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In vitro and in vivo activity of d-serine in combination with β-lactam antibiotics against methicillin-resistant Staphylococcus aureus

As d-amino acids play important roles in the physiological metabolism of bacteria, combination of d-amino acids with antibiotics may provide synergistic antibacterial activity. The aim of the study was to evaluate in vitro and in vivo activity of d-serine alone and in combination with β-lactams against methicillin-resistant Staphylococcus aureus (MRSA) strains, and to explore the possible sensitization mechanisms. The activity of d-serine, β-lactams alone and in combinations was evaluated both in vitro by standard MICs, time–kill curves and checkerboard assays, and in vivo by murine systemic infection model as well as neutropenic thigh infection model. An in vitro synergistic effect was demonstrated with the combination of d-serine and β-lactams against MRSA standard and clinical strains. Importantly, the combinations enhanced the therapeutic efficacy in the animal models as compared to β-lactam alone groups. Initial mechanism study suggested possible revision of d-alanine-d-alanine residue to d-alanine-d-serine in peptidoglycan by adding of d-alanine in the medium, which may cause decreased affinity to PBPs during transpeptidation. In conclusion, d-serine had synergistic activity in combination with β-lactams against MRSA strains both in vitro and in vivo. Considering the relatively good safety of d-serine alone or in combination with β-lactams, d-serine is worth following up as new anti-MRSA infection strategies.


Introduction
Staphylococcus aureus (S. aureus) is one of the most important clinical organisms among Gram-positive bacteria. It is a leading cause of skin and soft tissue infections (SSTIs), bacteremia and infective endocarditis 1,2 . Methicillin-resistant S. aureus (MRSA) strain has been a heavy burden worldwide and caused a range of serious infections with poor clinical outcomes [3][4][5] . However, grim scenario of drug discovery for new antibiotics has been presented for the last few decades as pharmaceutical companies lack interest in this field, owing to difficulty in recouping drug discovery costs from antibiotics which developed resistance within a decade or so 6 . Therefore, there is a critical need to develop new treatment strategies against these MRSA life-threatening infections.
D-Amino acids play important roles in bacterial physiology 7,8 . D-alanine (D-Ala) and D-glutamate (D-Glu) are components of bacterial peptidoglycan 9 . D-amino acids could also influence peptidoglycan composition, amount and strength, both via their incorporation into the polymer and by regulating enzymes that synthesize and modify it 8,10,11 . Up to date, researches mainly focused on the effects of D-amino acids on biofilm, finding that D-amino acids could not only prevent biofilm formation, but also disrupt existing biofilms [12][13][14][15] . In addition, D-amino acids were also able to enhance the activity of rifampin against biofilm formation in S. aureus, and to increase the efficacies of colistin, ciprofloxacin and amikacin against Pseudomonas aeruginosa 16,17 . However, less studies focused on the effects of D-amino acids on planktonic bacteria, except Tong et al. 18 showed that the application of D-cysteine (D-Cys), D-aspartic acid (D-Asp) and D-Glu could significantly improve the antibacterial activity of nisin against planktonic bacteria of S. mutans. D-Serine (D-Ser) was reported to be able to replace D-Ala residue of peptidoglycan stem peptides and increase susceptibility of methicillin in MRSA 19 . In order to determine whether D-Ser can improve susceptibility of β-lactams against MRSA strains, we investigated the activity of D-Ser, β-lactams (e.g., oxacillin and meropenem) alone and in combination against MRSA strains, including clinical and standard isolates, both in vitro and in vivo.

Bacterial strains
Three standard MRSA strains, 17 clinical MRSA strains and 1 standard methicillin-susceptible S. aureus (MSSA) strain, 18 clinical MSSA strains, randomly selected from our S. aureus strain collection from hospitals in China during 2005-2013 were included in the current study. MLST was performed as described by Enright et al. 20 previously. The seven housekeeping gene sequences were compared with known alleles from the MLST database (https://pubmlst.org/saureus/), and the allelic profiles and ST types were determined based on the database. The polymorphic X region of spa gene was amplified as previously described 21 , and the spa type was determined by submitting the sequencing data to the S. aureus type database (http://spaserver.ridom.de). The genotypic features of the isolates are shown in Supporting Information Table S1.

Antibiotics, D-amino acids and culture medium
D-Amino acids were purchased from Sigma-Aldrich (St. Louis, MO, USA). The stock solutions were prepared in water (for in vitro experiments) or 0.85% NaCl (for in vivo experiments), and sterilized by filtration after adjusting pH to 7.0. Antibiotics were purchased from National Institute for Food and Drug Control (National Institutes for Food and Drug Control, Beijing, China).

Laboratory animals
CD-1 (ICR) mice (female, 18-20 g for systemic infection model, and 24-26 g for neutropenic thigh infection model) were purchased from Vital River Laboratories (Beijing, China). All animals were housed under controlled humidity (30%-70%), temperature (22 7 3 1C) and a 12-h light-dark cycle. Animals had free access to food and water during the study. All the animal studies complied with the ARRIVE guidelines, and all experiments were approved by Animal Research Committee of the Institute of Medicinal Biotechnology (Beijing, China).

Minimal inhibitory concentration (MIC) determination
MICs were determined by broth microdilution method as recommended 22 . The final inoculum in each well was about 5Â10 5 CFU/mL. The microtiter plates were incubated at 35 1C for 24 h, and the results were recorded by naked eyes.

Checkerboard assay
The antimicrobial combination was defined to be synergistic when the FICI was r 0.5; indifferent when 0.5 o FICI o 4; antagonistic when FICIZ 4 23 . The experiments were performed in duplicate on different days.

Time-kill curves
Time-kill curve assays were performed with standard MRSA ATCC 43300 and N315, as well as clinical MRSA isolates (0603 and 0850), according to method described by Lu et al. 24 with minor modifications. Briefly, an overnight culture of each isolate was diluted with 3 mL CAMH broth to a final concentration of $ 10 6 CFU/mL. Then D-Ser (at 20 mmol/L), antibiotics (at the lowest concentrations that can show synergistic effects when combined with D-Ser) alone and in combinations were added. Viable cell counts were determined at 0, 2, 4, 6, 8 and 24 h after incubation at 35 1C by plating 10 μL serial diluted samples onto MH agar plates in triplicate. The results were recorded as log 10 CFU/mL. Synergy was defined as Z 2 log 10 CFU/mL decrease at 24 h incubation in the combination treatment in comparison to single antibiotic alone exposure 23 .

Murine systemic infection model
Representative MRSA strains, ATCC 43300, N315 and 0850, were used in this model. After intraperitoneal injection of 0.5 mL bacterial suspension (100% minimum lethal dose) in 5% mucin, mice were administered with D-Ser, antibiotics alone or in combinations through subcutaneous injection at 1 and 6 h after infection (8-10 animals/group). The doses of OXA were 2.5, 5, and 10 mg/kg for N315 strain and 5, 10, and 25 mg/kg for ATCC 43300; the doses of MEM were 0.5, 1, and 2.5 mg/kg for N315 and 25, 50, and 100 mg/kg for 0850. Animal deaths were recorded for 7 days and the survival rates were calculated.

Murine neutropenic thigh infection model
The experiment was carried out according to previously described methods with some modifications 25,26 . Briefly, CD-1 (ICR) female mice were rendered neutropenic by intraperitoneally dosed with cyclophosphamide on day 4 (150 mg/kg) and day 1 (100 mg/kg) prior to infection 27 28,29 . Mice were sacrificed at 24 h for MEM groups and 48 h for OXA groups after infection. Right thigh muscles were then aseptically excised, homogenized, serially diluted and plated on MH agar plates for CFU counts. Bacterial colony counts were expressed as mean log 10 CFU/ thigh (7SEM).

The docking assay
Penicillin-binding protein 2a (PBP2a), a protein exists in MRSA strains, can lead to resistance by its low affinity with β-lactams but normal affinity to natural substrate as well as existing activity on catalyzing cell wall synthesis. As we speculated that the effect of D-Ser might be related with PBP2a, we compared the binding activity of natural substrate with PBP2a as well as changed substrate after adding D-Ser with PBP2a by docking assay. Using crystal structure of PBP2a (PBD entry: 1VQQ) as receptor, we compared the binding activity of D-Ala-D-Ala and D-Ala-D-Ser by Discovery Studio 4.5. The binding modes for dipeptides towards the binding site of PBP2a were generated by MOE (Molecular Operating Environment) version 2009.10. The docking was performed through the "Dock" module and scored using LibDock scoring system. The higher the score, the better affinity of the dipeptide with the protein.

Statistical analysis
Statistical analysis was performed by SPSS v16.0. P values were calculated using one-way ANOVA to compare the differences between each pair of groups. P o 0.05 was considered statistically significant.

In vitro activity of D-Ser in combinations against MRSA and MSSA strains
The MICs of 12 different β-lactams alone and in combination with D-Ser at 20 mmol/L against MRSA ATCC 43300 are summarized in Table 1. The MICs of β-lactams against ATCC 43300 were from 8 to 4 1024 mg/L. Interestingly, the MICs of the tested β-lactams against the studied MRSA strain were significantly reduced (8 to 4128-folds) with addition of D-Ser at 20 mmol/L. MEM (meropenam, usually not used alone in MRSA infection) and OXA (oxacillin, traditionally considered "inactive" against MRSA) were then chosen as the representative antibiotics in further evaluation for the purpose of reusing them.
In contrast, D-Ser showed very limited sensitization effect on oxacillin and meropenem against MSSA strains. MICs of the  Information Table S2).

Checkerboard assay
The checkerboard assay was conducted using D-Ser and the representative β-lactams (MEM and OXA) against all studied MRSA strains and the results are summarized in Table 2

In vivo antibacterial activity of OXA/D-Ser and MEM/D-Ser combinations in murine neutropenic thigh infection model
As shown in Fig. 3A, D-Ser alone at 4 mmol/kg didn't reduce colony counts in thigh vs. the control group, OXA alone at   The test concentrations were D-Ser: 0, 2.

The docking results of dipeptides with PBP2a
As shown in Supporting Information Table S4, the LibDock_Score of D-Ala-D-Ala and D-Ala-D-Ser were 70.9502 and no poses respectively, hence we predicted that D-Ala-D-Ala had a good affinity with PBP2a, while D-Ala-D-Ser had no (or very weak) affinity with the same protein.
3.8. The sensitization activity of D-Ser with β-lactam antibiotics with relatively selective affinities for different PBPs Cefaclor and cephradine are representative compounds which have relatively selective affinities for PBP3, while cefoxitin for PBP4. Table S5, the level of sensitization activity of D-Ser varied with β-lactam antibiotics. Addition of 40 mmol/L D-Ser could enhance the antibacterial activity of all three cephalosporins, with MICs reduced by 2-128 times. Notably, MICs of cefoxitin in combination with 40 mmol/L D-Ser were reduced to larger degrees than cefaclor and cephradine. The reduced folds of cefoxitin with addition of 40 mmol/L D-Ser were generally 8-16 times larger than cefaclor, and 4-8 times larger than cephradine.

Discussion
MRSA has the ability to cause a range of serious problems since its first emergence and it now still maintains a high infection rate worldwide. β-Lactam antibiotics, widely used in the treatment of infections caused by S. aureus, have a limited effect in treating infections caused by MRSA strains. This highlights the need of development of novel treatments. Research institutes, including our laboratory, have been searching for novel targets 30 , potential alternatives to antibiotics 31 , as well as effective combinations of antibiotics with agents such as manuka honey, isoliquiritigenin, plant essential oils and plant extracts 24,30-34 . D-Ser was reported to be able to increase the susceptibility of MRSA strain to methicillin by replacing D-Ala residue of the peptidoglycan stem peptides 19   4 mmol/kg D-Ser, the antimicrobial activity of OXA at 20/50 mg/kg and MEM at 25/50 mg/kg showed no significant difference. Considering the potential resistance development in future, the lowest effective concentration is recommended, and more precise data are needed for future preclinical as well as clinical studies.
Besides, D-Ser also had sensitization activity in combination with ceftaroline. Ceftaroline, currently used as treatment of critical S. aureus infections, received FDA approval several years ago, now facing challenges that non-susceptible S. aureus is on the rise worldwide 35,36 . We studied and found that addition of 40 mmol/L D-Ser could enhance the antibacterial activity of ceftaroline against MRSA strains, with the MICs reduced by 2-16 times (data not shown). The results suggested that D-Ser might have a promising application in future.
β-Lactams inhibit bacterial growth by competing with D-Ala-D-Ala termini to bind with active sites of PBPs and interfering with cell wall assembly 37 . Methicillin resistance of S. aureus is associated with PBP2a 38 , which can mediate cell wall assembly with low β-lactam affinity 39 . Our results demonstrated that the sensitization effect of D-Ser in MSSA (Supporting Information Table S2) was much weaker than in MRSA strains (Table 1), suggesting the possible involvement of PBP2a in D-Ser sensitization in MRSA.
It was reported that addition of D-Ser could replace D-Ala in D-Ala-D-Ala termini of the peptidoglycans, and result in sensitization of the bacteria to methicillin 19 . In consistent with this report, our study showed that the sensitization effect of D-Ser on OXA and MEM against MRSA could be abolished with addition of the same concentration of D-Ala, while addition of D-Glu with even higher concentration had no effect (Supporting Information Table S3). Herein, the sensitization mechanism of D-Ser on β-lactams against MRSA may be related to the replacement of D-Ala residue in the peptidoglycans and the formation of D-Ala-D-Ser. D-Ala-D-Ser may not be recognized by PBPs, especially PBP2a, and result in unnormal transpeptidation process as well as cell wall assembly, leading to increased sensitivity of MRSA to β-lactams. Results by Thorsing et al. 40 also demonstrated that the exposure to thioridazine can lead to sensitization of MRSA strain to β-lactams through change of intracellular amino acids, which lead to unnormal peptidoglycan precursor formation. To verify the hypothesis that we made on PBP2a, docking study between protein and dipeptides (D-Ala-D-Ala or D-Ala-D-Ser) was conducted. The binding activity of D-Ala-D-Ala and D-Ala-D-Ser to PBP2a predicted by the dock module (Supporting Information Table S4) showed that compared with D-Ala-D-Ala, D-Ala-D-Ser had no detectable affinity to PBP2a.
PBP2a cannot accounts for the full mechanism of resistance present in MRSA strains, though it dose play an important role. Researches had been done that different β-lactam antibiotics had relatively selective affinities for PBPs 41 . Sieradzki et al. 42 found that cephradine, a relatively selective inhibitor of PBP3, was effective in reducing methicillin resistance, indicating that PBP3 might participate in methicillin resistance through some cooperative functioning with PBP2a. However, recent researches through genome sequencing and molecular genetic studies revealed that PBP4 could mediate S. aureus resistance of β-lactam antibiotics 43,44 . Our results (Supporting Information Table S5) demonstrated that although D-Ser had sensitization activity with all three antibiotics, D-Ser in combination with cefoxitin, a relatively selective inhibitor of PBP4, had a more profound sensitization activity than relatively selective inhibitors of PBP3, i.e., cefaclor and cephradine. We hypothesize that several PBPs might participate in β-lactam resistance, while PBP4 might act primarily as an efficient transpeptidase in addition to PBP2a. D-Ser is well known for its nephrotoxicity due to oxidative stress caused by hydrogen peroxide, a byproduct of D-amino acid oxidase (DAAO)-mediated metabolism of D-Ser 45,46 . However, in our initial safety experiment in mice, D-Ser alone and in combination with MEM or OXA didn't demonstrate obvious toxicity. Administration of D-Ser 10, 20, and 40 mmol/kg, MEM 100 mg/kgþD-Ser 4 mmol/kg or OXA 100 mg/kgþD-Ser 4 mmol/kg didn't cause any death in mice over 7 days. No significant body weight change was observed between treatment and control groups during the 7 days experiment period. And there was also no significant difference of urea nitrogen and creatinine in serum between the treatment and control groups at 24 and 48 h after dosing (Supporting Information Fig. S1). Co-administration of DAAO inhibitors with D-Ser was believed to be able to minimize its metabolism by DAAO, and hence to improve D-Ser bioavailability and reduce nephrotoxic effects. Indeed, oral administration of DAAO inhibitor CBIO in conjunction with D-Ser enhanced the plasma and brain levels of D-Ser in rats compared to the oral administration of D-Ser alone 47 . Hence, we expected that co-administration of DAAO inhibitors with D-Serþantibiotic combinations can reduce the effective dose of D-Ser and the possible toxicity caused by oxidation of D-Ser.

Conclusions
In summary, bacterial resistance is an increasingly serious problem. While new antibiotic development is time-consuming, combination therapy of bioactive molecules with existing antibiotics is a good way to solve this conflict. Considering the great in vitro and in vivo activity as well as relatively good safety of OXA/D-Ser and MEM/D-Ser combinations against MRSA, D-Ser/ β-lactam combinations may have the potential to be new treatment strategies against MRSA infections.