Fosfomycin Enhances the Inhibition Ability of Linezolid Against Biofilms of Vancomycin-Resistant Enterococcus faecium in vitro

Introduction

Enterococcus faecium is an important conditionally pathogenic bacteria of nosocomial infections in recent years which usually causes urinary tract, abdominal, and bloodstream infections.¹ Of particular concern is the increasing difficulty of treating E. faecium, due to its resistance to some antimicrobial agents, including vancomycin, the first-line treatment for Enterococcus. However, its inappropriate use has contributed to an increasing detection rate of vancomycin-resistant E. faecium (VREfm) and has caused high mortality and clinical failure rates.²

Considering rising incidences of invasive VREfm diseases, linezolid has become a vital compound to treat VREfm infections. VREfm strains are generally susceptible to linezolid. However, it had been reported that high variability of serum linezolid concentration was detected in critically ill patients who received standard dosing of 600 mg linezolid intravenously twice a day.³ These might lead to the development of resistance and drug-related toxicity. Moreover, prior exposure to linezolid proved to be an independent risk factor for drug-induced tolerance.⁴ A report by the German National Reference Center indicated an increasing prevalence of linezolid resistance among VRE, passing from a prevalence of <1% in 2008 to >9% in 2014.⁵

E. faecium is also a bacterial species most commonly capable of producing biofilms (a pivotal virulence factor in the pathogenesis).⁶ Biofilm is a microbial population of cells attached irreversibly to the surface of a wide variety of medical devices, these living tissues are encased in extracellular polymeric substances (EPSs) consisting of proteins, extracellular DNA, and polysaccharides.⁷ Bacteria associated with a biofilm are up to 1000 times more resistant to antibiotics in comparison to their planktonic counterparts and are insensitive to the host immune response, allowing them to persist and promote continued infection despite aggressive antibiotic therapy.⁸ In confronting VREfm infections (especially biofilm-forming E. faecium), monotherapy is often ineffective and may cause adverse drug reactions due to the need for long-term medication.⁹ Thus, it is urgent to find antibiotic combinations to treat VREfm infections.

Treatment choices should consider adverse effects, antibiotic penetration, and drug interactions. It has been proved that an increasing dose and treatment time of linezolid may lead to hematologic toxicity and the development of resistance during linezolid therapy.¹⁰ Considering the limitations of linezolid monotherapy, a combination of drugs may be a good approach. Fosfomycin is an old antibiotic, with activity against several bacteria, including multidrug-resistant Gram-positive and Gram-negative bacteria, by irreversibly inhibiting an early stage in cell wall synthesis.¹¹ However, fosfomycin monotherapy may raise the MIC (minimum inhibitory concentration) of the susceptible bacterium. Many studies have demonstrated synergistic effects on Enterococcus between fosfomycin and many antibiotics.¹² Our team also did some research in this area. We confirmed that linezolid combined with fosfomycin had a significant synergistic effect on vancomycin-susceptible and -resistant Enterococci treatment in vitro and the vivo via a time-kill curve study and the Galleria mellonella infection model, prevented Enterococcus resistance and weakened the virulence of fosfomycin-susceptible and -resistant Enterococcus strains.^13,14 We also got the conclusion that, for VRE strains, neither linezolid nor fosfomycin monotherapy inhibited amplification of the resistant sub-populations, and the development of fosfomycin resistance was at the expense of the virulence of VREfm.¹⁵ Fosfomycin plus linezolid or tigecycline showed synergism on VRE in time-kill studies of approximately 10% and 30%, respectively.¹⁶ The combination of oritavancin and fosfomycin increased drug susceptibility and showed a synergistic effect in 80% of isolates and an additive effect in the remaining isolates. In addition, the combination manifested a synergistic or additive effect in a biofilm assay.¹⁷

Fosfomycin is active against the adherent Enterococcus faecalis isolates.¹⁸ High-dose (16×MIC) fosfomycin combined with daptomycin demonstrated significantly more anti-biofilm activities than daptomycin or fosfomycin alone and effectively killed the adherent cells in the mature biofilms of linezolid-resistant isolates of E. faecalis.¹⁹ Linezolid could also inhibit E. faecalis biofilm formation and the addition of gentamicin significantly increased activity for linezolid.^20,21 However, whether linezolid combined with fosfomycin has a synergistic effect against VREfm biofilm is still unknown.

Biofilm formation goes through four steps.²² (1) Planktonic bacteria adhere to the surface and start to form biofilm; (2) Attached bacteria secrete EPSs resulting in a conglomeration of bacteria and matrix production; (3) Biofilms grow in multiple layers by forming micro-colonies and water channel structures; (4) Biofilms are basically mature and start releasing bacterial micro-colonies from the primary community to new sites and spreading the infection. Recent studies²³ focused on the eradication ability of reagents in mature biofilms, and few articles tried to investigate the effect of drugs against immature biofilms. Due to the growth property of the biofilms, even if the mature biofilms are eradicated, bacteria released from the biofilms can migrate to new sites and form biofilms again. So, if we can inhibit the formation of biofilms in the initial stage, it will drastically improve the cure rate of biofilm-related infections.

Therefore, in this study, we assessed the combination of linezolid with fosfomycin against VREfm immature and mature biofilms by biofilm biomass and the metabolic activity of bacteria in the biofilms. The difference between linezolid alone and linezolid-plus-fosfomycin against selected biofilm-formation-associated genes evaluated by RT-qPCR.

Materials and Methods

Bacterial Isolates

All eight clinical isolates of VREfm collected from patients’ urine at the First Affiliated Hospital of Anhui Medical University have been tested for the ability of biofilm formation, and four clinical isolates (No.2, No.4, No.5, and No.6) have been selected for the study. All strains were identified by the automated VITEK-2 system (BioMerieux, Marcy I’Etoile, France). Vancomycin-resistant Enterococcus ATCC 51299 was used as the quality control strain. These strains were part of the routine hospital laboratory procedure, which was approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University and was conducted in accordance with the principles outlined Declaration of Helsinki. According to the national guidelines in China, isolates were collected as part of the routine clinical management of patients. Therefore, informed consent was not required. The isolates used in the research were not specifically isolated for this research.

Antimicrobial and Media

Linezolid, fosfomycin, and vancomycin were purchased from the National Institute for Food and Drug Control of China (Beijing, China). Mueller‒Hinton broth (MHB) (Oxoid, England) and MH agar (MHA) (Oxoid, England) were used for all experiments, and Brain Heart Infusion agar (BHIA, Oxoid, United Kingdom) was only used for the susceptibility testing of vancomycin. In addition, all media with fosfomycin was added in 25 mg/L glucose-6-phosphate (Sigma-Aldrich).

In vitro Susceptibility Test and Fractional Inhibitory Concentration Index (FICI) Assay

Minimum inhibitory concentrations (MICs) of linezolid, fosfomycin and vancomycin were determined using the agar dilution method according to Clinical and Laboratory Standards Institute guidelines.²⁴ Briefly, Mueller–Hinton agar and Brain Heart Infusion agar plates containing a series of two-fold concentration antibiotics of each agent were prepared. The agar plates containing fosfomycin needed to add in glucose-6-phosphate with the final concentration of 25 mg/L. Then, 5×10⁵ colony forming units (CFU) of bacterial cells were inoculated with these plates and incubated at 37°C for 18–24 h. The MIC was defined as the lowest drug concentration without visible colony growth. According to the CLSI 2020 guidelines,²⁴ drug resistance (R) was defined as vancomycin MIC≥32 mg/L, linezolid MIC≥8 mg/L, and fosfomycin MIC≥256 mg/L. Vancomycin-resistant Enterococcus ATCC 51299 was used as the quality control strain for these experiments. MIC determinations were performed in triplicate for each strain.

A checkerboard assay was used to evaluate the synergistic effects of linezolid and fosfomycin at different concentrations. The final concentration of linezolid and fosfomycin ranged from 1/16×MIC to 2×MIC. Each well of a 96-well plate contained 10⁵ CFU/mL bacterial suspension. The plates were incubated under aerobic conditions at 37°C for 24 h.

The fractional inhibitory concentration index (FICI) was defined as follows: FICI = (drug A combined MIC/Drug A alone MIC)/(drug B combined MIC/Drug B alone MIC). The effect of FICI was explained as follows: FICI ≤ 0.5, synergistic effect; 1 < FICI ≤ 4, no difference; and FICI > 4, antagonistic effect.²⁵

Minimum Biofilm Eradication Concentrations (MBECs) in vitro

The in vitro minimum biofilm eradication concentrations were determined according to the aforementioned reference.²⁶ Suspensions of all strains at initial inocula of 5×10⁶ CFU/mL in MHB were added to the plates and incubated at 37◦C for 24 h to form the mature biofilms. The wells were then washed three times with PBS and MHB supplemented with serial dilutions of linezolid or fosfomycin, ranging from 2×MIC to 64×MIC, was added. After 24 h of exposure, the wells were washed three times with PBS again and a solution of 0.01% Alamar blue (Maokang Bio, China) in MHB was added. Alamar blue, also named resazurin, is a redox indicator that represents cell viability. After 3 h of incubation at 37◦C, the absorbance was recorded at 570_nm and 600_nm with a microplate reader (Nanodrop, USA). The% of metabolic activity (MA) when compared with positive (sample with bacteria suspended in pure MHB) and negative (pure MHB) controls, which were taken as 100% and 0%, respectively, was calculated according to the specification as following formula:

A570 was the 570_nm absorbance value of treated sample, and A600 was the 600_nm absorbance value of treated sample; P570 was the 570_nm absorbance value of untreated sample, and P600 was the 600_nm absorbance value of untreated sample.

The results presented as MBEC₉₀ and MBEC₅₀, which were regarded as the lowest concentrations of antibacterials that reduced the MA of bacteria by at least 90±5% and 50±5%, respectively. The MA taken for determining the MBEC was the mean of three results obtained on three different days.

Biofilm Biomass Assay

The effects of linezolid monotherapy or combined with fosfomycin against VREfm biofilm were analyzed by crystal violet staining as previously described.¹⁹ Briefly, VREfm isolates were inoculated into 96-well polystyrene microtiter plates with MHB containing antimicrobial for 48 h to evaluate the inhibition capability of antibiotics against biofilms. In order to compare the effects of drugs on immature and mature biofilms, antibiotics were added to the plates after incubating for 2 h and 24 h, respectively. After 24 h or 48 h of static incubation, with the medium containing antibiotics replaced daily, the supernatant liquid was discarded and the wells were gently washed with PBS thrice, the remaining biofilm biomass was determined by crystal violet staining and was measured at 590_nm with a microplate reader. Each assay was performed in triplicate three times.

Biofilm Metabolic Activity Assay

The Alamar blue cell viability assay was used as a reliable, reproducible, and recommended method to evaluate the bacterial activity of biofilm cells.²⁷ After 24 h or 48 h incubation as mentioned above, the supernatant liquid was removed, and the wells were gently washed with PBS thrice. Then, fresh MHB media containing 10% (v/v) Alamar blue staining reagent was added and incubated at 37°C for 3 h, as recommended by the manufacturer (Maokang Bio, China). The metabolic activity (MA) was calculated as above. Each assay was performed in triplicate on different days.

RT-qPCR to Determine the RNA Levels of VREfm Biofilm Formation-Related Genes

The RNA levels of six biofilm formation-related genes of No.5 and No.6 clinical isolates were determined by RT-qPCR based on published reports.²⁸ The VREfm clinical isolates were inoculated into 100 mm×20 mm nonpyrogenic polystyrene cell culture dishes with MHB containing 1/8×MIC linezolid and 1/2×MIC fosfomycin for 24 h to investigate the inhibition to the biofilms in initial stage and containing 2×MIC linezolid and 1×MIC fosfomycin for 48 h to test the eradication to the mature biofilms. After static incubation, total RNA was extracted from planktonic and biofilm cells for RT-qPCR. RT-qPCR is performed using SYBR^® Green Pro Taq HS Premix II (Rox Plus) (Agbio Co, Hunan, China) on a fluorescence quantitative PCR instrument (Roche LightCycler 96, Switzerland). The target gene expression levels are normalized to that of the housekeeping gene (16srRNA) and determined via the 2^–ΔΔCT calculation method, where CT is the threshold cycle. The primers used for RT-qPCR are listed in Table 1. Each assay was performed in triplicate.

Table 1 PCR Primers Used for Determining the Expression Levels of Selected Genes of Vancomycin-Resistant E. faecium Isolates by RT-qPCR

Statistical Analysis

All data were analyzed in SPSS version 20.0 (SPSS, Inc., Chicago, IL, United States). All figures were performed using GraphPad Prism, version 8.0 (GraphPad Software, Inc., San Diego, CA, United States). The data were analyzed using One-way ANOVA, and the significance was cited as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Results

MIC and FICI Results

The MICs of linezolid and fosfomycin against four isolates are shown in Table 2. The MICs of linezolid against the four strains were 2mg/L and that of fosfomycin was 128mg/L except the No.4 strain which MIC of fosfomycin was 256mg/L. By the way, all strains were resistant to vancomycin.

Table 2 MICs and FICI of Antimicrobial Reagents Against Four Strains

The FICI results represented the interaction between linezolid and fosfomycin are listed in Table 2, too. The FICI results of reagents against four strains were all below 0.5 meaning that linezolid and fosfomycin showed a synergistic effect on the VREfm strains.

Minimum Biofilm Eradication Concentrations (MBECs) in vitro

According to the MBECs listed in Table 3, VREfm biofilms showed a high level of resistance to the antibiotics compared with planktonic cells in Table 2. The biofilms of VREfm exhibited stronger resistance to fosfomycin than to linezolid. The MBEC₅₀ of four strains against fosfomycin were 8×MIC and 64×MIC respectively, in contrast, that of linezolid ranged from 4×MIC to 32×MIC. The situation of MBEC₉₀ was similar to that in MBEC₅₀. The biofilms formed by No.4 and No.5 strains showed a high level of resistance on account of the MBEC₉₀ of the two isolates against linezolid and fosfomycin were nearly higher than 64×MIC. The biofilm of No.6 isolate was the most susceptible to the two antibiotics, while the MBEC₉₀ against linezolid and fosfomycin was 8×MIC and 32×MIC, respectively.

Table 3 MBEC90 and MBEC50 of Antimicrobial Agents Against Four Strains

Biofilm Biomass Assay

First, we compared the inhibition potential of linezolid combined with fosfomycin and linezolid monotherapy against the four VREfm isolates’ biofilms in the initial stage. When we added the antibiotics, planktonic bacteria started to adhere to the surface and secrete EPSs to form a biofilm. Sub-minimum inhibitory concentrations of antibiotics inhibited the formation of biofilm in Figure 1. Linezolid (1/8×MIC or 1/4×MIC) combined with fosfomycin (1/4×MIC) efficiently inhibited VREfm-biofilm formation and to a greater extent than linezolid alone. This trend was observed in all four isolates. In the No.5 isolate, linezolid (1/4×MIC) combined with fosfomycin (1/4×MIC) showed inhibition potential better than linezolid (1/2×MIC).

Figure 1 Sub-minimum inhibitory concentrations (1/8, 1/4, or 1/2×MIC) of linezolid combined with 1/4MIC of fosfomycin inhibited VREfm-biofilm formation in the initial stage. Abbreviations: LIN, linezolid; FOS, fosfomycin; LIN+FOS, linezolid-fosfomycin combination. Notes: Data represent the average of three independent experiments (mean±SD). *P < 0.05, **P < 0.01.

We also compared high concentrations (2, 4, or 8×MIC) of linezolid combined with fosfomycin (1×MIC) eradicated VREfm-formed biofilm in the mature stage with linezolid alone. As shown in Figure 2, combined administration demonstrated excellent ability to clear mature biofilms. Even compared with the high concentration group at 8×MIC of linezolid, linezolid combined with fosfomycin eradicated formed biofilms effectively.

Figure 2 High concentrations (2, 4, or 8×MIC) of linezolid combined with 1×MIC of fosfomycin inhibited VREfm-biofilm formation in the mature stage. Abbreviations: LIN, linezolid; FOS, fosfomycin; LIN+FOS, linezolid-fosfomycin combination. Notes: Data represent the average of three independent experiments (mean±SD). *P < 0.05, **P < 0.01.

Biofilm Metabolic Activity Assay

In the initial stage, linezolid at medium and high concentration groups (1/4 and 1/2×MIC) inhibited bacterial metabolic activity in the biofilms above 50% in the four isolates. As shown in Figure 3, fosfomycin significantly enhanced the inhibitory effect of linezolid at low and medium concentration groups (1/8 and 1/4×MIC). In the No.6 isolate, the inhibition of linezolid at low and medium concentration groups (1/8 and 1/4×MIC) combined with fosfomycin (1/4×MIC) was stronger than linezolid at high concentration group (1/2×MIC).

Figure 3 Sub-minimum inhibitory concentrations (1/8, 1/4, or 1/2×MIC) of linezolid combined with 1/4MIC of fosfomycin inhibited cells in VREfm-biofilm in the initial stage. Abbreviations: LIN, linezolid; FOS, fosfomycin; LIN+FOS, linezolid-fosfomycin combination. Notes: Data represent the average of three independent experiments (mean±SD). *P < 0.05, **P < 0.01, ***P < 0.001.

Antibiotics exhibited weak inhibitory effects on bacterial metabolism in mature biofilms in Figure 4. In No.2, No.4, and No.5 isolates, whether linezolid was used alone or combined with fosfomycin, the inhibition rate of metabolism of bacteria in the mature biofilm was lower than 50%. Fosfomycin (1×MIC) enhanced the effect of linezolid in the four isolates, especially in the No.4 and No.5 isolates. We observed medium concentration (4×MIC) of linezolid plus fosfomycin (1×MIC) showed a stronger inhibitory effect than the high concentration group (8×MIC) of linezolid alone.

Figure 4 High concentrations (2, 4, or 8×MIC) of linezolid combined with 1×MIC of fosfomycin inhibited cells in VREfm-biofilm in the mature stage. Abbreviations: LIN, linezolid; FOS, fosfomycin; LIN+FOS, linezolid-fosfomycin combination. Notes: Data represent the average of three independent experiments (mean±SD). *P < 0.05, **P < 0.01, ***P < 0.001.

Relative Quantification of Biofilm Formation Gene Expression

Last, No.5 and No.6 isolates were selected to clarify the relative expression levels of six biofilm formation genes by RT-qPCR. In Figure 5, fosfomycin had no effect on the expression of asa1 and esp in two isolates, as well as atlA in the No.6 isolate. The gene expression levels of cylA, ebpA, and gelE showed a significant decrease when isolates were treated with 1/8×MIC linezolid and 1/4×MIC fosfomycin in the initial stage for 24 h.

Figure 5 Relative gene expression of biofilm formation-related genes of isolates No.5 and No.6 with linezolid alone or linezolid combining with fosfomycin treatment in the initial stage. Abbreviations: LIN, linezolid; FOS, fosfomycin; LIN+FOS, linezolid-fosfomycin combination. Notes: Data represent the average of three independent experiments (mean±SD). **P < 0.01, ***P < 0.001; No.5 strain (A); No.6 strain (B).

In Figure 6, six gene expressions showed a significant decline except for atlA in the No.5 isolate. The asa1 and gelE expression were markedly decreased both in two isolates when the isolates were treated with 2×MIC linezolid and 1×MIC fosfomycin for 48 h in the mature stage.

Figure 6 Relative gene expression of biofilm formation-related genes of isolates No.5 and No.6 with linezolid alone or linezolid combining with fosfomycin treatment in the mature stage. Abbreviations: LIN, linezolid; FOS, fosfomycin; LIN+FOS, linezolid-fosfomycin combination. Notes: Data represent the average of three independent experiments (mean±SD). **P < 0.01, ***P < 0.001; No.5 strain (A); No.6 strain (B).

Discussion

Biofilm-associated infections have caused increasing concerns, which include device-related infections, chronic infections in the absence of a foreign body, and even malfunction of medical devices. Enterococci prefer to survive as microbial colonies and cause 25% of all catheter-associated urinary tract infections. Enterococci are frequently isolated in wounds and increasingly found in infective endocarditis, and all of these infections are associated with biofilms.²⁹ Due to the properties of biofilms, biofilm-associated enterococcal infections are hard to eradicate and serve as a nidus for bacterial dissemination and as a reservoir for antibiotic resistance genes.³⁰

Multi-resistant bacteria increase the difficulty of biofilm-associated infections treatment. As a first-line treatment for VRE infections, many studies have indicated that linezolid suppressed the formation of enterococci biofilms, whether when administered alone or in combination with rifampicin or gentamicin.^21,31 Fosfomycin also has inhibitory effects on biofilms of various bacteria, including Enterococcus spp.³² In the study, we verified that linezolid and fosfomycin have synergistic antibacterial effects on VRE, which was consistent with the conclusion of the previous study of our research group.¹⁴ Fosfomycin has also been proven to have either a synergistic or additive effect with linezolid against 32 VREfm urinary isolates in a time-kill study.³³ It was worth noting that most of these strains collected from urine. Another investigation in 26 cases of VRE-infected patients from a hospital in Thailand found a synergistic effect of fosfomycin combined with linezolid.³⁴ These isolates in the research were collected from different infection sites, like the bloodstream, urine, and gastrointestinal system. In general, most articles evaluating the effectiveness of the combination of fosfomycin and linezolid on VRE were conducted on clinical isolates collected from the urinary tract, and more research on Enterococcus from different infection sites is needed.

The results of the biofilm biomass assay showed that fosfomycin enhanced the inhibitory effect of linezolid on VRE biofilm, especially in the mature stage. In Figure 2, the inhibition against the biofilm grown 24 h was about fold increased in the co-administration group over the linezolid alone, especially in No.4 and No.5 isolates. What was important was that the OD590_nm in the combination group was lower than the high-dose linezolid alone group (8×MIC), which was very meaningful for clinical treatment. That suggested that combining with fosfomycin may substitute for the high-dose linezolid alone in VRE infection treatment because high-dose linezolid was liable to cause adverse drug reactions such as lactic acidosis.³⁵ In the initial adhesion stage, the inhibitory effect of the drugs was not as strong as the effect on the mature biofilm, the combination treatment and the linezolid monotherapy group reduced the biofilm biomass by about 50% and 30%, respectively.

In the biofilm metabolic activity assay, linezolid showed bacterial killing ability against bacteria in the biofilm, fosfomycin enhanced the bactericidal capacity of linezolid. The bactericidal effect in different isolates was not the same, in the No.6 isolate, the combination of linezolid and fosfomycin resulted in a higher degree of bacterial kill than monotherapy no matter in the initial biofilm or in the mature biofilm. The situation in the No.2 isolate was that only low dose linezolid (1/8×MIC or 2×MIC) combined with fosfomycin killed more bacteria in the biofilm than linezolid alone, there was no significant difference between combination treatment at 1/4×MIC, 1/2×MIC, or 4×MIC, 8×MIC linezolid and combination treatment (Figures 3 and 4). This was inconsistent with the results of the biofilm inhibition experiment, suggesting that besides directly killing bacteria to reduce biofilm formation, drugs may also have other ways to reduce biofilm formation. This was confirmed by the results of the RT-qPCR experiments. Many genes are involved in the formation of enterococci biofilm. We chose six biofilm formation-related genes in the RT-qPCR experiments. The gelE gene, part of the gelE-sprE operon, encodes gelatinase which plays an important role in the initial stage of biofilm formation via the ability to degrade the collagen adhesion protein (Ace).³⁶ Gelatinase helps the bacteria adhere to surfaces and contributes to dissemination and colonization by degrading Ace. Gelatinase also functions as a stimulant for N-acetylglucosaminidase (AtlA) release, which is a vital autolysin that prompts lysis of a bacterial subpopulation (autolysis) and releases extracellular DNA (eDNA). eDNA acts as an adhesive and is significant for biofilm attachment and stability.³⁷ In Figure 5, linezolid and fosfomycin reduce gelatinase production in order to lessen bacterial adhesion at the surface by inhibiting gelE expression in the initial stage of biofilm formation. After 24 h stationary incubation, the biofilm has basically formed, and gelatinase secretion decreases which may reduce the release of eDNA due to the reduction of autolysis. The adhesion ability and stability of the biofilm decrease, which makes the biofilm easy to destroy (Figure 6). Linezolid combined with fosfomycin can inhibit the level of cylA expression to decrease biofilm formation. The cylA, a gene related to toxin structure and function, composes one of the cytolysin operon promoters, the P_L promoter, together with cylLL, cylLS, cylM, cylB, and cylI.³⁸ The cytolysin can compromise target cell membranes leading to lysis, which is necessary for eDNA formed.

When the biofilm matures, linezolid combined with fosfomycin also reduced aggregation substance (AS) production, a hair-like glycoprotein on the bacterial surface from the asa1 gene (Figure 6). Linezolid-combined-fosfomycin can reduce the expression level of the asa1 gene compared with linezolid alone. The expression of AS led to bacterial clumping and higher antibiotic resistance, helping in biofilm formation.³⁹ It is consistent with our observation of a decline of the biofilm biomass and MIC in the co-administration group.

The esp gene that encodes the surface-associated enterococcal surface protein (Esp) is located on a 153-kb pathogenicity island, and its expression significantly increases the bacterial cell surface hydrophobicity and attachment on a substratum.⁴⁰ It has been reported the esp gene of E. faecalis was critical for biofilm formation, but not indispensable.⁴¹ However, there is no doubt that Esp can enhance the formation of biofilm.⁴² Esp has also been demonstrated that it can highly enhance primary binding to polystyrene and polyvinyl chloride plastic from urine collection bags,⁴³ and esp expression enhanced in vitro binding to bladder and kidney epithelial cells in mice.⁴⁴ Therefore, the reduced level of esp expression in combination treatment may be beneficial to reduce the occurrence of VRE-associated urinary tract infections in clinical. Endocarditis- and biofilm-associated pili (Ebp), cotranscribed at the ebpABC locus, are composed of 3 subunits, EbpA, EbpB, and EbpC, and play a role in biofilm formation as well as in endocarditis. The tip adhesin EbpA mediates attachment to host fibrinogen and collagen and contributes to urinary tract infections, catheter-associated urinary tract infections, and endocarditis.⁴⁵ We observed a substantial reduction of ebpA expression in combination treatment, especially in the initial stage of biofilm formation (Figure 5). These may be because the genes ebpA to srtC were transcribed as a polycistronic operon, and the ebpA disruption mutant showed highly reduced transcription of downstream ebpB and ebpC genes. Furthermore, ebpA, ebpB, ebpC, and srtC mutant strains showed significant differences with the wild type in the initial attachment step of biofilm formation using phase-contrast microscopy, indicating that these genes played a role in the early cell-surface interactions of the multistep biofilm formation process.⁴⁶

However, there were some limitations in the study. First, only four clinical isolates were studied in the assays and were all collected from patients’ urine. These isolates might not be representative of all VREfm. To be more universal and representative, more isolates from different infection sites with dissimilar MIC values should be studied. Second, all assays were performed in vitro and failed to take account of the host immune response. Therefore, more animal studies may be needed to assess the role of the immune system further. Furthermore, we quantitatively examined six biofilm formation-related genes expressions in RT-qPCR but ignored the remaining genes and other potential influencing factors.

Conclusion

In conclusion, linezolid combined with fosfomycin had a synergistic effect on VREfm. The combination inhibited the biofilm biomass of four isolates of VREfm and reduced the metabolic activity of live bacteria in the biofilm. The results of RT-qPCR informed linezolid combined with fosfomycin had a significant decrease in the expression of cylA, ebpA, and gelE, which played an important role in promoting biofilm formation. For the mature biofilm, the transcription of asa1, atlA, and esp was also inhibited by the combination. This suggested that linezolid combined with fosfomycin may be a viable therapeutic option for the treatment of VREfm biofilm-associated infections.