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Year : 2022  |  Volume : 11  |  Issue : 3  |  Page : 200-207

Phenotypic detection of ESBL-producing Enterobacteriaceae using combined disk diffusion, ESBL HiCrome agar, and E-test: A comparative study

1 Department of Microbiology, MKCG Medical College, Berhampur, Odisha, India
2 Department of Microbiology, SCB Medical College, Cuttack, Odisha, India

Date of Submission28-Jul-2021
Date of Decision05-Oct-2021
Date of Acceptance03-Dec-2021
Date of Web Publication26-Dec-2022

Correspondence Address:
Dr. Swetalina Dash
Department of Microbiology, MKCG Medical College, Berhampur, Odisha
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jdrntruhs.jdrntruhs_105_21

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Background: Extended-spectrum beta-lactamases (ESBLs) are in a rising trend in recent years, creating confusion for physicians to choose appropriate antimicrobials for treatment.
Aim: The aim of the study is to detect ESBL-producing Enterobacteriaceae by using rapid detection tests such as combined disk diffusion, ESBL E-test strips (based on cefotaxime and cefotaxime+clavulanate), and ESBL HiCrome agar and compare the efficacy of these tests.
Materials and Methods: Samples were processed using conventional methods. Bacterial antibiotic susceptibility testing was done on Mueller-Hinton agar according to the Clinical and Laboratory Standards Institute guidelines. All Enterobacteriaceae isolates were subjected to ESBL HiCrome agar, combined disk diffusion, and E-test.
Results: Out of 5299 samples, 2097 (39.57%) were culture-positive, and 200 (9.5%) Enterobacteriaceae isolates were obtained. The majority of the isolates were Escherichia coli (67.5%), followed by Klebsiella pneumoniae (25%), Proteus mirabilis (3.5%), Proteus vulgaris (2%), and Citrobacter freundii (2%). 29.5% of all Enterobacteriaceae isolates were found to be ESBL producers by combined disk diffusion, ESBL HiCrome agar, and E-test methods, which showed 100% concordance.
Conclusion: It is important to identify ESBL-producing Enterobacteriaceae from clinical samples for the judicious use of antibiotics. For early detection of ESBL-producing Enterobacteriaceae isolates, combined disk diffusion, ESBL HiCrome agar, and E tests were found to be equally effective in detecting ESBL production.

Keywords: ESBL, E-test, HiCrome agar

How to cite this article:
Dash S, Sahu SK, Paty BP. Phenotypic detection of ESBL-producing Enterobacteriaceae using combined disk diffusion, ESBL HiCrome agar, and E-test: A comparative study. J NTR Univ Health Sci 2022;11:200-7

How to cite this URL:
Dash S, Sahu SK, Paty BP. Phenotypic detection of ESBL-producing Enterobacteriaceae using combined disk diffusion, ESBL HiCrome agar, and E-test: A comparative study. J NTR Univ Health Sci [serial online] 2022 [cited 2023 Feb 7];11:200-7. Available from: https://www.jdrntruhs.org/text.asp?2022/11/3/200/365006

  Introduction Top

Extended-spectrum beta-lactamases (ESBLs) are plasmid-mediated beta-lactamases capable of hydrolyzing and inactivating ESBLs with an oxyimino side chain such as cephalosporins (cefotaxime, ceftriaxone, and ceftazidime) and oxyimino-monobactam (aztreonam).[1] They have no detectable activity against cephamycins and carbapenems. The Clinical and Laboratory Standards Institute (CLSI) issued national guidelines for the laboratory detection of  Escherichia More Details. coli, Proteus mirabilis, and Klebsiella spp. with ESBLs.[2] Previous studies in India have reported that the incidence of ESBLs ranges from 6 to 87%.[3],[4],[5],[6],[7] Increase in infections due to ESBL-producing Enterobacteriaceae is now on the rise. ESBL-producing Enterobacteriaceae are important members of antibiotic-resistant bacteria causing hospital and community-acquired infections.[8] Gram-negative bacteria of the family Enterobacteriaceae cause mostly urinary tract infections, bloodstream infections, intra-abdominal infections, and hospital-associated pneumonia.[9] Infections caused by ESBL-producing organisms are associated with higher morbidity and mortality.[10] To date, there are >350 natural ESBL variants known, and they have been classified into 9 structural and evolutionary families based upon the amino acid sequences such as TEM, SHV, CTX-M, PER, VEB, GES, BES, TLA, and OXA.[11] Nowadays, treating ESBL-producing strains is a major challenge for physicians. Rampant use of β-lactam antibiotics such as penicillin, cephalosporins, monobactam, and carbapenems led to the emergence of their resistance.[12] Some authors suggested the use of β-lactam/β-lactam inhibitors in combination as empirical therapy for ESBL-producing Gram-negative bacteria.[13] ESBLs may not always be detected in routine susceptibility tests, and selection of antibiotic or antibiotic combination becomes difficult, and because no data regarding the prevalence of ESBLs is available in our area, the aim of this study is to detect ESBL-producing Enterobacteriaceae by using rapid detection tests such as combined disk diffusion, ESBL E-test strips (based on cefotaxime and cefotaxime+clavulanate) and ESBL HiCrome agar and compare the efficacy of these tests.

  Materials and Methods Top

Study design

The present study was a prospective, observational, and hospital-based study which was conducted over a period of two years from October 2018 to September 2020 in the Department of Microbiology, M.K.C.G. Medical College and Hospital, Berhampur, India, in collaboration with different clinical departments of the same hospital.

Study group

The study was carried out on 5299 patients attending outpatient or admitted to various departments such as the Department of Surgery, Obstetrics and Gynaecology, Medicine, Paediatrics, Orthopaedics, and I.C.U. of M.K.C.G Medical College and Hospital, Berhampur, India.

Inclusion criteria

Clinical samples, that is, urine, pus/wound swabs, blood, and body fluids were collected from patients and transferred to the laboratory without delay for further processing.

Exclusion criteria

  • Escherichia. coli isolated from the stool of adult patients where it is a commensal.
  • All other bacteria isolated other than Enterobacteriaceae.

Data collection

Patients' demographic details, brief clinical history, details of diagnosis (as made by the consultant-in-charge of the concerned department), the date of admission to the hospital, duration of stay in the hospital, and antibiotic treatment were taken.

Ethical approval

Informed consent was taken from all the patients concerned, and institutional ethical committee clearance was obtained.

Sample processing

All of the samples collected were cultured aerobically on pre-dried solid media, that is, blood and MacConkey agar. They were properly labeled with the patient ID. The cultured plates were incubated at 37°C for 48 hours and examined for growth.

Identification of the organism

Organisms were identified by colony morphology, Gram staining, biochemical reactions, and motility tests after inoculating them on routine media used in our microbiology laboratory. An antimicrobial susceptibility test was performed for all Enterobacteriaceae isolates with positive cultures according to Kirby Bauer's disk diffusion method.[14] All Enterobacteriaceae isolates were subjected to the combined disk test, ESBL HiCrome agar, and E-test for the detection of ESBL producers.

Combined disk test

Disks containing 30 μg of cefotaxime or ceftazidime and disks containing a combination of the two drugs plus 10 μg of clavulanic acid (HiMedia, Mumbai, India) were placed independently, 30 mm apart, on a lawn culture of 0.5 McFarland opacity of the test isolate on a Mueller-Hinton agar plate and incubated for 18–24 hours at 35°C. Isolates were considered ESBL-positive if the inhibition zone measured around one of the combination disks after overnight incubation was at least 5 mm larger than that of the corresponding cephalosporin disk [Figure 1]. As per CLSI 2019,[2]  Escherichia coli Scientific Name Search 22 strains and Klebsiella pneumoniae Scientific Name Search  ATCC 700603 strains were taken as the appropriate negative and positive controls, respectively.
Figure 1: Combined disk diffusion test showing a positive result for ESBL production

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Epsilometer test

The Enterobacteriaceae isolates which were positive in the confirmatory combined disk diffusion test were subjected to the E-test for the measurement of minimum inhibitory concentration (MIC) of ceftazidime and ceftazidime + clavulanic acid and cefotaxime and cefotaxime + clavulanic acid. A MIC-determining paper strip (HiMedia E strip) was coated on two sides, one side with a single drug (cefotaxime or ceftazidime) and on the other side with a combination of these drugs with clavulanic acid in a concentration gradient manner, capable of showing MICs in the range of 0.25–128 μg/ml (ceftazidime [CAZ]) and 0.25/4–128/4 μg/ml (ceftazidime + clavulanic acid [CAZ+]) and other MICs in the range of 0.25–64 μg/ml (cefotaxime [CTX]) and 0.25/4–64/4 μg/ml (cefotaxime + clavulanic acid [CTX+]) on testing against the test organism.

  • Muller-Hinton agar plates were prepared.
  • A sterile non-toxic cotton swab on a wooden applicator was dipped into the standardized inoculum and rotated firmly against the upper inside wall of the tube to express excess fluid.
  • The entire agar surface of the plate was streaked with the swab three times, turning the plates at a 60° angle between each streaking.
  • The Ezy MIC strip was removed from the container from cold and kept at room temperature for 15 minutes before opening.
  • One applicator was removed from the self-sealing bag stored at room temperature.
  • The applicator was held in the middle and gently pressed on its broader sticky side on the center of the Ezy MIC™ strip.
  • The applicator was lifted along with the attached Ezy MIC strip.
  • The strip was placed at the desired position on the agar plate swabbed with the test culture.
  • The plates were transferred into the incubator under appropriate conditions.

The isolate showing MIC reduction of ceftazidime or cefotaxime by 3 two-fold dilutions in the presence of clavulanic acid is considered as an ESBL producer [Figure 2] and [Figure 3].
Figure 2: Confirmatory E-test using CTX/CTX + E strips showing >3 2-folds decrease in the MIC of cefotaxime after adding clavulanate

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Figure 3: Confirmatory E-test using CAZ/CAZ + E strips showing >3 2-folds decrease in MIC of ceftazidime after adding clavulanate

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ESBL HiCrome agar

The test organisms were inoculated onto CHROMagar using the spread-plate technique of direct streaking and then incubated at 37°C for 18–24 hours. Colonies of ESBL producers develop species-specific colors.

The colony morphology on ESBL HiCrome agar is as follows [Figure 4] and [Figure 5]
Figure 4: HiCrome agar showing ESBL-producing blue-colored Klebsiella colonies and pink-colored Escherichia coli colonies

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Figure 5: HiCrome agar showing ESBL-producing blue-colored Klebsiella colonies and pink-colored Escherichia coli colonies and yellowish-brown colonies of Proteus spp

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ESBL-producing E coli: pink to reddish colonies.

ESBL-producing Klebsiella spp.: metallic blue colonies.

ESBL-producing Proteus spp.: brown halo colonies.

ESBL non-producers: inhibited.

  Results Top

Out of 5299 samples, 2097 (39.57%) were cultured positive and 200 (9.5%) Enterobacteriaceae isolates were obtained. A majority of the isolates were Escherichia coli (67.5%), followed by Klebsiella pneumoniae (25%), Proteus mirabilis (3.5%), Proteus vulgaris (2%), and Citrobacter freundii (2%).

The antibiotic sensitivity pattern of the Enterobacteriaceae showed that a maximum number of Enterobacteriaceae isolates were resistant to cefotaxime (82%), followed by levofloxacin (63.5%) and amoxiclav (57.5%). Most of the isolates were sensitive to piperacillin tazobactam (71.5%) and imipenem (50%) [Table 1].
Table 1: Antibiotic resistance among enterobacteriaceae isolates (n=200)

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Out of 200 Enterobacteriaceae isolates, 59 (29.5%) were found to be ESBL-positive by the combined disk diffusion method [Figure 1]. The majority of ESBL producers were found to be Escherichia coli [42 (71.1%)], followed by Klebsiella pneumoniae [13 (22%)] and Proteus mirabilis [4 (6.8%)].

All 59 ESBL isolates were subjected to the E-test [Figure 2] and [Figure 3] and HiCrome agar [Figure 4] and [Figure 5] test, and it was found that all 59 isolates showed a positive result in both tests (100%) [Table 2].
Table 2: Comparison of different phenotypic methods for detection of ESBL

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  Discussion Top

The frequency of use of antibiotics and even the dosages and period of administration vary greatly from country to country, region to region, and to some degree even in a locality. This has led to large differentials in the emergence of resistant patterns. The detection of ESBL production is of paramount importance both in hospital and community isolates. Infection-control practitioners and clinicians need the clinical laboratory to rapidly identify and characterize different types of resistant bacteria. This in turn is required to minimize the spread of these bacteria and help select appropriate antibiotics. Acquisition of efficient mobile elements results in acceleration of the transfer of various antibiotic resistance genes. Probably, a ''super bug'', resistant to relatively all licensed antibiotics, may arise in the future.[15]

In our present study, out of 5299 samples, 2097 (39.57%) were cultured positive. Out of the 2097 cultured positive isolates, 200 (9.5%) isolates were Enterobacteriaceae, which was lower in comparison to Lohani et al.'s study,[16] where, from 550 clinical samples, 343 (62.36%) were culture-positive and 157 (28.54%) Enterobacteriaceae isolates were obtained.

In our study, Escherichia coli (67.5%) was the major isolate, followed by Klebsiella pneumoniae (25%), Proteus mirabilis (3.5%), Proteus vulgaris (2%), and Citrobacter freundii (2%), similar to those reported in other studies.[10],[17],[18],[19],[20],[21],[22] However, in Chandel et al.'s study,[23] Klebsiella pneumoniae was the predominant isolate, and in Giriyapur et al.'s[24] study, Klebsiella oxytoca was the major isolate.

In this study, the maximum number of Enterobacteriaceae isolated were from urine (55%) followed by pus (30%), blood (8.5%), sputum (5.5%), and throat swab (1%), similar to those in Emmanuel et al.'s[25] and Moglad et al.'s[26] studies, where the maximum isolates were from urine (68.7% and 60.4%, respectively).

In our study, the maximum number of Enterobacteriaceae isolates were resistant to cefotaxime (82%) followed by levofloxacin (63.5%) and Amoxclav (57.5%). Escherichia coli followed by Klebsiella pneumoniae were the predominant multidrug-resistant bacteria found to be similar to those reported in Moglad et al.'s study.[26] In general, the increase in the resistance of isolated organisms to penicillin, fluoroquinolones, macrolides, and cephalosporins in this study might be due to the increase in the usage of these antibiotic classes in the hospital. We infer that a maximum number of isolates were resistant to cefotaxime (82%), which is in line with Alfola et al.'s[27] and Iroha et al.'s[28] studies, where isolates showed high resistance to cefotaxime.

Fluoroquinolones, such as ofloxacin, ciprofloxacin, levofloxacin, and others, have increasingly been used clinically because of their potent and broad antibacterial activity. In the present study, levofloxacin resistance was seen in 63.5% of isolates, which is found to be close to the finding of Mahamat et al.,[29] where it was reported as 55.34%.

The prevalence of ESBL-producing Enterobacteriaceae is found to be 29.5%, which is close to those reported in other studies carried out at different locations such as 33.2% in Nepal by Lohani et al.,[16] 38.4% in Jimma by Siraj and colleagues,[30] 36% in Jimma by Mulualem and colleagues,[31] 33.3% in Harar,[32] and 25% in Adama.[33] However, the incidence of ESBL producers such as in Bahir Dar, Ethiopia (57.6%),[34] Burkina Faso (58.0%),[35] Uganda (62.0%),[36] Ghana (49.3%),[37] and Karnataka, India (57.5%)[38] were reported to be much higher as compared to our present results. The possible reason for the high magnitude of ESBL incidence could be due to the lack of antibiotic surveillance, antibiotic misuse, and weak infection control measures.

According to the CLSI, three phenotypic methods were used in the present study, which are the combined disk diffusion test, E-test, and HiCrome agar test. It was seen that all the 200 Enterobacteriaceae were screened positive, out of which 59 isolates were confirmed to be ESBL-positive. Our present finding is in accord with that of Shakya et al.'s study,[39] where the screening test was positive for 168 isolates, out of which 36 were ESBL producers, and also with that of the observation by Yadav K et al.[40] that reported 43 confirmed ESBL-positive isolates out of 69 suspected ESBL producers.

In the present study, all phenotypic methods were 100% in concordance with each other. Earlier, a discrepancy was seen in the detection of ESBL producers in different phenotypic methods.[41] The E-test was found to be 100% sensitive comparable with Hassan et al.'s[42] report that indicated 97% sensitivity. Interestingly, it was observed that the combined disk test using cefotaxime and ceftazidime disks with and without clavulanate was 100% sensitive, whereas in other earlier studies, it was seen that using cefotaxime drug alone showed the same result.[43]

Specifically, out of 59 ESBL isolates, 42 (71.1%) were Escherichia coli, 13 (22%) were Klebsiella pneumoniae, and 4 (6.8%) were Proteus mirabilis. Escherichia coli was the predominant ESBL producer which is close to the finding of Al-Garni et al.,[44] where ESBL-producing E. coli (62.7%) were the most common, followed by K. pneumoniae (23.6%), P. mirabilis (10.8%), and others with lower rates (2.8%). Similar results were reported by Elhassan et al.,[45] who revealed that E. coli and K. pneumoniae were detected at high rates of 77.6%, and 22.4%, respectively. However, ESBL producers were reported to be different in an earlier study[16] which reported that ESBL-E. coli (52.8%) were more prevalent, followed by ESBL-K. oxytoca (27.3%) and ESBL-K. pneumoniae (14.6%), whereas only one each of Providencia spp. (1/3) and C. koseri (1/3) was ESBL-positive. Our studies showed a sensitivity of 100% in detecting ESBL producers by ESBL HiCrome agar, as observed by Prabha et al.[46] and is close to another report with a sensitivity of 94.4%.[47]

  Conclusions Top

ESBL-producing Enterobacteriaceae have disseminated worldwide, with very high prevalence in countries of low and middle income and a roughly 10% prevalence in the community and at hospital admission in high-income countries. For the judicious use of antibiotics, detection of ESBL producers is important. In this study, it is observed that for rapid detection of ESBL producers, combined disk, E-, and HiCrome agar tests are equally efficient. Restriction of extended-spectrum cephalosporin use is accompanied by switching empirical therapy for serious infections to other classes of antibacterials. The two most studied alternatives are imipenem and piperacillin/tazobactam.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

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Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2]


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