candidiasis is one of the most prevalent and important opportunistic fungal infections of the oral cavity caused by candida yeast species like candida albicans , c. glabrata , and c. krusei .
different forms of candidiasis , in acute and chronic forms , affect various parts of the body , such as the oral and genital mucosa , skin , bronchus , gastrointestinal tract , and lungs .
these infections usually diffuse in immunosuppressant status and afflict the internal organs , such as the kidneys and liver ( 1 - 3 ) .
antifungal drugs , in various formulations , are being used topically ( like nystatin and clotrimazole ) and systemically ( azoles and amphotericin b ) for the treatment of candidiasis . however , in recent years , numerous studies have reported the failure of treatment in patients with different clinical types of candidiasis .
the long - term consumption of antifungal agents caused adverse effects and drug resistance , thereby limiting the use of these therapeutics ( 4 , 5 ) .
the number of infections caused by candida species is increasing , and these microorganisms now respond poorly to azole treatments , such as fluconazole , which is the best - in - class therapy for the treatment of fungal infections .
moreover , concomitant side effects of the common antifungal agents include nausea , vomiting , hepatic dysfunction , arrhythmias , and neuropathies , among others ( 1 , 6 ) .
therefore , recent researches are directed to find more effective antifungal agents with natural origins and fewer side effects . among herbal extracts , the inhibitory effect of ginger ( zingiber officinale ) extract on microorganisms has been well documented .
previous studies revealed the antimicrobial effects of ginger extract against staphylococcus aureus , pseudomonas aeruginosa , and escherichia coli ( 7 , 8) .
furthermore , it has been demonstrated that ginger extract has potent antifungal properties against fluconazole - resistant c. albicans species isolated from patients with genital candidiasis ( 9 ) .
indeed , in traditional medicine , ginger is administrated to cure movement inabilities , nausea , and vomiting during pregnancy .
more importantly , apart from sedation and drowsiness , there is no report of any side effects for ginger ( 10 , 11 ) .
multiple lines of evidence favor the antimicrobial , antifungal , and anti - biofilm effects of ginger extract against some bacterial and fungal species , particularly against c. albicans , and there are a few studies on other candida species like c. glabrata and c. krusei , but none of them have evaluated this effect in mouthwash form .
this study aimed to evaluate the antifungal , antimicrobial , and anti - biofilm property of a ginger extract prepared in mouthwash form against c. albicans , c. krusei , and some bacterial pathogens as well as its effect on their biofilm formation .
ginger ethanolic extract as a potential mouthwash was provided from commercially available ethanolic extract of ginger ( rojin cosmetic co , tabriz , iran ) with an 80 mg / ml concentration . in a 96-well cell culture microplate , 100 l of sabouraud dextrose medium ( 2x for fungi strains ) (
merck kgaa , darmstadt , germany ) and muller hinton broth ( 2x for bacterial strains ) ( merck kgaa , darmstadt , germany ) were added to each well ( a medium quantity was measured to reach the expected optimal concentration ) .
subsequently , 80 l of ginger extract was added to the first well and later dilutions were prepared in subsequent wells .
then , 20 l of a fungal or bacterial specimen ( with 0.5 mcfarland concentrations ) was added to each well and after 18 - 24 hours of incubation at 37c , wells were evaluated with respect to their turbidity .
the well before the first turbid well was considered to have the minimum inhibitory concentration ( mic ) .
this measurement for mics and minimum bactericidal concentrations ( mbcs ) was done as recommended by the clinical and laboratory standards institute ( clsi ) m27-a3 and clsi m100-s22 ( 12 - 14 ) .
different concentrations of ginger extract ( 80 mg / ml - 0.625 mg / ml ) were used .
mic values were estimated using a visual and spectroscopic method by absorbance measurement at 620 nm ( od620 optical density reading at 620 nm ) ( 15 ) .
control tubes with the muller hinton agar ( merck ) ( without ginger extract ) were used as negative controls and 70% ethanol as positive controls . for the determination of mbcs
, sterile swabs were used to inoculate concentrations higher than the mics into the blood agar plate for 24 hours ( 16 ) .
all investigated strains were obtained from the iranian persian type culture collection in iranian research organization for science and technology ( irost ) .
investigated strains were e. coli atcc 25922 , p. aeruginosa atcc 27853 , k. pneumoniae atcc 700603 , s.aureus atcc 25923 , a. baumannii atcc 19606 , b. cereus atcc 11778 , c. krusei dms 70079 , c. albicans atcc 0231 , and two oral isolates of c. albicans .
biofilm assays were conducted based on a previously described method ( 17 ) . for each strain ,
a few colonies were suspended in physiological saline to 0.5 mcfarland and vortexes for 1 minute .
96 well polystyrene microtiter plates ( greiner cellstar flat - bottomed sterile cell - culture nr .
655180 ) were filled with 180 l of trypticase soy broth ( tsb ) ( merck kgaa , darmstadt , germany ) + 0.5% glucose ( merck kgaa , darmstadt , germany ) and 20 l of bacteria suspension was added to each well ( with changing medium dilution final concentrations that were optimized ) .
four wells per strain were incubated and their mean of absorbance was considered the final absorbance .
negative controls ( blank ) were tsb + 0.5% glucose alone and were dispensed into 8 wells per tray .
after stationary aerobic incubation for 24 hours at 37c and 5% co2 , the broth was carefully drawn off and the wells were washed 3 times with 300 l of sterile phosphate buffered saline ( pbs , room temperature ) .
biofilms were fixed with 150 l of methanol for 20 minutes , flicked , and air dried in an inverted position in the warm room ( about 30 minutes ) .
biofilms were stained with 150 l of crystal violet solution in water ( 2% ) for 15 minutes at room temperature and the wells were rinsed by placing the plate under running tap water .
to quantify biofilm production , 150 l of 33% acetic acid was added to each well to destain the biofilms and lidded plates were placed at room temperature for 30 minutes without shaking .
thereafter , the optical density of the resolubilized crystal violet was measured at 570 nm ( od570 ) using a microtiter plate reader ( multiskan fc microplate photometer , thermo scientific , nr 89087 - 320 ) .
the cut - off optical density ( od ) for biofilm formation by isolates was defined as the optical density upper than od570 = 0.524 was defined as the cut off optical density for biofilm formation ( 17 ) .
mtt assay is an essential method for evaluating the cytotoxicity of biomaterials in vitro condition .
the cytotoxicity effects of ginger extract mouthwash in effective antifungal mics was investigated by mtt assay on the human gingival fibroblasts cell line .
mtt assaytest was evaluated in 24 , 48 and 72 hours after adding the mouthwash to the media ( figure 1 ) .
spss version 22 ( ibm spss statistics , new york , usa ) was used for statistical analysis .
ginger ethanolic extract as a potential mouthwash was provided from commercially available ethanolic extract of ginger ( rojin cosmetic co , tabriz , iran ) with an 80 mg / ml concentration . in a 96-well cell culture microplate , 100 l of sabouraud dextrose medium ( 2x for fungi strains ) (
merck kgaa , darmstadt , germany ) and muller hinton broth ( 2x for bacterial strains ) ( merck kgaa , darmstadt , germany ) were added to each well ( a medium quantity was measured to reach the expected optimal concentration ) .
subsequently , 80 l of ginger extract was added to the first well and later dilutions were prepared in subsequent wells .
then , 20 l of a fungal or bacterial specimen ( with 0.5 mcfarland concentrations ) was added to each well and after 18 - 24 hours of incubation at 37c , wells were evaluated with respect to their turbidity .
the well before the first turbid well was considered to have the minimum inhibitory concentration ( mic ) .
this measurement for mics and minimum bactericidal concentrations ( mbcs ) was done as recommended by the clinical and laboratory standards institute ( clsi ) m27-a3 and clsi m100-s22 ( 12 - 14 ) .
different concentrations of ginger extract ( 80 mg / ml - 0.625 mg / ml ) were used .
mic values were estimated using a visual and spectroscopic method by absorbance measurement at 620 nm ( od620 optical density reading at 620 nm ) ( 15 ) .
control tubes with the muller hinton agar ( merck ) ( without ginger extract ) were used as negative controls and 70% ethanol as positive controls . for the determination of mbcs
, sterile swabs were used to inoculate concentrations higher than the mics into the blood agar plate for 24 hours ( 16 ) .
all investigated strains were obtained from the iranian persian type culture collection in iranian research organization for science and technology ( irost ) .
investigated strains were e. coli atcc 25922 , p. aeruginosa atcc 27853 , k. pneumoniae atcc 700603 , s.aureus atcc 25923 , a. baumannii atcc 19606 , b. cereus atcc 11778 , c. krusei dms 70079 , c. albicans atcc 0231 , and two oral isolates of c. albicans .
biofilm assays were conducted based on a previously described method ( 17 ) . for each strain ,
a few colonies were suspended in physiological saline to 0.5 mcfarland and vortexes for 1 minute .
96 well polystyrene microtiter plates ( greiner cellstar flat - bottomed sterile cell - culture nr .
655180 ) were filled with 180 l of trypticase soy broth ( tsb ) ( merck kgaa , darmstadt , germany ) + 0.5% glucose ( merck kgaa , darmstadt , germany ) and 20 l of bacteria suspension was added to each well ( with changing medium dilution final concentrations that were optimized ) .
four wells per strain were incubated and their mean of absorbance was considered the final absorbance .
negative controls ( blank ) were tsb + 0.5% glucose alone and were dispensed into 8 wells per tray .
after stationary aerobic incubation for 24 hours at 37c and 5% co2 , the broth was carefully drawn off and the wells were washed 3 times with 300 l of sterile phosphate buffered saline ( pbs , room temperature ) .
biofilms were fixed with 150 l of methanol for 20 minutes , flicked , and air dried in an inverted position in the warm room ( about 30 minutes ) .
biofilms were stained with 150 l of crystal violet solution in water ( 2% ) for 15 minutes at room temperature and the wells were rinsed by placing the plate under running tap water .
to quantify biofilm production , 150 l of 33% acetic acid was added to each well to destain the biofilms and lidded plates were placed at room temperature for 30 minutes without shaking .
thereafter , the optical density of the resolubilized crystal violet was measured at 570 nm ( od570 ) using a microtiter plate reader ( multiskan fc microplate photometer , thermo scientific , nr 89087 - 320 ) .
the cut - off optical density ( od ) for biofilm formation by isolates was defined as the optical density upper than od570 = 0.524 was defined as the cut off optical density for biofilm formation ( 17 ) .
mtt assay is an essential method for evaluating the cytotoxicity of biomaterials in vitro condition .
the cytotoxicity effects of ginger extract mouthwash in effective antifungal mics was investigated by mtt assay on the human gingival fibroblasts cell line .
mtt assaytest was evaluated in 24 , 48 and 72 hours after adding the mouthwash to the media ( figure 1 ) .
spss version 22 ( ibm spss statistics , new york , usa ) was used for statistical analysis .
to investigate the effects of antibiotics against the study strains , the mics for nystatin against c. albicans and c. krusei were 32 g / ml and 16 g / ml .
the mics for fluconazole against c. albicans and c. krusei were 16 g / ml and 16 g / ml , which suggested that these microorganisms respond poorly to fluconazole and nystatin . to remove the ginger extract background from the wells , a serial dilution without bacteria and fungi
was used , and its results were minus from all the results to normalize the final results .
the mic for s. aureus , k. pneumoniae , b. cereus , and a. baumannii was 20 mg / ml and for p. aeruginosa and e. coli it was 40 mg / ml ; however , for fungi isolates it was 5 mg / ml for c. krusei and 10 mg / ml for c. albicans .
the lowest rates of the anti - biofilm effect of ginger extract were observed against k. pneumoniae , s. aureus , p. aeruginosa , and e. coli respectively , but ginger successfully inhibited the biofilm formation in a. baumannii , b. cereus , c. krusei , and c. albicans ( figure 2 ) . in particular , c. albicans strains of the present study were strong biofilm formers , and ginger extract successfully reduced the biofilm formation from 3 od to zero or -2od ( in comparison with no microbial load extract ) .
there were fluctuations seen in high concentrations due to high concentrations of the extract ; therefore , we used dead / alive staining .
the dead / alive staining results indicated that with increasing ginger extract concentration , live cells in biofilm form reduced significantly ( figure 3 ) .
mtt assay at 24 , 48 , and 72 hours with a concentration range of 1 mg / ml revealed no significant reductions in cell viability after 24 hours .
a , p. aeroginosa atcc 27853 ; b , k. pneumoniae atcc 700603 ; c , a. baumannii atcc 19606 ; d , b. cereus atcc 11778 ; e , s.aureus atcc 25923 ; f , c. krusei dms 70079 ; g , c. albicans atcc 0231 , and two oral isolates of c. albicans ; h , for ginger extract concentrations ranging from 80 mg / ml to 0.625 mg / ml .
kolmogorov - smirnov analysis revealed non - parametric data distribution ( p > 0.05 ) .
the kruskal - wallis test indicated that the means of mics of the tested materials were statistically significant in the case of both fungal species ( p = 0.000 ) .
the mann whitney u - test showed that all pair - wise comparisons were statistically significant ( p = 0.000 ) .
in other words , the mean mibc of ginger extract for fungi strains ( c. krusei and c. albicans ) was greater than fluconazole and nystatin .
in this study , the antifungal and antimicrobial effects of ginger extract were respectively investigated against disease - causing bacteria and fungi and the effects were compared with two commonly used antifungal agents ( fluconazole and nystatin ) . according to the results of the present study , ginger has a greater antifungal than antimicrobial effect and it had an acceptable anti - candida effect against strains isolated from patients .
in addition , a lower mic ( 5 mg / ml ) was found against c. krusei than c. albicans ( 10 mg / ml ) .
ginger extract had high antibiofilm effect against candida strains compared to bacterial strains ( figure 2 ) .
biofilm reduction against c. albicans started at a concentration of 0.625 mg / ml , and at 40 mg / ml there was no sign of biofilm formation .
these results indicate that concentrations between 0.625mg / ml and 5mg / ml can be used successfully against candida colonization in the oral cavity .
moreover , mtt assay showed no adverse effects of ginger extract after 24 hours ; however , the mouthwash was used for less than a minute .
ginger extract also had powerful effect against bacterial agents , particularly p. aeruginosa and s. aureus .
the antifungal effects in the present study were the same for all investigated fungi strains .
a previous study revealed the antifungal effects of the protein in ginger rhizome and its inhibitory effect on some fungi , such as fusarium oxysporum ( 18 ) .
( 19 ) isolated a material called cmuac 130 from ginger , which had an inhibitory effect on phytopathogenic fungi growth , like fusarium .
( 20 ) indicated that ginger extract could prevent the proliferation of f. moniliforme , aspergillus flavus , and a. fumigatus in vitro .
evaluated the antifungal properties of 36 herbal extracts on 13 human fungal pathogens and reported that among these extracts , ginger and jipijapa extracts had inhibitory effects on various fungal species .
more importantly , it was revealed that ginger extract precluded the growth of fungi that were resistant to amphotericin b and ketoconazole ( 21 , 22 ) . moreover , agarwal et al .
other researchers have evaluated the antifungal effects of ginger s rhizome and assigned it as an effective extract on aspergillus and phytopathogens ( 24 ) .
mohammadi and moatar ( 9 ) assessed ginger s antifungal properties against clinical isolates of fluconazole - resistant c. albicans .
their findings indicated that ginger extract had an inhibitory effect on all tested species and they declared ginger as an effective agent on c. albicans in a laboratory setting .
there have been no studies on the antifungal effect of ginger extract on non - albicans candida species ; our findings indicated a significant antifungal effect of ginger extract against candida species other than c. albicans . in agreement with previously published studies
, our findings also suggested that ginger s antifungal properties are stronger than fluconazole and nystatin . although the antimicrobial effects of common antimicrobial agents on different microorganisms are compared to the standards deducted by clsi m27-a3 and clsi m100-s22 , there are no reference standards for other materials , such as herbal extracts .
former studies have merely reported the antifungal properties of these extracts descriptively without comparing them with any references .
herein , we compared the antifungal effects of ginger extract with two commonly used antifungal agents , fluconazole and nystatin , and underscored that its antifungal activity is much greater than fluconazole and nystatin against c. albicans . because p. aeruginosa and s. aureus have an important role in oral cavity infections , our findings indicate powerful effect of ginger extract against biofilm formation by these strains ; however it did not have a significant protective effect against the biofilm formation of e. coli and k. pneumoniae . by using dead / alive staining
we found that an increase in absorbance in 80 mg / ml was not related to biofilm formation , and there were no signs of live cells ( figure 3 ) . in conclusion ,
the present study s findings indicate that ginger extract has good antifungal and antibiofilm formation by fungi against c. albicans and c. krusei .
concentrations between 0.625 mg / ml and 5 mg / ml have the highest antibiofilm and antifungi effect , and it had no adverse effect on mtt assay , which indicates the safety of this extract for use as a mouthwash .
in addition , ginger extract was effective against biofilm formation by p. aeruginosa and a. baumannii .
perhaps , the use of herbal extracts such as ginger represents a new era for antimicrobial therapy after developing antibiotic resistance in microbes .
 background : candidiasis is one of the most prevalent and important opportunistic fungal infections of the oral cavity caused by candida yeast species like candida albicans , c. glabrata , and c. krusei . 
 in addition , several bacteria can cause oral infections . 
 the inhibition of microbial biofilm is the best way to prevent oral infections.objectives:the aim of the present study is to evaluate the antifungal , antimicrobial , and anti - biofilm properties of ginger ( zingiber officinale ) extract against candida species and some bacterial pathogens and the extract s effects on biofilm formation.materials and methods : ginger ethanolic extract as a potential mouthwash was used to evaluate its effect against fungi and bacteria using the microdilution method , and biofilm was evaluated using the crystal violet staining method and dead / alive staining . 
 mtt assay was used to evaluate the possible cytotoxicity effects of the extract.results:the minimum inhibitory concentrations ( mics ) of ginger extract for evaluated strains were 40 , 40 , 20 , 20 , 20 , 20 , 10 , and 5 mg / ml for pseudomonas aeruginosa , escherichia coli , staphylococcus aureus , klebsiella pneumoniae , bacillus cereus , acinetobacter baumannii , c. albicans , and c. krusei , respectively . 
 ginger extract successfully inhibited biofilm formation by a. baumannii , b. cereus , c. krusei , and c. albicans . 
 mtt assay revealed no significant reduction in cell viability after 24 hours . 
 the minimum inhibitory biofilm concentrations ( mibcs ) of ginger extract for fungi strains ( c. krusei and c. albicans ) were greater than those of fluconazole and nystatin ( p = 0.000).conclusions : the findings of the present study indicate that ginger extract has good antifungal and antibiofilm formation by fungi against c. albicans and c. krusei . 
 concentrations between 0.625 mg / ml and 5 mg / ml had the highest antibiofilm and antifungal effects . perhaps , the use of herbal extracts such as ginger represents a new era for antimicrobial therapy after developing antibiotic resistance in microbes . 
1. Background
2. Objectives
3. Materials and Methods
3.1. Study Design
3.2. Biofilm Assays
3.3. Cell Cytotoxicity Effect
3.4. Statistical Analysis
4. Results
5. Discussion