the term oral cavity refers to lips , buccal mucosa , alveolar ridges , retro molar 
 trigone , hard palate , floor of the mouth and anterior two - thirds of the tongue . oral 
 cancer or oral cavity cancer , a subtype of head and neck cancer , is any cancerous tissue 
 growth located in the oral cavity . the most common oral cancer is squamous cell carcinoma ( scc ) that affects the tissue 
 lining of the oral cavity . oral 
 cancer is the eleventh most common cancer in the world with an estimated 267,000 cases 
 and 128,000 deaths in around 2000 , two - third of which occur in developing countries . the 
 incidence of oral cancer is increasing in several parts of the world , particularly in 
 australia , japan and parts of europe . oro - pharyngeal cancer is a significant part of the 
 global burden of cancer . incidence 
 rates for oral cancer vary in men from 1 to 10 cases per 100,000 
 populations in many countries . the immune compromised patients after treatment of oral cancer may have a chance of 
 infection by drug - resistant opportunistic microbes . such microbes that originate in the 
 oral cavity , representative microorganisms include staphylococcus aureus , 
 pseudomonas aeruginosa , and candida albicans . one of the most worrying 
 characteristics of p. aeruginosa is its low antibiotic 
 susceptibility . this low 
 susceptibility is attributable to a concerted action of multidrug efflux pumps with 
 chromosomally encoded antibiotic resistance genes . most of these organisms have become drug - resistant , which has 
 resulted in difficulties in curing the related infectious diseases . the 
 candida species educe the infectious disease candidiasis , which 
 causes infections such as oral thrush and vaginitis as well as life - threatening 
 diseases , known as candidemia . 
 c. albicans are yeast that normally inhabits the human mouth and 
 skin , where it generally uneventfully coexists with a variety of other microorganisms . an infection occurs when the balance of bacteria in the body is disrupted , especially in 
 immunocompromised situations , allowing drug - resistant candida species 
 to proliferate and overcome other healthy microorganisms . immunocompromised situations are frequently seen in 
 older individuals , infants , people infected with hiv , and individuals with cancer ; oral 
 cancer can reduce immunity in the maxillofacial region . the principal 
 treatments for oral cancer are surgical excision , radiotherapy , and chemotherapy , 
 employed alone or in combination . currently , the systemic applications of antibacterial drugs have shown better results on 
 curing diseases than local application , which could induce drug - resistant bacteria in 
 the particular area . in this study , we have investigated the reason behind 
 the frequent cause of oral infection after the treatment of oral cancer in elderly 
 chinese . out of several elderly patients ( 60 - 95 years old ) , 128 patients who had undergone 
 treatment for oral - cavity related problems participated in the study at ninth 
 people 's hospital , shanghai jiao tong university . we have excluded patients with 
 other systemic diseases like autoimmune disease or diabetes to avoid misleading of 
 our parameter . the participants were divided into two groups : group i - the patients 
 undergone oral cancer treatment ( n=93 ; 41 men , 52 women ; average age 68.18.3 years ) 
 ranging from 1 month to 7 years after the initial treatment , 43 members were involved 
 in species identification , remaining 50 members involved in drug - resistant test . these patients have been treated with surgery , chemo and/or radiotherapy ; group ii - 
 the control ( n=35 ; 15 men , 20 women ; average age 70.210.1 years ) , who had received 
 treatment for oral cavities or with no history of any cancer treatment . the 
 institution review board of affiliated ninth people 's hospital of shanghai jiao tong 
 university , according to helsinki declaration ii , approved the study . written 
 informed consent microbes were collected from the areas of surgery ( group i ) , tongue , gingiva , and 
 palate by using sterilized dry cotton swabs . after wetting , the cotton swab was 
 immediately put into an airtight sterilized test tube . to collect anaerobic microbes 
 samples , about 1 ml of saliva samples was collected from each subject for the 
 analysis . the microorganisms were cultured on nalidixic acid / cetrimide agar ( sigma - aldrich 
 chemie gmbh , switzerland ) for the first screening of pseudomonas 
 species , while mannital salt agar ( acumedia manufacturers , usa ) was used for the 
 first screening of staphylococcus species and brilliance 
 candida agar ( thermo fisher scientific inc . ) was used for the 
 first screening of candida species . the nalidixic acid / cetrimide 
 agar and mannital salt agar plates were incubated at 37c under aerobic conditions 
 for 2 days , while candida - gs agar plates were incubated at 30c for 
 3 days . the morphological assessment was carried out from the colonies on the plates , and 
 those with different shapes were collected , gram stained and observed under a light 
 microscope for species detection . genomic dna from each colony was obtained using a 
 wizard genomic extraction kit ( promega , madison , wi , usa ) . for 
 staphylococcus species , cells were treated with 1 mg / ml of 
 lysostaphin in tris - edta buffer ( te ; 10 mm tris - cl , 1 mm edta , ph 8.0 ) at 37c for 1 
 h before using the kit . final identification was done by polymerase chain reaction 
 ( pcr ) amplification with bacterial universal primers for 16s rdna and 26s rdna for 
 fungus , followed by dna sequencing . dna sequencing was performed using an abi prism 
 3100 genetic analyzer ( applied biosystems , tokyo , japan ) with a big dye cycle 
 sequencing reaction kit ( ab applied biosystems ) . identification of experimentally 
 determined nucleotide sequences using sequence databases was performed by basic local 
 alignment search tool ( blast ) . the antimicrobial activity was tested with respective 
 antibiotics used commonly in clinical practice ( figure 1 ) . antibiotics used to test the antimicrobial activity statistical analyses were conducted using spss 15.0 ( spss inc . , u.s.a . ) and any 
 differences at p<0.05 level were considered as statistically significant . out of several elderly patients ( 60 - 95 years old ) , 128 patients who had undergone 
 treatment for oral - cavity related problems participated in the study at ninth 
 people 's hospital , shanghai jiao tong university . we have excluded patients with 
 other systemic diseases like autoimmune disease or diabetes to avoid misleading of 
 our parameter . the participants were divided into two groups : group i - the patients 
 undergone oral cancer treatment ( n=93 ; 41 men , 52 women ; average age 68.18.3 years ) 
 ranging from 1 month to 7 years after the initial treatment , 43 members were involved 
 in species identification , remaining 50 members involved in drug - resistant test . these patients have been treated with surgery , chemo and/or radiotherapy ; group ii - 
 the control ( n=35 ; 15 men , 20 women ; average age 70.210.1 years ) , who had received 
 treatment for oral cavities or with no history of any cancer treatment . the 
 institution review board of affiliated ninth people 's hospital of shanghai jiao tong 
 university , according to helsinki declaration ii , approved the study . written 
 informed consent microbes were collected from the areas of surgery ( group i ) , tongue , gingiva , and 
 palate by using sterilized dry cotton swabs . after wetting , the cotton swab was 
 immediately put into an airtight sterilized test tube . to collect anaerobic microbes 
 samples , about 1 ml of saliva samples was collected from each subject for the 
 analysis . the microorganisms were cultured on nalidixic acid / cetrimide agar ( sigma - aldrich 
 chemie gmbh , switzerland ) for the first screening of pseudomonas 
 species , while mannital salt agar ( acumedia manufacturers , usa ) was used for the 
 first screening of staphylococcus species and brilliance 
 candida agar ( thermo fisher scientific inc . ) was used for the 
 first screening of candida species . the nalidixic acid / cetrimide 
 agar and mannital salt agar plates were incubated at 37c under aerobic conditions 
 for 2 days , while candida - gs agar plates were incubated at 30c for 
 3 days . the morphological assessment was carried out from the colonies on the plates , and 
 those with different shapes were collected , gram stained and observed under a light 
 microscope for species detection . genomic dna from each colony was obtained using a 
 wizard genomic extraction kit ( promega , madison , wi , usa ) . for 
 staphylococcus species , cells were treated with 1 mg / ml of 
 lysostaphin in tris - edta buffer ( te ; 10 mm tris - cl , 1 mm edta , ph 8.0 ) at 37c for 1 
 h before using the kit . final identification was done by polymerase chain reaction 
 ( pcr ) amplification with bacterial universal primers for 16s rdna and 26s rdna for 
 fungus , followed by dna sequencing . dna sequencing was performed using an abi prism 
 3100 genetic analyzer ( applied biosystems , tokyo , japan ) with a big dye cycle 
 sequencing reaction kit ( ab applied biosystems ) . identification of experimentally 
 determined nucleotide sequences using sequence databases was performed by basic local 
 alignment search tool ( blast ) . the antimicrobial activity was tested with respective 
 antibiotics used commonly in clinical practice ( figure 1 ) . statistical analyses were conducted using spss 15.0 ( spss inc . , u.s.a . ) and any 
 differences at p<0.05 level were considered as statistically significant . the samples were collected from surgical scar , saliva , gingiva , and palate of 78 
 elderly chinese ( 43 cases of group i and 35 cases of group ii ) . the graphical representation for the 
 percentage of species identification from one species in one isolates or two species 
 in same isolates were presented in two sets of group i & ii in each species 
 ( figure 2 ) . statistical analysis revealed no 
 significant difference between groups in the number of isolated microorganisms or the 
 number of participants with these isolates . graphical representation of the percentage of species identification , 
 identified as one species in on isolates or two species in same isoltates were 
 presennted in two sets of group i & ii in each species the universal primers for fungi , 26s rdna sequencing was used to identify the 
 candida species ( figure 3 ) . the candida species isolated from 78 participants ( both groups ) are 
 identified as c. albicans ( 43.6% ) , c. glabrata 
 ( 37.2% ) , c. krusei ( 23.1% ) , c. africana 
 ( 9% ) and c. guilliermondii ( 6.4% ) . the number of 
 candida species isolated from group i were greater than that of 
 group ii . on comparison between the groups , the c. albicans were 
 found to be the dominant species in both group i ( 55.8% ) and group ii ( 28.6% ) . 
 however , there is a significant difference ( p<0.05 ) found between groups . similarly , c. glabrata were found to be the next dominant species 
 with significant difference between group i ( 48.8% ) and group ii ( 22.8% ) participants 
 ( table 1 ) . however , no significant 
 difference was found for c. africana and c. guilliermondii species , 
 rather very less percentage of the species were identified in both groups ( table 1 ) . graphical representation of the overall percentage of identified candida 
 species identification of candida species and their percentage of 
 occurrence there were significant differences between the two groups ( p<0.05 ) the universal primers for bacteria , 16s rdna sequencing was used for species 
 identification ( figure 4 ) . the bacterial 
 species isolated from the 78 participants ( both groups ) are identified as 
 staphylococcus ( 59% ) , pseudomonas ( 53.8% ) , 
 streptococcus ( 44% ) , neisseria ( 37.2% ) , 
 actinomyces ( 29.5% ) and veillonella ( 25.6 ) , 
 which are mostly found in the surgical scar and saliva . on comparison between two 
 groups of isolates , the streptococcus , staphylococcus , pseudomonas 
 and neisseria showed significant differences ( p<0.05 ) in 
 percentage of these species between groups i and ii ( table 2 ) . in group i , the numbers of positive isolates of bacterial 
 species were greater than that of the control group using traditional oral care 
 methods . for veillonella and actinomyces , no 
 significant differences were shown between the two groups ( table 2 ) . graphical representation of the overall percentage of identified bacterial 
 species identification of bacterial species and their percentage of occurrence there were significant differences between the two groups ( p<0.05 ) the species identification reveals that there are higher percentages of dominant 
 microorganisms found in group i than those of group ii . to investigate the reason 
 behind this dominant character , we studied the occurrence of microorganisms against 
 the antimicrobial agents by categorizing the remaining group i participants ( 50 
 cases ) with their history of treatment with commonly practiced antimicrobial agents 
 ( table 1 ) after the treatment for oral 
 cancer . these 50 participants were divided as 25 cases for the study against 
 antifungal agents and remaining 25 against antibacterial agents . the participants after the treatment of oral cancer were taking commonly practiced 
 antifungal agents such as itraconazole , miconazole , fluconazole , 5-fluorocytosine , 
 amphotericin b , voriconazole and micafungin . the isolates were tested for the 
 presence of candida species . except for the c. 
 glabrata and c. krusei species , all other species were 
 susceptible to the antifungal agents . interestingly , itraconazole had 100% resistance 
 by c. glabrata and 60% resistance by c. krusei 
 species . the c. glabrata showed resistance to miconazole ( 20% ) , 
 fluconazole ( 20% ) , 5 fluorocytosine ( 8% ) . however , c. krusei showed 
 44% , 32% and 28% of resistance respectively . the other drugs were susceptible to all 
 candida species ( table 
 3 ) . prevalence of candida species against respective antibiotics the antibacterial agents were tested for the drug - resistant activity of staphylococci 
 subspecies , particularly on methicillin - resistant staphylococcus 
 aureus ( mrsa ) , s. epidermidis and s. 
 haemolyticus . the tested agents are commonly practiced antibiotics such 
 as oxacillin , arbekacin , vancomycin , teicoplanin , linezolid , imipenem / cilastatin , 
 amikacin and ciprofloxacin . surprisingly , mrsa showed drug - resistance to almost all 
 antibiotics with variation in percentage of resistance . the mrsa showed the highest 
 percentage of resistance to oxacillin ( 96% ) and arbekacin . however , the lowest 
 percentage ( 4% ) of resistance was shown in relation to amikacin and ciproflaxacin 
 ( table 4 ) . the other species , s. 
 epidermidis and s. haemolyticus , showed resistance to 
 oxacillin with 60 and 40% , respectively . however , s. haemolyticus 
 showed the lowest percentage ( 4% ) of resistance in relation to arbekacin . the samples were collected from surgical scar , saliva , gingiva , and palate of 78 
 elderly chinese ( 43 cases of group i and 35 cases of group ii ) . the graphical representation for the 
 percentage of species identification from one species in one isolates or two species 
 in same isolates were presented in two sets of group i & ii in each species 
 ( figure 2 ) . statistical analysis revealed no 
 significant difference between groups in the number of isolated microorganisms or the 
 number of participants with these isolates . graphical representation of the percentage of species identification , 
 identified as one species in on isolates or two species in same isoltates were 
 presennted in two sets of group i & ii in each species the universal primers for fungi , 26s rdna sequencing was used to identify the 
 candida species ( figure 3 ) . the candida species isolated from 78 participants ( both groups ) are 
 identified as c. albicans ( 43.6% ) , c. glabrata 
 ( 37.2% ) , c. krusei ( 23.1% ) , c. africana 
 ( 9% ) and c. guilliermondii ( 6.4% ) . the number of 
 candida species isolated from group i were greater than that of 
 group ii . on comparison between the groups , the c. albicans were 
 found to be the dominant species in both group i ( 55.8% ) and group ii ( 28.6% ) . 
 however , there is a significant difference ( p<0.05 ) found between groups . similarly , c. glabrata were found to be the next dominant species 
 with significant difference between group i ( 48.8% ) and group ii ( 22.8% ) participants 
 ( table 1 ) . however , no significant 
 difference was found for c. africana and c. guilliermondii species , 
 rather very less percentage of the species were identified in both groups ( table 1 ) . graphical representation of the overall percentage of identified candida 
 species identification of candida species and their percentage of 
 occurrence there were significant differences between the two groups ( p<0.05 ) the universal primers for bacteria , 16s rdna sequencing was used for species 
 identification ( figure 4 ) . the bacterial 
 species isolated from the 78 participants ( both groups ) are identified as 
 staphylococcus ( 59% ) , pseudomonas ( 53.8% ) , 
 streptococcus ( 44% ) , neisseria ( 37.2% ) , 
 actinomyces ( 29.5% ) and veillonella ( 25.6 ) , 
 which are mostly found in the surgical scar and saliva . on comparison between two 
 groups of isolates , the streptococcus , staphylococcus , pseudomonas 
 and neisseria showed significant differences ( p<0.05 ) in 
 percentage of these species between groups i and ii ( table 2 ) . in group i , the numbers of positive isolates of bacterial 
 species were greater than that of the control group using traditional oral care 
 methods . for veillonella and actinomyces , no 
 significant differences were shown between the two groups ( table 2 ) . graphical representation of the overall percentage of identified bacterial 
 species identification of bacterial species and their percentage of occurrence there were significant differences between the two groups ( p<0.05 ) the species identification reveals that there are higher percentages of dominant 
 microorganisms found in group i than those of group ii . to investigate the reason 
 behind this dominant character , we studied the occurrence of microorganisms against 
 the antimicrobial agents by categorizing the remaining group i participants ( 50 
 cases ) with their history of treatment with commonly practiced antimicrobial agents 
 ( table 1 ) after the treatment for oral 
 cancer . these 50 participants were divided as 25 cases for the study against 
 antifungal agents and remaining 25 against antibacterial agents . the participants after the treatment of oral cancer were taking commonly practiced 
 antifungal agents such as itraconazole , miconazole , fluconazole , 5-fluorocytosine , 
 amphotericin b , voriconazole and micafungin . the isolates were tested for the 
 presence of candida species . except for the c. 
 glabrata and c. krusei species , all other species were 
 susceptible to the antifungal agents . interestingly , itraconazole had 100% resistance 
 by c. glabrata and 60% resistance by c. krusei 
 species . the c. glabrata showed resistance to miconazole ( 20% ) , 
 fluconazole ( 20% ) , 5 fluorocytosine ( 8% ) . the other drugs were susceptible to all 
 candida species ( table 
 3 ) . prevalence of candida species against respective antibiotics the antibacterial agents were tested for the drug - resistant activity of staphylococci 
 subspecies , particularly on methicillin - resistant staphylococcus 
 aureus ( mrsa ) , s. epidermidis and s. 
 haemolyticus . the tested agents are commonly practiced antibiotics such 
 as oxacillin , arbekacin , vancomycin , teicoplanin , linezolid , imipenem / cilastatin , 
 amikacin and ciprofloxacin . surprisingly , mrsa showed drug - resistance to almost all 
 antibiotics with variation in percentage of resistance . the mrsa showed the highest 
 percentage of resistance to oxacillin ( 96% ) and arbekacin . however , the lowest 
 percentage ( 4% ) of resistance was shown in relation to amikacin and ciproflaxacin 
 ( table 4 ) . the other species , s. 
 epidermidis and s. haemolyticus , showed resistance to 
 oxacillin with 60 and 40% , respectively . however , s. haemolyticus 
 showed the lowest percentage ( 4% ) of resistance in relation to arbekacin . in this study , a prevalence of drug - resistant microorganisms in the oral cavity after 
 treatment of oral cancer was performed in aged chinese . the study was carried out in 
 ninth people 's hospital , school of medicine , shanghai jiao tong university , shanghai . the participants were the patients from this hospital who had undergone oral cancer 
 treatment and others without any history of cancer but that could be patients who had 
 undergone treatment for other oral diseases . this study was aimed to investigate the 
 reason behind the frequent cause of oral infection after the treatment of oral cancer in 
 elderly individuals . several lines of evidence support our views that there is a possibility of 
 drug - resistant microbes present in immunocompromised patients , particularly after the 
 treatment of oral cancer . in general , infections are commonly found in oral cancer 
 patients after surgical excision of the tumor . this might be due to 
 wound exposure during and after the operation , even if sutured , when microorganisms may 
 infect oral regions , oropharynx , nasal cavity , and paranasal sinuses areas . patients 
 after oral cavity surgery often appear to have complications after bacterial infections , 
 as well . colonization of pathogenic bacteria in oral cavity is thought to increase the 
 risk of infections such as pneumonia and bacteraemia . it is therefore of high 
 importance the prevention from or cure for the infections . during the development of 
 tumor , the tumor cells or soluble products produced by tumor cells the high percentages of microbial species identified from the isolates of participants 
 were candida , staphylococcus , streptococcus and 
 pseudomonas species ( figure 
 2 ) . the highest numbers of these species were found in patients after the 
 treatment of oral cancer in comparison with the non - cancer patients ( tables 1 and 2 ) . similar results were obtained in a phase 1 clinical trial with the 
 application of an antibacterial dressing spray in the prevention of post - operative 
 infection in oral cancer patients . the abuse of 
 antimicrobial drugs brings severe adverse effects to people , for example , allergy , toxic 
 reaction , and opportunistic infections . as a result , a new and ideal preventive method is needed for people 
 who have surgery to reduce the chances of bacterial infections . in this study , after revealing the high percentage of microorganisms found particularly 
 in post - treatment of oral cancer , we started to focus on screening or identification of 
 drug - resistant microbes within the species of already identified microbes . the 
 antimicrobial agents ( figure 1 ) , which are 
 common in clinical practice , were taken for this study . the isolates were tested for the 
 presence of candida species and subspecies of staphylococci , 
 particularly on mrsa , s. epidermidis and s. 
 haemolyticus . except for the c. glabrata and c. 
 krusei species , all other candida species were susceptible 
 to the antifungal agents ( table 3 ) . the mrsa 
 showed the highest percentage of resistance to oxacillin ( 96% ) and arbekacin . the other 
 species , s. epidermidis and s. haemolyticus , showed 
 resistance to oxacillin with 60 and 40% , respectively . all other antibiotics were found 
 susceptible to these species ( table 4 ) . 
 according to a study performed in the united kingdom , 13 ( 41.9% ) of 31 s. 
 aureus isolates from the oral mucosa and pockets of patients with another study , in the united states , 
 reported that the prevalence of mrsa organisms in the nasal and oral cavities of nursing 
 home residents was 20 - 35% . furthermore , a japanese group investigated mrsa colonization in neonatal intensive care 
 units and found that 207 ( 49.9% ) of 415 newborns had mrsa organisms . when compared with these results , the 
 drug - resistance ( 96% ) of mrsa against oxacillin was extremely high , possibly due in part 
 to the tendency to prescribe long - term , high - dose antibiotic treatment in 
 japan . our study revealed a group of opportunistic - microorganisms such as c. glabrata , 
 c. krusei and subspecies of staphylococci , particularly 
 methicillin - resistant s. aureus ( mrsa ) , s. 
 epidermidis and s. haemolyticus . nevertheless , anti - tumor 
 drugs used in tumor treatment will also lead to immune and bone marrow suppression . continuous monitoring 
 and a basic infection control strategy , including standard precautions , are important 
 for older individuals , especially those receiving follow - up care for oral cancer . there 
 is always a risk that they may become immunocompromised hosts easily susceptible to oral 
 infections . clinicians must also pay careful attention to opportunistic - microorganisms 
 during the treatment of oral cancer , and to realize the fact of immune compromise in 
 elderly patients .
objectivethe immune compromised patients after treatment of oral cancer may have a chance 
 of infection by drug - resistant opportunistic microbes . 
 we investigated the 
 occurrence of opportunistic microorganisms in aged individuals receiving follow - up 
 examinations after treatment of oral cancer in china . 
 material and methodsthese patients were used as test group and the respective age grouped healthy 
 individuals as control group . in this study , the oral cavity microorganisms such 
 as bacteria and yeast were taken for the analysis . 
 after the screening of 
 representative microorganisms , their aptitude of pervasiveness against drugs was 
 studied . here , we used antimicrobial agents which are common in clinical practice . 
 we also performed studies to investigate the presence of toxin genes in 
 methicillin - resistant s. aureus ( mrsa ) . 
 resultsthe results indicate that the prevalence of drug - resistant microbes was more 
 pronounced in oral cancer patients after initial treatment above 70 years old . 
 the 
 oxacillin resistance of s. aureus isolate confirms that the 
 prevalence of mrsa is increasing in accordance to age - factor and immune compromise 
 in elderly patients . 
 conclusionsthis study reveals the occurrence of drug - resistant opportunistic microorganisms 
 in oral cavity after treatment for oral cancer in aged individuals . 
 special 
 attention should be directed to mrsa during the treatment of oral cancer , and to 
 realize the fact of immune compromise in elderly patients .