ESBL and Multidrug-Resistance Profile of Gram-Negative Urinary Isolates in a Tertiary-Care Microbiology Lab

This project is a laboratory-based cross-sectional study of the antibiotic-resistance profile of Gram-negative bacilli isolated from urine samples received in the microbiology laboratory of a tertiary-care teaching hospital. Urinary tract infection is the commonest bacterial infection seen in Indian OPDs, and Escherichia coli and Klebsiella pneumoniae producing extended-spectrum β-lactamases (ESBLs) now make empirical treatment with third-generation cephalosporins unreliable. The Indian Council of Medical Research's antimicrobial-resistance surveillance network has repeatedly reported high cephalosporin and fluoroquinolone resistance in these organisms.

The two-member team collects 100 consecutive, non-duplicate significant isolates, identifies them by colony morphology, Gram stain and a standard biochemical panel, and tests susceptibility by Kirby–Bauer disc diffusion interpreted with the current CLSI M100 breakpoints. Isolates resistant on screening are confirmed as ESBL producers by the combined-disc test (ceftazidime vs ceftazidime–clavulanate). Each isolate is classified as MDR, XDR or PDR using the international expert definitions (Magiorakos et al., 2012). Data are entered in WHONET and summarised in Excel/SPSS as frequencies, cumulative antibiogram and chi-square comparisons (OPD vs IPD, E. coli vs Klebsiella).

The output is a local antibiogram that a hospital infection-control committee can actually use, and a hands-on demonstration of the QC discipline — ATCC control strains, McFarland standards, zone reading — that separates a reliable AST report from a guess.

Abstract

A cross-sectional laboratory study of 100 consecutive, non-duplicate Gram-negative urinary isolates will be carried out over eight weeks of sample collection. Isolates will be identified by conventional methods, tested by Kirby–Bauer disc diffusion as per CLSI M100, screened and confirmed for ESBL production by the combined-disc method, and classified as MDR/XDR/PDR by standard definitions. Results will be presented as a cumulative antibiogram with 95% confidence intervals and compared across patient settings by the chi-square test.

Introduction

Urine is the single largest specimen category in most hospital microbiology labs. Clinicians frequently start empirical antibiotics before culture reports arrive, so the choice depends on local resistance data. ESBLs hydrolyse penicillins, third-generation cephalosporins and aztreonam, but are inhibited by clavulanic acid — the basis of phenotypic confirmation. Because ESBL genes travel on plasmids that often carry resistance to aminoglycosides, fluoroquinolones and co-trimoxazole, ESBL producers are frequently multidrug-resistant.

Literature gap

Published Indian studies report ESBL prevalence among urinary E. coli ranging roughly from one-third to over two-thirds depending on region and setting. Resistance changes with local prescribing, so a hospital's own antibiogram, updated regularly, is recommended by CLSI M39 and by the national AMR action plan. The study hospital has no published cumulative antibiogram for urinary Gram-negatives in the last three years.

Proposed work

• Semi-quantitative culture on CLED agar with a calibrated 1 µL loop; significant bacteriuria as ≥ 10⁵ CFU/mL.
• Identification by Gram stain, oxidase, catalase, indole, methyl red, Voges–Proskauer, citrate, urease, TSI and motility.
• AST on Mueller–Hinton agar with a panel selected from CLSI M100 for Enterobacterales.
• ESBL screening and combined-disc confirmation; MDR/XDR/PDR classification.

Feasibility

• Technical: all methods are routine in a NABL-style hospital lab; discs and media are standard stock.
• Economic: extra consumables (clavulanate combination discs, ATCC strain maintenance) cost under ₹6,000.
• Time: eight weeks of collection yields well over 100 isolates in a 500-bed hospital.
• Ethical: anonymised residual laboratory isolates; the Institutional Ethics Committee is requested to grant a waiver of consent.

Empirical treatment of urinary tract infection in Indian hospitals is increasingly failing because Gram-negative uropathogens — mainly E. coli and Klebsiella — produce extended-spectrum β-lactamases and carry co-resistance to fluoroquinolones, aminoglycosides and co-trimoxazole. Clinicians choose first-line drugs based on general guidelines or habit, while the laboratory's own susceptibility data sit in individual reports and are never summarised.

The study hospital lacks a current, CLSI-compliant cumulative antibiogram for urinary Gram-negative isolates that states ESBL prevalence and the proportion of multidrug-resistant strains. Without it, the antimicrobial-stewardship and infection-control teams cannot recommend evidence-based empirical choices for OPD and ward patients. This project generates that data set with documented quality control, so that its percentages can be trusted and repeated.

In scope

• Urine samples (midstream clean-catch and catheter) received in the hospital microbiology lab during the eight-week collection period.
• Gram-negative bacilli only; first isolate per patient.
• Phenotypic identification, disc-diffusion AST, ESBL screening and confirmation, MDR classification.
• Descriptive and comparative statistics, WHONET antibiogram.

Out of scope

• Gram-positive uropathogens and Candida.
• MIC determination by broth microdilution (except where the lab's routine automated system already reports it).
• Molecular detection of blaCTX-M, blaTEM, blaSHV or carbapenemase genes.
• Clinical outcome of patients.

Study design: laboratory-based, descriptive cross-sectional study.

Setting: Department of Microbiology of a 500-bed teaching hospital (fictional: Sri Venkateshwara Institute of Medical Sciences), eight weeks of sample collection within a 14-week project.

Sample size: using n = Z²·p·(1−p)/d² with an expected ESBL prevalence of 40% among urinary Gram-negatives (Indian literature), Z = 1.96 and absolute precision d = 10%, n = 92; rounded up to 100 isolates to allow for exclusions.

Inclusion criteria: significant growth (≥ 10⁵ CFU/mL) of a single Gram-negative bacillus, or two organisms each ≥ 10⁵ in catheter samples; first isolate per patient.

Exclusion criteria: mixed growth of three or more organisms (contamination), repeat isolates from the same patient, Gram-positive and yeast isolates, samples delayed > 2 h without refrigeration.

Procedure

1. Inoculate 1 µL urine on CLED agar and MacConkey agar; incubate at 37 °C for 18–24 h; count colonies.
2. Identify by Gram stain and the biochemical panel; record in a coded case sheet (lab number only — no patient identifiers).
3. Prepare a 0.5 McFarland suspension, lawn on Mueller–Hinton agar, apply discs (ampicillin, amoxicillin–clavulanate, cefazolin, ceftriaxone, ceftazidime, cefepime, piperacillin–tazobactam, imipenem/meropenem, gentamicin, amikacin, ciprofloxacin, co-trimoxazole, nitrofurantoin, fosfomycin for E. coli), incubate 16–18 h at 35 ± 2 °C.
4. Measure zones with a vernier caliper and interpret with CLSI M100 (edition in force).
5. ESBL screen: reduced zones to ceftazidime, cefotaxime/ceftriaxone, cefpodoxime or aztreonam. Confirm: an increase of ≥ 5 mm with ceftazidime–clavulanate (30/10 µg) over ceftazidime (30 µg).
6. QC daily: E. coli ATCC 25922 (negative control) and K. pneumoniae ATCC 700603 (ESBL-positive control); results outside CLSI QC ranges invalidate that day's run.

Statistical analysis: WHONET for antibiogram; Excel/SPSS for frequencies, percentages with 95% CI, and chi-square (or Fisher's exact) tests; p < 0.05 significant.

Ethics: proposal approved by the guide and HOD; Institutional Ethics Committee approval with waiver of informed consent because only anonymised residual lab isolates are used, consistent with the ICMR National Ethical Guidelines (2017). Biomedical waste handled as per the BMW Management Rules, 2016.