Original Paper

Caries Res 2003;37:261–266DOI: 10.1159/000070868

Non-Mutans Streptococci in PatientsReceiving Radiotherapy in the Head andNeck Area

H.C. Tonga X.J. Gaoa X.Z. Dongb

aDepartment of Cariology, Endodontology and Operative Dentistry, School of Stomatology, Peking University, andbState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences,Beijing, PR China

Received: November 1, 2002Accepted after revision: February 24, 2003

X.J. GaoDepartment of Cariology, Endodontology and Operative DentistrySchool of Stomatology, Peking University, No. 22Zhongguancun Nan Dajie Haidian, Beijing, 100081 (PR China)Tel. +86 10 6217 9977/ext. 2523, Fax +86 10 6217 3402, E-Mail xjgao@95777.com

ABCFax + 41 61 306 12 34E-Mail karger@karger.chwww.karger.com

© 2003 S. Karger AG, Basel0008–6568/03/0374–0261$19.50/0

Accessible online at:www.karger.com/cre

Key WordsHigh-caries-risk individual W Non-mutans streptococci W

Radiotherapy

AbstractObjective: To study mutans and non-mutans streptococ-ci in patients after radiotherapy of the head and neck.Methods: Oral rinse samples collected from nasopharyn-geal carcinoma patients before and after radiotherapywere diluted and cultured on nonselective and selectivemedia for enumeration of total cultivable plaque flora,mutans and non-mutans streptococci and lactobacilli.Non-mutans streptococci were identified biochemicallyand by 16S rDNA sequence homology analysis. Results:

After irradiation, mutans streptococci were not isolated;the levels of Streptococcus mitis and lactobacilli in-creased significantly. The level of Streptococcus saliva-rius increased, but the significance was the borderline.The level of Streptococcus sanguis decreased signifi-cantly after irradiation. The abundance of other oralstreptococci species showed no significant changes.Conclusions: S. mitis and S. salivarius are the predomi-nant non-mutans streptococci in the high-caries-risk oralflora following radiotherapy.

Copyright © 2003 S. Karger AG, Basel

Oral streptococci comprise part of the normal micro-flora of the oral cavity and upper respiratory tracts ofhumans. Mutans streptococci (MS) have been consideredas some of the most important cariogenic bacteria[Krasse, 1966; Loesche, 1986; Alaluusua and Renkonen,1983], but there is evidence that dental caries can occurwithout the presence of MS [Hardie et al., 1977; Mikkel-sen and Poulsen, 1976; Mikkelsen et al., 1981]. Recently,groups of ‘low-pH’ non-mutans streptococci (non-MS)have been implied to be associated with dental caries.They appear at higher levels in the high-caries individuals[van Houte et al., 1991; Sansone et al., 1993], produceorganic acid at low pH (!4.4), induce caries in rodents[Willcox et al., 1990] and are also prevalent in ‘white-spot’caries [van Ruyven et al., 2000]. To clarify the relation-ship between non-MS and dental caries further, it isimportant to study the status of this group of bacteriaprior to the onset of clinical lesions.

Dental caries can happen at a higher frequency inpatients who have received radiotherapy of the head andneck, and caries lesions can be detected clinically within afew months. Radiotherapy is the first choice of treatmentfor nasopharyngeal carcinoma patients. However, ioniz-ing irradiation also causes a pronounced reduction in sali-va flow rate and the onset of clinical xerostomia [Schubert

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262 Caries Res 2003;37:261–266 Tong/Gao/Dong

and Izutsu, 1987; Stephens et al., 1986]. Irradiation-induced xerostomia is always accompanied by pro-nounced shifts in specific microbial components of theoral microflora [Brown et al., 1975; Llory et al., 1972;Keene et al., 1994], and severe, rampant caries oftenbreaks out within a short time [Epstein et al., 1999; Laca-tusu et al., 1996]. Hence, we studied this group of subjectsto observe the microflora change before appearance ofclinical lesions and help clarify the role of non-MS in den-tal caries.

Materials and Methods

Study GroupsTwelve nasopharyngeal carcinoma resident patients (9 men and 3

women) in the Tumor Hospital of Beijing, China, voluntarily partici-pated in the saliva rinse. Their ages ranged from 25 to 68 years, witha mean age of 45.3 years. Participant selection was restricted topatients who had natural dentitions, no other systemic diseases, noprevious radiotherapy, were not receiving antibiotics, and would bescheduled to receive a minimum of 7,000 cGy of ionizing irradiationat a level of 1,000 cGy/week for 7 consecutive weeks. The irradiationfields included complete coverage of the three major salivary glands.The patients were given dental examination, cleaning, and oralhygiene instruction before the irradiation and at the time of salivasampling, but no special fluoride remedy was added until the lastsampling. Reducing sugar consumption was always advised but therewere no strict restrictions. The mean DMFT of the patient group was1.50 B 2.47 before the start of irradiation.

For controls, 12 healthy subjects were selected to pair with thepatient group in age, sex and oral health conditions. They had notreceived antibiotics within 2 weeks before sampling. The meanDMFT was 0.75 B 1.42.

SamplingOral rinse samples were collected from each subject in the patient

group at 3 time points: before radiotherapy, immediately after thelast radiotherapy and 1 month after radiotherapy, respectively, whilesamples from the control group were taken just once. The sampleswere obtained between 09:00 and 11:00 a.m., using the method ofSamaranayake et al. [1986] with slight modifications. In brief, eachsubject was asked to rinse the mouth with 5 ml of sterilized phos-phate-buffered saline (PBS, pH 7.2) for 60 s, and spit the oral rinseinto a sterilized test tube. The tubes were preserved on ice and takento laboratory within 2 h.

Isolation and Counting of BacteriaThe samples were dispersed with a Vortex-Genie mixer, and 10-

fold serial dilutions were made to 10–4. A dilution of 0.1 ml was spreadon the following duplicate media for counting different groups of bac-teria: (1) brain-heart infusion (Oxoid, Hampshire, UK) agar supple-mented with 5% (v/v) defibrinated sheep blood for enumeration of thetotal cultivable bacterial flora; (2) mitis-salivarius agar (Difco, Sparks,USA) supplemented with 0.1% (v/v) potassium tellurite for enumera-tion and isolation of oral streptococci; (3) tryptone-yeast extract-cys-tine agar (Bury, UK) supplemented with 0.2 units/ml bacitracin and15% sucrose (TYCSB) for the specific enumeration and isolation

of MS, and (4) Rogosa agar [Rogosa et al., 1951] for enumeration oflactobacilli. All the agar plates were incubated in an atmosphere of95% N2, 5% CO2 in GasPak jars at 37°C for 72 h. The plates withabout 30–300 bacterial colonies were counted. The bacterial countswere recorded as colony-forming units per milliliter (CFU/ml) ofrinse medium. MS and lactobacilli were enumerated according tocolonial morphology on the selective culture media.

Biochemical Identification of Oral StreptococciColonies in different morphology on mitis-salivarius agar were

selected and subcultured. Morphological and physiological charac-terizations were carried out according to Hardie [1986], and bio-chemical tests were performed according to methods and criteriadescribed previously [Kilian et al., 1989; Beighton et al., 1991].

16S rDNA Sequencing of Oral StreptococciBrown et al. [1975] investigated the influence of radiotherapy in

the head and neck area on oral streptococci flora, but the identifica-tion of oral streptococci mainly relied on sugar fermentation. The16S rRNA has been demonstrated to be a good tool for bacterialidentification and phylogenetic study [Woese, 1987], and Kawamuraet al. [1995] reclassified oral streptococci into four phylogeneticgroups on the basis of 16S rRNA sequence homology analysis.

The genome DNA was extracted and purified using a modifiedmethod of Marmur [1961; Dong et al., 2000]. The oligonucleotideprimers used in this study were synthesized by Sangon (Shanghai,China). Two primers, 27f (5)-AGAGTTTGATCC/ATGGCTCAG-3)) and 1541r (5)-AAGGAGGTGATCCAGCC-3)), were comple-mentary to positions 8–27 and 1525–1541 of 16S rDNA of Escheri-chia coli, respectively [Winker and Woese, 1991]. Each PCR mixture(25 Ìl) contained the following reagents: 80 ng DNA template;0.25 mmol/l each of dATP, dCTP, dGTP, dTTP; 2.5 mmol/l Mg2+;1 Ìmol/l each of the two primers and 1 U Taq DNA polymerase (Ta-KaRa Co., Japan). The thermal cycling conditions were initial dena-turation of DNA template at 94°C for 5 min, followed by 30 cyclescomprising (1) denaturation at 94°C for 30 s, (2) annealing at 55°Cfor 1 min, (3) elongation at 72°C for 1.5 min. When 30 cycles werecompleted, the reaction mixture was maintained at 72 °C for 10 min.The PCR amplification was performed with a Thermolyne Ampli-tron (Barnstead Thermolyne Corporation, USA). After amplifica-tion, the reaction mixtures were electrophoresed through 0.7% w/vagarose gel (Promega, USA) in 1 ! TAB buffer (50 mmol/l Tris-acetate, 1 mmol/l EDTA, pH 8.0) and visualized with ethidium bro-mide under UV light.

The PCR product bands about 1.5 kb in length were cut and puri-fied using UNIQ-5 column DNA purification kit (Sangon, PR China,and NSBC) as recommended by the manufacturer. Sequencing of the16S rDNA was performed by TaKaRa Co. using ABI PRISM BigDye Terminator Cycles Sequencing Ready Reaction Kits (PerkinElmer, USA) and ABI PRISM 377XL DNA Sequencer.

The complete 16S rDNA sequences were submitted to GenBankand matched with the closest relatives, based on the sequence simi-larities using program BLAST (version 2.2.1).

Analysis of DataThe isolation frequencies of individual species were compared

using Fisher’s exact test. The abundance of species in the oral rinsesamples of nasopharyngeal carcinoma patients before radiotherapywere compared with healthy controls using t test. The abundance ofspecies in the oral rinse samples of nasopharyngeal carcinoma

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Non-Mutans Streptococci afterRadiotherapy

Caries Res 2003;37:261–266 263

Table 1. Comparison of the biochemical characteristics of 5 strains of oral streptococci isolated from post-radiotherapy samples with those ofthe corresponding type strains

S. salivarius

isolate 1 ATCC13419T

S. sanguis

isolate 2 ATCC10556T

S.mitis

isolate 3 ATCC49456T

S. constellatus

isolate 4 ATCC27823T

S. parasanguis

isolate 5 ATCC15912T

Acid produced fromArbutin + + – + – – + + – +Mannitol – – – – – – – – – –Sorbitol – – – – – – – – – –Lactose + + – + + + – + + +Amygdalin – – – + – – – + – +Raffinose – + – + – + + – + +Inulin – + – + – – – – – –Melibiose – – – + – + – + + –Trehalose – + – + – + + + – –

Hydrolysis ofArginine – – – + – + – + +w +Esculin + – – + – – – – – +

Species identification was on the basis of 16S rDNA-sequencing analysis.+ = Positive reaction; – = negative reaction; w = weak reaction.

patients before and after radiotherapy were compared using paired-sample t test. All statistical analyses were performed using SPSS soft-ware, Release 10.0.1 (SPSS Inc., USA).

Results

Identification of Oral StreptococciTo count particular species of the oral streptococci, col-

onies of different morphology on mitis-salivarius agarwere picked up for species identification. In total 240 rep-resentative colonies from 48 oral rinse samples were iden-tified based on their biochemical characteristics. Amongthem, 150 colonies were identified as strains of Strepto-coccus mutans, Streptococcus sanguis, Streptococcus gor-donii, Streptococcus oralis, Streptococcus anginosus,Streptococcus salivarius and Streptococcus mitis. How-ever, the remaining 90 colonies, mostly isolated frompost-radiotherapy samples, could not be assigned to anexisting Streptococcus species. According to their bio-chemical features, these 90 colonies could be grouped intofive biotypes, and a strain was chosen from each of thefive biotypes for 16S rDNA sequencing. Based on homol-ogy analysis, they were identified as 5 known Streptococ-cus species, namely S. salivarius, S. sanguis, S. mitis, S.constellatus and S. parasanguis, although they did not

match the biochemical characteristics of the correspond-ing strains (table 1).

Comparison of Pre-Radiotherapy and Control SamplesThe isolation frequencies and abundance of lactobacil-

li and oral streptococci species in patients before radio-therapy were compared with those in healthy controls.There were no significant differences between the twogroups (table 2).

Changes following RadiotherapyThe isolation frequencies and abundance of oral strep-

tococci and lactobacilli in the rinse samples from thepatient group were compared at three time points: beforeradiotherapy, immediately after the last radiotherapy and1 month after radiotherapy.

The results showed that, after radiotherapy, the isola-tion frequency of S. sanguis decreased while that of lacto-bacilli increased. The isolation frequencies of other oralstreptococci showed no significant change (table 2).

The counts of S. mitis in the rinse samples immediate-ly after the last time and 1 month after radiotherapyincreased significantly (p ! 0.05) compared with beforeirradiation. The abundance of lactobacilli also increasedsignificantly after radiotherapy (p ! 0.01). The abundanceof S. salivarius increased as well after radiotherapy, but

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Table 2. Isolation frequencies and abundance (log10 CFU/ml, means B SD) of oral streptococci and lactobacilli before and after radio-therapy

Bacteria Control group(n = 12)

abundance frequency

Patient group beforeradiotherapy (n = 12)

abundance frequency

Immediately afterradiotherapy (n = 12)

abundance frequency

One month afterradiotherapy (n = 12)

abundance frequency

Total anaerobes 4.83B0.84 12/12 4.97B1.02 12/12 5.42B0.39 12/12 5.49B0.39 12/12Lactobacilli ND ND 0.22B0.59 2/12 1.46B1.12** 8/12a 1.49B1.14** 8/12a

Streptococcus spp. 3.85B0.85 12/12 4.28B0.76 12/12 4.30B0.88 12/12 4.16B0.80 12/12S. mutans 0.13B0.33 2/12 0.32B0.75 2/12 ND ND ND NDS. salivarius 0.85B1.57 2/12 1.25B1.88 6/12 2.66B1.50 10/12 2.81B1.36 11/12S. mitis 2.00B1.63 8/12 1.38B1.58 7/12 2.91B1.71* 9/12 2.61B2.01* 8/12S. parasanguis ND ND ND ND ND ND 0.23B0.79 1/12S. oralis 0.31B0.77 2/12 0.91B1.68 3/12 0.19B0.66 1/12 ND NDS. sanguis 2.01B1.72 10/12 2.22B1.72 10/12 0.25B0.87** 1/12a 0.14B0.49** 1/12a

S. gordonii 0.48B1.11 2/12 0.76B1.38 3/12 0.08B0.29 1/12 ND NDS. constellatus ND ND ND ND 0.25B0.87 1/12 ND NDS. anginosus 0.72B1.45 2/12 0.22B0.75 1/12 ND ND 0.22B0.78 1/12

ND = Not detected.Significantly different from before radiotherapy: * p ! 0.05; ** p ! 0.01.

a Significantly different from before radiotherapy by Fisher’s exact test (p ! 0.05).

the significance was borderline (p = 0.063). On the otherhand, the abundance of S. sanguis decreased significantly(p ! 0.01). The abundance of other oral streptococcishowed no significant changes (table 2).

Discussion

When nasopharyngeal carcinoma patients receive ra-diotherapy, three major salivary glands can be involved inthe radiation field. The irradiation often causes degenera-tion and necrosis of the salivary glands. When the irradia-tion dose exceeds 5,000 cGy, the damage to the salivaryglands is permanent [Fox, 1998]. Ship et al. [1997] foundthat when parotid glands received an average dose of5,745 cGy, unstimulated and stimulated parotid flowrates decreased dramatically after the initiation of radio-therapy, and were significantly lower 1 year after radio-therapy than at baseline. So in this study sample collec-tion was selected at three time points: before radiothera-py, immediately after the last time of radiotherapy and 1month after radiotherapy.

The results showed that the isolation frequency andquantity of oral streptococci and lactobacilli in the rinsesamples before radiotherapy was similar to healthy people(table 2), indicating that pharyngeal carcinoma itself had

no influence on oral ecology in the patient group, so thatthe subjects in the patient group before radiotherapycould be on a similar level of caries susceptibility tohealthy controls.

The patients could be assumed to be a high-caries-riskpopulation after radiotherapy. Lacatusu et al. [1996]reported that rampant caries had been found in 89% ofpatients who received prolonged or repeated cervico-facial radiotherapy. A major reason could be the change ofmicroflora upon irradiation since bacteria had been as-sumed to be the initial factor of dental caries. Hence,studying bacterial changes in the oral flora of this patientgroup could identify the predominant oral streptococciprior to the clinical lesions and assist the prevention oframpant caries.

The abundance of total anaerobic bacteria and totalstreptococci did not show a significant change after radio-therapy, but the predominant species of microflorachanged. While S. sanguis, S. mitis and S. salivarius werethe predominant oral lactic acid-producing bacteria be-fore irradiation, S. mitis, S. salivarius and lactobacillibecame the main ones in the post-irradiation samples (ta-ble 2). Lactobacilli are considered to have stronger aci-dogenic and aciduric abilities, and can grow and produceacid in medium with an initial pH of 4.5. That lactobacillisubstituting S. sanguis became one of the main compo-

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Caries Res 2003;37:261–266 265

nents of the microflora of the oral cavity after irradiationthus indicates that the oral microflora was more acidicand aciduric.

Kilian et al. [1989] found that S. salivarius and S. mitisgenerated a final pH of 4.1 and 4.4 in TPYG liquidmedia, respectively. The increased abundance of S. mitisand S. salivarius in the post-radiotherapy samples indi-cated that the oral environment became more acidifiedupon radiotherapy. Whether this is a signal of the break-out of rampant caries needs to be proven, yet the results ofthis study are consistent with those of van Houte et al.[1991], Sansone et al. [1993] and van Ruvyen et al.[2000]. They believe that the emergence of MS in plaqueis often preceded by non-MS. However, Llory et al. [1971]observed an increase in the amount of S. mitis and a dis-appearance of S. salivarius after radiotherapy, in contrastto the observations in this study. The reason might be theidentification method used. Irradiation could affect someenzyme-encoding genes [Eugene, 1981], so that some col-onies recovered from the samples after radiotherapycould not be identified as any Streptococcus species basedon biochemical tests. Since the 16S rDNA gene is notexpressed as a protein product, its sequence might beinfluenced less by the irradiation and could give moreaccurate identification by homology analysis (table 1).

Because of the decrease of salivary flow rate andincrease of S. salivarius, S. mitis and lactobacilli, oralenvironment becomes more acidic, leading to inhibitionof acid-sensitive species such as S. sanguis. Like Brown etal. [1975], we found that the level of S. sanguis decreasedsignificantly after radiotherapy (table 2). According toMarsh’s [1994] ecological plaque hypothesis, fewer S. san-guis in post-radiotherapy rinse samples might suggest thatthe normal coordinated relationship in the oral microflorahad broken down. Under this circumstance, the propor-

tion of oral microflora shifted towards to a more cariogen-ic status of increasing caries risk.

MS have long been considered the main cariogenicbacteria; epidemiological studies demonstrated that MSpresented in substantial numbers in most active cariouslesions [Kristofferson et al., 1985]. Recently, however,van Ruyven et al. [2000] found a low proportion of S.mutans (!0.1%) in white-spot dental caries, and consid-ered that non-MS might become predominant when thehosts ingested dietary carbohydrates at a lower level thanthat necessary for the exuberant growth of the MS. Cariesdevelopment would be at a lower rate when carbohydratewas ingested at a lower level and the more weakly acidog-enic non-MS predominated in the plaque. The irradiatedpatients were routinely given the advice of reducing inges-tion of carbohydrates, which may be the reason for notdetecting MS after radiotherapy in our study (table 2),further supporting the conclusion of van Ruvyen et al.[2000]. Changes in the abundance of S. oralis, S. parasan-guis, S. anginosus, S. constellatus and S. gordonii afterradiotherapy were investigated in this study. The similarlevels of isolation frequencies and abundance of thesebacteria in both patient and control groups indicated thatthey are not affected by the irradiation.

This study demonstrated that non-MS such as S. mitisand S. salivarius had increased following the irradiationwhile S. sanguis decreased. However, further studies areneeded to clarify whether these changes can be taken asrisk factors for a new caries breakout after irradiation.

Acknowledgments

This study was supported by the Peking University ResearchGrant 985.

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