Introduction

Antimicrobial resistance is a top priority worldwide in healthcare system. In 2017, WHO published a list of global priority pathogens, including carbapenem-resistant Enterobacterales, carbapenem-resistant Pseudomonas aeruginosa, and carbapenem-resistant-Acinetobacter baumannii under the category of critical organisms. (1,2) The available antibiotics have either lost their ability to treat bacterial infections or are going to lose very quickly. The increasing prevalence of antimicrobial resistance in Gram-negative bacteria is a matter of serious concern, as the acquisition of antimicrobial resistance in gram-negative bacteria is more rapid than that in Gram-positive bacteria. (3-6)

The β-lactam group of antibiotics is the most prevalently prescribed antibiotic. The widespread use of antibiotics in the community as well as in hospital practices has induced selection pressure toward resistant strains and favoured the evolution of antimicrobial resistance.(7) Gram-negative bacteria have inherent abilities to become resistant to antimicrobials and also facilitate other bacteria to become drug-resistant.(8,9) Spontaneous random mutations and the transfer of genes either horizontally or vertically can evolve resistant strains.(10)

Enzymatic drug inactivation of β-lactam antibiotics by β-lactamases (e.g., AmpC, ESBLs, carbapenemases) is the primary cause of resistance to β-lactam antibiotics, especially in gram-negative bacteria.(11) These β-lactamases differ structurally as well as characteristically from each other. Various widespread β-lactamase-mediated resistance mechanisms can be detected in the microbiology laboratory following CLSI (Clinical and Laboratory Standards Institute) guidelines and other standard references without the involvement of any molecular biology techniques and can help in the assessment of the prevalence of β-lactamases in the region. During the COVID-19 pandemic which lasted from December, 2019-21, there was a widespread use of broad-spectrum antibiotics in COVID-19 patients, which was considered responsible for further escalation of antimicrobial resistance. (12,13)

We conducted this study to find out the local prevalence of β-lactamases in Gram-negative bacteria in our region.

Methods

This cross-sectional study was conducted in a multi-specialty hospital located in Central India from 01st January 2021 to 31st December 2023. In this study, all clinical specimens submitted in microbiology department from in-patient and outpatient departments of hospital were included.

Preliminary identification of organisms isolated from clinical samples was performed by Gram staining, colonial characteristics, and biochemical characterization of the bacteria. (14) Confirmed identification was done by automated system.

The antimicrobial susceptibility of all 691 gram-negative isolates to antimicrobials was determined by both the broth microdilution method and manually by the Kirby Bauer disk diffusion method. Selection of antimicrobials and interpretation of MIC (minimum inhibitory concentration) and zone diameter results were checked in accordance with CLSI guidelines. An additional colistin broth disk elution test was performed by using colistin (10 μg) discs to evaluate the MIC of colistin against the isolates. (15)

Analysis of antimicrobial resistance due to β-lactamase production in gram-negative bacteria was detected by phenotypic methods. The basic β-lactamases detected were ESBL, AmpC BL and metallo-β-lactamase by standard manual methods described in CLSI. The methods that we used were simple and performed manually on a routine basis.

ESBL and AmpC production in the gram-negative isolates was checked by performing the double disc synergy test recommended by CLSI. The antibiotic disks containing cefotaxime (30 μg), cefotaxime clavulanic acid (30/10 μg), ceftazidime(30 μg), ceftazidime clavulanic acid (30/10 μg), cefoxitin (30 μg), imipenem (10 μg), imipenem-EDTA (10/750), and aztreonam (30 μg) were used for resistance detection. Bacteria were classified as ESBL positive when the zone of inhibition around the third-generation cephalosporin and clavulanic acid-containing combination disk was 5 mm higher than the zone obtained by only the third-generation cephalosporin disk.

Complete resistance against β-lactams and β-lactamase inhibitor combinations, cephalosporins up to third generation and cefoxitin were used to predict a high level of AmpC production. (20)

Phenotypic detection of metallo β-lactamase production was checked by using the imipenem EDTA disk method. An increase in zone diameter around the Imipenem EDTA disk by ≥ 7 mm compared to the Imipenem -containing disk confirms the presence of MBL in the isolate tested. (17, 22, 23)

Statistically, significance was considered at p value less than 0.05 by using Chi-square test. Data analyses were conducted using the Excel software.

Result

In this study, we included a total of 3710 clinical samples received in the Microbiology Department for culture tests between 2021-2023. Out of 3710 cultures, 991 (27%) cultures were positive with significant microscopic findings.

The percentage of gram-negative bacilli was 65% of the total positive samples, followed by gram-positive cocci (32%) and fungi (2%). E. coli (41%) was found to be the most prevalent pathogen followed by Pseudomonas aeruginosa (15%), Salmonella spp. (15%) and, Klebsiella pneumoniae (11%). Our findings suggest that 74% of the total gram-negative bacilli belong to the family Enterobacterales.

Here, we studied 285 cultures of E. coli isolated from various specimens, such as blood, urine and pus. Total of 77% were found to be associated with urinary tract infections.

Klebsiella was the third most predominant organism isolated, that belongs to the family Enterobacterales. We obtained 78 isolates identified as Klebsiella pneumoniae, of which 54% were from urine samples, 26% were from pus samples, 13% were from respiratory samples, and the remaining 7% were from blood cultures.

Pseudomonas is a known pathogen associated with various infections, especially nosocomial infections. In our study, the prevalence of Pseudomonas was 15% of the total Gram-negative isolates.

Figure 1. Comparative analysis of antimicrobial resistance in E. coli

Antimicrobial susceptibility testing and antimicrobial resistance profiling of E. coli isolates revealed that more than 85% of isolates are resistant to the most often used third-generation cephalosporin class of antibiotics.

The extended spectrum β-lactamase resistance mechanism was the leading antimicrobial resistance detected in 53% of total E. coli isolates, followed by metallo β-lactamases (14%). Overall, 28% of the isolates were found to be resistant to even carbapenems, which is an alarming situation. Our study revealed a 6% prevalence of MBL in E. coli in the year 2021, which has now increased to 21% by 2022. By comparing the data from 2021 and 2022, we found an overall increase in antimicrobial resistance, showing a significant 15% increase in metallo β-lactamases (Figure 1). The therapeutic choice suggested for the infections caused by ESBL-producing E. coli was found to be meropenem with susceptibility (79%) and amikacin (91%). In the case of lower urinary tract infections, fosfomycin (99% susceptibility) and nitrofurantoin (87% susceptibility) have emerged as alternatives to β-lactam antibiotics.

Antimicrobial susceptibility testing of Klebsiella showed an overall increasing trend in antimicrobial resistance. It is also evident from the resistance trend that the highest rise in resistance was found against amikacin. The amikacin resistance was found to increase by 30% with a statistically significant p-value of 0.02325 (at p<0.05). The current study revealed 72% of the clinical isolates to be resistant to third-generation cephalosporins. In addition to the drastic drop in cephalosporin susceptibility, susceptibility to carbapenems has also decreased. Comparative analysis of resistance levels in 2021 and 2022 revealed an increase in the prevalence of MBL resistance by 33% which is statistically significant with p-value of 0.009937.

Figure 2: Comparative analysis of antimicrobial resistance in Klebsiella pneumoniae

Figure 3: Comparative analysis of antimicrobial resistance in P. aeruginosa

Among the range of drugs analysed in the current study, we observed that 53% of the Pseudomonas aeruginosa isolates were susceptible to piperacillin tazobactam, 50% were susceptible to aztreonam, 49% were susceptible to meropenem and 51% were susceptible to gentamicin. The matter of major concern in our study is that the prevalence of MBL increased by 51% from 2021 to 2022 which is statistically significant with p value of 0.009937 (Figure 3).

Discussion

Out of 3710 samples received in microbiology laboratory between 2021-23, 990 (27%) were culture positive with 65% being Gram-negative bacilli, 25% Gram-positive cocci and 2% were fungus. Among Gram-negative bacilli, 41% were E. coli being most prevalent followed by Pseudomonas aeruginosa (15%), Salmonella Typhi (15%) and Klebsiella pneumoniae (11%). In the neighbouring country of India, Nepal, a study was conducted that showed nearly the same prevalence of organisms viz. E. coli (46.7%), followed by Klebsiella and Pseudomonas spp. which is in concordance with our study. (16)

Our findings suggest that 74% of the total gram-negative bacilli belong to the family Enterobacterales which is similar to study conducted by Erdem et al., where 75-95% of community-acquired lower urinary tract infections are due to E. coli. (17) Shreshtha et al. reported the presence of 53% ESBL-producing E. coli in their study conducted in Nepal which is in accordance with our study, that revealed 53% of E. coli isolates to be ESBL resistant. (16) Our findings were consistent with a study stating that ESBL genes such as CTX-M, TEM and SHV families are most frequently found in Enterobacterales with ESBL CTX-M type as the most prevalent resistance phenotype. (16,18)

In our study, we observed the rising trend of metallo β-lactamases similar to other various studies that reported the widespread presence of metallo β-lactamases in the Indian subcontinent. Clinical isolates may exhibit the presence of one or more carbapenem resistance genes, such as MBL blaNDM-1, OXA-48, or KPC. (19) Data published by Kamble D. from Pune in 2015 stated a 5% prevalence of MBL in E. coli. (20) In case of E. coli mediated urinary tract infections Fosfomycin and Nitrofurantoin are found to be effective as non β-lactam options available.

In our study, we found that the susceptibility of Klebsiella pneumoniae to amikacin in 2021 was 83%, and in 2022 it was 53%. As per study by Kuti et al., in 2018 found 83.7% susceptibility of Klebsiella pneumoniae to amikacin. (21) As per study by Bora et al., in 2014 there was 21.08% prevalence of MBL-producing Klebsiella pneumoniae in different clinical samples. (22) The results of our study have also put forward an alarming situation for carbapenem resistance. Total of 57% of the clinical isolates in the current study were carbapenem resistant, and 28% of these isolates exhibited MBL-mediated resistance.

In our study, we observed that 59% of the Pseudomonas isolates were carbapenem resistant, and out of that 43% were MBL producers. Among all the isolates of P. aeruginosa 31% showed inducible AmpC β-lactamase-mediated resistance. Pseudomonas exhibits intrinsic resistance against most commonly used antibiotics, resulting in fewer available options to treat infections. Colistin and polymyxin B are considered the most effective antipseudomonal drugs. The colistin broth disk elution (CBDE) method was used to confirm the colistin MIC. According to Kuti et al.’s findings in 2018, the susceptibility of Pseudomonas aeruginosa to ciprofloxacin was 83.9%, that to ceftazidime was 81.3%, and that to cefepime was 80.4%. When compared to our findings, the data from Kuti et al. indicated a significant decrease in susceptibility to ciprofloxacin, ceftazidime, and cefepime of 29%, 42%, and 52%, respectively, for Pseudomonas aeruginosa. The same research reported interesting findings on amikacin resistance. Amikacin susceptibility was studied in carbapenem-resistant and carbapenem-susceptible strains. A total of 91.1% carbapenem-sensitive strains were found to be susceptible to amikacin, whereas only 80.9% carbapenem-resistant strains were susceptible to amikacin. (21) We found 53% Pseudomonas aeruginosa susceptible to amikacin.

The current study is based on phenotypic expression of β-lactamases in pathogens. There might be many other β-lactamases exhibited by the isolates that might be unexpressed, which can be further detected with the help of molecular methods. This study was conducted to learn more about the microorganisms that possess potentially widespread antimicrobial resistance mechanisms. The findings of the study will help in preparing the region-specific empirical antibiotic policy, which in turn will help in limiting the injudicious use of antibiotics.

The study findings revealed that the three most common gram-negative clinical isolates E. coli, K. pneumoniae and P. aeruginosa, have a significantly higher incidence of MBL. The ability of Pseudomonas to develop resistance against available antibiotics by following various mechanisms makes it a dreadful pathogen. The increasing carbapenem resistance in Pseudomonas may develop following chromosomal mutations that decrease permeability while also causing the overexpression of efflux pumps and the production of β-lactamases. (23) The rise in MBL points towards excessive use of carbapenems in our healthcare, leading to selection pressure as well as rapid transmission of resistance across patients due to inefficient infection control practices.

In such scenario carbapenems have become an inefficient option and thus it becomes difficult to treat infections caused by these resistance-carrying pathogens. Use of carbapenems as empiric therapy should strongly discouraged with the help of effective antibiotic stewardship programmes, and antibiotics particularly higher ones should be prescribed only with undeniable bacterial infections in which case the empiric antibiotic therapy should be immediately switched to targeted therapy based on the results of culture and sensitivity testing. There is also a need to implement diagnostic stewardship practices in healthcare, so as to reduce the incidence of inappropriate diagnosis of infections resulting in irrational use of broad-spectrum antibiotics.

Conflicting Interests: The authors declared no potential conflicts of interest.

Funding sources: None

Acknowledgements

The authors would like to thank the management and colleagues of institution for providing us the necessary support to conduct the study. We would like to thank Parvati Birla and Murli Patel for the technical support.

References

1. WHO. Global Antimicrobial Resistance and Use Surveillance System (GLASS) Report. 2020.
2. Asokan GV, Ramadhan T, Ahmed E et al. WHO global priority pathogens list: A bibliometric analysis of Medline-Pubmed for knowledge mobilization to infection prevention and control practices in Bahrain. Oman Med J. 2019;34(3):184–93.
3. Exner M, Bhattacharya S, Christiansen B. Antibiotic resistance: What is so special about multidrug-resistant Gram-negative bacteria? GMS Hyg Infect Control. 2017 Apr 10;12:Doc05.
4. Miller SI. Antibiotic resistance and regulation of the Gram-negative bacterial outer membrane barrier by host innate immune molecules. mBio. 2016 Sep 1;7(5).
5. Gupta V, Datta P. Next-generation strategy for treating drug resistant bacteria: Antibiotic hybrids. Indian Journal of Medical Research. 2019;149:97–106.
6. Breijyeh Z, Jubeh B, Karaman R. Resistance of gram-negative bacteria to current antibacterial agents and approaches to resolve it. Molecules. 2020 Mar 16;25(6):1340.
7. Kwapong AA, Soares S, Teo SP et al. Myristica lowiana Phytochemicals as Inhibitor of Plasmid Conjugation in Escherichia coli. Evidence-based Complementary and Alternative Medicine. 2020 Jun 19;2020:1604638. Available at https://pmc.ncbi.nlm.nih.gov/articles/PMC7321511/
8. Nordmann P, Poirel L, Walsh TR. et al. The emerging NDM carbapenemases. Trends in Microbiology. 2011 Dec;19(12):588-95.
9. Munoz-Price LS, Poirel L, Bonomo RA, et al. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. The Lancet Infectious Diseases. 2013 Sep;13(9):785–796. Available at https://pmc.ncbi.nlm.nih.gov/articles/PMC4673667/
10. Wellington EMH, Boxall ABA, Cross P, et al. The role of the natural environment in the emergence of antibiotic resistance in Gram-negative bacteria. The Lancet Infectious Diseases. 2013 Feb;13(2):155-65.
11. Queenan AM, Bush K. Carbapenemases: The versatile β-lactamases. Clinical Microbiology Reviews. 2007 Jul;20(3):440-58. Available  at https://pmc.ncbi.nlm.nih.gov/articles/PMC1932750/
    
12. Malik SS, Mundra S. Increasing Consumption of Antibiotics during the COVID-19 Pandemic: Implications for Patient Health and Emerging Anti-Microbial Resistance. Antibiotics. 2022 Dec 28;12(1):45. Available at https://pmc.ncbi.nlm.nih.gov/articles/PMC9855050/
13. Clancy CJ, Buehrle DJ, Nguyen MH. PRO: The COVID-19 pandemic will result in increased antimicrobial resistance rates. JAC Antimicrob Resist. 2020 Sep 1;2(3).
14. Cheesbrough Monica. District Laboratory Practice in Tropical Countries. Second, 2006. Vol. 2. Cambridge University Press;
15. CLSI. CLSI M100-ED33:2023 Performance Standards for Antimicrobial Susceptibility Testing, 33rd Edition. 2023.
16. Shrestha A, Acharya J, Amatya J, et al. Detection of Beta-Lactamases (ESBL and MBL) Producing Gram-Negative Pathogens in National Public Health Laboratory of Nepal. Int J Microbiol. 2022 Oct 6:2022:5474388. Available at https://pmc.ncbi.nlm.nih.gov/articles/PMC9560861/
17. Erdem I, Kara Ali R, Ardic E, et al. Community-acquired lower urinary tract infections: Etiology, antimicrobial resistance, and treatment results in female patients. J Glob Infect Dis. 2018 Jul 1;10(3):129–32.
18. Paterson DL, Bonomo RA. Extended-spectrum β-lactamases: A clinical update. Clinical Microbiology Reviews. 2005;18:657–86.
19. Boyd SE, Livermore DM, Hooper DC, et al. Metallo-β-lactamases: Structure, function, epidemiology, treatment options, and the development pipeline. Antimicrob Agents Chemother. 2020 Sep 21;64(10):e00397-20. Available at https://pmc.ncbi.nlm.nih.gov/articles/PMC7508574/
20. Kamble D. Phenotypic detection of ESBL and MBL in Gram-negative bacilli isolated from clinical specimens. Int J Med Res Rev. 2015;3(8):866-870. doi: 10.17511/ijmrr.2015.i8.163.
21. Kuti JL, Wang Q, Chen H, et al. Defining the potency of amikacin against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii derived from Chinese hospitals using CLSI and inhalation-based breakpoints. Infect Drug Resist. 2018 May 25;11:783–90.
22. Bora A, Sanjana R, Jha BK, et al. Incidence of metallo-beta-lactamase producing clinical isolates of Escherichia coli and Klebsiella pneumoniae in central Nepal. BMC Res Notes. 2014 Aug 21;7:557.Available at https://bmcresnotes.biomedcentral.com/articles/10.1186/1756-0500-7-557
23. Brink AJ. Epidemiology of carbapenem-resistant Gram-negative infections globally. Current Opinion in Infectious Diseases. 2019 Dec;32(6):609-616.