In Vitro Synergy Evaluation of Trimethoprim/Sulfamethoxazole Combined with Levofloxacin and Ceftazidime Against Stenotrophomonas maltophilia: A Comparative Study Using Checkerboard and Gradient Diffusion Methods
1
Department of Medical Microbiology, Faculty of Medicine, Bursa Uludağ University, Bursa 16000, Turkey
2
Department of Infectious Diseases and Clinical Microbiology, Faculty of Medicine, Bursa Uludağ University, Bursa 16000, Turkey
*
Author to whom correspondence should be addressed.
Acta Microbiol. Hell. 2025, 70(3), 37; https://doi.org/10.3390/amh70030037
Submission received: 23 June 2025
/
Revised: 12 September 2025
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Accepted: 13 September 2025
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Published: 22 September 2025
Abstract
Stenotrophomonas maltophilia is a nosocomial pathogen that is resistant to many broad-spectrum antibiotics. This study aimed to evaluate the in vitro synergy of trimethoprim/sulfamethoxazole (SXT) combined with levofloxacin (LEV) or ceftazidime (CAZ) using checkerboard and gradient diffusion methods. Between 2016 and 2021, 20 S. maltophilia strains (five SXT-resistant and 15 SXT-susceptible strains) were collected from various clinical settings. Their susceptibility to SXT, LEV, and CAZ was assessed using both checkerboard and gradient diffusion synergy tests. The gradient diffusion method was performed using commercial strip-based tests (Liofilchem®). It should be noted that the gradient diffusion method has not been standardized by the Clinical and Laboratory Standards Institute (CLSI) for synergy testing and is considered for research purposes only. In the checkerboard method, the SXT + LEV combination showed synergy in one strain and an additive effect in 19 strains; the SXT + CAZ combination exhibited synergy in eight strains and an additive effect in 12 strains. In the gradient diffusion method, the SXT + LEV combination showed synergy in one strain and an additive effect in 19 strains; the SXT + CAZ combination exhibited synergy in five strains, an additive effect in 14 strains, and antagonism in one strain. A correlation between the two methods was observed in 90% of SXT + LEV combinations and 65% of SXT + CAZ combinations. Both checkerboard and gradient diffusion methods yielded similar results, indicating their reliability in determining antibiotic combinations. Given the observed synergy, CAZ combinations may be effective for treating SXT-resistant strains.
1. Introduction
Stenotrophomonas maltophilia is a bacterium that causes nosocomial infections, exhibits multidrug resistance (MDR), is increasingly isolated due to the intensive use of carbapenems to which it is intrinsically resistant, and causes high morbidity and mortality in infections [1,2,3]. There is a limited number of antibiotics that can be used against S. maltophilia. The problem is compounded when these difficulties are combined with the methodological problems associated with susceptibility testing. According to recent surveillance data, the prevalence of S. maltophilia in clinical samples is increasing globally, particularly in intensive care units and immunocompromised patient populations. In the United States, it is reported as the third most common non-fermenting Gram-negative bacillus isolated from hospitalized patients [4]. Resistance rates to trimethoprim/sulfamethoxazole (SXT), the first-line treatment, vary between 5% and 20% depending on the region, while levofloxacin (LEV) and ceftazidime (CAZ) resistance can exceed 30% in some areas [5,6]. These rising resistance rates highlight the need for alternative therapeutic approaches. The resistance of S. maltophilia to SXT and other agents is largely attributed to specific genetic mechanisms. The sulfonamide resistance genes sul1 and sul2, which encode alternative dihydropteroate synthases, and the dfrA gene family, encoding dihydrofolate reductase variants, are commonly found in resistant strains. These genes are often carried on mobile genetic elements such as plasmids or class 1 integrons, facilitating horizontal gene transfer. In addition, efflux pump systems, biofilm formation, and decreased outer membrane permeability contribute significantly to the intrinsic and acquired MDR [7,8,9,10]. In recent years, combined treatment approaches have gained importance with the use of antibiotic combination testing in MDR bacteria [11]. In the presence of resistance to SXT, which is the first choice for S. maltophilia, or in cases where it cannot be evaluated, it remains unclear which antibiotics or combinations can be considered as an alternative, as studies are insufficient and the methods are not standardized.
According to the Clinical and Laboratory Standards Institute (CLSI) 2024 guidelines, although SXT remains the recommended first-line therapy, its use as monotherapy is discouraged in severe infections due to the potential for resistance development and treatment failure [12,13,14,15]. Several in vitro studies have explored combinations of SXT with LEV or CAZ, reporting varying degrees of synergistic or additive activity. However, clinical evidence supporting their efficacy is still limited to small-scale studies and case reports, indicating a need for further clinical research to validate their use.
The aim of this study was to determine the in vitro synergistic status of combinations of SXT with LEV or CAZ antibiotics and to compare the in vitro efficacy results of gradient diffusion and checkerboard methods.
2. Materials and Methods
This study was approved by the Clinical Research Ethics Committee of Bursa Uludag University Faculty of Medicine (decision number: 2021-6/64) and supported by the Scientific Research Projects of Bursa Uludag University (Project number: TYL-2021-608).
Samples analyzed using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS; Bruker Daltonik, Bremen, Germany) at the Department of Medical Microbiology, Bacteriology Laboratory, Bursa Uludag University Health Application and Research Center, and collected from various intensive care units and outpatient clinics between 2016 and 2021, were included in the study. Among these, 20 strains identified as S. maltophilia were selected and tested for antibiotic susceptibility using the Phoenix M50 automated system (Becton Dickinson, Franklin Lakes, NJ, USA). The strains tested for antibiotic susceptibility were selected from isolates stored at −20 °C in bead-based cryovials (Pro-Lab, Wirral, UK). These isolates were obtained from blood, respiratory tract (including sputum, bronchoalveolar lavage, and pleural fluid), urine, and peritoneal fluid samples. The strains were not selected consecutively; all SXT-resistant isolates obtained during the study period were included; therefore, the five resistant strains represent all available resistant isolates. If the same patient grew S. maltophilia more than once, only the first isolated strain was included in the study. Fresh cultures of the strains to be included were prepared, revived on 5% sheep blood agar, and identification was confirmed by MALDI-TOF MS.
Antimicrobial susceptibility testing and MIC determination were performed by the broth microdilution method according to the CLSI guidelines [14]. Checkerboard and gradient diffusion methods were then used to assess synergy. The susceptibility of the strains to the antibiotics SXT (Sigma Aldrich, St. Louis, MO, USA), LEV (Sigma Aldrich, St. Louis, MO, USA), and CAZ (Sigma Aldrich, St. Louis, MO, USA), and the in vitro evaluation of the combinations between these antibiotics, were performed using checkerboard and gradient diffusion tests. The combinations were prepared as SXT/LEV and SXT/CAZ.
2.1. Checkerboard Method
The efficacy of the combinations was evaluated by combining the two antibiotics used in the combinations (SXT/LEV, SXT/CAZ) at different concentrations in the horizontal and vertical axes on sterile 96-well polystyrene microplates with U-bottom. Solvents, dilution ratios, and bacterial inoculum of the antibiotics in combination were prepared according to the CLSI guidelines [14,15]. The number of bacteria in 100 µL of the antibiotic combination solution in one well was distributed in the microplates to a level of 5 × 105 CFU/mL. Escherichia coli ATCC 25922 was used as a quality control strain.
The MIC values of SXT, LEV, and CAZ were interpreted according to the Stenotrophomonas maltophilia criteria in the 2024 Clinical Breakpoint Guide of the Clinical and Laboratory Standards Institute (CLSI).
For combination testing, Fractional Inhibitory Concentration (FIC) values were calculated using the following formulas:
FICLEV = MICLEV (+SXT)/MICLEV (alone)
FICSXT = MICSXT (+LEV)/MICSXT (alone)
FICCAZ = MICCAZ (+SXT)/MICCAZ (alone)
FICSXT = MICSXT (+CAZ)/MICSXT (alone)
The FIC index (∑FIC) was calculated by summing the individual FIC values:
where
∑FIC = FICSXT + FICLEV for the SXT + LEV combination
∑FIC = FICSXT + FICCAZ for the SXT + CAZ combination
- MICLEV (+SXT) is the MIC of levofloxacin in the presence of sulfamethoxazole-trimethoprim (SXT);
- MICLEV (alone) is the MIC of levofloxacin alone;
- MICSXT (+LEV) is the MIC of SXT in the presence of levofloxacin, and so forth.
Alternatively, the ∑FIC can be generalized as:
where MICA and MICB are the MICs of drugs A and B alone, respectively, and CA and CB are the concentrations of the drugs in combination.
∑FIC = CA/MICA + CB/MICB
When evaluating the combination wells, the well with the lowest Fractional Inhibitory Concentration Index (FICI) value was considered for analysis. The FIC and FICI values were interpreted as follows: if ∑FICI ≤ 0.5, the effect was considered synergistic; if 0.5 < FICI ≤ 4, additive; and if FICI > 4, antagonistic [14,15,16]. Each combination experiment using the checkerboard method was performed in two technical replicates to ensure the reliability of the results.
2.2. Gradient Diffusion Method
Bacterial suspensions prepared from S. maltophilia colonies conforming to the 0.5 McFarland standard were homogeneously inoculated onto Mueller–Hinton agar (MHA) medium, and SXT, LEV, and CAZ (Liofilchem, Roseto degli Abruzzi, Italy) antibiotic gradient diffusion strips were used as a single test; then, combination tests were performed by first applying SXT and then other antibiotic strips to measure the combination.
The quality control strain E. coli ATCC 25922 was used for the quality control of the test.
After a 24 h incubation period at 35 ± 2 °C, the media were evaluated according to the criteria outlined in the clinical breakpoint table for S. maltophilia in the CLSI (2016) guidelines. The FIC and FICI values were calculated, and ∑FICI ≤ 0.5 was interpreted as a synergistic effect, 0.5 < FICI ≤ 4 as an additive effect, and FICI > 4 as an antagonistic effect.
2.3. Statistical Analysis
The sensitivities of the antibiotics used in the study, the results of the combinations, and the differences between the two methods are stated as descriptive statistics, frequencies, and percentages for qualitative data. Pearson chi-square, Fisher–Freeman–Halton, and Fisher’s Exact Chi-square tests were used to analyze the categorical data. The significance level was set at p = 0.05. Statistical analysis of the data was performed using the statistical package program IBM SPSS 28.0 (IBM Corp., released 2021. IBM SPSS Statistics for Windows, Version 28.0., Armonk, NY, USA: IBM Corp.).
3. Results
A total of 20 S. maltophilia strains were obtained from 20 different patients. Of these, six strains (30%) were isolated from the respiratory tract (including one from sputum, one from bronchoalveolar lavage, and four from pleural fluid), one strain (5%) from urine, two strains (10%) from peritoneal fluid, and 11 strains (55%) from blood samples. The antimicrobial susceptibility results, interpreted according to the CLSI (2016) clinical breakpoints, are presented in Table 1.
When examining the FIC and FICI results of the antibiotics tested using the checkerboard method (Table 2), the following observations were made: with the SXT + LEV combination, synergy was detected in one strain and additive effects were observed in 19 strains; with the SXT + CAZ combination, synergy was observed in eight strains and additive effects were observed in 12 strains.
Using the gradient diffusion method (Table 3), the SXT + LEV combination showed synergy in one strain and an additive effect in 19 strains. The SXT + CAZ combination exhibited synergy in five strains, additive effects in 14 strains, and antagonism in one strain.
Furthermore, when the results from both the checkerboard and gradient diffusion methods were evaluated together, a correlation was observed in 18 strains (90%) for the SXT + LEV combination and in 13 strains (65%) for the SXT + CAZ combination (Table 4).
These findings indicate a high degree of concordance between the two methods, supporting the reliability of the observed synergistic and additive interactions among the tested antibiotic combinations.
For the SXT + LEV combination in the five SXT-resistant strains examined in our study, a 100% additive effect was detected using the checkerboard method. In contrast, the gradient diffusion method revealed 80% additive and 20% synergistic effects. For the SXT + CAZ combination in five SXT-resistant strains, the checkerboard method identified 80% synergistic and 20% additive effects, while the gradient diffusion method showed 60% synergistic, 20% additive, and 20% antagonist effects. (Table 5).
4. Discussion
MDR S. maltophilia infections, particularly in intensive care unit (ICU) patients, contribute significant to morbidity and mortality while narrowing the therapeutic options. Effective management is further complicated by the lack of standardized antibiotic susceptibility testing protocols, which makes selecting appropriate treatment regimens challenging [2,3].
In terms of susceptibility testing, EUCAST recommends testing only SXT, while CLSI guidelines list SXT as the first-line therapy and LEV and CAZ as second-line options [15,16]. In various studies, resistance rates in S. maltophilia strains have been reported to range from 1% to 48.2% for SXT and from 3.8% to 43.4 for LEV [17,18,19,20,21,22,23]. In Turkey, the resistance rates reported range from 0% to 56% for SXT and 6% to 24% for LEV [24,25,26,27,28,29,30,31,32,33]. In our study, levofloxacin resistance was detected at 35% by the microdilution method and 25% by the gradient diffusion method. These relatively high rates may be associated with the inclusion of SXT-resistant strains in our sample. Similarly, several studies have reported that LEV resistance tends to be higher among S. maltophilia isolates that are also resistant to SXT [23,24,25,26,27,28,29,30,31,32,33].
CAZ resistance also varies widely between countries and institutions, with reported rates ranging from 34.9% to 85% globally [17,18] and 30% to 88.8% in Turkey [24,25,26,27,28,29,30,31,32,33]. Our study found a CAZ resistance of 80% by microdilution and 65% by gradient diffusion among the 20 selected isolates. This suggests that using CAZ alone in S. maltophilia infections would not be an appropriate choice.
Several factors can influence in vitro CAZ susceptibility results. For instance, Hamdi et al. found that the MIC values obtained in nutrient-limited media may yield more accurate readings than those in rich media, suggesting that some strains labeled as CAZ-resistant might, in reality, still be susceptible [34]. Recently, interpretive criteria for ceftazidime against S. maltophilia were removed by both CLSI and FDA due to insufficient clinical data to validate existing breakpoints. Clinical data have been mixed, with some studies reporting treatment success while others report failure. Notably, Matthew C. Phillips et al. demonstrated that ceftazidime retains in vivo activity against S. maltophilia strains that are predicted to be resistant by conventional testing methods [35]. We believe antimicrobial testing is inadequate and that advanced testing strategies, such as using zinc-limited media for cultures, may better model in vitro ceftazidime activity. Improved susceptibility testing could better discriminate truly resistant isolates from those incorrectly identified as resistant by conventional methods. This implies CAZ may remain a viable treatment in cases of MDR S. maltophilia, although CLSI and EUCAST have both discontinued breakpoints for CAZ, which complicates interpretation in clinical labs.
Numerous studies have assessed the efficacy of antibiotic combinations against S. maltophilia, yielding variable outcomes—synergy, additivity, or antagonism [36,37,38,39]. Notably, the 2024 Infectious Diseases Society of America (IDSA) guideline recommends combination therapies including cefiderocol, minocycline, SXT, and LEV. As an alternative, a ceftazidime–avibactam (CAZ-AVI) plus aztreonam regimen is suggested. The guideline recommends SXT dosing based on the trimethoprim component at 10–15 mg/kg, but that clinicians should consider its bacteriostatic nature [38]. In Turkey, cefiderocol is not yet available, and the removal of CAZ breakpoints by CLSI/EUCAST complicates CAZ-based decision-making, making this guideline especially valuable.
Previous studies have reported varying degrees of synergy for antibiotic combinations against S. maltophilia. Specifically, synergy rates for the SXT + CAZ combination ranged from 56% using the checkerboard method to 72% with gradient diffusion, while the SXT + LEV combination showed synergy in 23.6%, additivity in 68.5%, and antagonism in 7.9% of isolates [36]. In our study, the SXT + CAZ combination exhibited 40% synergy and 60% additivity by the checkerboard method, compared to 25% synergy, 70% additivity, and 5% antagonism by the gradient diffusion method. The SXT + LEV combination consistently demonstrated 95% additivity and 5% synergy with both methods. Notably, synergy was more pronounced in SXT-resistant strains treated with the SXT + CAZ combination.
Statistical analyses revealed no significant differences between microdilution and gradient diffusion for synergy detection. Given its practical advantages—speed and ease of use—gradient diffusion may be better suited for routine laboratory practice. However, only in vitro data are currently available; in vivo and time–kill studies are required to confirm clinical relevance [39,40,41,42].
LEV’s generally low to moderate MIC values suggest it could be considered for empirical therapy, although confirmation from further in vitro and in vivo research is needed. CAZ, with prevalent intermediate or resistant MIC readings, may not be appropriate for empirical use. These combination data suggest LEV- or CAZ-based regimens may have a role in treating SXT-resistant strains, but additive effects do not guarantee clinical success and, thus, warrant cautious interpretation [43,44].
Our previous work showed no significant mortality difference between SXT and LEV regimens, and early appropriate therapy initiation and central venous catheter removal improved outcomes [45]. This underscores that treatment success is multifactorial and not solely tied to antimicrobial synergy.
In conclusion, this study evaluated the in vitro synergy of SXT + LEV and SXT + CAZ. The observed synergy in some CAZ-resistant isolates suggests that SXT + CAZ may be a viable alternative in scenarios with limited therapeutic options. Furthermore, reports of synergy with SXT + LEV in selected cases support the potential role of combination therapies, which should be tailored to specific clinical contexts.
5. Limitations
This study has several limitations. First, the relatively small number of isolates (n = 20) restricts the generalizability of the findings. Additionally, only two antibiotic combinations (SXT + LEV and SXT + CAZ) were evaluated, while other potentially synergistic regimens were not tested. All synergy assessments were conducted in vitro, and no in vivo or clinical outcome data were available to support direct application in clinical practice. Furthermore, the CLSI M26 document highlights the lack of standardized protocols for synergy testing, contributing to potential variability between laboratories. Lastly, only two testing methods (checkerboard and gradient diffusion) were used, and time–kill assays, which could provide more dynamic and comprehensive data, were not performed.
6. Conclusions
Although the number of strains and antibiotics tested in combination was limited, our study provides valuable preliminary insights into the treatment of multidrug-resistant S. maltophilia strains. Due to notable regional and institutional variability in resistance patterns, it is essential for individual healthcare facilities to perform similar susceptibility and synergy testing to tailor optimal treatment strategies.
Given the exploratory nature and relatively small sample size of our study, these findings should be interpreted with caution. Nonetheless, the demonstrated in vitro synergy of SXT + CAZ—especially among CAZ-resistant isolates—highlights its potential as an alternative therapy in cases with limited options. Additionally, the consistent additive effects observed with SXT + LEV suggest a supportive role for this combination in clinical practice.
Further investigations with larger cohorts, including in vivo and clinical outcome studies, are warranted to validate these results and to better understand the clinical relevance of combination therapies against S. maltophilia. Such research will help refine the therapeutic guidelines and improve patient management in the face of rising multidrug resistance.
Author Contributions
M.P. and R.B. design, study, and analyzed the work; M.P. and H.A. prepared the draft of the study; E.K. contributed to writing the article. All authors have read and agreed to the published version of the manuscript.
Funding
This study was supported by the Scientific Research Projects of Bursa Uludag University (Project number: TYL-2021-608).
Institutional Review Board Statement
This study was approved by the Bursa Uludağ University Faculty of Medicine Clinical Research Ethics Committee (decision number: 2021-6/64, approved on 29 September 2021).
Informed Consent Statement
The authors certify that all experiments were performed in accordance with the relevant guidelines and regulations. This study was conducted prospectively on strains obtained from cultural materials, and, therefore, informed consent forms were not required in accordance with national ethics committee regulations. Our article does not contain data belonging to any person.
Data Availability Statement
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Acknowledgments
We would like to thank all laboratory technicians who supported the laboratory studies of this research.
Conflicts of Interest
The authors declare that they have no financial, personal, or professional interests that could be perceived as influencing the work reported in this paper. There are no known conflicts of interest or biases related to this study.
Abbreviations
The following abbreviations are used in this manuscript:
| CLSI | Clinical and Laboratory Standards Institute |
| MIC | Minimum Inhibitory Concentration |
| SXT | Sulfamethoxazole–Trimethoprim |
| LEV | Levofloxacin |
| CAZ | Ceftazidime |
| FIC | Fractional Inhibitory Concentration |
| FICI | Fractional Inhibitory Concentration Index |
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Table 1.
Susceptibility of S. maltophilia strains to antibiotics.
| Antibiotic | Method | CLSI Clinical Breakpoint | S | I | R | |||
|---|---|---|---|---|---|---|---|---|
| n | % | n | % | n | % | |||
| SXT | checkerboard method | S ≤ 2/38 | 15 | 75 | - | - | 5 | 25 |
| I - | ||||||||
| R ≥ 4/76 | ||||||||
| LEV | S ≤ 2 | 11 | 55 | 2 | 10 | 7 | 35 | |
| I 4 | ||||||||
| R ≥ 8 | ||||||||
| CAZ | S ≤ 8 | 3 | 15 | 1 | 5 | 16 | 80 | |
| I 16 | ||||||||
| R ≥ 32 | ||||||||
| SXT | gradient diffusion method | S ≤ 2/38 | 13 | 65 | 2 | 10 | 5 | 25 |
| I - | ||||||||
| R ≥ 4/76 | ||||||||
| LEV | S ≤ 2 | 13 | 65 | 2 | 10 | 5 | 25 | |
| I 4 | ||||||||
| R ≥ 8 | ||||||||
| CAZ | S ≤ 8 | 4 | 20 | 3 | 15 | 13 | 65 | |
| I 16 | ||||||||
| R ≥ 32 | ||||||||
n: Number of S. maltophilia strains; SXT: trimethoprim-sulfamethoxazole, LEV: levofloxacin, CAZ: ceftazidime; S: susceptible, I: susceptible, increased exposure, R: resistant.
Table 2.
MIC and FICI results of antibiotic combinations against S. maltophilia strains using the checkerboard method.
Table 2.
MIC and FICI results of antibiotic combinations against S. maltophilia strains using the checkerboard method.
| Strain No | SXT | LEV | CAZ | LEV + SXT | SXT + LEV | FICI SXT + LEV | CAZ + SXT | SXT + CAZ | FICI SXT + CAZ | Interaction SXT + LEV | Interaction SXT + CAZ |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | ≥16/304 | 0.5 | 64 | 0.5 | 0.5/9.5 | 1.03125 | 8 | 0.5/9.5 | 0.15625 | Additive | Synergy |
| 2 | 0.5/9.5 | 4 | 32 | 0.25 | 0.5/9.5 | 1.0625 | 1 | 0.25/4.75 | 0.5312 | Additive | Additive |
| 3 | 0.5/9.5 | 2 | 32 | 0.25 | 0.25/4.75 | 0.625 | 1 | 0.5/9.5 | 1.03125 | Additive | Additive |
| 4 | 0.5/9.5 | 1 | ≥64 | 0.5 | 0.125/2.375 | 0.75 | 1 | 0.25/4.75 | 0.515625 | Additive | Additive |
| 5 | ≥16/304 | 2 | 32 | 1.0 | 0.125/2.375 | 0.5078125 | 4 | 1/19 | 0.1875 | Additive | Synergy |
| 6 | 2/38 | 16 | ≥64 | 4.0 | 1/19 | 0.75 | 4 | 1/19 | 0.5625 | Additive | Additive |
| 7 | 0.5/9.5 | 0.5 | ≥64 | 0.5 | 0.25/4.75 | 1.5 | 4 | 0.125/2.375 | 0.3125 | Additive | Synergy |
| 8 | 16/304 | 2 | 32 | 1.0 | 0.125/2.375 | 0.5078125 | 2 | 0.25/4.75 | 0.078125 | Additive | Synergy |
| 9 | 2/38 | 8 | ≥64 | 0.5 | 1/19 | 0.5625 | 2 | 2/38 | 1.03125 | Additive | Additive |
| 10 | 0.5/9.5 | 2 | ≥64 | 0.25 | 0.25/4.75 | 0.625 | 8 | 0.125/2.375 | 0.375 | Additive | Synergy |
| 11 | 0.5/9.5 | 0.5 | 16 | 0.25 | 0.125/2.375 | 0.75 | 1 | 0.125/2.375 | 0.3125 | Additive | Synergy |
| 12 | 0.5/9.5 | 2 | ≥64 | 1.0 | 0.25/4.75 | 1.0 | 2 | 0.125/2.375 | 0.28125 | Additive | Synergy |
| 13 | 0.25/4.75 | 2 | 8 | 0.25 | 0.25/4.75 | 1.125 | 1 | 0.25/4.75 | 1.125 | Additive | Additive |
| 14 | 2/38 | ≥16 | 64 | 0.25 | 4/76 | 2.015625 | 16 | 1/19 | 0.75 | Additive | Additive |
| 15 | 0.5/9.5 | 16 | 32 | 0.5 | 0.5/9.5 | 1.03125 | 1 | 0.25/4.75 | 0.53125 | Additive | Additive |
| 16 | 0.5/9.5 | 8 | 8 | 0.5 | 0.125/2.375 | 0.3125 | 1 | 0.25/4.75 | 0.625 | Synergy | Additive |
| 17 | 0.5/9.5 | 4 | 64 | 2.0 | 0.5/9.5 | 1.5 | 2 | 0.25/4.75 | 0.53125 | Additive | Additive |
| 18 | ≥16/304 | ≥16 | ≥64 | 16.0 | 16/304 | 2.0 | 64 | 0.125/2.375 | 1.0078125 | Additive | Additive |
| 19 | ≥16/304 | ≥16 | ≥64 | 16.0 | 16/304 | 2.0 | 16 | 2/38 | 0.375 | Additive | Synergy |
| 20 | 0.5/9.5 | 0.5 | 1 | 0.5 | 0.125/2.375 | 1.25 | 1 | 0.125/2.375 | 1.25 | Additive | Additive |
SXT: trimethoprim/sulfamethoxazole; LEV: levofloxacin; CAZ: ceftazidime; MIC: Minimum Inhibitory Concentration; FICI: Fractional Inhibitory Concentration Index. Interpretation of FICI values: FICI ≤ 0.5: synergy; 0.5 < FICI ≤ 4.0: additive; FICI > 4.0: antagonism.
Table 3.
MIC and FICI results of SXT, LEV, and CAZ alone and in combination against S. maltophilia strains using the gradient diffusion method.
Table 3.
MIC and FICI results of SXT, LEV, and CAZ alone and in combination against S. maltophilia strains using the gradient diffusion method.
| Strain No | SXT | LEV | CAZ | LEV + SXT | SXT + LEV | FICI SXT + LEV | CAZ + SXT | SXT + CAZ | FICI SXT + CAZ | Interaction SXT + LEV | Interaction SXT + CAZ |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | ≥32 | 0.19 | 24 | 0.19 | 0.19 | 1.0059 | ≥256 | ≥256 | 18.66 | Additive | Antogonist |
| 2 | 0.5 | 4 | 8 | 0.5 | 0.5 | 1.125 | 0.5 | 0.5 | 1.06 | Additive | Additive |
| 3 | 0.5 | 0.75 | 32 | 0.25 | 0.38 | 1.09 | 0.5 | 0.38 | 0.77 | Additive | Additive |
| 4 | 0.38 | 0.38 | ≥256 | 0.19 | 0.19 | 1 | 0.38 | 0.25 | 0.658 | Additive | Additive |
| 5 | ≥32 | 0.75 | 96 | 0.25 | 0.25 | 0.34 | 4 | 0.75 | 0.06 | Synergy | Synergy |
| 6 | 3 | 24 | ≥256 | 3 | 6 | 2.125 | 3 | 2 | 0.67 | Additive | Additive |
| 7 | 0.38 | 0.5 | ≥256 | 0.25 | 0.25 | 1.15 | 0.38 | 0.38 | 1.0014 | Additive | Additive |
| 8 | ≥32 | 0.5 | 12 | 0.25 | 0.5 | 0.515 | 1 | 0.5 | 0.09 | Additive | Synergy |
| 9 | 2 | 6 | ≥256 | 2 | 2 | 1.33 | 1.5 | 0.75 | 0.380 | Additive | Synergy |
| 10 | 0.38 | 0.5 | 96 | 0.19 | 0.25 | 1.03 | 0.38 | 0.125 | 0.323 | Additive | Synergy |
| 11 | 0.38 | 0.25 | 24 | 0.25 | 0.25 | 1.65 | 0.5 | 0.5 | 1.33 | Additive | Additive |
| 12 | 0.75 | 1 | ≥256 | 1 | 0.75 | 2 | 1.5 | 0.75 | 1.005 | Additive | Additive |
| 13 | 0.125 | 0.38 | 6 | 0.19 | 0.19 | 2.02 | 0.19 | 0.19 | 1.55 | Additive | Additive |
| 14 | 4 | ≥32 | 48 | 6 | 6 | 1.68 | 2 | 2 | 0.54 | Additive | Additive |
| 15 | 0.75 | 8 | ≥256 | 1 | 0.5 | 0.785 | 0.5 | 0.5 | 0.661 | Additive | Additive |
| 16 | 0.25 | 0.38 | 6 | 0.19 | 0.19 | 1.26 | 0.125 | 0.125 | 0.52 | Additive | Additive |
| 17 | 0.5 | 1.5 | 48 | 0.38 | 0.5 | 1.25 | 0.75 | 0.38 | 0.77 | Additive | Additive |
| 18 | ≥32 | ≥32 | 128 | ≥32 | ≥32 | 2 | 6 | 8 | 0.29 | Additive | Synergy |
| 19 | ≥32 | ≥32 | 128 | ≥32 | ≥32 | 2 | 96 | 24 | 1.5 | Additive | Additive |
| 20 | 0.38 | 1 | 1 | 0.5 | 0.38 | 1.5 | 0.5 | 0.5 | 1.81 | Additive | Additive |
SXT: trimethoprim/sulfamethoxazole; LEV: levofloxacin; CAZ: ceftazidime; MIC: Minimum Inhibitory Concentration; FICI: Fractional Inhibitory Concentration Index. Interpretation of FICI values: FICI ≤ 0.5: synergy; 0.5 < FICI ≤ 4.0: additive; FICI > 4.0: antagonism.
Table 4.
Comparison of checkerboard and gradient diffusion methods.
| SXT + LEV Checkerboard Method | ||||
|---|---|---|---|---|
| Synergy | Additive | Antagonist | ||
| SXT + LEV gradientdiffusion method | Synergy | - | 1 | - |
| Additive | 1 | 18 | - | |
| Antagonist | - | - | - | |
| SXT + CAZ Checkerboard method | ||||
| Synergy | Additive | Antagonist | ||
| SXT + CAZ gradientdiffusion method | Synergy | 3 | 4 | 1 |
| Additive | 2 | 10 | - | |
| Antagonist | - | - | - | |
SXT: trimethoprim + sulfamethoxazole; LEV: levofloxacin; CAZ: ceftazidime.
Table 5.
Distribution of synergy results in SXT-sensitive and resistant strains.
| Strain No | SXT | LEV | CAZ | SXT + LEV (Checkerboard Method) | SXT + LEV (Gradient Diffusion Method | SXT + CAZ (Checkerboard Method) | SXT + CAZ (Gradient Diffusion Method |
|---|---|---|---|---|---|---|---|
| 1 | R | S | R | Additive | Additive | Synergy | Antagonist |
| 2 | S | I | R | Additive | Additive | Additive | Additive |
| 3 | S | S | R | Additive | Additive | Additive | Additive |
| 4 | S | S | R | Additive | Additive | Additive | Additive |
| 5 | R | S | R | Additive | Synergy | Synergy | Synergy |
| 6 | S | R | R | Additive | Additive | Additive | Additive |
| 7 | S | R | R | Additive | Additive | Synergy | Additive |
| 8 | R | R | R | Additive | Additive | Synergy | Synergy |
| 9 | S | R | R | Additive | Additive | Additive | Synergy |
| 10 | S | S | R | Additive | Additive | Synergy | Synergy |
| 11 | S | S | R | Additive | Additive | Synergy | Additive |
| 12 | S | S | R | Additive | Additive | Synergy | Additive |
| 13 | S | S | R | Additive | Additive | Additive | Additive |
| 14 | S | R | R | Additive | Additive | Additive | Additive |
| 15 | S | R | R | Additive | Additive | Additive | Additive |
| 16 | S | S | R | Synergy | Additive | Additive | Additive |
| 17 | S | S | R | Additive | Additive | Additive | Additive |
| 18 | R | R | R | Additive | Additive | Additive | Synergy |
| 19 | R | R | R | Additive | Additive | Synergy | Additive |
| 20 | S | S | R | Additive | Additive | Additive | Additive |
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Payaslioğlu, M.; Başkiliç, R.; Kazak, E.; Akalin, H. In Vitro Synergy Evaluation of Trimethoprim/Sulfamethoxazole Combined with Levofloxacin and Ceftazidime Against Stenotrophomonas maltophilia: A Comparative Study Using Checkerboard and Gradient Diffusion Methods. Acta Microbiol. Hell. 2025, 70, 37. https://doi.org/10.3390/amh70030037
AMA Style
Payaslioğlu M, Başkiliç R, Kazak E, Akalin H. In Vitro Synergy Evaluation of Trimethoprim/Sulfamethoxazole Combined with Levofloxacin and Ceftazidime Against Stenotrophomonas maltophilia: A Comparative Study Using Checkerboard and Gradient Diffusion Methods. Acta Microbiologica Hellenica. 2025; 70(3):37. https://doi.org/10.3390/amh70030037
Chicago/Turabian StylePayaslioğlu, Melda, Reyhan Başkiliç, Esra Kazak, and Halis Akalin. 2025. "In Vitro Synergy Evaluation of Trimethoprim/Sulfamethoxazole Combined with Levofloxacin and Ceftazidime Against Stenotrophomonas maltophilia: A Comparative Study Using Checkerboard and Gradient Diffusion Methods" Acta Microbiologica Hellenica 70, no. 3: 37. https://doi.org/10.3390/amh70030037
APA StylePayaslioğlu, M., Başkiliç, R., Kazak, E., & Akalin, H. (2025). In Vitro Synergy Evaluation of Trimethoprim/Sulfamethoxazole Combined with Levofloxacin and Ceftazidime Against Stenotrophomonas maltophilia: A Comparative Study Using Checkerboard and Gradient Diffusion Methods. Acta Microbiologica Hellenica, 70(3), 37. https://doi.org/10.3390/amh70030037
