Introduction

The emergence of multidrug-resistant (MDR) bacteria worldwide is a serious public health problem. Antimicrobial-resistant (AMR) bacteria cause over 2.8 million infections and 35,000 fatalities annually in the U.S. alone1. Globally, bacterial AMR led to 4.95 million deaths in 2019. It is estimated that 10 million people will die due to bacterial AMR by 20502. The WHO reported that in Europe, AMR directly leads to 133, 000 deaths annually and is indirectly associated with 541, 000 deaths3. Moreover, AMR causes a serious financial burden, and the U.S. CDC reported that health care costs exceed $4.6 billion annually to treat the six most alarming AMR bacteria4. In the European Union, losses reach approximately €11.7 billion each year for health expenditures5and economic losses reach approximately €5.1 billion because of reduced participation in the workforce. AMR bacteria affect not only human health but also animal and plant health. To date, drug-resistant microbes have been found in humans, animals, food, and the environment. Moreover, new resistance genes and mechanisms are emerging around the world. In summary, AMR bacteria threaten every country in the world and affect global food security and health. With the spread of drug-resistant bacteria, the world faces the crisis of inadequate research and development on new antibiotic pipelines, as the development of new antibiotics is obviously lagging behind the emergence and spread of drug-resistant bacteria. Therefore, new methods to address the harm caused by drug resistance are needed. These tactics include the reintroduction and revival of “old” antibiotics6the use of synergistic antibiotic pairs7and the synergistic combination of antibiotics with nonantibiotic drugs8,9.

Dimetridazole is a significant veterinary medicine that is commonly employed to treat infections caused by bacteria and protozoa in swine, poultry, and honeybees9. It has also been used as a growth promoter in animals10. However, the misuse of dimetridazole may lead to residues in food10. Dimetridazole residues may result in adverse effects11,12. With respect to safety concerns, dimetridazole (MET) has been prohibited for feed production and can only be used to treat infections caused by protozoa in China; however, dimetridazole substances should not be detectable in animal food13,14.

Cefotaxime is a third-generation cephalosporin antibiotic with a wide-ranging antimicrobial spectrum that is commonly used to treat infections caused by bacteria (including gram-positive, gram-negative, and anaerobic bacteria) resistant to first-line antibiotics15. With the widespread use of antibiotics, cefotaxime resistance in bacterial isolates is becoming a growing concern16,17.

In this context, we rediscovered the anthelmintic drug dimetridazole to explore the potential synergistic action of its combination with cefotaxime. Currently, there are no studies on the combination of dimetridazole and cefotaxime to revive the activity of cefotaxime against E. coli. Therefore, the objective of this study was to explore the activity of cefotaxime in combination with cefotaxime against multidrug-resistant E. coli isolates.

Materials and methods

Bacterial strains and drugs

We used the multidrug-resistant strain E. coli NX400 for the experiments. It was isolated from cows carrying the resistance genes blaTEM−1, blaCTX−M and Tet(A). Cefotaxime and dimetridazole were purchased from Solarbio (Beijing, China) and Mackin (Shanghai, China), respectively.

Assay of minimum inhibitory concentration (MIC)

The MICs of cefotaxime and dimetridazole against E. coli were evaluated by broth microdilution according to the CLSI method18. Briefly, cefotaxime and dimetridazole were serially diluted twofold with Mueller‒Hinton broth in 96-well plates to achieve the required final concentrations. In each well, 100 µL of bacterial suspension (106 CFU/mL) and 100 µL drug dilution were added, and the mixture was incubated at 37 °C for 16 h, after which the OD600 was measured. The MICs were determined in triplicate.

FICI calculations

The synergistic interaction of cefotaxime in combination with dimetridazole was assessed by checkerboard fractional inhibitory concentration index (FICI) testing. The FICI was measured in 96-well microtiter plates with twofold dilution steps. FICI = (MIC of drug A in combination / MIC of drug A alone) + (MIC of drug B in combination / MIC of drug B alone). ‘Synergy’ between antibiotics was defined as FICI ≤ 0.520.

Determination of growth curves

Growth curves were evaluated according to a previous study19. Based on the results of FICI, bacterial suspensions of E. coli were treated with 1/8 MIC cefotaxime (CEF), 1/16 MIC dimetridazole (MET), and a combination of 1/8 MIC CEF + 1/16 MIC MET (COM) at 37 °C with continuous shaking (160 rpm). At various time points, the OD600 of a 200 µL aliquot was measured using a spectrophotometer (Thermo Fisher, Vantaa, Finland).

LIVE/DEAD BacLight bacterial viability assay

A Live/Dead™ BacLight™ bacterial viability kit (Invitrogen, USA) was used to test E. coli viability after treatment with CEF, MET, and COM. Fresh E. coli (108 CFU/.

mL) were incubated with different drugs (CEF, MET, and COM) for 4 h at 37 °C. The treated E. coli was suspended and subsequently washed with PBS twice. The bacterial suspension was stained with Syto 9 and propidium iodide (PI) for 15 min at room temperature in the dark. The bacteria were subsequently centrifuged at 4500 rpm for 5 min to remove unreacted dye and then washed twice with PBS. The tagged cells were then subjected to laser scanning confocal microscopy (LSCM) (Carl Zeiss Microscopy GmbH, Jena, Germany). The signals from the red channel (kex = 535 nm; kem = 617 nm) and the green channel (kex = 485 nm; kem = 498 nm) were recorded using a confocal microscope.

Morphological observation by scanning electron microscopy (SEM)

The morphological alterations of E. coli after incubation with CEF, MET, and COM were examined using SEM to elucidate the mechanism of drug combination activity. E. coli was centrifuged at 4,500 × g for 10 min, and the resulting bacterial precipitates were washed twice with PBS. The precipitate cells were fixed for 2 h at room temperature with 2.5% glutaraldehyde and then incubated overnight at 4 °C. These cells were sequentially washed three times with phosphate buffer. The methods previously outlined were used to carry out the pretreatment steps for scanning electron microscopy analysis21.

Analysis of fatty acid composition changes in the cell membrane

E. coli treated with CEF, MET, or COM and E. coli without drug treatment composed the negative control group. Each group was cultivated at 37 °C for 4 h. Cultures were subsequently centrifuged at 4,500 × g for 10 min at 4 °C and washed twice. Following drying at 65 °C for 10 h, the harvested bacterial pellets were subjected to lipid extraction according to the Bligh and Dyer method22. Methyl esterification and dehydration were performed using a Hewlett-Packard 5890 gas chromatograph, as described previously23. The fatty acid content is expressed relative to the total amount of fatty acids.

qPCR verification of fatty acid biosynthesis-related genes

Total RNA was extracted using an E.Z.N.A. Bacterial RNA Kit (Omega, Georgia, USA) according to the manufacturer’s instructions. A Nanodrop One ultraviolet spectrophotometer (Thermo Scientific, WI, USA) was used to measure the purity and concentration of the RNA at the OD260 and OD280 wavelengths. The PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TakaRa, Dalian, China) protocol was followed to perform the qPCR. The sequences of primers used for real-time qPCR, as previously described24 are listed in Table 1. To assess the changes in the expression levels of each sample, 16 S rRNA (housekeeping gene) was used as an endogenous control. Independent biological replicates were performed in triplicate.

Table 1 Primers used in real-time qPCR.
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Galleria Mellonella infection model

The synergy between CEF and MET was assessed using the G. mellonella infection model. G. mellonella larvae were purchased from Tianjin Huiyude Biotech Company.

Larvae with any indications of melanization or deformity were discarded to prevent sampling bias. The G. mellonella were divided into four groups at random (n = 12 per group), and the right posterior gastropoda was injected with E. coli NX400 suspension (1.0 × 107 CFU). One hour post infection, the larvae were administered PBS, CEF, MET or COM at the left posterior gastropoda. The larvae were then kept at 37 °C in complete darkness after the injections. The larvae were observed and categorized as either dead or alive every 24 h. If there were no signs of movement in response to external stimulation and if the larvae exhibited dark pigmentation due to melanization, they were considered dead. The survival rates of G. mellonella were monitored over 5 days.

Statistical analysis

Statistical analysis was performed using Omicshare Online tools (https://www.omicshare.com/). Data from three biological replicates are shown as means ± standard deviation (SD). Unless specified otherwise, an unpaired two-tailed Student’s t-test for two groups or one way analysis of variance for multiple groups was employed to calculate p-values (* p < 0.05; ** p < 0.01).

Results

MIC assay and combination effect of CEF and MET on E. coli

The MICs of CEF and MET against E. coli NX400 were 512 µg/mL and 256 µg/mL, respectively. The checkerboard assay results revealed that 16 µg/mL (1/16 MIC) dimetridazole had a synergistic effect when combined with 64 µg/mL (1/8 MIC) CEF, resulting in an eightfold reduction in the MIC of cefotaxime. The FIC index was 0.188.

Growth inhibitory effects

The growth inhibitory effects of CEF, MET, and COM against E. coli strain NX400 were evaluated. The growth curves revealed that CEF and MET did not influence the growth of E. coli NX400 (Fig. 1). However, COM prolonged the logarithmic phase, increasing the time to reach the stationary phase.

Fig. 1
figure 1

The growth curve of E. coli.

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Evaluation of the integrity of the cell membrane and Inhibition of E. coli growth

The antibacterial activity is represented by the severity of cell membrane damage. Previous research has demonstrated the important role of membrane integrity in bacterial growth, as membrane damage directly impedes bacterial proliferation, ultimately leads to bacterial cell death25. We assumed that COM might damage the integrity of the E. coli cell membrane. To verify this hypothesis, SEM was used to evaluate the morphology of the cells subjected to different treatments. As shown in Fig. 2, the cells in the control group exhibited clear, intact, smooth edges, and the same short rod shape (Fig. 2A). Morphological changes, including elongated and slightly wrinkled cells, were observed in the group treated with CEF or MET alone (Fig. 2B and C). In contrast, in the group of E. coli exposed to COM, the cells presented clear deformation, caveolae, shriveling, and leakage of intracellular compounds, ions, and genetic materials (Fig. 2D). These results indicated that the antibacterial activity of COM was better than that of CEF and MET. Moreover, the cell morphology was strikingly different in the COM group compared with the CEF and MET groups. Based on the results, we speculate that COM employs a unique mechanism rather than a simple superposition of the two drugs acting alone. Wambaugh MA et al. have reported similar results — combined drug use activates a stress circuit not triggered by the individual drugs7.

Fig. 2
figure 2

Representative SEM images of E. coli untreated (A), showing clear edges, the same short rod, smooth cell membrane of a normal cell; E. coli treated with cefotaxime(B) and metronidazole (C), cells were elongated; E. coli treated with cefotaxime and metronidazole combination (D), cells deformation, severely crinkled, surface collapse and fusion.

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The effects of CEF, MET, and COM on the viability of E. coli were evaluated by Syto9/PI double staining. Normally, green fluorescent Syto9 stains cells with an intact membrane. In contrast, red fluorescent PI selectively identifies bacteria with damaged membranes. The results revealed that the amount of E. coli treated with COM was significantly lower than that of the control group, as well as the CEF-treated group and the MET-treated group, indicating that COM had a stronger inhibitory effect on E. coli. Moreover, the morphological changes were also investigated in E. coli after incubation with CEF and MET. In the CEF and MET groups, the cell morphologies were elongated, whereas in the COM group, the cell morphology was similar to that of the control. These results were also supported by SEM observations, indicating that the COM condition employs a unique antibacterial mechanism against E. coli, in addition to damaging the cell membrane. Based on the above experimental results, we concluded that the COM activity goes beyond the additive effect of each individual drug on its own26. Therefore, synergy is a promising strategy to overcome antimicrobial resistance.

Effects of COM on membrane permeability and fluidity

The above results clearly indicate that COM can damage the integrity of cell membranes, thereby exerting inhibitory effects on cell growth. To understand the mechanism in detail, we examined the effects of COM on cell membrane permeability and fluidity.

Cell membrane permeability can be evaluated by PI staining and fluorescence microscopy. Cells with compromised membranes that are dead or dying stain red with PI, whereas cells with intact membranes remain unstained. E. coli was treated with CEF, MET, COM, and then stained with PI. As depicted in Fig. 3, cell membrane damage in the COM-treated strain increased significantly compared to that in the CEF- and MET-treated strains. These findings further confirm the capacity of COM to cause cell membrane damage, thus increasing cell membrane permeability.

Fig. 3
figure 3

Bacterial viability assay by Styo9/PI staining. (A) E. coli treated with PBS; (B) E. coli treated with CEF. No inhibitory effect of CEF on E. coli; (C) E. coli treated with MET. No inhibitory effect of MET on E. coli; (D) E. coli treated with COM. Most bacteria were inhibited.

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The variation in the fluorescence intensity of the samples after Syto9 and PI staining allowed researchers to pinpoint the damage to the cell membrane. Green fluorescent Syto9 stains cells with an unbroken membrane, while red fluorescent PI selectively identifies bacteria with damaged membranes27. As shown in Fig. 4, the fluorescence intensity (red/green ratio, R/G) of E. coli treated with COM was greater than that of the other groups. Specifically, the fluorescence intensities of E. coli exposed to COM were 4.03 and 6.15 times greater than those of the CEF and control groups, respectively. These findings indicate that cell membrane damage was more severe in the COM group. Overall, these results suggest that the antibacterial mechanism is related to the cell membrane28.

Fig. 4
figure 4

Fluorescence intensity (red/green ratios, Ratio R/G) of E. coli treated with COM, CEF and MEF.

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Effect of COM on the composition and fluidity of the cell membrane

The cell membrane plays a key role in controlling cell structure and function and is composed mainly of lipids and proteins. Disturbance of membrane lipid composition can have a profound effect on cell fluidity and growth. To fully understand the effect of COM on membrane integrity, GC‒MS was performed to analyze the fatty acid composition and content. A total of 17 fatty acids were identified in the E. coli cell membrane after the relative percentages of membrane fatty acids were determined. These fatty acids included C11:0, C12:0, C14:0, C14:1, C15:1, C16:0, C16:1, C17:1, C18:0, C18:1n9t, C18:1n9c, C18:2 n6t, C18:2n6c, C18:3n6, C20:0, C20:2 and C21:0 (Fig. 5). Compared with those in the control group, the relative amounts of C14:1, C16:1, C18:1n9t, C18:2n6t, C18:2n6C and C20:2 was significantly greater. Saturated fatty acids (SFAs) and unsaturated fatty acids (UFAs) were separated into two groups from the 17 known fatty acids. Compared with the control, the relative content of UFAs significantly increased when E. coli was treated with COM. C14:1 (from 0 to 0.43%), C16:1 (from 0.44 to 0.83%), C181n9t (from 0.6 to 5.2%), C18:2n6t (from 1.59 to 2.23%), C18:2n6c (from 6.74 to 12.94%), and C20:2 (from 0 to 0.77%). The UFA contents were positively correlated with membrane fluidity29. Our study confirmed that COM treatment caused changes in the type and content of fatty acids in the cell membrane, which eventually led to increased membrane rigidity, diminished mobility, and increased susceptibility to cell death.

Fig. 5
figure 5

Composition of fatty acid in each group.

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Expression of fatty acid biosynthesis-associated genes following exposure to CEF, MET and COM

The fatty acid biosynthesis genes fab D, cfa, fab A and fab G were examined by real-time qPCR to evaluate their transcriptional levels in order to better investigate the molecular activity of COM against E. coli cells. After exposure to COM, noticeable variations in the expression levels of the genes involved in fatty acid production were observed, as shown in Fig. 6. Considerable down-regulation in fab A expression was noted following exposure to COM (Fig. 6A) compared to the control, CEF-treated and MET-treated groups. Similarly, the expression of fab D was significantly lower (Fig. 6B) in COM group than in the other groups. Compared with the control, CEF and MET treatments significantly inhibited the expression of fab G (Fig. 6C). After treatment with COM, the expression levels of cfa were also significantly decreased (p < 0.05) (Fig. 6D). According to our research, COM dramatically modifies the expression of genes involved in fatty acid production in E. coli. A series of enzymatic reactions are involved in bacterial fatty acid production. Several antibacterial compounds have been shown to target the key genes and enzymes involved in fatty acid production30. In this study, the fatty acid synthesis genes fab D, cfa, fab A and fab G were strongly downregulated by COM treatment, indicating that antibacterial activity is associated with reduced expression levels of genes involved in fatty acid biosynthesis.

Fig. 6
figure 6

Relative gene expression levels of fatty acids biosynthesis-associated genes in E. coli exposed to MET, CEF and COM. (A) relative expression of fabA; (B) relative expression of fabD; (C) relative expression of fabG; (D) relative expression of cfa.

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Galleria Mellonella infection model

In the G. mellonella killing model, larvae treated with the combination of CEF and MET exhibited markedly increased survival rates at 5 days after infection. As shown in Fig. 7, all the larvae that were injected with the PBS control remained alive throughout the 5-day period. In contrast, in the positive control group, the NX400-infected larvae all died, resulting in 100% mortality. Survival curves correlated with drug type, with a greater number of deaths observed for CEF and MET alone (Fig. 7). However, in the combination group of CEF and MET, the survival rate of larvae was higher than those in the single drug administration groups.

Fig. 7
figure 7

Survival rates of G. mellonella larva. Infected larvae (n = 12) with E. coli NX400 (1.0 × 105 c.f.u.) at the right posterior gastropoda under the treatment of MET or CEF alone, and in the combination of CEF with MET at the left posterior gastropoda.

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Discussion

Drug-resistant bacterial infections pose a severe threat to public health globally; however, few effective medications are currently available because the rate of antibiotic discovery is far behind the demand. In the past 50 years, only a few novel compounds effective against gram-negative bacteria have been discovered31. In the absence of new medications, repurposing old medications or broadening the antibacterial spectrum must become viable options to treat MDR gram-negative bacteria32. In this manner, synergistic antibiotic combination therapies have been developed against pathogens that are difficult to treat33.

In this study, the antiparasitic medication dimetridazole was combined with the antibiotic cefotaxime to investigate the synergistic effect. The synergy and underlying mechanism were evaluated. The results indicated that COM had a significant synergistic effect. The synergy results were supported by the FICI and membrane integrity assay results. PI fluorescence significantly increased as a result of COM, indicating severe outer membrane damage34. This finding is in consistent with the SEM observations that the COM caused severe damage to the cell membrane.

Antibacterial compounds alter the levels of fatty acids in the plasma membrane35. For example, exposure of gram-negative bacteria to essential oils and other antimicrobials elevates the level of UFAs36 whereas fatty acids affect bacterial fitness and the integrity of the membrane37. The findings of this study indicate that the fatty acid composition of E. coli is altered when the bacteria are exposed to COM. This finding is in line with previous research.

The fab A and cfa genes in E. coli are responsible for encoding ACP dehydratase (Fab A) and cyclopropane fatty acyl phospholipid synthase (CFA), which are associated with the production of UFAs and CFAs, respectively30. Fab G is an NADPH-dependent 3-ketoacyl-ACP reductase that plays critical roles in fatty acid biosynthesis and elongation cycles in bacteria, while fab D catalyzes the trans-thioesterification of malonate from CoA to ACP. In this study, COM significantly reduced the expression levels of fatty acid synthesis-related genes, including fab G, fab A, fab D and cfa. These results are consistent with those of previous studies. It is noteworthy that, although the expression of these genes was down-regulated, only the synthesis of C17:1 and C18:1n9c was significantly decreased among all UFAs, while the levels of other UFAs (C14:1, C16:1, C18:1n9t, C18:2n6c, C20:2) increased. This phenomenon warrants further comprehensive investigation in order to provide a more reasonable explanation.

This study has several limitations. First, the evaluation of CEF/MET effectiveness may be impacted by the limited sample size; more strains should be evaluated to strengthen these findings. Second, despite the in vitro identification of synergistic CEF/MET combinations, dosage regimens for this combination have not been logically optimized.

Conclusion

In this study, we explored the synergistic effect of COM against E. coli and its impact on the cell membrane properties and composition of E. coli. Our results indicated that COM has a synergistic effect on E. coli. After combination of the CEF and MET, the antibacterial activity of CEF and dimetridazole increased 8 times and 16 times, respectively. The growth curve showed COM prolonged the logarithmic phase, increasing the time to reach the stationary phase. COM has the ability to disrupt cell membrane integrity and induce alterations in other membrane functions. COM regulates specific key genes associated with cell membrane synthesis. In summary, our study provides evidence for the inhibitory effect of COM on growth.