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1、1Vol.:(0123456789)Scientific Reports|(2021)11:7134|https:/doi.org/10.1038/s41598-021-86647- properties of Chinese Herbal Formula SanHuang decoction on biofilm formation by antibioticresistant Staphylococcal strainsShaoe Zhang,Peizhao Wang,Xiaotao Shi&Honglue Tan*The aim of this study was to explore
2、the effect of Chinese herbal SanHuang decoction(SH)on biofilm formation of antibioticresistant Staphylococci on titanium surface,and to explore its mechanism.Biofilmforming ATCC 35984,ATCC 43300 and MRSE 287 were used in this study.The MICs of SH and vancomycin against Staphylococci were determined
3、by the broth microdilution method.Six groups were designed,namely control group(bacteria cultured with medium),1/8MIC SH group(1MIC SH was diluted by 1/8 using TSB or saline),1/4MIC SH group,1/2MIC SH group,1MIC SH group and vancomycin group(bacteria cultured with 1MIC vancomycin).The inhibitory eff
4、ect on bacterial adhesion and biofilm formation were observed by the spread plate method,CV staining,SEM,and CLSM.Realtime PCR was performed to determine the effect of SH on the expression levels of ica AD and ica R gene in ATCC 35984 during the biofilm formation.The strains were found to be suscept
5、ible to SH and vancomycin with MIC of 38.75 mg/ml and 2.5 g/ml,respectively.SH with 1 MIC and 1/2 MIC could inhibit the bacteria adhesion,showing only scattered adhesion from SEM.CLSM showed that SH with 1 MIC and 1/2 MIC inhibited the biofilm formation.The quantitative results of the spread plate m
6、ethod and CV staining showed that there was significant differences between the SH groups(P 0.05).Further,with an increase in SH concentration,the inhibitory effect became more obvious when compared with control group.Among the groups,vancomycin had the strongest inhibitory effect on bacterial adhes
7、ion and biofilm formation(P 0.01).With an increase in SH concentration,the expression levels of ica AD decreased,and the expression of ica R increased correspondingly(P 0.05).In conclusions,SH can inhibit the biofilm formation of antibioticresistant Staphylococci.Its probable mechanistic activity ma
8、y be through the inhibition of polysaccharide intercellular adhesin synthesis by downregulating the expression of ica AD gene.Postoperative infection is one of the serious complications impacting the clinical results of orthopedic surgery,and its inadequate management will lead to nonunion of fractu
9、re,loss of limb function and even amputa-tion,which will inevitably impose an economic burden to the patients families and social medical insurance1.According to the literature,the incidence of postoperative infections for closed fracture is about 12%,but the incidence of an open fracture is signifi
10、cantly as high as 30%1,2.Studies have shown that 80%of clinical infec-tions are related to bacterial biofilm formation,which plays an important role in the pathogenesis of orthopedic infections3.Firstly,bacteria in a biofilm are very resistant to antibiotics,and their antibiotic tolerance is 1000-fo
11、ld higher than the MIC(minimum inhibitory concentration)of the planktonic form3,4.Secondly,the biofilm and its colonized pathogens can successfully defend the hosts immune system,so that they cannot be phagocytized and cleared by polymorphonuclear leukocytes5.Titaniumand its alloys have superb bioco
12、mpatibility,low elastic modulus,and favorable corrosion resistance.These exceptional properties lead to its wide use as a medical implant material for orthopedic surgery6.Titanium itself does not have antibacterial properties,so bacteria can gather and adhere to its surface resulting in biofilm6.OPE
13、NHenan Orthopedic Institute,Henan Luoyang Orthopedic-Traumatological Hospital(Henan Orthopedic Hospital),Qiming Southern Road,Luoyang 471002,Henan,Peoples Republic of China.*email:2Vol:.(1234567890)Scientific Reports|(2021)11:7134|https:/doi.org/10.1038/s41598-021-86647- to inhibit biofilm formation
14、 on the surface of titanium alloy becomes important in the prevention and treatment of orthopedic infections.For intractable orthopedic infections,in addition to local debridement,an important option is a long-term application of systemic intravenous antibiotics7.In clinical practice,a very large pr
15、oportion of implant-related infections are caused by Staphylococci,and two Staphylococcal species,Staphylococcus aureus and Staphylococcus epidermidis,together account for two out of three infection isolates8.As for biofilm-related infections,however,antibiotic-resistant Staphylococcus,especially MR
16、SE(meticillin-resistant Staphylococcus epidermidis)and MRSA(methicillin-resistant Staphylococcus aureus),makes the treatment of implant-associated infections with antibiot-ics still one of the challenges for orthopedic surgeons2,4,7.Hence,discovering alternative treatment methods for orthopedic infe
17、ctions linked to multi-antibiotic-resistant bacterial biofilm is becoming more and more relevant.For a long time,traditional Chinese medicine(TCM)has been widely used in the treatment of various infec-tious diseases in China,and many traditional or empirical prescriptions have been proved to have sa
18、tisfactory clinical therapeutic effects912.The SanHuang decoction(abbreviated as SH)is one of the treatment methods widely used in our hospital for treatment of limb infections with bone and implant exposure.This decoction is composed of Scutellaria baicalensis Georgi,Coptidis rhizoma,Cortex Phellod
19、endri chinsis and other Chinese herbs.According to the theory of traditional Chinese medicine,it has the heat-clearing and detoxing functions.Through external soaking and washing of the locally infected wound with SH,it was found that this decoction can effectively control the infections1012.However
20、,there has been no study to date that has explored the antibi-ofilm formation properties of the SH invitro.Therefore,based on previously published clinical studies and the widely used titanium implant material for orthopedic surgery6,1012,the aim of this study was to investigate the effect of SH dec
21、oction on adhesion and biofilm formation of MRSE and MRSA on titanium surfaces,and also to explore the probable mechanism behind its effect on biofilm formation.Materials and methodsPreparation of SH decoction.SH decoction contains Scutellaria baicalensis Georgi(25g),Coptidis rhi-zoma(25g),Cortex Ph
22、ellodendri chinsis(25g),Sophora flavescens(20g),Lonicera Japonica(20g),Forsythia suspensa(20g)and Taraxacum mongolicum Hand.-Mazz.(20g)(Table1).All the herbs were provided by the TCM pharmacy of Henan Luoyang Orthopedic-Traumatological Hospital following the requirement standards of the section one
23、of PharmacopoeiaofthePeoplesRepublicofChina(2015 edition).The procedure of prepar-ing the SH aqueous extract is as follows:firstly,all herbs were tipped into a ceramic pot with 1000ml pure water and soaked for 30min.Secondly,the concoction was boiled at high heat for 30min,and was maintained at a lo
24、w flame until the aqueous extract reduced to 250ml.Finally,aqueous extract was filtered with filter paper,and water extraction of the liquid was performed through 22m filter.Based on the original herb dose,the initial liquid concentration of the extract solution was about 0.62g/ml,which was stored i
25、n the refrigerator at 20C for the subsequent experiments.Only one batch of SH were used in this study.Preparation of bacterial strains.Two standard strains,the positive biofilm-forming S.epidermidis ATCC 35984(American Type Culture Collection ATCC,MRSE)and S.aureus ATCC 43300(MRSA),and one clinical
26、isolate,methicillin-resistant S.epidermidis(MRSE 287)which was isolated from one patient with an orthopaedic implant-related infection,were used in this study13,14.The strains frozen in a 80C refrigerator were rapidly thawed and inoculated on a Tryptone Soy Agar(TSA)plate and cultured in an incubato
27、r at 37C for 24h.A single colony was selected from the TSA and inoculated in a 50ml centrifuge tube containing 10ml TryptoneSoyBroth(TSB),the tube was incubated for 10h at 37C with agitation at 100rpm.Then,25l of bacterial suspension was transferred to another sterile centrifuge tube containing 10ml
28、 TSB and incubated at 37C for 14h,after which 1ml suspension was transferred into a 1.5ml centrifuge tube and the bacteria in the suspension was harvested by centrifugation for 5min at 4C and 1000rpm(Sorvall TC6 centrifuge,Du Pont,Bad Nauheim,Germany).The precipitate was then washed three times with
29、 0.15M PBS(phosphate buffered saline)to remove the remaining TSB,and resuspended in sterile PBS to an optical density of 0.490 at 600nm using Synergy HT multi-detection microplate spectrophotometer(Bio-tek,Winooski,VT),which corresponded to 109 CFUs(Colony forming units)/ml.Table 1.The composition o
30、f SH decoction.Chinese nameLatin namePart usedProportion(g)Huang QinScutellaria baicalensis GeorgiRoot25Huang LianCoptidis rhizomaRoot25Huang BaiCortex Phellodendri chinsisBark25Ku ShenSophora flavescensRhizome20Jin Yin HuaLonicera JaponicaBud20Lian QiaoForsythia suspensaFruit20Pu Gong YingTaraxacum
31、 mongolicum Hand.-MazzRoot203Vol.:(0123456789)Scientific Reports|(2021)11:7134|https:/doi.org/10.1038/s41598-021-86647- inhibitory concentration(MIC)determination.The broth microdilution method was used to determine the MIC of SH,gentamicin,penicillin and vancomycin as recommended by the National Co
32、mmit-tee for Clinical Laboratory Standards Institute15.An overnight culture of the strains were diluted tenfold in TSB and incubated at 37C until they reached exponential growth phase.Serial twofold dilutions of SH(155mg/ml to 4.84mg/ml),gentamicin(1024g/ml to 0.5g/ml),penicillin(1024g/ml to 0.5g/ml
33、)and vancomycin(8g/ml to 0.0625g/ml)in Mueller Hinton(MH)broth were prepared in a 96-wells plate with 190l per well.Ten microlitres of bacterial inocula of each strain containing 5 106 CFUs/ml was added in all wells.A number of wells with only MH broth(without inoculum and drugs)in each plate were d
34、esigned as blank control,and a number of wells with MH broth and inoculum(without drugs)were designed as negative control.The plates were incubated for 24h at 37C.The MIC was detected following addition of 50l of INT(2-4-Iodophenyl-3-4-nitrophenyl-5-phenyl-2H-tetrazolium chloride)at a final concentr
35、ation 0.2mg/ml in all the wells and incu-bated for 30min at 37C.Bacterial growth was determined by observing the color change of INT in the wells.Biologically active bacterial cells will reduce the colourless tetrazolium salt which act as an electron acceptor to a red-coloured formazan product16.Inh
36、ibition of bacterial growth is observed when the solution in the well remained clear after incubation with INT.MIC was defined as the lowest extract concentration that completely inhibits the growth of microorganisms.Each experiment was performed three times in duplicate.Experimental grouping.In tot
37、al,our study had six experimental groups,namely control group(bacteria cultured with only TSB or saline),1/8MIC SH group(1MIC SH was diluted by 1/8 using TSB or saline,SH con-centration was 4.85mg/ml),1/4MIC SH group(SH concentration was 9.69mg/ml),1/2MIC SH group(SH con-centration was 19.38mg/ml),1
38、MIC SH group(SH concentration was 38.75mg/ml)and vancomycin group(bac-teria cultured with TSB or saline containing vancomycin with 1MIC,vancomycin concentration was 2.5g/ml).Effect of different SH concentration on bacterial adhesion and biofilm formation.Titanium discs of 10mm in diameter and 1mm in
39、 thickness were placed into a 48-well plate(Costar 3548,Corning,NY,USA),and six replicates were used for each group.All disks were autoclaved at 121C for 20min before used.S.epidermidis ATCC 35984,S.aureus ATCC 43300 and MRSE 287 were diluted to a density of 106 CFUs/ml with 0.9%saline,saline-vancom
40、ycin,or different sub-MICs of SH(dilution with saline),and 1ml of the cell suspen-sion was added to the well containing the discs.The plates were incubated in aerobic conditions for 4h.Then,the bacterial adhesion on the titanium surface were analyzed with the following methods.The spread plate metho
41、d.The discs were gently washed three times with saline to remove loosely adherent bacteria,and then transferred into a 10ml glass tube containing 0.5ml saline.The tubes were then placed in an ultrasonic bath(B3500S-MT,Branson Ultrasonics Co.,Shanghai,China)and the bacteria attached on the disc were
42、dislodged by ultrasonication(5min)at a frequency of 50Hz.Ultrasonication was followed by rapid vortex mixing(Vortex Genie 2,Scientifific Industries,Bohemia,NY,USA)at maximum power for 1min to remove bac-teria that had adhered to the material.This method is known to be effective for removing biomater
43、ial-adherent bacteria17.The vortexed solutions were serially diluted tenfold and the final three dilutions were plated in tripli-cate onto TSA and then incubated at 37C for 24h.Finally,the number of CFUs on the TSA were counted,and the number of bacteria adhering to titanium surface was calculated a
44、nd is expressed relative to the surface area(CFUs/mm2).SEM assay.A sterile pipette was used to carefully remove the saline from each well.The discs were transferred with forceps into another fresh 48-well plate and gently washed with saline three times to remove loosely adher-ent bacteria.The discs
45、were fixed in 2.5%glutaraldehyde for 2h at 4C,then washed with PBS and dehydrated through a series of graded ethanol solutions(25,50,75,95 and 100%).The samples were subsequently freeze-dried,sputter coated with gold,and observed using SEM(Joel JSM-6310LV,JEOL Ltd.,Tokyo,Japan).After incubation for
46、24h withTSB,TSB-vancomycin,or different sub-MICs of SH(dilution with TSB).Biofilm formation on the titanium surface were analyzed by the following methods.Crystal violet(CV)stain-ing assay:A sterile pipette was used to carefully remove the TSB from each well.The discs were transferred into another 4
47、8-well plate and gently washed three times with PBS.Then,1ml of 2.5%glutaraldehyde solution was added into each well for 10min to allow fixation.The glutaraldehyde solution was removed and the wells were washed with PBS.Subsequently,PBS was removed and 2ml of 0.1%(w/v)aqueous CV solution was added t
48、o each well followed by incubation for 20min at room temperature.CV solution was discarded,and the wells were washed with PBS again and air-dried for 12h in the dark.Next,the quantity of biofilm was analyzed by adding 30%acetic acid in a volume of 1ml to each well to dissolve dye for 30min,after whi
49、ch 200l of the dye solution were transferred into a 96-well microtiter plate,one group equaling one vertical row of the plate.The optical density was read at a wavelength of 492nm using a plate reader(Synergy HT,BioTEK)18.CLSM assay.After 24h incubation,the TSB medium were removed carefully from eac
50、h well,and the discs were transferred into another 48-well plate and gently washed with PBS three times.Then the wells contain-ing the titanium discs were added with 500l combination dye(LIVE/DEAD Baclight bacteria viability kits,L13152;Molecular Probes,Eugene,OR,USA)in a dark environment at room te