Original article / research
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Early Detection of Drug Resistant Enterobacteriaceae in Urinary Tract Infections using Chromogenic Agar Medium in a Tertiary Care Hospital, Kakinada, Andhra Pradesh, India |
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Neerajakshi Reddi, Sobharani Sanapala, Radhika Budumuru 1. Assistant Professor, Department of Microbiology, Rangaraya Medical College, Kakinada, Andhra Pradesh, India. 2. Civil Assistant Surgeon, Department of Microbiology, ESI Hospitals, Visakhapatnam, Andhra Pradesh, India. 3. Assistant Professor, Department of Microbiology, Andhra Medical College, Visakhapatnam, Andhra Pradesh, India. |
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Correspondence
Address : Dr. Neerajakshi Reddi, Assistant Professor, Department of Microbiology, Rangaraya Medical College, Kakinada, Andhra Pradesh, India. E-mail: drneerajakshi@gmail.com |
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ABSTRACT | |||||||||||||||||||||||||||||||||||
: The irrational and inappropriate use of beta lactam antimicrobial drugs has led to the advent of Extended Spectrum Beta-Lactamase (ESBL) resistant strains. ESBL producing Enterobacteriaceae strains are frequent causative agents both in community and in acquired nosocomial infections and Urinary Tract Infections (UTI). The phenotypic confirmatory tests rarely identify all ESBLs. Chrom ID (Chromogenic identification Media) ESBL – Bx (bioMerieux) is a completely new and innovative chromogenic medium designed specifically for the screening of ESBL producing Enterobacteria directly from urine samples. It is a ready to use selective media which is sensitive and specific for rapid and presumptive identification of ESBL producing Enterobacteriaceae. Aim: To detect ESBL producing Enterobacteriaceae directly from urine samples on chromogenic medium (Chrom ID-ESBL- Bx) and confirmation of ESBL producing Enterobacteria using Disc Potentiation Test (DPT). Materials and Methods: The present cross-sectional study was conducted in the Department of Microbiology, Rangaraya Medical College, Kakinada, Andhra Pradesh, India from November 2019 to March 2020. The study was done on 70 urine samples from patients with UTI. All samples were subjected to wet mount, inoculated directly for culture on Chrom ID ESBL- Bx agar and MacConkey agar. Antibiotic Susceptibility testing of ceftazidime and cefotaxime was done by Kirby-Bauer disc diffusion method and confirmation of ESBL production by DPT using Clinical and Laboratory Standards Institute (CLSI) method. The Statistical Package for the Social Sciences (SPSS) Statistical package version (18.0) was used. Results: A total of 56 (80%) isolates were obtained from 70 urine samples, out of them 28 (50%) were Escherichia coli, 21 (37.5%) were Klebsiella spp., 7 (12.5%) were Proteus spp., 23 (82.14%) isolates of Escherichia coli, 15 (71.43%) of Klebsiella spp., 6 (85.71%) of Proteus spp., isolated were screened positive using Chrom ID ESBL-Bx agar. About 44 (78.57%) of total Enterobacteria (56) were screened for ESBL production. Total 20 (86.96%) of Escherichia coli, 11 (73.33%) of Klebsiella spp., and 5 (83.33%) of Proteus spp., were screened positive using Chrom ID ESBL agar and was confirmed (by DPT) as ESBL producers and 2 (16.6%) of total (12) isolates that were screened negative by Chrom ID ESBL agar were confirmed as ESBL producers when screened and confirmed by DPT. So sensitivity and specificity CHRO Magar was 94.73% and 55.5%. Conclusion: ESBL continues to become a serious public health threat. Results from present study showed that CHROMagar ESBL has a high sensitivity and a convenient method for making provisional diagnosis of drug resistant Enterobacterial infections in 24 hours. Chrom ID ESBL-Bx agar medium allows easy differentiation of different bacteria based on colony colouration. | |||||||||||||||||||||||||||||||||||
Keywords : Chromogenic identification media, Disc potentiation test, Extended spectrum beta-lactamase | |||||||||||||||||||||||||||||||||||
INTRODUCTION | |||||||||||||||||||||||||||||||||||
The Urinary Tract Infection (UTI) is a serious threat to human health resulting in high morbidity and mortality. UTI is common in males and females but females are more susceptible than males. Enterobacteriaceae species like Escherichia coli, Klebsiella pneumoniae, Proteus spp., Enterobacter aerogenes, Citrobacter freundii are the most common uropathogens resulting in bacteraemia and hospital acquired infections and are the common pathogens producing ESBL (1). The injudicious and misuse of beta lactam antimicrobial drugs has led to the emergence of ESBL resistant strains worldwide. ESBL producing Enterobacteriaceae strains are frequent causative agents both in community and in acquired nosocomial infections. The phenotypic confirmatory tests rarely identify all ESBLs. Chrom ID ESBL-Bx (bioMerieux) is a completely new and innovative chromogenic medium designed specifically for the screening of ESBL producing enterbacteria directly from urine samples. It is a ready to use selective media which is sensitive and specific for rapid and presumptive identification of ESBL producing Enterobacteriaceae (2). Chrom ID ESBL agar is a rich nutrient medium with a mixture of antibiotics, including cefpodoxime which is recognised as being the marker of choice for this resistance mechanism. There is incorporation of chromogenic enzyme substrate as a detection system. Chromogenic substrates consist of chromophor linked to an enzyme recognising part such as carbohydrate, aminoacids or phosphate. Specific enzymes produced by the target microorganisms will cleave to the chromogenic substrate liberating the chromophor which highlight the microorganism by coloration of the grown colony. The addition of antibiotics to the chromogenic media has been a revolution for the explicit detection of ESBL from clinical specimen. This media selectively inhibits gram positive bacteria and yeasts. It allows early identification bacteria depending on colony colour (3). This media are increasingly being used as versatile tools in early differentiation and identification of gram positive and gram negative isolates from clinical specimens (4). Chromogenic culture media have wide range of uses in diagnostic clinical microbiology and it is also being used to found carbapenamases and have a role in molecular diagnostics (5). The very good sensitivity (97%) of chrom ID ESBL agar in ESBL detection represents a convenient method for the identification and recovery of ESBL producing Enterobacteriaceae (6). The sensitivity of chrom ID ESBL agar can be increased at 48 hours of incubation and its selectivity are particularly useful in specimens containing resident associated flora (7). The aim of the present study was to detect ESBL producing Enterobacteria directly from the urine samples inoculated on chromogenic agar medium and confirmation of ESBL producing Enterobacteria using DPT among multidrug resistant pathogens causing UTI. | |||||||||||||||||||||||||||||||||||
MATERIAL AND METHODS | |||||||||||||||||||||||||||||||||||
The present study was a cross-sectional study carried out for a period of five months duration from November 2019 to March 2020 in the Department of Microbiology, Rangaraya Medical College, Kakinada, Andhra Pradesh, India. Informed consent was obtained from all the patients before collecting the sample. Ethics clearance was not required as only urine samples were taken as routine testing which was a non inversion procedure. Inclusion criteria: Urine samples from patients with UTI, clinical symptoms and cell count of more than 8 pus cells per high power field by wet mount. Exclusion criteria: Urine samples of patients who were already on antibiotic treatment were excluded from the study. Procedure Sample size: A total of 126 samples were collected from patients admitted in hospital and subjected for wet mount, 56 samples did not showed any pus cells and so were excluded for culture and total 70 samples were included in the study. Sample collection: Midstream clean catched urine samples were collected and samples were processed without delay. Direct plating on Chrom ID ESBL agar was performed in parallel on to MacConkey agar and nutrient agar. The isolates from these media were identified using various biochemical reactions (8) and then subjected to screening test for ESBL production and positive screened isolates were subjected to confirmatory test for routine confirmation of ESBL production. Identification of ESBL- producing isolates: Chrom ID ESBL agar is a rich nutrient medium with a mixture of antibiotics, including cefpodoxime which is recognised as being the marker of choice for this resistance mechanism. After inoculation on both media all culture plates were interpreted. Any colored colonies on ESBL- Bx (bioMerieux) were considered as presumptive ESBL producers. For Chrom ID ESBL, the colour and intensity of the colonies were recorded according to the colour chart provided by the manufacturer. All media were incubated at 37°C for 18-24 hours (Table/Fig 1). Escherichia coli- pink /burgundy Klebsiella/Enterobacter/Serratia- blue /green. Proteus spp.- light to dark brown. Acinetobacter spp.- cream colour Then the colonies were subjected (those screened positive) for antibiotic susceptibility testing of ceftazidime and cefotaxime and confirmation of ESBL production by DPT using CLSI method [9,10]. The isolates from MacConkey agar and nutrient agar were identified using various biochemical reactions and then were subjected to screening test (Kirby-Bauer disc diffusion test showed evidence of resistance that zone of inhibition of <22 mm with ceftazidime and cefotaxime) for ESBL production and the screened positive isolates were subjected to confirmatory test. Screening test for screened negative isolates on CHROMagar by Kirby Bauer disc diffusion test: Isolates from MacConkey agar and CHROMagar seperately prepared in suspension equivalent to 0.5 MacFarland standards were used for antibiotic susceptibility testing with following antibiotic discs. The test was conducted in accordance with Kirby-Bauer disc diffusion method. Zones of inhibition were interpreted according to CLSI guidelines (9). Controls were utilised as endorsed by CLSI. Isolates which had diameter of zone of inhibition of <17 mm with cefpodoxime and <22 mm with ceftazidime and cefotaxime were suspicious for producing ESBL and thus subjected to confirmatory test by DPT (Table/Fig 2). • Cefpodoxime [10 μg) <17 mm • Ceftazidime [30 μg) <22 mm • Aztreonam [30 μg) <27 mm • Cefotaxime [30 μg) <27 mm • Ceftriaxone [30 μg) <25 mm Confirmation test (10) by DPT: Muller Hinton agar was inoculated with an overnight culture of test strain, previously adjusted to 0.5 McFarland standard turbidity using broth or saline according to CLSI recommendations (9), ceftazidime and ceftazidime-clavulanic acid discs were placed at a distance of 10 mm from one another in the centre of the plate. The plates were examined after 18-24 hours of incubation at 37°C. The organism is said to be ESBL producer when the zone of inhibition around the ceftazidime-clavulanic acid disc is more than 5 mm compared to zone of inhibition around ceftazidime disc alone (Table/Fig 3). Escherichia coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as the control strains obtained from the department. Statistical Analysis The Statistical Package for the Social Sciences (SPSS) Statistical package version (18.0) was used. The data was entered in Microsoft excel and results were expressed in terms of frequency and percentage. | |||||||||||||||||||||||||||||||||||
RESULTS | |||||||||||||||||||||||||||||||||||
Total 56 (80%) isolates were obtained from 70 urine samples. Out of them 28 (50%) were Escherichia coli, 21 (37.5%) were Klebsiellla spp., 7 (12.5%) were Proteus spp. Total 23 (82.14%) of E. coli, 15 (71.43%) of Klebsiella, 6 (85.71%) of Proteus spp. isolated were screened positive using Chrom ID ESBL agar and 44 (78.57%) of total Enterobacteria isolated were screened positive for ESBL production (by chrom ID ESBL agar) (Table/Fig 4). Total 86.96% of E.coli, 73.33% of Klebsiella and 83.33% of Proteous spp. that were screened positive using Chrom ID ESBL agar were confirmed as ESBL producers. Total 36 (81.8%) of total isolates that were screened positive by chrom ID ESBL were confirmed as ESBL producers when screened and confirmed using disc diffusion test (Table/Fig 5). Two (16.6%) of total isolates that were screened negative by Chrom ID ESBL were confirmed as ESBL producers when screened and confirmed using disc diffusion test. (Table/Fig 6). Sensitivity and specificity of CHROM agar was 94.73% and 55.5% repectively. | |||||||||||||||||||||||||||||||||||
DISCUSSION | |||||||||||||||||||||||||||||||||||
Urinary Tract Infection (UTI) are most common hospital acquired infections and are life threatening with drug resistant microorganisms due to ESBL production. Enterobacteriaceae group of organisms are most important causative agents of drug resistant UTIs. UTIs are a serious threat to human health and affecting millions of people every year results in high morbidity and mortality (1). So the present study highlights that early detection of ESBL producing enterobacters from urine samples by directly inoculating on new Chrom ID agar can give the quick report to the patient. Methods to detect ESBL producing organisms from clinical samples should have high sensitivity and specificity (2). The most common isolates in the present study were Escherichia coli (50%), followed by Klebsiella spp. (37.5%) and Proteus spp. (12.5%) which was similar to study done by Ahmed NF et al., (3) (E.coli- 48.3%, Klebsiella 10%, Proteus-11.7%) and also with the study of Sharmin S et al., (4). In the present study, 78.57% of Enterobacteria were screened positive for ESBL production using Chrom ID agar when compared to study of Prabha R et al., (1) where 35% of ESBL producers, study by Glupczynski Y et al., (2) (29.7% of ESBL producers), study by Blane B et al., (11) (39% of ESBL producers) and 35% of ESBL producers were seen in the study by Uyanga FZ et al., (12) so more percent (78.5%) of Enterobacteria were screened positive for ESBL production when compared to the other studies. In the present study 86.96% of E.coli, 73.33% of Klebsiella spp. and 83.33% of Proteus spp. that were screened positive using Chrom ID ESBL agar were confirmed as ESBL producers. So 81.82% of ESBL producers were confirmed positive with DPT correlating with the study of Teklu DS et al., (10) (84.5% of screened ESBL positive Enterobacteria were confirmed as ESBL producers) and two of total isolates that were screened negative by CHROMagar were confirmed as ESBL producers when screened and confirmed by CLSI guidelines. So sensitivity is 94.73% and specificity is 55.5%. Sensitivity (94.73%) in the present study coincide with the study of Ahmed NF et al., (91.7%) (3), Glupczynski Y et al., (97.7%) (2), Tuchilus C et al., (97%) (6), and Uyanga FZ et al., (98%) (12). Specificity (55.5%) in the present study correlating to study of Tuchilus C et al., (6) (66%) and lesser than (may be due to other mode of resistance mechanism in enterobacteria, regional variation and concerning the low specificity, all suspected ESBL producing strains should be verified with additional tests) the study of Glupczynski Y et al., (89%) (2), Ahmed NF et al., (100%) (3). In current study, ESBL producers were isolated more from inpatients were 86% (samples collected before the use of antibiotics ) whereas, in the study of Davoodabadi A et al., (13), 68.16% of urinary isolates of Escherichia coli were from communities. Chrom ID ESBL medium permitted the differential growth of many with ESBL and carbapenemases and is a potential medium to detect many other resistant bacteria (14). In recent studies, HiCromUTI agar is also using as a primary urine culture media (15). CHROMagar TM Serratia Chromogenic medium is also available detection of carbapenemases producing Serratia marcescens and given an advantage of its implementation in nosocomial outbreaks (16). The highest sensitivity showed that chrom ID ESBL agar is a suitable method for making provisional diagnosis of drug resistant enterobacterial infections in 24 hours. Concerning the low specificity, all suspected ESBL producing strains should be verified with additional tests. Limitation(s) One of the limitation of CHROM agar included lack of difficulty in distinguishing Candida species and other enteric microbes like Shigella spp. and small sample size of the study. | |||||||||||||||||||||||||||||||||||
CONCLUSION | |||||||||||||||||||||||||||||||||||
Extended Spectrum Beta-Lactamase (ESBL) continues to become a serious public health threat and accounts for high morbidity and mortality. Results from this study showed that CHROMagar ESBL has a high sensitivity and a convenient method for making provisional diagnosis of drug resistant Enterobacterial infections in 24 hours and so appropriate treatment can be given to the patients by which we can reduce the drug resistant strains. | |||||||||||||||||||||||||||||||||||
ACKNOWLEDGEMENT | |||||||||||||||||||||||||||||||||||
Authors are thankful to the Department of Microbiology, Government General Hospital, Kakinada, for providing financial and necessary facilities to complete this study. | |||||||||||||||||||||||||||||||||||
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