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BRIEF COMMUNICATION
Year : 2015  |  Volume : 33  |  Issue : 2  |  Page : 282-285
 

Frequency of extended spectrum β-lactamase producing urinary isolates of Gram-negative bacilli among patients seen in a multispecialty hospital in Vellore district, India


1 Sri Sakthi Amma Institute of Biomedical Research, Sri Narayani Hospital and Research Centre, Sripuram, Vellore, Tamil Nadu, India
2 Department of Microbiology, Sri Narayani Hospital and Research Centre, Sripuram, Vellore, Tamil Nadu, India
3 Sri Sakthi Amma Institute of Biomedical Research, Sri Narayani Hospital and Research Centre, Sripuram, Vellore, Tamil Nadu; Department of Microbiology, Sri Narayani Hospital and Research Centre, Sripuram, Vellore, Tamil Nadu, India
4 Department of Urology, Sri Narayani Hospital and Research Centre, Sripuram, Vellore, Tamil Nadu, India
5 Department of General Surgery, Sri Narayani Hospital and Research Centre, Sripuram, Vellore, Tamil Nadu, India

Date of Submission10-Jan-2014
Date of Acceptance02-Sep-2014
Date of Web Publication10-Apr-2015

Correspondence Address:
S Sankar
Sri Sakthi Amma Institute of Biomedical Research, Sri Narayani Hospital and Research Centre, Sripuram, Vellore, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.153563

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 ~ Abstract 

Extended-spectrum beta-lactamase (ESBL) producing strains of Coliform bacilli are on the rise and present a major threat especially in India. We assessed the frequency of ESBL producers among urinary isolates from patients presenting urinary tract infections. ESBL screening was done using Double Disk Synergy Test (DDST) and confirmed using E-test and Polymerase Chain Reaction (PCR). With E-test, 92.2% were positive for ESBL. In PCR, 100% strains were positive for any of the three gene targets tested. CTX-M was positive in majority of the strains followed by TEM and SHV. Two (3.22%) strains were positive for all the three genes; 21% strains were positive for both TEM and CTX-M genes. There was no statistically significant difference in the findings of E-test and PCR testing in the determination of ESBL producers (Fisher exact test P = 0.15). The strength of agreement between them was 'fair' (k = 0.252). Continuous monitoring of ESBL producers among Indian strains is important to rationalize the antibiotic policy to be followed.


Keywords: E. coli, extended spectrum β-lactamase, resistance, susceptibility, uropathogens


How to cite this article:
Nandagopal B, Sankar S, Sagadevan K, Arumugam H, Jesudason M V, Aswathaman K, Nair A. Frequency of extended spectrum β-lactamase producing urinary isolates of Gram-negative bacilli among patients seen in a multispecialty hospital in Vellore district, India. Indian J Med Microbiol 2015;33:282-5

How to cite this URL:
Nandagopal B, Sankar S, Sagadevan K, Arumugam H, Jesudason M V, Aswathaman K, Nair A. Frequency of extended spectrum β-lactamase producing urinary isolates of Gram-negative bacilli among patients seen in a multispecialty hospital in Vellore district, India. Indian J Med Microbiol [serial online] 2015 [cited 2019 May 19];33:282-5. Available from: http://www.ijmm.org/text.asp?2015/33/2/282/153563



 ~ Introduction Top


Extended-spectrum beta-lactamase (ESBL) producing strains of Gram-negative bacilli are on the rise and present a major threat especially in India. Urinary tract infection (UTI) is one of the most common bacterial infections encountered as both hospital- and community-acquired infections. [1] The course and outcome of an infection is affected with the limited therapeutic options available. [2] Uropathogens differ in terms of the virulence factors and pathogenic mechanisms that allow them to colonize and infect the urinary tract leading to hospital and community acquired infections. [3] To the best of our knowledge, only few studies exist in India on the evaluation of E-test along with Polymerase Chain Reaction (PCR) assay in the detection of ESBL producers. The present study was undertaken to assess the frequency of ESBL producers among urinary isolates to firstly confirm the validity of disk diffusion findings, secondly, to ascertain the genetic basis of ESBL production among strains tested and thirdly, to obtain information on the correlation between E-test and PCR.


 ~ Materials and Methods Top


The study was carried out at Sri Narayani Hospital and Research Centre between January 2013 and March 2013. The study was approved by Institutional ethical committee and Institutional Review Board.

Patients with suspected urinary tract infections (UTI) were recruited for the study, and clean catch mid-stream urine samples were obtained. Patients' demographic details and hospitalization status (In- and outpatient) were obtained. The urine samples were processed immediately or within two hours of collection. E-test and PCR were carried out on culture isolates in batches.

In all, 348 urine specimens were collected from the same number of patients. The samples were processed and antimicrobial susceptibility testing was performed using standard microbiology laboratory procedures. [4] Isolates (n = 100) recovered from 92 patients gave either significant or probably significant growth and were resistant to cephalosporins. ESBL detection in uropathogens was carried out by using the Kirby-Bauer disk diffusion technique as a screening test. E-test was carried out to ascertain the MIC value of the antibiotics on the ESBL strains. PCR was performed to identify the ESBL type.

ESBL testing

Screening was done by using Double Disk Synergy Test (DDST), as per CLSI guidelines (2010). Ceftazidime (30 μg) disks with and without clavulanic acid (10 μg) were used. E-test was performed on MH agar according to the manufacturer's instructions (Etest ESBL, AB Biodisk, Solna, Sweden).

Polymerase chain reaction

The plasmid DNA was extracted from the ESBL-producing  Escherichia More Details coli isolates using plasmid mini kit (HiYield plasmid mini kit, Real Biotech Corporation, Banqiao, Taiwan). The extracted Plasmid DNA was stored at –20°C until use.

All the plasmid DNA samples were subjected to uniplex PCR using primer sets each specific for TEM, SHV and CTX-M genes (George et al. In Press). The PCRs were carried out in an Eppendorf thermal cycler (Mastercycler ® personal 5332, Hamburg, Germany). Negative controls replacing the template with nuclease free water were included in every assay. An aliquot of 5 μL amplicon was analyzed by gel electrophoresis: TEM-1080 bp, SHV-471 bp and CTX-M-544 bp. Statistical analysis was carried out using Epi Info 7.1.2.


 ~ Results Top


Information on patients in relation to age and length of hospitalization (in-patients) is shown in [Table 1]. Among the 100 isolates, 64 (64%) isolated from 62 patients were positive for ESBL, majority were E. coli. The age of the patients (n = 92) ranged from 1 year to 89 years and the median was 54.5. Among the patients, 27 were females and 65 were males; the difference in ESBL-positive status between them was not statistically significant (χ2 = 1.88, P = 0.17). Out of 92 patients, 40 were inpatients and 52 were outpatients. The difference in ESBL-positive rate of the strains between them was not significant (χ2 = 0.18, P = 0.67).
Table 1: Information on patients who were harboring ESBL positive strains


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Among the inpatients, four were from high dependency units (ICU and CCU), of which three were positive for ESBL by E-test and PCR. Clinical information of the patients with uropathogens in mid-stream urine samples was shown in [Table 2]. The ESBL-positive strains were also found resistant to other antimicrobials tested and this includes ciprofloxacin (n = 57), gentamicin (n = 41), cefoperazone-sulbactum (n = 6) and nitrofurantoin (n = 12). Among the antimicrobials tested, ESBL-producing strains showed highest susceptibility (9.68%) to cefoperazone-sulbactum. The 64 phenotypically ESBL-positive strains were further tested by E-test ESBL strips for confirmation and by uniplex PCR using primers specific to CTX-M, TEM and SHV genes.
Table 2: Clinical information of the patients with uropathogens in mid - stream urine samples


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Among 64 strains tested, 55 (85.9%) were E. coli, 4 (6.2%) were Enterobacter spp., 3 (4.7%) were Klebsiella spp. and one each of Proteus mirabilis (1.6%) and Pseudomonas aeruginosa (1.6%). With E-test ESBL strips, 59 (92.2%) were positive for ESBL. PCR results indicated positive for CTX-M in 60 (93.7%) strains followed by TEM in 15 (23.4%) and SHV in 3 (4.7%) strains. Two (3.1%) strains (one E. coli and one Enterobacter spp.) were positive for all the three genes, 13 (20.3%) strains were positive for both TEM and CTX-M genes. In all, 62 (96.9%) strains were positive for any of the three gene targets tested. Among the five E-test negative strains, 3 were positive by PCR for CTX-M, one positive for TEM and the other was negative by PCR as well.

There was no statistically significant difference in the findings of E-test and PCR testing in the determination of ESBL producers (Fisher exact test - two-tailed P = 0.15). The kappa coefficient between the two test was 0.252; the chance expected agreement and the observed agreement were 0.89 and 0.92 (SE = 0.11; z = 2.26; P = 0.01), respectively. The confidence interval (95%) ranged between -0.189-0.694, and the strength of agreement was considered to be 'fair'.


 ~ Discussion Top


Sixty-four strains were positive for ESBL on screening with ceftazidime and ceftazidime-clavulanic acid by Double Disk Synergy test. Majority of the patients (33.7%) presented with UTI alone (no underlying abnormalities); of these, 87.1% were positive for ESBL-producing strains. This was followed by urethretic/ureteric/prostate-related abnormalities, Urethral/ureteric/renal calculi and diabetes as underlying complications. With E-test ESBL strips, 59 (92.2%) were positive for ESBL. PCR results indicated CTX-M in 96.8% of the strains followed by TEM in 24.2% and SHV in 4.8% of the strains. Two (3.22%) strains were positive for all the three genes; 13 (21%) strains were positive for both TEM and CTX-M genes. In PCR, all 62 (96.87%) strains were positive for any of the three gene targets tested.

To the best of our knowledge, only a few studies exist in India on the evaluation of E-test along with PCR assay in the detection of ESBL producers. Using E-test to detect the presence of ESBL resulted in a false-positive outcome in 14% of the cases resulting in unnecessary isolation of patients (barrier nursing) that could have been omitted by using a genotypic method. Our study demonstrates the usefulness of E-test because it detected almost all ESBL positives compared to PCR outcome.

Kaur and Aggarwal [5] reported 59% of their E. coli strains as possessing CTX-M genes. Many of the Klebsiella isolates were positive for 2 or all 3 genes. In our study, CTX-M was positive in 98.2% (54/55) of E. coli strains. TEM was positive in 15 strains including 12 E. coli, 2 Klebsiella and 1 Enterobacter spp. Occurrence of SHV was low (4.7%) and found in two Enterobacter spp. and an E. coli strain. Two of these SHV positive strains co-occurred with CTX-M and the other strain was positive for all three genes tested. Manoharan et al. [6] reported TEM and CTX-M to be predominant in E. coli (39.2%) while, among the Klebsiella spp., TEM, SHV and CTX-M occurred together in 42.6% of the isolates.

Among our in-patients, out of four E. coli strains recovered from high dependency units (ICU and CCU), three were ESBL positive. This indicates the need of intense surveillance among critical care patients and institution of appropriate measures to contain the spread of resistant pathogens causing hospital-acquired infections.

Compared with the DDST, the inhibitory potentiated disk diffusion and E-strip tests were found to be preferable methods for detecting ESBLs, with better sensitivity (100%) and specificity (97.6%) and positive predictive values (97.3%).

The British Society of Antimicrobial Chemotherapy (http://bsac.org.uk/) and the Health Protection Agency of the United Kingdom (http://www.hpa.org.uk/) suggest testing all isolates of E. coli or Klebsiella spp. with ceftazidime (the best indicator for TEM and SHV-derived ESBLs) and cefotaxime (the best indicator for CTX-M types) or with cefpodoxime (a good indicator for all ESBL types) as a first screening test. Giriyapur et al. [7] showed ceftazidime to have a better sensitivity and specificity as a screening agent for efficient detection. In our study, only ceftazidime disks were used both for screening by DDST and E-test.


 ~ Conclusions Top


E-test ESBL strips, 92.2% were found positive for ESBL. PCR results indicated a majority to be positive for CTX-M (93.7%) followed by TEM (23.4%) and SHV (4.7%). In all, 62 (96.9%) strains were positive for any of the three gene targets tested. There was no statistically significant difference in the findings of E-test and PCR testing. Any one of these methods may be used to verify the findings of disk diffusion test. Monitoring for ESBL producing strains of coliform bacteria would reveal information on the magnitude of the problem and is important to rationalize the antibiotic policy to be followed in the country.

 
 ~ References Top

1.
Saleem M, Daniel B. Prevalence of urinary tract infection among patients with diabetes in Bangalore city. Int J Emerg Sci 2011;1:133-42.  Back to cited text no. 1
    
2.
Fennell J, Vellinga A, Hanahoe B, Morris D, Boyle F, Higgins F, et al. Increasing prevalence of ESBL production among Irish clinical Enterobacteriaceae from 2004 to 2008: An observational study. BMC Infect Dis 2012;12:116.  Back to cited text no. 2
    
3.
Tabibian JH, Gornbein J, Heidari A, Dien SL, Lau VH, Chahal P, et al. Uropathogens and host characteristics. J Clin Microbiol 2008;46:3980-6.  Back to cited text no. 3
    
4.
Myers RM, Koshi G. Manual of diagnostic procedures in medical microbiology and immunology/serology. Vellore: Christian Medical College and Hospital; 2000. p. 137-62.  Back to cited text no. 4
    
5.
Kaur M, Aggarwal A. Occurrence of the CTX-M, SHV and the TEM genes among the extended spectrum ß-lactamase producing isolates of enterobacteriaceae in a tertiary care hospital of North India. J Clin Diagn Res 2013;7:642-5.  Back to cited text no. 5
    
6.
Manoharan A, Premalatha K, Chatterjee S, Mathai D, SARI Study Group. Correlation of TEM, SHV and CTX-M extended-spectrum beta lactamases among Enterobacteriaceae with their in vitro antimicrobial susceptibility. Indian J Med Microbiol 2011;29:161-4.  Back to cited text no. 6
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7.
Giriyapur RS, Nandihal NW, Krishna BV, Patil AB, Chandrasekhar MR. Comparison of disc diffusion methods for the detection of extended-spectrum Beta lactamase-producing enterobacteriaceae. J Lab Physicians 2011;3:33-6.  Back to cited text no. 7
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