Synergistic activity of meropenem and sulbactam against carbapenem-resistant Acinetobacter baumannii

Abstract

OBJECTIVE:

The purpose of this study was to determine the in vitro synergistic effect of meropenem and sulbactam drug combinations through checkerboard assay against Carbapenem-resistant Acinetobacter baumannii (CRAB) to establish potential treatment options.

MATERIALS AND METHODS:

CRAB was cultured from the clinical samples of Endotracheal aspirate, blood, pus, throat swab, and urine specimens from 80 patients with suspected nosocomial infection in 1 year. Minimum inhibitory concentration (MIC) was determined for meropenem and sulbactam drugs individually. Synergism was determined for meropenem + sulbactam combinations using checkerboard assay.

RESULTS:

All the 80 CRAB isolates were found to be resistant (>16 MIC) for Meropenem drug and 64 out of 80 showed resistant (>16 MIC) to sulbactam, 14 isolates displayed intermediate (8 MIC), and 2 of them were found to be sensitive (<2 MIC). The checkerboard assay showed 66.25% of synergism between meropenem and sulbactam followed by 25% of additivity and 8.75% of them were found to be indifferent.

CONCLUSION:

In the current checkerboard assay, we observed potential synergistic activity between meropenem and sulbactam against CRAB isolates which indicates that this combination can be an appealing strategy in the treatment of nosocomial infections caused by CRAB isolates.

Introduction

With the emergence of the nosocomial infections and multidrug-resistant species associated with it, Acinetobacter species, especially Acinetobacter baumannii has been determined. Besides its significant taxonomical modification, the ability to upregulate multidrug resistance justifies its clinical importance.

A. baumannii is a nonfastidious, aerobic Gram-negative coccobacillus organism that exploits the variety of growth requirements. These properties contribute to the organism persistence and growth in hospital environment, thereby contributing to the transmission and development of intrinsic resistance to many antimicrobial agents.

Acinetobacter infections are commonly linked with the usage of a medical ventilator or any invasive devices. The risk factors include neurosurgery, acute respiratory distress syndrome, and head trauma. With the rise in the number of intensive care facilities and the increased use of invasive clinical procedures, the prevalence of Acinetobacter infections is anticipated to increase.[1]

The use of preventive measures, early detection, and treatment are critical to reducing the incidence of these infections. With the increase in the resistance due to various mechanisms (e.g.: carbapenamase production), it has limited the treatment option for clinicians.[2]

Hence, combination antibiotic therapy can potentially have a beneficial effect on Acinetobacter infections. This is known as synergy. The combination of ampicillin-sulbactam or sulbactam alone with carbapenems has been proven to improve the action of β-lactams against Carbapenem-resistant A. baumannii (CRAB) isolates significantly (Park et al., 2004; Wang et al., 2004).[3,4] However, it is important to note that synergy is not a guaranteed outcome and needs to be evaluated on a case-by-case basis. There are several methods that can be used to detect synergy between antibiotics, but the most commonly used tests are the checkerboard and time-kill tests. The checkerboard test is an easy way to measure inhibitory activity, while the time-kill test evaluates bactericidal activity.

This study particularly thrives on drawing an effective treatment by determining combinations in vitro antimicrobial synergy of meropenem-sulbactam by micro-broth dilution checker-board method.

Materials and Methods

Data and bacterial isolates collection

CRAB was selected from the clinical samples of endotracheal aspirate, blood, pus, throat swab, and urine specimens from 80 patients with suspected nosocomial infection during 1 year of duration.

Isolation and susceptibility testing of A. baumannii was done as performed in the microbiology laboratory. Specimens were streaked on 5% sheep blood agar and MacConkey agar plates which was incubated overnight at 37°C for the growth and identification of A. baumannii. When exhibiting positive growth, the bacterial Gram-positive and Gram-negative organisms recovered were identified to species level, and susceptibility testing was performed using automated VITEK 2 system.

Antibacterial agents and Micro broth dilution (minimum inhibitory concentration assay)

The antimicrobial drugs used in the combinations were meropenem (carbapenem) and sulbactam (β lactamase inhibitor) procured from Sigma-Aldrich.

Preparation of antimicrobial stock solutions was carried out using solvents and diluents according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, 2024. The prepared antimicrobial primary stock solutions of individual drug were stored in − 80°C as per the CLSI guidelines. The working stock solutions of each drug (Meropenem and sulbactam) were made by serial dilution using primary stock solution with concentrations that covered 3–4 dilutions above and below the minimum inhibitory concentration (MIC) breakpoint range (CLSI 2023 breakpoints).

MIC of individual drug is determined using micro-broth dilution method with standardized inoculum. The protocol according to the CLSI guidelines per say, is to first prepare broth suspension of CRAB isolated colonies from MacConkey Agar (pure culture) plate to match 0.5 McFarland. This suspension is then diluted in the ratio of 1:75 using cation-adjusted Muller Hinton broth to achieve the final concentration of 5 × 10⁴ CFU/ml. Following this step, microtiter wells were inoculated with their respective working stock solutions (25 µl) that contained desired concentrations (0.5 µg/ml–256 µg/ml for both drugs) from column 1 to 10, standardized inoculum 25 µl to column 1–11 and sterile CaMHB 50 µl to columns 1–10, and 75 µl to column 11 (growth control) and 100 µl to column 12 (Media control). After incubation for 24 h at 37°C, the microtiter wells are read visually. Escherichia coli ATCC 25922 was used as an internal quality control strain to validate the assay performed.

Checkerboard assay

The synergy testing of two drug combinations in various different concentrations was determined by checkerboard assay. Sulbactam (in powder form) and Meropenem were used in the checkerboard approach. Using a broth microdilution, the MIC for meropenem and sulbactam was predetermined and recorded. Following the introduction of 50 μl of a standardized inoculum into each well, Meropenem was serially diluted from 256 µg/ml to 0.5 µg/ml along the ordinate. Sulbactam was diluted twice serially by taking concentrations along the abscissa that were less than, equal to, and greater than their previously obtained MIC values. Rows 1 through 10 had 25 μl of each drug distributed across them; the 11^th well served as a growth control, and the 12^th as a media control. A checkerboard is made up of rows with the same quantity of drug diluted on the y-axis and wells with the same amount of the drug diluted along the x-axis.[5] Every well has a distinct mix of both drugs tested.[5] The microtiter wells are incubated at 37°C for 22–24 h after being sealed. The row and column with the lowest concentrations, where no turbidity (visual growth) was seen across the row and column, served as the basis to compute the fractional inhibitory concentration index (FICI) for each drug. The following formula was used to interpret the result as FIC:

FIC A = MIC of meropenem in combination/MIC of meropenem alone

FIC B = MIC of sulbactam in combination/MIC of sulbactam alone

FICI = FIC A + FIC B

Moreover, the interpretation is as follows: An FIC ≤ 0.5 indicated synergy, 0.5 to ≤2 indicated additive effect, and combination was said to be antagonistic if FIC >4.

Results

Patient clinical characteristics

From September 2013 to March 2024, 80 CRAB isolates were obtained for the study. Clinical characteristics such as age, co-morbidities, gender, residence in an intensive care facility, cases of malignancy and transplant recipient state within the mentioned duration were identified. A total of 80 isolates (53 Endotracheal aspirate, 15 from sputum, and 12 from pus) from 80 patients were isolated, sub-cultured and included for synergy studies.

Antimicrobial susceptibility testing by microbroth dilution

The individual MIC values of both meropenem and sulbactam against study isolates are depicted in Table 1.

Table 1.

Effect of combined meropenem and sulbactam against Acinetobacter baumannii isolates (n=80)

Isolate number	MIC of meropenem alone (μg/mL)	MIC of sulbactam alone (μg/mL)	MIC of meropenem with sulbactam (μg/mL)	MIC of sulbactam with meropenem (μg/mL)	FICI	Interpretation
1	32	64	2	8	0.185	Synergistic
2	32	256	16	32	0.625	Additive
3	128	64	32	8	0.375	synergistic
4	16	128	8	16	0.625	Additive
5	32	32	4	4	0.25	Synergistic
6	16	64	8	8	0.625	Additive
7	32	32	2	8	0.31	Synergistic
8	16	64	8	16	0.75	Additive
9	32	128	32	32	1.25	Indiffeerent
10	32	128	8	16	0.37	Synergistic
11	64	128	16	32	0.5	Synergistic
12	64	64	64	16	1.25	Indiffeerent
13	16	64	4	16	0.5	Indiffeerent
14	128	8	8	2	0.31	Synergistic
15	64	32	8	8	0.37	Synergistic
16	64	64	32	16	0.75	Additive
17	32	8	64	2	2.25	Indiffeerent
18	64	16	4	4	0.31	Synergistic
19	128	16	2	4	0.26	Synergistic
20	32	32	16	8	0.75	Additive
21	64	128	2	32	0.28	Synergistic
22	64	16	32	4	0.75	Additive
23	64	8	8	2	0.375	Synergistic
24	32	32	16	8	0.75	Additive
25	128	8	16	2	0.375	Synergistic
26	64	64	16	16	0.5	Synergistic
27	32	32	4	8	0.375	Synergistic
28	32	128	4	32	0.375	Synergistic
29	64	64	16	16	0.5	Synergistic
30	32	16	16	4	0.75	Additive
31	64	16	2	4	0.28	Synergistic
32	64	8	8	2	0.375	Synergistic
33	128	32	32	8	0.5	Synergistic
34	32	32	32	8	1.25	Indiffeerent
35	64	16	32	4	0.75	Additive
36	128	8	2	2	0.26	Synergistic
37	64	8	8	2	0.375	Synergistic
38	128	64	16	16	0.375	Synergistic
39	128	32	32	8	0.5	Synergistic
40	64	128	8	32	0.375	Synergistic
41	128	16	64	4	0.75	ADDITIVE
42	32	16	16	4	0.75	ADDITIVE
43	64	16	16	4	0.5	Synergistic
44	64	32	8	8	0.375	Synergistic
45	128	8	64	2	0.75	Additive
46	64	32	64	s	1.25	Indiffeerent
47	128	32	32	8	0.5	Synergistic
48	128	16	16	4	0.375	Synergistic
49	32	64	16	16	0.75	Additive
50	64	16	8	4	0.375	Synergistic
51	32	8	2	2	0.31	Synergistic
52	64	16	2	4	0.28	Synergistic
53	128	32	8	8	0.31	Synergistic
54	64	8	16	4	0.5	Synergistic
55	128	8	64	4	0.75	Additive
56	32	4	32	1	1.25	Indiffeerent
57	64	4	8	1	0.375	Synergistic
58	128	16	2	4	0.26	Synergistic
59	32	32	2	8	0.31	Synergistic
60	128	32	32	8	0.5	Synergistic
61	64	16	32	4	0.75	Additive
62	64	16	4	4	0.31	Synergistic
63	128	8	8	2	0.31	Synergistic
64	128	16	8	4	0.31	Synergistic
65	64	16	2	4	0.28	Synergistic
66	32	32	32	8	1.25	Indiffeerent
67	32	32	16	8	0.75	Additive
68	32	8	2	2	0.3	Synergistic
69	128	32	32	8	0.5	Synergistic
70	32	32	4	8	0.375	Synergistic
71	128	32	64	8	0.75	Additive
72	128	16	2	4	0.28	Synergistic
73	64	8	4	2	0.28	Synergistic
74	64	32	32	8	0.75	Additive
75	64	32	32	8	0.75	Additive
76	64	16	4	4	0.31	Synergistic
77	64	32	16	8	0.5	Synergistic
78	64	32	8	8	0.375	Synergistic
79	128	16	2	4	0.26	Synergistic
80	32	16	4	4	0.375	Synergistic

MIC=Minimum inhibitory concentration, FIC=Fractional inhibitory concentration

The MIC values of meropenem (Carbapenem) against all the CRAB isolates were within the resistance range (>16) which means all the test CRAB isolates exhibited resistance to meropenem drug. However, among 80 isolates, 22 of them showed the highest resistance concentration of 128 µg/ml, 31 isolates showed 64 µg/ml concentration of resistance, 23 of them showed 32 µg/ml, and 4 isolates displayed least resistance of 16 µg/ml.

Table 1 also enlists the MIC values of sulbactam which were obtained in diverse concentrations. Majority of the isolates were in the resistance range of concentration (>16 µg/ml) i.e. 256 µg/ml concentration of isolates being the highest, 7 isolates showed 128 µg/ml concentration of resistance, whereas 21 isolates showed 16 µg/ml of resistance. Fourteen isolates exhibited intermediate MIC value (8 µg/ml) concentration and two isolates were found sensitive to the sulbactam drug, concentration being 4 µg/ml.

Synergy testing

After obtaining the MIC values and interpreting each isolate breakpoint drug concentration, synergy of meropenem in combination with sulbactam and sulbactam in combination of meropenem was examined and the values obtained are represented in Table 1. Two drugs (meropenem and sulbactam) with different concentration were put up in the unwanted microtiter wells for each isolate according to the synergy testing procedure[6] in reference to their individual MIC obtained previously.

Although all the isolates selected were not only carbapenem resistant also multi-drug resistant, to keep a track on the sensitivity pattern of all the isolates participated becomes necessary objective to not neglect any potential treatment options.[7] Our study found out that out of 80 isolates, most of them 46 (57.5%) were sensitive to tigecycline following the combination drug cefoperazone/sulbactam 26 isolates (32.5%). Quarter of the total number was found to be sensitive to minocycline, whereas 21.25% of sensitivity was shown to cephalosporin class of drug (ciprofloxacin). The sensitivity pattern to other class of drugs such as aminoglycosides (gentamycin - 15% and amikacin - 7%), co-trimoxazole (15%) kept decreasing with the least sensitive rate being 6.25% for levofloxacin [Graph 1].[7]

Graph 1.

Antimicrobial susceptibility Percentage of Carbapenem resistant Acinetobacter baumannii[7]

Graph 2 highlights the various interactions between the two drugs. The interpretation is based on the reference table obtained from Ozseven et al. 2012.[2] Synergistic interaction was detected among 66% of isolates (53 isolates of 80). About 25% of them displayed additivity. Only 7 isolates, i.e. 9% showed indifferent interaction and none of the isolates demonstrated antagonistic interaction.

Graph 2.

Synergy testing data comparison

Discussion

A. baumannii is commonly identified from illnesses related to healthcare, particularly pneumonia linked with ventilator use. Globally, A. baumannii carbapenem resistance has been rising over the past 10 years, and there are currently very few treatment choices for infections that pose a serious risk to human life.[6] Hence, the selection of antimicrobials capable of penetrating the cell at high concentrations and appropriate therapy duration are necessary for successful treatment. Patients must closely follow th treatment guidelines to avoid problems and relapses. If medication is not administered as prescribed, the phagocytosed germs will infect the host again. Patients’ quality of life will be impacted by relapses and chronic cases, which might result in financial losses.[8]

Consistent with our results, numerous prior investigations exploring the “in vitro synergistic actions of carbapenems”, with sulbactam-containing drugs have documented substantial synergy rates (Anandan et al., Özseven et al., Abolfazl vahhabi et al.).[2,9] In addition, Anandan et al.’ s 2016 clinical study published in J Infect Dis Ther, one of the few that was undertaken, found positive clinical outcomes of the meropenem + ampicillin/sulbactam combination, demonstrating a greater rate of synergy (94.1%). Furthermore, the checkerboard assay showed a 52% synergy between carbapenem and sulbactam.[9] Because of the high rates of synergism between carbapenems and sulbactam-containing drugs, we consider that these combinations could be useful as alternatives for treating multidrug-resistant A. baumannii infections.

Although sulbactam as a drug has nominal antibacterial action, its ability to bind to penicillin-binding protein 2 is exceptional along with its anti-β lactamase activity.[10] On the other hand, due to their strong binding ability to penicillin-binding protein, imipenem and meropenem have significant action against the majority of Gram-negative bacteria.[11] Hence, when meropenem and sulbactam are taken together, synergism is assumed to happen owing to the improved accessibility of meropenem to its designated site causing cell damage.

While there are several ways to test for synergism, the checkerboard synergy testing has the upper-hand of allowing the concentration of antibiotics to be changed, unlike the Epsilometer-test strip, which only offers fixed antibiotic concentrations. Time-kill testing provides data on how quickly synergistic action occurs, but it is not practical to test numerous isolates at once because it is laborious and time-consuming.[12] The E-test method is the simplest, but it has less standardization and is limited by the antibiotic’s fixed concentration on the strips. Therefore, in the current study, checkerboard assay is the choice of method for testing synergy where meropenem and Sulbactam are the choice of drug combinations to be tested.

MIC (according to the CLSI guidelines 2020) was determined for the individual drug for various concentrations and the breakpoint susceptibility for each CRAB isolate that were included in the study was noted. Since the choice of the test organism is already found to be carbapenem resistant, the concentration range of susceptibility was found to be higher than the breakpoint susceptibility range in reference to the CLSI breakpoints. The highest susceptibility drug concentration of meropenem was found to be 128 µg/ml for 22 isolates and least being 16 µg/ml for 4 isolates. The susceptibility range of sulbactam was found to be much higher compared to meropenem, highest being 256 µg/ml for the single isolate and lowest being 4 µg/ml for two isolates. Considering the susceptibility range, checkerboard assay was performed combining Meropenem and sulbactam in various concentrations to determine the synergy, indifferent or antagonistic interaction of respective CRAB isolate.

Synergy determination using the checker board assay involves a formula and a table that provides range of values that corresponds to various interactions (synergistic, additive, indifferent, and antagonistic) of drugs employed. Many studies that have employed synergy testing is found to have different FICI. For instance, Le Minh et al.[13] took a different tack, considering FICI ≤ 0.5 as synergistic, indifferent 0.5 < FICI <4, and antagonistic when FICI ≥4. Bonapace et al.[14] considers synergism if the values are ≤0.5, while values of > 0.5–4 considered to be additive/indifferent, and antagonism at >4. However, we used the similar combination of antibiotics (meropenem and Sulbactam) on the Carbapenem resistant A. baumannii isolates as Anandan et al.[8] had done in a comparable study in 2016.

The purpose of conducting synergy testing is to determine whether combining antibiotics can enhance the clinical outcomes in patients whose in vitro synergy analysis of antibiotics demonstrated synergism to extend the empiric therapy and postpone the development of resistance during antimicrobial treatment. Out of 80 isolates, 66.25% of them showed synergism FICI ≤ 0.5 which proves that Meropenem and Sulbactam can be an effective combination which could be considered as a potential therapeutic option against CRAB isolates.

Conclusion

In the current checkerboard method study, we observed potential synergistic activity between meropenem and sulbactam against CRAB A. baumannii isolates which indicates that this combination can be an appealing strategy in the treatment of nosocomial infections caused by CRAB isolates. Nevertheless, since in vitro settings cannot fully replicate in vivo settings, more clinical trials are needed to determine their efficacy and to investigate their therapeutic potential.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

JSSAHER Institute.