The rising bacterial resistance to various antimicrobial agents is becoming a serious public health concern. This problem often leaves little or no effective treatment options to combat infections caused by pathogenic organisms. Antimicrobial resistance has escalated globally, affecting both Gram-positive and Gram-negative bacteria more widely. ¹ Establishing standardized definitions and classifications for multidrug-resistant (MDR) bacteria is essential for consistently collecting and comparing epidemiological data across diverse healthcare settings and countries. ² MDR organisms are pathogens that display in vitro resistance to at least two distinct classes of antimicrobial agents. Infections caused by these organisms frequently result in delayed or inadequate antimicrobial therapy, contributing to poorer patient outcomes, prolonged hospital stays, and increased healthcare costs. ³ These resistant organisms present a significant challenge in hospital and community settings, leading to increased mortality rates, particularly among critically ill patients. ⁴

Recent advancements highlight the importance of global initiatives, such as the World Health Organization’s (WHO) Global Antimicrobial Resistance and Use Surveillance System (GLASS), to monitor and combat this growing threat (WHO, 2022). Furthermore, research continues to explore innovative treatment approaches, such as combining therapies and utilizing supplementary agents, to effectively manage multidrug-resistant (MDR) infections. ⁵ Addressing this issue requires implementing robust infection control measures, responsible antibiotic use, and developing new antimicrobial drugs to ensure public health protection. Studies also show that inadequate antimicrobial therapy in critical infections, such as bloodstream infections, significantly impacts patient outcomes, making timely and appropriate treatment essential. ⁴

Escherichia coli ( E. coli) is a gram-negative, facultative anaerobic, rod-shaped bacterium commonly found in the intestines of warm-blooded animals. ^{6, 7} Although the majority of E. coli strains are harmless, specific serotypes, such as Enteropathogenic E. coli (EPEC) and Enterotoxigenic E. coli (ETEC), can cause severe infections. EPEC is a leading cause of urinary tract infections, while ETEC is commonly responsible for traveler’s diarrhea and can lead to significant foodborne illnesses. ⁸ Pathogenic strains occasionally cause food contamination events, which can lead to product recalls. ⁹ The main treatment strategy focuses on addressing dehydration and restoring fluids and electrolytes. Although antibiotic treatment has been shown to reduce the duration of infection and the shedding of ETEC, especially in adults in endemic regions and travelers, the growing resistance to commonly used antibiotics has raised concerns. As a result, antibiotics are often discouraged unless necessary. ¹⁰ The prevalence of multidrug-resistant E. coli strains in Iraq has grown, creating significant public health challenges. Many research studies have been carried out to meet the critical need to control infections caused by these resistant strains. ^{11, 12}

Klebsiella pneumoniae ( K. pneumoniae) is a gram-negative, non-motile, encapsulated, lactose-fermenting, facultative anaerobic, rod-shaped bacterium. ¹³ When inhaled, it can cause significant damage to the lungs of humans and animals, particularly impacting the alveoli and producing sputum that may be bloody, brownish, yellow, or jelly-like. ^{14, 15} Recently, Klebsiella species have emerged as significant pathogens responsible for nosocomial infections, posing a considerable challenge to healthcare settings. ¹⁶ These bacteria have developed multiple mechanisms of multidrug resistance (MDR), including the formation of plasmids and the production of extended-spectrum beta-lactamase (ESBL) enzymes, further complicating treatment options. ⁸ The rise and transmission of multidrug-resistant (MDR) Klebsiella pneumoniae in Iraq is becoming an increasing concern, especially in healthcare environments, where it presents serious threats to patient outcomes. Klebsiella pneumoniae has developed resistance to several commonly used antibiotics, including extended-spectrum beta-lactams (ESBLs), which complicates treatment strategies and leads to prolonged hospital stays, increased healthcare costs, and higher mortality rates. ^{12, 17}

Ethylenediaminetetraacetic acid (EDTA) is a chelating agent that binds divalent cations, such as magnesium (Mg ²⁺) and calcium (Ca ²⁺), essential for maintaining bacterial cell wall integrity and biofilm formation. EDTA destabilizes bacterial membranes by chelating vital ions, which compromises membrane integrity, increases permeability, and enhances both antimicrobial and antibiofilm properties. ^{16, 18, 19} Studies have demonstrated that EDTA significantly improves the efficacy of various antibiotics, particularly in combating biofilm-associated infections caused by multidrug-resistant (MDR) bacteria. ^{20, 21} For example, EDTA, when combined with indocyanine green, enhanced photodynamic therapy (PDT) in diabetic foot infections, although the reduction in bacterial viability was modest, with bacterial counts decreasing by less than tenfold at 5 mM concentrations. ²² The combination of EDTA with antibiotics, such as ceftriaxone, sulbactam, and gentamicin, has also proven to be effective against resistant Gram-negative infections isolated from clinical samples, including respiratory, blood, and urine cultures. ^{23– 25}

This experimental study aims to investigate the effects of combining EDTA with Meropenem, Amikacin, and Piperacillin-tazobactam on multidrug-resistant Escherichia coli and Klebsiella pneumoniae isolates from hospital settings. The study will evaluate changes in bacterial susceptibility to these antibiotics after the addition of EDTA compared to their susceptibility before the combination. In addition, the research will examine the extent of antibiotic resistance at Ghazi Al-Hariri Surgical Specialties Hospital in Baghdad, Iraq, shedding light on the present status of antimicrobial resistance in the region.

The results about the prevalence of resistance provide essential insights into the antimicrobial resistance patterns of E. coli and K. pneumoniae isolates in an essential Iraqi hospital setting like Ghazi Al-Hariri Surgical Specialties Hospital which occurs in the center of Baghdad and receives a large number of patients daily. The findings draw attention to the significance of growing antibiotic resistance and the challenges in treating infections caused by multidrug resistant pathogens. In Table 1 the case of E. coli, Meropenem remains practical mainly, with 86% of the isolates susceptible. The presence of 13% resistant and 1% intermediate strains points out the importance of continuous monitoring to control resistance spread. The results of K . pneumoniae show a susceptibility to meropenem of 56%, while 42% were resistant isolates. The high significance of resistance reflects a growing thread, lowering the available treatment options, and underscoring the need for more advanced strategies to overcome resistance spread. Resistance to meropenem can be developed primarily via the production of carbapenemases, enzymes that hydrolyze the β-lactam ring of carbapenems, making them ineffective. The most common types of carbapenemases in these organisms include KPC (Klebsiella pneumoniae carbapenemase) and NDM (New Delhi metallo-β-lactamase). ³⁵

When tested against both pathogens, amikacin shows susceptibility rates of 67% for E. coli and 64% for K. pneumoniae. There is a rising resistance rates (31% for E. coli and 35% for K. pneumoniae), which suggest that these organisms increasingly acquire resistance mechanisms. Resistance to amikacin is often due to drug enzymatic modification, which includes acetylation and adenylation mediated by aminoglycoside-modifying enzymes (AMEs). ³⁶ Bacterial ribosomal RNA mutations can also lead to decreased drug binding efficiency. Piperacillin/Tazobactam findings against E. coli shows 70% susceptible isolates, and a lower percentage against K. pneumoniae with 41%. The resistance then for K. pneumoniae was 56% of resistant isolates. Resistance mechanisms against piperacillin/tazobactam typically involve the production of β-lactamases, including ESBLs (extended-spectrum β-lactamases) and carbapenemases, that can inactivate piperacillin. Moreover, alterations in porin channels can prevent drug penetration into the bacterial cell. ³⁷

Looking after good antibiotic stewardship programs and optimizing the use of antibiotics is essential to improve clinical outcomes and reduce treatment failure among the growing antimicrobial resistance. ³⁸ The use of novel agents, in addition to combination therapies, is an important search area to combat resistant pathogens. Moreover, the high resistance rates observed in this study highlight the necessity to control and monitor the use of antibiotics and reflects the rising antimicrobial resistance in Iraqi healthcare settings. This deals with the global trend of antibiotic resistance and calls for coordinated efforts to address the issue effectively in Iraq. ¹

The antimicrobial activity results in Tables 1– 3 highlights on the mean diameter of growth inhibition zones for three antimicrobial agents—Meropenem, Amikacin, and Piperacillin/Tazobactam—both alone and in combination with EDTA at varying concentrations on E. coli and K. pneumoniae isolates this was also clarified in Figures 1 and 2. The trend observed suggests that as the concentration of the antibiotics increases (C1 < C2 < C3), the effectiveness of the antibiotics improves, as shown by the larger inhibition zones. Notably, the addition of EDTA enhances the antimicrobial activity of each antibiotic, as the inhibition zones are consistently larger when EDTA is combined with the antibiotics compared to when each antibiotic used alone in a range of 2-5 mm. This suggests that EDTA can play a role in potentiating the effectiveness of these antibiotics.

EDTA is known to chelate divalent metal ions like calcium and magnesium, which are essential for bacterial cell wall integrity and function. By sequestering these ions, EDTA may increase bacterial permeability or make the bacteria more susceptible to the antibiotics, thus enhancing their overall antimicrobial action ³⁹ and this was summarized in Table 4. This finding is consistent with other studies, such as one published in 2011 in the Iraqi Journal of Medical Sciences which found that EDTA, when combined with antibiotics, significantly increased the antimicrobial activity against Pseudomonas aeruginosa isolates. ⁴⁰

These results also are in agreement with a 2022 study published in Antimicrobial Agents and Chemotherapy, which examined the pharmacodynamics of Piperacillin-Tazobactam/Amikacin combinations versus Meropenem on ESBL-producing K. pneumoniae strains and found that EDTA enhanced antimicrobial activity. ¹⁶

The overall findings are especially significant in the context of the growing global thread of antimicrobial resistance. Studies continue to explore strategies to combat resistant bacterial strains, and the use of EDTA as an adjunct to antibiotics appears promising. Research by Al-Jumaili and Ahmed (2024) highlighted the growing issue of antimicrobial resistance in Iraq, emphasizing the need for effective control measures using a One Health approach. ⁴¹ A study by Shamsee and Ibrahim (2024) also found that K. pneumoniae isolates exhibited significant resistance to various antibiotics, highlighting the need for novel solutions like combining EDTA with antibiotics. ⁴² Additionally, Al-Jumaili and Almawla (2023) ⁴³ pointed out the importance of evidence-based antibiotic prescribing practices to combat multi-antibiotic resistance. Alkarkhi, Zeiny, and Ali (2016), Khan and Khan (2023), Khan et al. (2022) observed high antibiotic resistance in microorganisms isolated from surgical floors in hospitals, further reinforcing the need for new antimicrobial strategies. ^{44– 46}

In conclusion, escalating antimicrobial resistance is a growing challenge. The data indicates that EDTA enhances the antimicrobial activity of Meropenem, Amikacin, and Piperacillin/Tazobactam against multidrug-resistant E. coli and K. pneumoniae isolates. This suggests that combining EDTA with these antibiotics could be an effective strategy for combating infections caused by multidrug-resistant pathogens. The larger inhibition zones observed reflect EDTA’s potential to boost antibiotic efficacy. Future studies should focus on understanding the mechanisms behind EDTA’s enhancement of antibiotic activity and its clinical applications.

Ethical Approval was obtained from the deanship of Baghdad college of medicine no. 23 in 4 ^th of January 2025 and the Department of (Pharmacology), College of Medicine, University of Baghdad has approved the study protocol from the clinical and ethical points of view. Written informed consent was obtained from each patient involved in the study, and copyright was also obtained in regard to assessment tools involved.