Synergistic antibacterial activity between penicillenols and antibiotics against methicillin-resistant Staphylococcus aureus

Penicillenol A2 (isolated from deep-sea fungus Penicillium biourgeianum DFFSCS023) has good antibacterial activity against methicillin-sensitive Staphylococcus aureus and in combination with beta-lactam antibiotics it could significantly decrease methicillin-resistant Staphylococcus aureus (MRSA) survival, which provides a novel treatment consideration for MRSA-caused infections.


Introduction
Staphylococcus aureus (S. aureus) is a ubiquitous pathogen that commonly colonizes in the nose, respiratory tract and also on the skin. Staphylococcus aureus usually survives in a commensal condition with other strains, and it often presents weak pathogenicity [1,2]. However, there were also identified pathogenic S. aureus strains, which produce virulence factors that cause a wide variety of diseases from skin infections (abscesses, folliculitis and scalded skin syndrome) to life-threatening disease (meningitis, osteomyelitis, endocarditis, sepsis and toxic shock syndrome) [3,4].

Fermentation and extraction
The fungus strain DFFSCS024 was isolated from a deep-sea sediment collected at a depth of 2226 m from the South China Sea, Sansha City (17°60' N, 111°48' E), Hainan Province, China. It was identified as Penicillium biourgeianum based on the sequence (Genbank accession no. JX156370) of the internal transcribed spacers region of rDNA. Spores were inoculated into 1000 ml Erlenmeyer flasks containing 300 ml of liquid medium (glucose 1%, maltose 2%, monosodium glutamate 1%, KH 2 PO 4 0.05%, MgSO 4 · 7H 2 O 0.003%, corn steep liquor 0.05%, yeast extract 0.3%, dissolved in seawater, pH 6.5). After 35 days of cultivation at 28°C, the broth cultures (a total of 24 l used) were filtered through cheesecloth. Sterilized XAD-16 resin (20 g l −1 ) was added into the previous liquor, and shaken at 200 r.p.m. for 30 min to absorb the organic products. The resin was then washed with distilled water to remove medium residues and eluted with methanol. The methanol solvent was removed under vacuum and subsequently produced a brown residue (approx. 21 g). The mycelium portion was smashed and extracted twice with 80% acetone. The acetone soluble fraction was dried in vacuo to yield 20 g of residue. The residues of liquor and mycelium extracts were combined together based on thin-layer chromatography instruction.

Preparation of the test solutions
The powder of each compound (4, 5, 6, 8 and 9) was dissolved in a small volume of dimethylsulfoxide (DMSO) to a final concentration of 8 mg ml −1 stock solution, and they were sterilized through a 0.22 μm pore membrane filter. The sterilized stock solutions of each compound were subsequently diluted with TSB buffer to different concentrations for following experimentations. To avoid physiological toxicity, DMSO concentrations were strictly below 0.5% (v/v) [22].

Strains and growth condition
Standard ATCC strain of MSSA ATCC 25923 was used in this study, and MRSA strain was isolated from a patient in Queen Mary Hospital as previously described [23]. Bacteria were grown on Tryptone Soya Agar (TSA; Oxoid, UK), incubated at 37°C overnight. Tryptone Soya Broth (TSB; Oxoid, UK) was used for bacterial broth culture, incubated at 37°C with agitation at 200 r.p.m.

Bauer-Kirby disc diffusion method
Antimicrobial susceptibility discs (Oxoid, UK) containing a standard amount of antibiotics were used as positive control, and a negative control with no antibiotics was also set up. To evaluate the antibacterial activities of compounds 4, 5, 6, 8 and 9, 5 µl or 10 µl of 8 mg ml −1 stock solution of each compound were added onto the blank disc (without any chemicals or antibiotics), and dried at 37°C to generate the compound-containing (40 or 80 µg) discs. Similarly, the discs containing a combination of each compound and antibiotics were prepared by adding 5 or 10 µl of the corresponding 8 mg ml −1 stock solution onto each previously prepared antibiotics-carrying discs, and dried at 37°C. Single colonies of MSSA and MRSA grown on the TSA plate were, respectively, inoculated into 10 ml of fresh TSB medium, and incubated overnight at 37°C with shaking at 200 r.p.m. A 100 µl aliquot of the overnight bacterial culture suspension (adjusted to a 0.5 McFarland standard) was uniformly spread onto the TSA plates, and the above-prepared discs were each placed on the agar surface. Plates were incubated at 37°C overnight for further antibiotic resistance measurement, and the diameter of inhibition zones was measured through the BIOMIC V3 Microbiology System. data between groups, and p < 0.05 was considered as significant; p < 0.01 indicated a very significant difference between groups; p > 0.05 was considered as no significance.
We have chosen 5 of the above compounds (4, 5, 6, 8 and 9) to investigate their antibacterial activities on MSSA and MRSA. Other compounds, including the new-found compound (compound 1) and penicillenol A1 (compound 3), have almost no antibacterial activities against MSSA and MRSA (these results were not given). The Kirby-Bauer disc diffusion susceptibility test and the in vitro time-kill curve assay were performed according to CLSI guidelines. Bauer-Kirby disc diffusion is one of the most commonly used methods of antimicrobial susceptibility assay [40]. The paper discs (6 mm diameter) without antibiotics but containing 40 µg of the compounds were tested, and the zone diameters (ZDs) were read by the BIOMIC V3 Microbiology System. The results (electronic supplementary material, figure S1) revealed that most of the ZDs were too small (ZD ≤ 6 mm) to measure except the ZDs of compound 4 (ZD = 6.75 ± 0.25 mm) and 5 (ZD = 7.5 ± 0.5 mm) against MSSA. Results indicated that these compounds presented weak antibacterial activities that could be caused by less diffusion [41,42]. Furthermore, the in vitro time-kill curve assay was necessary to explore the antibacterial activity. As shown in figure 3a, all the viable bacteria counts of the supplied compound groups at each time point                  compounds. The antibacterial efficiencies were in the order of compound 4 > compound 5 > compound 6 > compound 8 > compound 9.
In addition, compounds 4, 5 and 6 were selected for the following investigation of the antimicrobial susceptibility to MRSA as described above. There were no significant (p > 0.05) differences found  between the treating groups and the control groups in MRSA (figure 3b). This could be caused by MRSA-produced lactamases, which destroyed the lactam ring of compounds [43].
Moreover, the synergistic effects of compound 4 associated with penicillin G sodium (Pen), cefotaxime sodium (Ctx) and oxacillin sodium (Oxa) were investigated by plate counting and the Kirby-Bauer disc diffusion method. The synergistic antibacterial effect was defined as the decrease of more than or equal to 2 log CFU/ml between the combination and the single treatment [44,45]. As shown in figure 4, the antibacterial capacity of Pen (10 U ml −1 ), Ctx (15 U ml −1 ) and Oxa (1 U ml −1 ) were all less than 1 log of bacterial CFU per ml when compared with the viable bacteria counts of controls. All the combinations groups between compound 4 and each of the antibiotics (Pen, Ctx and Oxa) presented a decrease of more than or equal to 2 log CFU/ml bacterial count when compared with compound 4 used alone. It can be concluded that the combination of compound 4 with beta-lactam antibiotics presented a synergistic effect. As a result, compound 4 could be used in beta-lactam antibiotic treatment in MRSA to enhance susceptibility.
Additionally, the results of the disc diffusion assay revealed that the ZD values of drug combination groups were shown to be larger than each of the ZD values of either penicillenols (compounds 4, 5 and 6) or antibiotics (Pen, Ctx and Oxa) used alone, respectively (figure 5). The disc diffusion assay results supported the above outcomes of the synergistic effect between compound 4 and beta-lactam antibiotics.
Previous research had demonstrated the antibacterial activities or cytotoxicity of penicillenols and their derivatives [30,39,46], and some penicillenols exhibit antibiofilm activities [47]. However, so far there is no correlative literature about the synergistic effect between penicillenols and antibiotics. So our finding will reveal an avenue for the treatment of MRSA by combining penicillenols and betalactam antibiotics. Besides, we suspect that the synergistic effect is probably because penicillenols can competitively bind to the beta-lactamase or PBP2a of MRSA, or inhibit their biological activities. However, this inference needs further investigation and findings.

Conclusion
In summary, penicillenols from deep-sea-derived fungus Penicillium biourgeianum DFFSCS023 presented antibacterial activity to MSSA, but lower antibacterial capacity against MRSA. A significant synergistic antibacterial effect was found for the combination of penicillenol A2 with either penicillin G sodium, cefotaxime sodium or oxacillin sodium in MRSA treatment. Our discoveries provided a novel choice for clinicians to use penicillenols in combination with beta-lactam antibiotics in MRSA infection treatment.
Data accessibility. The supporting data for this article have been uploaded as part of the electronic supplementary material.
Authors' contributions. X.X. and S.Q. conceived and designed the research. S.L. performed and wrote the paper. All the authors contributed to the discussion and review, and gave their approval for publication.
Competing interests. We declare we have no competing interests.