Pharmacological Evaluation of Combination Antibiotic Therapy Against Resistant Pathogens

Introduction

AMR has become one of the greatest global health, food security, economic stability and sustainable development threats. If microorganisms develop or are able to express mechanisms which make them less susceptible to the action of antimicrobial agents such that they can still persist despite contact with antimicrobial agents which would normally stop the spread of infection. Resistance can also occur naturally as a result of genetic mutation and microbial evolution but has been facilitated by inappropriate use of antimicrobials by people, unnecessary empirical prescriptions, non-completion of courses of treatment, easy access to antimicrobials, poor infection control, substandard medicines, environmental contamination, and widespread antimicrobial use in animal production and in agriculture. The effects of treatment failures are not just the treatment itself, but risks of increased hospitalisation, increased greater monitoring and use of more expensive or potentially poisonous reserve antibiotics due to resistant infections. AMR is a public health and development problem acknowledged by the World Health Organization (WHO) due to AMR misuse and overuse in the human, animal and plant sectors. Recent surveillance indicates that approximately 1 in 6 laboratory confirmed bacterial infections that resulted in common human diseases was be resistant to currently available antibiotics in 2023. Worldwide the proportion of resistant pathogens to the antibiotic has increased for more than 40% of combinations among monitored pathogens and antibiotics, by an average of 5% – 15% annually from 2018-2023. Resistance was highest in the WHO South-East Asia and Eastern Mediterranean regions, with almost one third of reported infections demonstrating resistance. Clinical relevance of bacterial AMR is elucidated. According to a comprehensive global study, 1.27 million people died in 2019 due to bacterial resistance and it has been identified as a cause in almost 4.95 million deaths. There were over 1.5 million deaths attributable to resistance and the burden of the RRTI was the smallest of the infectious syndromes. This effect differed from region to region, age to age and health care setting to health care setting. Why? Much of the poor countries had high rates of infectious disease and because they have less access to second line antibiotics, a lack of sanitation, too few hospital beds to be able to isolate patients and patients were not being diagnosed, because the lack of blood culture, urine culture etc. facilities. Low access to basic medicines is also a significant determinant of preventable deaths in many resource-poor settings worldwide, meanwhile. AMR is a two-part issue – suboptimal (or inappropriate) antimicrobial use and access to good quality treatment – and must be addressed. AMR impact is also evaluated by an impact on the current healthcare. In surgeries, transplanted organs, intensive care, cancer chemotherapy and neonatal, or immunocompromised patients, high demands are set for the reliable antibacterial prophylaxis and treatment. The resistance is higher, which also means there is a higher risk associated with these procedures, as they make it more difficult to achieve infection prevention, and fewer treatment options should an infection occur afterwards or during healthcare delivery. Resistant infections increase illness duration, make complications more likely and increase the utilization of drugs with sub-optimal pharmacokinetic properties and increase the kidney, nervous system and blood toxicity. The economic impacts consist of greater cost of medicines, longer hospital stays, lost productivity and strain on already limited healthcare systems. The pipeline for antibacterials has not been large enough to offset a loss of activity of older products. Although there was a slight increase in the number of “potentially new” antibacterial agents in clinical development from 80 in 2021 to 97 in 2023, WHO’s research demonstrates that innovation is still lacking to the extent that, by contrast, there was no progress in the development of agents to work against important pathogens.

The rise of Pathogens that are resistant to multiple antibiotics

Pathogens exhibiting poly-resistance to multiple drugs have emerged as a result of microbial adaptations, selection pressure of antimicrobial use, horizontal gene transfer, and due to global travel of humans, animals, foodstuffs, and microbes. In general terms, multidrug resistance (MDR) means resistance to at least one antimicrobial agent from two or more different antimicrobial classes; this definition varies with the organism and surveillance systems. E. coli PDR are resistant to almost all clinically used antimicrobials and those that are XDR are resistant to multiple classes. These phenotypes can be acquired by bacteria in several different ways: enzymatic destruction of the drug, modification of antibiotic targets, decreased membrane permeability, active drug efflux and metabolic bypass or protection of crucial cellular processes. Some of these mechanisms can be integrated within an individual strain and transferred via mobile genetic units like plasmids, integrons and transposons. Resistance determinants can be transferred horizontally, between species and genera and can form complex resistance phenotypes which are hard to overcome by increasing dosage of a single antibiotic. Gram negative bacteria are particularly problematic because they have an outer membrane which impedes the entry of antibiotics and which is coupled to the presence of efflux systems, loss of porins and acquired resistance enzymes. There are many penicillins, cephalosporins and carbapenems (e.g. KPC, NDM, VIM and OXA-type carbapenems) that contain extended spectrum β-lactamases. In the list of most important bacterial threats covered are carbapenem-resistant Acinetobacter baumannii (CRA), carbapenem-resistant Enterobacterales (CRE) and third generation cephalosporin-resistant Enterobacterales (3GC). Resistant Pseudomonas aeruginosa is difficult to treat and has the specific features of low intrinsic membrane permeability, good resistance to induction during treatment (therapy), and inducible chromosomal β-lactamases. Of the Gram positive microbes, resistant pneumococci, Vancomycin resistant enterococci and MRSA are significant community and hospital associated infections. The WHO Bacterial Priority Pathogens List 2024 lists of 44 bacterial species, split into 24 critical, high and medium priority pathogens of 15 families. It is effective against resistant gram negative bacteria, rifampicin resistant Mycobacterium tuberculosis and high burden pathogens such as Salmonella, Shigella, Neisseria gonorrhoeae, P. aeruginosa and S. aureus. MultiDrug Resistant organisms (MDROs) tend to be particularly well ‘grown’ and spread within health care settings. Accept almost any kind of antibiotic, may have invasive devices put in place, may be hospitalized for a long time and be exposed to multiple hospital contacts with contaminated surfaces. Mechanical ventilation, central venous catheterization or urinary catheterization and surgical wounds can allow opportunities for the entry of opportunist pathogens in breach of normal protective barriers. If there are no measures implemented to prevent adequate hand, environmental or antimicrobial stewardship, there may be opportunity to get resistant strains from one patient to another. Biofilm is another contributing factors to the persistence on medical devices and host tissues. Bacteria in a biofilm are surrounded by an extracellular matrix that can limit the ability of AMs to reach the bacteria, and alter their metabolism to make certain sub-populations more tolerant of the AM and better able to survive. The organism may be “susceptible” in planktonic laboratory conditions and may lead to a recurrent or chronic infection in the case of a Biofilm. Hospital setting is not the only arena for resistance. Urinary, blood (septicemias) and gastrointestinal infections with resistant Enterobacterales have been reported in community settings more frequently with resistant gonococci posing potential risk for effective treatment of a common sexually transmitted infection. Antibiotics may be used in livestock, and may lead to the development of bacteria resistant to the drugs, which can be passed directly onto humans, or through the food chain or environment. Antimicrobial residues from pharmaceutical and hospital effluents can lead to antimicrobial residues and resistant bacteria being transferred to the environment, creating the environment for the exchange of resistance genes. These are the trans-cutting pathways which allow a One Health approach to AMR that takes into account human health, animal health, agriculture and environmental integrity. With the increasing geographic spread of resistance, the antibiograms, recent healthcare exposure, travel history, colonization, and prior antimicrobial treatment should be taken into account when making treatment decisions.

The use of antibiotic Monotherapy is restricted

For many infections, particularly those identified by a known organism, antibiotic activity proven from in vitro studies and adequate exposure to antibiotic achieved at site of infection, consider antibiotic monotherapy. It reduces unnecessary antibiotic use and most of the time, also reduces the risk of cumulative toxicity, drug interactions and disruptions of normal flora. Effectiveness of monotherapy, however, is reduced as the severity of infection progresses along with susceptibility which can be uncertain, resistance mechanisms that become more numerous or can be more sophisticated or the bacteria can carry a high burden or have high resistance acquisition capability during the course of infection. A single drug is only able to target a small section of the cellular pathway and therefore not kill or tolerate any subpopulation of resistant bacteria. In the environments where antibiotics are used sensitive microorganisms die and survivors of the resistant strains survive and multiply. One of the main value of this process is that it is the process that counts in the presence of high number of bacteria, low concentration of drugs, or narrow mutant prevention range (MPR) of the antibiotic. Differences in pharmacokinetics and pharmacodynamics could further avert the use of monotherapy. Influences on drug distribution, such as elevated or decreased renal clearance, hypoalbuminaemia and tissue perfusion changes, may occur in a critical setting and can cause inappropriate and sometimes even confusing doses. The antibiotic could be active in vitro, but not active in vivo due to inability to achieve adequate free-drug levels at the infection site. In the lungs, central nervous system, bone, abscess cavities or biofilm associated infections penetration can be limited. Many bacteria may make the population inactive because of its “inoculum effect”, while inactive cells such as stationary-phase and persister cells may be less susceptible to agents that act on active cell division. In addition, resistance can exist in a heterogenous distribution and traditional susceptibility testing may fail to detect small resistant sub-populations that can also lead to failure of monotherapy. The use of older drugs, such as the polymyxins, has been reintroduced as there are limited options for available active drug against extremely resistant organisms. But, if the antibiotic in the toxic reserve class is the only one available to be used, there could be an undesirable risk/benefit ratio. There is a risk of dosage insufficiency, resulting in insufficient killing of bacteria, if the dosage is reduced and a risk of neurotoxicity or nephrotoxicity if the dosage is increased without consideration of which side-effect might occur. Critically, combination therapy works successfully for patients only if monotherapy has failed—this does not mean that combination therapy in general is an effective treatment. A randomized study of colistin alone or combined with meropenem for treating severe infections with resistant Gram-negative bacteria has shown that there is no superiority to combination therapy compared to colistin alone, especially when infection is most often caused by resistant A. baumannii. In another randomized trial, for the treatment of pneumonia and bacteremia associated with EDRGNs, the combination of colistin and meropenem did not improve efficacy compared to colistin alone. The results provide an example of the rational use of combination therapy rather than using combination therapy due to a resistant or severe infection. Combination antibiotic therapy is the use of two or more antimicrobial medications at the same time, usually to enhance the microbiological and/or clinical results. The rationale behind it is based on a number of potential benefits namely: wider empirical coverage, synergistic bacterial killing, resistance break, reduced resistance and reduced exposure to the toxic part. In septic shock and other life-threatening infections, for which the delay in giving at least one effective drug may inevitably lead to greater morbidity or mortality, the duration of empirical coverage is an important problem. When two agents are complementary in their spectra, the chances of the initial therapy having a high success rate until culture and susceptibility testing are completed are enhanced. If the cause of infection is identified, and one safe and active agent can be used to treat successfully, treatment should be re-evaluated and de-escalated.

Materials and Methods

Study Design

The purpose of this study was to elicit a synergistic effect from combinations of the antibiotics used to treat the MDS bacterial pathogens in an extensive in vitro experimental design. The study has been done in a sequential manner; first clinical bacterial isolates have been collected and their identification performed, the second was determination of resistance pattern. After that, a tight testing program selection for the best resistant isolates was implemented. The main part of the study comprised of a series of standardized microbiological assays, such as the minimum inhibitory and bactericidal concentrations (MIC and MBC), checkerboard assays and time-kill assays for synergy, along with biofilm formation and eradication assays. Finally, there was a test for resistance (cytotoxicity test) for the most promising combinations, to confirm their safety. Three experiments were conducted with each experiment repeated to give statistical strength and reproducibility. Standard microbiological methods such as Gram staining, colony morphology on selective and differential media (MacConkey agar, Blood agar) as well as a battery of traditional biochemical tests (catalase, coagulase, oxidase, IMViC) were used mainly to identify the isolates. A sub-sample of isolates was used for the confirmation of species level identification by testing them using the Vitek 2 automated system (bioMérieux) and/or 16S rRNA gene sequencing. All isolates were preserved in a glycerol stock at -80℃ for transfer in the future and reseeded on nutrient media on nutrient agar plates at 37° C for 18-24 hrs for purity and viability to be confirmed for future experiments.

Resistant pathogens selection

The initial screening was done using the Kirby – Bauer disk diffusion technique on Mueller Hinton Agar (MHA) as per the Clinical and Laboratory Standards Institute (CLSI) guidelines. An antibiotic disk panel used which contained clinically relevant antibiotics of different classes, including penicillins, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones and tetracyclines. After 18-24 hours of incubation at 37°C, zones of inhibition were measured and isolates were determined as susceptible, intermediate or resistant according to the CLSI (M100) interpretive criteria. The isolates that showed resistance to three or more classes of antibiotics were called multidrug-resistant (MDR) and were used for further experimentation from this initial pool. Clinical and resistant strains especially for medical humans including MRSA and CRE were targeted.

Antibiotics or antimicrobial agents

In this study only those antibiotics were selected which have clinical significance and exhibit diverse mechanisms of action and used to assess for potential synergy. The main antibiotics used contained: [e.g., Colistin sulfate, Meropenem trihydrate, Vancomycin hydrochloride, Ciprofloxacin hydrochloride]. All the antibiotic powders were commercial with a purity grade of 98% or above (supplier, e.g., Sigma-Aldrich). Stock solutions for each antibiotic were prepared according to manufacturer’s instructions and CLSI recommendations. They were dissolved in with the solvents suitable for the compound (sterile distilled water, phosphate buffered saline ( PBS, pH 7.2) or very small amount of dimethyl sulfoxide ( DMSO) where necessary so that the final concentration of the solvent was not more than 1% (v/v) and hence caused no solvent induced toxicity in the assays.

Antibiotic stock solutions

The stock solution s were made at a stock concentration of 10240µg/mL (or 10240 IU/mL for the polymyxins) for the adequate preparation of two-fold serial dilutions. For example, if the antibiotic powder is used to make a stock solution, exactly the right amount of antibiotic powder was dissolved in a known amount of sterile solvent to produce a 10,240 µg/mL solution. A vortex was used to ensure complete solution of the dispersions. The stock solutions were subsequently syringe filtered to ensure that there was no bacterial contamination (0.22-µm filter, Millipore). Each aliquot of the sterile stock solutions was placed in the sterile microcentrifuge tubes and stored at -20℃ until they were used, and were stable and active for up to 2 weeks. For the all experiments working solutions were prepared to avoid degradation.

Antimicrobial susceptibility testing

Initially the broth microdilution method as described in the CLSI document (M07) was used to check the pattern of resistance of the selected isolates. All antibiotics were diluted in a 96-well U-bottom sterile microtiter plate using twofold serial dilutions in cation-adjusted Mueller-Hinton broth (CAMHB) and the resulting concentrations ranged from 0.125 to 1024 µg/mL. A bacterial suspension to a 0.5 McFa rland (~1.5 x 1000 fewer than CFU/mL) was prepared and then 1:100 diluted in CAMHB that gave a final inoculum of ~5 x 1000 fewer than CFU/mL for each well. Plates were capped and then grown at 37 degrees for 18-24 hours. The MIC was determined as the lowest antibiotic level which inhibited visible growth of the organism.

Determine the lowest dose of an antibiotic that inhibits the growth of bacteria

The CLSI broth microdilution method for determining the Minimum inhibitory concentration (MIC) was used. Positive growth control (bacteria and media only) and negative sterility control (no bacteria, only media) wells were placed in each plate to assure the desired accuracy. The plates were then placed under the light source on the black background for incubation and then inspected visually. The MIC was determined by the smallest concentration of the antibiotic for which no turbidity was noted. When the antibiotic was not readily identifiable (such as in cases where it had a slight precipitate) a resazurin dye 0.01%w/v was introduced into each well, and the plates were incubated again for a further 2 hours. The endpoint of the MIC was the colour change from purple (resazurin) to pink (resorufin) which is an indicator of  bacterial growth.

The ability of an antimicrobial agent to kill the microorganism and inhibit its growth can be determined in the laboratory

The MBC was determined after the MIC has been obtained, for the testing of the bactericidal activity of the antibiotics. Blank wells (at and above the MIC) and the m/z were determined using the 10 µL growth seen in the positive control. The aliquots were then transferred to the MHA plates using the spread plate method to pick up the subculture. The plates were allowed to grow at 37ºC for 18-24 hours. The smallest amount of antibiotic concentration where the original bacterial inoculum was reduced by ≥99.9% (i.e. no visible colonies were noticed) was identified by the MBC. Experiments were conducted twice for each experiment.

Fractional inhibitory concentration (FIC) index means

The result of the checkerboard assay was used to calculate the fractional inhibitory concentration index (FIC) to quantify the interaction between the two antibiotics. The fractional inhibitory concentration (FIC) index (FICI) was calculated as the sum of the fractional inhibitory concentrations (FICs) of the individual drugs (fICI = fICA + fICB = (MIC of drug A in combination / MIC of drug A alone) + (MIC of drug B in combination / MIC of drug B alone). It was defined by the interaction based on the criteria used previously, such as those used by Abo-Schiri et al. (2010): Synergy: FICI ≤ 0.5, Additive effect: 0.5 < FICI ≤ 1.0, Indifference: 1.0 < FICI < 4.0, and Antagonism: FICI > 4.0. The synergistic combination was one in which the FICI was < 0.5, which means a substantial decrease in the MICs of both drugs.

Laser beams can be used for time – kill kinetic assays

Time – kill kinetic assays were carried out for the most promising synergistic combinations as confirmation of the synergistic bactericidal activity found in checkerboard assays. After overnight growth in CAMHB, bacterial cultures were transferred to and diluted in fresh CAMHB to give an inoculum of ~1 x 10⁶ CFU/mL. Occupants of each of these cultures were then inoculated with each given antibiotic used either alone at doses of 1× MIC, ½× MIC, and ¼× MIC of the antibiotic or at the synergistic doses of the antibiotic combination utilized (e.g., ½× MIC + ½× MIC). Growth control (no antibiotic) treatment also was provided. Growth of cultures was carried out at 37C on a shaking incubator (150 rpm). Aliquots were taken and serially diluted in sterile saline at predetermined time points (0, 1, 2, 4, 6, 8 and 24 hours) and then plated on to MHA. After incubating for 24 hours the colonies were counted. The definition of synergy was ≥ 2 log10 CFUs/mL reduction in viable count after 24 hours by the combination compared to the most active single agent.

Biofilm formation assay

The ability of these clinical isolates to form biofilms was determined by crystal violet standard microtiter plate procedure. The bacterial cultures were diluted in Tryptic Soy Broth (TSB)- 1% glucose 1:100 and cultured overnight to ensure the growth of biofilm. This bacterial suspension was subsequently diluted to 200µL and subsequently placed in a sterile microtiter plate flat bottom 96 well, after incubating for 24-48 hours at 37°C. After the incubation, the planktonic cells were washed out from the wells three times with sterile PBS. After that the cells were fixed for 15 minutes with 99% methanol, then stained for 15 minutes with 1% (w/v) crystal violet to result in adherent cells. The unbound dye was washed off with water and the crystal violet was made soluble with 33% glacial acetic acid. Optical density (OD) was done at 595 nm on micro plate reader.

Statical Analysis

Each experiment was conducted 3 times with a minimum of triplicate repetition with the values expressed as mean ± standard deviation (SD). One way analysis of variance (ANOVA) and Tukey’s post hoc test for multiple comparisons were used to assess the significance of differences between treatment groups. The unpaired Student’s t-test was used for comparing two groups. The p value of < 0.05 (p < 0.05) was taken as statistically significant. Data were analyzed using the GraphPad Prism software (version 8.0 or above; GraphPad Software, San Diego, CA, USA).

Conclusion

The growing problem of antimicrobial resistance calls for new and fresh ideas on therapy. This study offers compelling in vitro results justifying combination antibiotic treatment with resistant pathogens. Systematic evaluation of 50 clinical isolates confirmed that certain combinations of antibiotics tested were shown to have substantial synergy, resulting in significant reductions in MIC, improvements in killing and removal of biofilms responsible for persistent infection. Finding synergistic combinations with favourable safety profiles is an important step to increase the extremely limited armamentarium against MDR pathogens. We found an important characteristic of synergy: it does not necessarily occur and depends on the mechanism of action of the antibody and the characteristics of the pathogen. These antibiotics—such as colistin-meropenem and vancomycin-rifampicin—in particular looked promising, and should be examined in clinical trials.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The author(s) do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

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