ahmed et al  reported frequency rates among Klebsiella species (40%) followed by Escherichia coli (30%), Proteus spp. (16%) and Enterobacter species (14%). Khurana et al  reported Klebsiella species prevalence of 38.5% followed by 24.7% of Escherichia coli in urinary isolates from hospitalised patients, while Mathur et al  reported 80% prevalence of Klebsiella species as a most frequent ESBL producing organism. In a study concluded by Shobha et al,  she states that Citrobacter spp. was the third most common urinary pathogen and 30% of the isolates were extended spectrum beta lactamase (ESBL) producers. The prevalence of ESBL producers was found to vary greatly in different areas of India. ESBL producing strains often arise in focal outbreaks. Regional and local estimates are probably more useful than are more global assessments in clinical decision making and for infection control purpose.  All isolates were applied for antibiotics sensitivity testing and nitrofurantoin was found to be the most effective drug. It showed 15.95% resistance to all isolates, whereas Amikacin was the second most effective drug which showed 25% resistance. Resistance to ampicillin was 98.4% followed by cotrimoxazole (93%), norfloxacin (69.14%), gentamicin (58.51%), ciprofloxacin (55.31%) and levofloxacin (40.95%). Garcia A et al  reported nitrofurantoin was a most effective drug, it showed 10.9% resistance. This study also reported 62%, 84.8% and 37% resistance to cotrimoxazole, ciprofloxacin and gentamicin respectively. Shashwati N et al  showed 50% resistance against gentamicin, 87.5% to ciprofloxacin and 94.65% to trimethoprim/ sulfamethoxazole. This study suggests that the use of these antibiotics for common illness should be avoided and the drug should be reserved as a second line drug. Nitrofurantoin, which is excreted primarily in urine and used also to treat UTI in pregnancy had also been found as the most effective drug against these isolates. Among other antibiotics, resistant isolates against norfloxacin and ciprofloxacin were found to be more than the aminoglycosides antibiotics. Therefore, aminoglycosides antibiotics can be used to treat the urinary infections on the empirical basis. ESBL producing Enterobacteriaceae are on rise and there are various factors that attribute to such kind of resistance. In this study, ESBL producing Enterobacteriaceae were studied in urinary samples and factors that can lead to rise in this type of resistance in urinary tract infections include old age, prolong bladder catheterisation, recurrent infection, long hospital stay, irrational use of antimicrobial agents, non-compliance of patient and presence of comorbidities.
Multidrug resistance was significantly higher in ESBL positive isolates. Knowledge of the prevalence of ESBLs and resistance pattern of bacterial isolates in a geographical area is of utmost importance. The sensitivity pattern of microorganisms to various antibiotics varies over time and among different geographical locations. Therefore, continuous analysis of the antibiotic resistance pattern acts as a guide in initiating the empirical treatment of UTI and the therapy must be started. Only urine culture and sensitivity have been done. It helps in avoiding the treatment failure.
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Hitendra Singh (1), Umesh (2)
(1) Assistant Professor, Department of Microbiology, Government Medical College, Haldwani, Uttarakhand.
(2) Professor, Department of Microbiology, Government Medical College, Haldwani, Uttarakhand.
'Financial or Other Competing Interest': None.
Submission 08-03-2018, Peer Review 01-04-2018, Acceptance 07-04-2018, Published 16-04-2018.
Dr. Hitendra Singh, Flat No. 301, Type 3, Government Doon Medical College Campus, Dehrakhas, Patel Nagar, Dehradun-248001, Uttarakhand.
Caption: Figure 1. A Photograph showing Production of ESBL by Phenotypic Confirmatory Disc Diffusion Test, Ceftazidime Clavulanic Acid (CAC) and Ceftazidime (CAZ)
Caption: Figure 2. A Photograph showing Production of ESBL by E-Test
Table 1. Screening of ESBL producing Enterobacteriaceae Isolates Organisms Enterobacteriaceae ESBL Producing Isolates (n) Enterobacteriaceae E. coli 355 144 Klebsiella pneumoniae 35 23 Klebsiella oxytoca 8 3 Proteus mirabilis 23 10 Proteus vulgaris 12 2 Citrobacter freundii 17 5 Providencia rettgeri 4 1 Total 454 188 Percentage Organisms (Enterobacteriaceae Isolates/ Gram Negative Bacilli) E. coli 40.56% Klebsiella pneumoniae 65.71% Klebsiella oxytoca 37.5% Proteus mirabilis 43.47% Proteus vulgaris 16.66% Citrobacter freundii 29.41% Providencia rettgeri 25.0% Total 41.4% Table 2. Antibiotic Resistance Pattern of ESBL producing Enterobacteriaceae Isolates Antibiotics Resistance Percentage (%) Isolates (n) Ampicillin 185 98.4% Gentamicin 110 58.51% Amikacin 47 25% Levofloxacin 77 40.95% Ciprofloxacin 104 55.31% Norfloxacin 130 69.14% Nitrofurantoin 30 15.95% Cotrimoxazole 175 93.08%
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|Title Annotation:||Original Research Article|
|Author:||Singh, Hitendra; Umesh|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||Apr 16, 2018|
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