Antimicrobial resistance (AMR) has emerged as one of the most serious global health challenges of the 21st century, causing significant morbidity, mortality, prolonged hospitalization, and increased healthcare costs. Among bacterial pathogens, Escherichia coli and Klebsiella pneumoniae are particularly important due to their ability to acquire and disseminate multiple resistance mechanisms, including Extended Spectrum β-Lactamases (ESBLs) and AmpC β-lactamases. These enzymes confer resistance to broad-spectrum β-lactam antibiotics, traditionally considered first-line therapy for Gram-negative infections.¹,²

ESBL-producing Enterobacterales hydrolyze third-generation cephalosporins such as cefotaxime, ceftazidime, and ceftriaxone and are inhibited by clavulanic acid.³ ESBLs are commonly plasmid-mediated and associated with co-resistance to other antibiotic classes, including aminoglycosides, fluoroquinolones, and trimethoprim-sulfamethoxazole, leading to multidrug-resistant (MDR) phenotypes.⁴,⁵ These plasmids often carry additional resistance genes, facilitating rapid spread both in hospitals and in the community.⁶

AmpC β-lactamases represent another clinically important resistance mechanism. They confer resistance to cephamycins (cefoxitin and cefotetan) and are not inhibited by β-lactamase inhibitors such as clavulanic acid.⁷ AmpC enzymes may be chromosomal or plasmid-mediated; plasmid-mediated AmpC (pAmpC) is particularly concerning due to its ability to spread between species, including E. coli and K. pneumoniae.⁸,⁹ The presence of AmpC enzymes often leads to treatment failure with cephalosporins, masking ESBL production and complicating laboratory detection.¹⁰

The prevalence of ESBL and AmpC producers has increased worldwide, particularly in Asia, Africa, and Latin America.¹¹,¹² India is recognized as a hotspot for antimicrobial resistance due to extensive antibiotic usage, overcrowding in healthcare settings, and limited antimicrobial stewardship practices.¹³ Several studies from India have reported ESBL rates ranging from 40–70% in E. coli and K. pneumoniae, and AmpC rates from 10–25%, highlighting the growing threat.¹⁴,¹⁵

Clinically, ESBL and AmpC producing E. coli and K. pneumoniae cause a wide range of infections including urinary tract infections, wound infections, septicemia, pneumonia, and intra-abdominal infections.¹⁶ Their increasing prevalence limits treatment options, often necessitating the use of carbapenems, which in turn drives the emergence of carbapenem-resistant Enterobacterales (CRE), further complicating management.¹⁷,¹⁸

Accurate laboratory detection of ESBL and AmpC β-lactamases is essential for appropriate antimicrobial therapy and infection control. Phenotypic tests such as combined disc diffusion, double-disc synergy testing (DDST), cefoxitin screening, and AmpC disk methods are the most commonly used, economical, and reliable diagnostic tools in resource-limited settings.¹⁹,²⁰ Regular surveillance of resistance mechanisms is crucial for guiding empirical therapy and preventing the spread of resistant organisms.²¹

Given the increasing burden of β-lactamase mediated resistance and its clinical implications, the present study was undertaken to determine the prevalence and antimicrobial susceptibility patterns of ESBL and AmpC β-lactamase producing Escherichia coli and Klebsiella pneumoniae isolated from various clinical samples at a tertiary care hospital.

MATERIALS AND METHODS

Study Setting

This study was conducted in the Department of Microbiology, Navodaya Medical College & Research Centre, Raichur, from July 2024 to June 2025.

Sample Collection and Processing

Specimens were collected aseptically and transported to the laboratory promptly. Samples not processed immediately were stored at room temperature.

A total of 200 non-repetitive isolates (140 Escherichia coli, 60 Klebsiella pneumoniae) were recovered from urine, pus, blood, sputum/ETA, and body fluids.

Samples were inoculated onto:

• MacConkey agar
• Blood agar
• Chocolate agar

Plates were incubated aerobically at 37°C for 24 hours. Identification of isolates was performed using standard microbiological procedures (1).

Antimicrobial Susceptibility Testing

Antibiotic susceptibility was tested on Mueller–Hinton agar using the Kirby–Bauer disc diffusion method. Interpretation followed CLSI guidelines.

• Screening for ESBLs and AmpC β-lactamases

As per CLSI recommendation, isolates showing resistance (zone ≤ 22 mm for ceftazidime and ≤ 25 mm for ceftriaxone) by disc diffusion method were considered potential ESBL producers and further preceded for confirmation.9

Isolates showing resistance to cefoxitin (inhibition zone < 18 mm) by disc diffusion method were considered potential AmpC producers and further tested for presence of AmpC β-lactamase enzyme by AmpC disk test.(1)

• Detection of ESBLs and AmpC β-lactamases
• The Phenotypic Confirmatory Disc Diffusion Test (PCDDT)

All strains that were potential ESBL producers were subjected to confirmation using the PCDDT as recommended by CLSI.(13) disc of cefotaxime (30 μg) and ceftazidime (30 μg) alone and a disc of cefotaxime/clavulanic acid (30 μg/10 μg) and ceftazidime/clavulanic acid (30 μg/10 μg) were placed independently 30 mm apart center to center on a lawn culture of 0.5 McFarland turbidity of the test isolate on Mueller-Hinton Agar (MHA) plate and incubated for 18-24 hours at 350 C. A ≥5 mm increase in zone diameter for either antimicrobial tested in combination with clavulanic acid versus its zone when tested alone confirmed ESBL production .

• Double Disc Synergy Test

A 0.5 McFarland suspension of the test isolate was swabbed on MHA plate and 30 μg antibiotic discs of ceftazidime, ceftriaxone and cefotaxime were placed on the plate 15 mm (center to center) from the amoxicillin/clavulanate (20 μg/ 10 μg) (augmentin) disc and incubated at 370C for 18-24 hrs. Clear extension of the edge of the inhibition zone of any of these cephalosporin discs towards the augmentin disc was interpreted as positive for ESBL production

• AmpC Disk Test

Lawn cultures of ATCC E. coli 25922 were prepared on MHA plate and a 30 μg cefoxitin disc was placed on the inoculated surface of the agar. A sterile plain disc moistened with sterile saline (20 μL) and inoculated with several colonies of the test organism was placed besides the cefoxitin disk almost touching it. After overnight incubation at 35°C, the plates were examined for either an indentation or a flattening of the zone of inhibition indicating enzymatic inactivation of cefoxitin (positive result) or the absence of a distortion indicating a negative result.14

• Quality Control

Every batch of media prepared was checked for sterility for 24 hours. CLSI reference strains of ESBL positive K. pneumoniae ATCC 700603 and ESBL negative E. coli ATCC 25922 were included in the study.

STATISTICAL ANALYSIS: All collected data were entered into Microsoft Excel and analyzed using SPSS version 20.0. Results were expressed as percentages (%), frequencies, and proportions. Comparison of ESBL and AmpC production between E. coli and K. pneumoniae was performed using the Chi-Square (χ²) test. Antibiotic resistance patterns of ESBL vs. non-ESBL and AmpC vs. non-AmpC producers were compared using cross-tabulation and chi-square tests. Confidence intervals (95% CI) were calculated wherever necessary. p-value < 0.05 was considered statistically significant.

RESULTS

A total of 200 non-repetitive clinical isolates were included in the present study, consisting of 140 (70%) Escherichia coli and 60 (30%) Klebsiella pneumoniae. E. coli constituted the majority of isolates (70%), indicating its predominance as a clinical pathogen across different samples as shown in Table 1

Table 1: Distribution of Clinical Isolates (n = 200)

ESBL production was more common in E. coli (42.8%) compared to K. pneumoniae (36.6%). The difference was statistically not significant (p > 0.05), although E. coli showed a slightly higher ESBL rate as shown in Table 2

Table 2: Prevalence of ESBL Producers

Statistical Test: χ² = 0.72, p > 0.05 → Not significant

AmpC production was significant in both organisms, with K. pneumoniae showing slightly higher AmpC positivity (21.6%) than E. coli (18.5%). The difference was not statistically significant as shown in Table 3

Table 3: Prevalence of AmpC Producers

Statistical Test: χ² = 0.28, p > 0.05 → Not significant

Combined ESBL and AmpC production was detected in both organisms. Co-production was slightly higher in K. pneumoniae (11.6%) compared to E. coli (8.5%) as shown in Table 4

Table 4: ESBL + AmpC Co-production

Urine yielded the highest number of ESBL producers.Pus/wound samples had the highest AmpC positivity as shown in Table 5

Table 5: Distribution of ESBL and AmpC Producers Based on Sample Type

Highest resistance to cephalosporins (80–90%). Moderate resistance to fluoroquinolones (60%). High sensitivity to carbapenems (98%). Nitrofurantoin was highly effective against urinary E. coli isolates as shown in Table 6

Table 6: Antimicrobial Susceptibility Pattern of E. coli (n = 140)

Klebsiella isolates showed higher resistance than E. coli for most antibiotics, reflecting their stronger resistance mechanisms (AmpC, ESBL, efflux pumps) as shown in Table 7

Table 7: Antimicrobial Susceptibility Pattern of K. pneumoniae (n = 60)

ESBL producers showed significantly higher resistance to fluoroquinolones and aminoglycosides (p < 0.05) as shown in Table 8

Table 8: Antibiotic Resistance in ESBL vs. Non-ESBL Isolates

*Significant at p < 0.05

AmpC producers showed significantly higher resistance to cefoxitin and aminoglycosides as shown in Table 9

Table 9: Resistance in AmpC vs Non-AmpC Producers

DISCUSISON

In the present study, E. coli (70%) emerged as the predominant isolate, followed by K. pneumoniae (30%), which is consistent with previous reports identifying E. coli as the leading cause of urinary and soft-tissue infections in both community and hospital settings (22,23). Its widespread prevalence is likely due to its natural gastrointestinal colonization and its enhanced ability to acquire resistance determinants (24).

ESBL Prevalence

The overall ESBL prevalence of 41% aligns with earlier findings from India and other developing nations, where rates between 35–60% are commonly observed (23,24). ESBL production was slightly higher in E. coli (42.8%) than K. pneumoniae (36.6%), though the difference was not statistically significant. Similar patterns have been reported by Paterson and Bonomo, who documented higher ESBL occurrence in E. coli across various clinical settings (25). Shaikh et al. also highlighted E. coli as a major ESBL producer globally, reinforcing our results (26).

The high ESBL rate is concerning because these enzymes hydrolyze third-generation cephalosporins and frequently coexist with resistance to other antibiotic classes.

AmpC Prevalence

AmpC producers constituted 19.5% of isolates, comparable to earlier Indian data showing prevalence between 15–25% (27). Although AmpC positivity was slightly higher in K. pneumoniae (21.6%), the difference from E. coli (18.5%) was not significant. K. pneumoniae is known for harboring plasmid-mediated and chromosomal AmpC genes, contributing to its multidrug-resistant nature (28,29). AmpC producers often remain undetected in routine labs, which increases the risk of therapeutic failure (30).

Co-production of ESBL and AmpC

Co-production of ESBL and AmpC was found in 9.5% of isolates, with marginally higher rates in K. pneumoniae (11.6%). Previous studies report co-production rates of 5–15% in Enterobacterales, particularly in hospital-acquired infections (30,31). Co-expressing strains demonstrate broader resistance profiles, making therapeutic decision-making more complex.

Sample-wise Distribution

Urine samples were the predominant source of ESBL-producing isolates, reflecting the dominance of uropathogenic E. coli in clinical practice (22). Pus/wound samples had the highest AmpC positivity, likely due to selective pressure from prolonged antibiotic exposure and higher prevalence of hospital-associated resistant strains (32). These findings reinforce the need for sample-specific antimicrobial stewardship.

Antimicrobial Susceptibility Trends

Both E. coli and K. pneumoniae showed very high resistance to third-generation cephalosporins (80–90%), directly correlating with the high prevalence of ESBL and AmpC enzymes. Similar resistance patterns have been documented in multiple Indian studies (33).

Fluoroquinolone resistance (55–65%) was also high, consistent with global trends of widespread quinolone misuse (34).

Aminoglycoside susceptibility was moderate, with amikacin showing better activity than gentamicin; this is consistent with studies showing that amikacin is less susceptible to enzymatic modification (34).

Carbapenems remained the most effective agents, with >95% sensitivity, underscoring their continued role as the drug of choice for severe ESBL/AmpC infections (35). However, the emergence of a small proportion of carbapenem-non-susceptible isolates is a warning signal for possible early carbapenemase dissemination.

Nitrofurantoin showed excellent activity (92%) against urinary E. coli isolates, in agreement with existing literature supporting its continued use in uncomplicated UTIs (36).

Resistance Among ESBL and AmpC Producers

ESBL producers demonstrated significantly higher resistance to fluoroquinolones and aminoglycosides (p < 0.05), which is explained by the co-location of multiple resistance genes on transferable plasmids (37). AmpC producers showed complete resistance to cefoxitin and higher resistance to gentamicin and ciprofloxacin, similar to earlier findings indicating multidrug resistance associated with AmpC production (38). These results highlight the need for early detection to guide therapy.

CONCLUSION

This study demonstrates a considerable prevalence of ESBL and AmpC production among E. coli and K. pneumoniae, accompanied by high resistance to cephalosporins and fluoroquinolones. Co-production of ESBL and AmpC further contributes to multidrug resistance. Carbapenems remained the most effective agents, while nitrofurantoin showed excellent activity against urinary E. coli isolates.

Continuous surveillance, prudent antibiotic use, and strengthened infection control measures are essential to contain the spread of these resistant pathogens and ensure effective clinical management.

REFERENCES

1. World Health Organization. Global antimicrobial resistance surveillance system (GLASS) report. Geneva: WHO; 2022.
2. Paterson DL, Bonomo RA. Extended-spectrum β-lactamases: a clinical update. Clin Microbiol Rev. 2005;18(4):657–686.
3. Rawat D, Nair D. Extended-spectrum β-lactamases in Gram Negative Bacteria. J Glob Infect Dis. 2010;2(3):263–274.
4. Jacoby GA, Munoz-Price LS. The new β-lactamases. N Engl J Med. 2005;352:380–391.
5. Shaikh S, Fatima J, Shakil S, Rizvi SM, Kamal MA. Antibiotic resistance and extended spectrum β-lactamases: types, epidemiology and treatment. Saudi J Biol Sci. 2015;22(1):90–101.
6. Cantón R, Coque TM. The CTX-M β-lactamase pandemic. Curr Opin Microbiol. 2006;9(5):466–475.
7. Philippon A, Arlet G, Jacoby GA. Plasmid-mediated AmpC-type β-lactamases. Antimicrob Agents Chemother. 2002;46(1):1–11.
8. Hanson ND. AmpC β-lactamases: what do we need to know for the future? J Antimicrob Chemother. 2003;52(1):2–4.
9. Bauernfeind A, Chong Y, Schweighart S. Extended broad-spectrum β-lactamase in Klebsiella pneumoniae including resistance to cephamycins. Infection. 1989;17(5):316–321.
10. Black JA, Moland ES, Thomson KS. AmpC detection in Enterobacteriaceae: comparison of phenotypic methods. J Clin Microbiol. 2005;43(1):311–318.
11. Woerther PL, Burdet C, Chachaty E, Andremont A. Trends in human fecal carriage of ESBL-producing Enterobacteriaceae. J Antimicrob Chemother. 2013;68(4):727–730.
12. Gupta N, Limb L, Sharma M. Trends in ESBL production among Enterobacteriaceae in India. J Infect Dev Ctries. 2013;7(6):478–483.
13. Laxminarayan R, Chaudhury RR. Antibiotic resistance in India: drivers and opportunities for action. PLoS Med. 2016;13(3):e1001974.
14. Rodrigues C, Shukla U, Jog S, Mehta A. Extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae: a study from a tertiary care hospital in India. Indian J Med Microbiol. 2004;22(3):161–164.
15. Singhal S, Mathur T, Khan S, Upadhyay DJ, Chugh S. Evaluation of methods for AmpC β-lactamase detection in Enterobacteriaceae. Indian J Med Microbiol. 2005;23(2):120–124.
16. Pitout JD, Laupland KB. Extended-spectrum β-lactamase-producing Enterobacteriaceae: an emerging public health concern. Lancet Infect Dis. 2008;8(3):159–166.
17. Nordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae. Lancet Infect Dis. 2012;12(9):823–836.
18. Tamma PD, Simner PJ. Phenotypic detection of carbapenemase-producing organisms from clinical isolates. Clin Microbiol Rev. 2018;31(1):e00079-17.
19. Performance Standards for Antimicrobial Susceptibility Testing. 33rd ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2023.
20. Thomson KS. Extended-spectrum β-lactamase, AmpC, and carbapenemase issues. J Clin Microbiol. 2010;48(4):1019–1025.
21. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: NDM-1. Lancet Infect Dis. 2010;10(9):597–602
22. Gupta K, Hooton TM, Naber KG, et al. International clinical practice guidelines for the treatment of acute uncomplicated urinary tract infection. Clin Infect Dis. 2011;52(5):e103–e120.
23. Rodrigues C, Joshi P, Jani S, et al. Detection of ESBL producers among Gram-negative isolates in a tertiary care hospital. Indian J Med Microbiol. 2004;22:247–250.
24. Mathur P, Kapil A, Das B, et al. Prevalence of ESBL-producing Gram-negative bacteria in a tertiary care hospital. Indian J Med Res. 2002;115:153–157.
25. Paterson DL, Bonomo RA. Extended-spectrum β-lactamases: a clinical update. Clin Microbiol Rev. 2005;18(4):657–686.
26. Shaikh S, Fatima J, Shakil S, et al. Antibiotic resistance and ESBLs: a global threat. Iran J Med Sci. 2015;40(4):313–323.
27. Singhal S, Mathur T, Khan S, et al. Evaluation of methods for AmpC β-lactamase detection. J Infect Dev Ctries. 2005; 4:177–181.
28. Coudron PE. Prevalence and detection of plasmid-mediated AmpC β-lactamases. Clin Microbiol Rev. 2005;18(4):657–686.
29. Jacoby GA. AmpC β-lactamases. Clin Microbiol Rev. 2009;22(1):161–182.
30. Black JA, Moland ES, Thomson KS. AmpC β-lactamases in Enterobacteriaceae. Antimicrob Agents Chemother. 2005;49(10):4037–4043.
31. Giske CG, Sundsfjord A, Kahlmeter G. Co-expression of ESBLs and AmpC in Enterobacteriaceae. Clin Microbiol Infect. 2009;15:697–708.
32. Kaur M, Aggarwal A. Occurrence of multidrug-resistant E. coli and K. pneumoniae in wound infections. J Lab Physicians. 2018;10(3):283–289.
33. Mandal J, Acharya NS, Buddhapriya D, et al. Antibiotic resistance pattern of ESBL producers. J Clin Diagn Res. 2012;6(2):278–280.
34. Poole K. Aminoglycoside resistance mechanisms in Gram-negative bacteria. Antimicrob Agents Chemother. 2005;49:479–487.
35. Nordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae: mechanisms and clinical impact. Clin Microbiol Rev. 2012;25(4):722–789.
36. Huttner A, Verhaegh EM, Harbarth S, et al. Nitrofurantoin revisited: a systematic review of its activity. J Antimicrob Chemother. 2015;70:2456–2464.
37. Canton R, Coque TM. The CTX-M β-lactamase pandemic. J Antimicrob Chemother. 2006;56:52–59.
38. Pérez-Pérez FJ, Hanson ND. Detection of plasmid-mediated AmpC β-lactamase genes. J Clin Microbiol. 2002;40:2153–2162.