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Preventive measures in mothers influence the establishment of the bacterium Streptococcus mutans in their infants
AlxIl\
OUII
Bid. Vol 1x,
PrInted it1C;re:ltBritam
vo. 3. pp.
0003-9961) 83 030225-07iK3.00,0
225 131. IYX.:
CopyrIght
All rlghrs reserved
PREVENTIVE
MEASURES
IN MOTHERS
ESTABLISHMENT STREPTOCOCCUS
OF
THE
MUTANS
c
I%?
INFLUENCE
Pergamon
Press Ltd
THE
BACTERIUM
IN THEIR
INFANTS
BIRC~ITTAKijHLER. D. BRATTHALLand B. KRAN Department
of Cariology.
Faculty
of Odontology.
University
of GGteborg.
S-400 33 GBteborg.
Sweden
Summary~First-time mothers who had a high salivary number of Strep. ~7t~f~f7.~ [ 2 1Oh colonyforming-unils (c.f.u.) per ml] were selected. Every second mother was given a special preventive programme to reduce her salivary level below 3 x IO5 c.f.u. per ml. Where a reduction of Strep. 177ufu17swas achieved in the mother. the establishment of Strep. 117ut~17sin her infant was prevented or delayed. Thus. 28 mothers were successfully treated until their infants were 23 months old ,.md only 3 of their infants (I I per cent) were infected with Strep. 117ut~t7.s, compared with I7 out of 38 infants in the control group (45 per cent). In both groups, the percentage of infected infants increased with increasing age, although at all ages fewer infants were infected with Strep. ,77utu77s in the test group than in the control group. Sixteen infants of successfully treated mothers had reached the age of 36 months. Three were infected (19 per cent) compared with I7 out of 27 in the control group (63 per cent). These findings show that the spread of Strep. rutrtar1.s can be delayed or prevented by measures directed against the main source of infection. an approach which is successful in the prevention of other infectious diseases.
INTRODlJCTIOiY
Strrptoumus mutuc.s is an important pathogen in the development of dental caries (reviewed by Gibbons and van Houte. 1975; Hamada and Slade, 1980: van Houte, 1980). This microorganism is either not detected at all or is only transiently present in the mouths of infants before emergence of teeth (Berkowitz, Jordan and W:lite, 1975; Carlsson. Grahnkn and Jonsson. 1975; Catalanotta, Shklair and Keene. 1975; Stiles et ~11.. 1976; Berkowitz. Turner and Green, 1980). The mother seems to be the major source of infection (Berkowitz and Jordan, 1975; Berkowitz et (I/.. 1975; Hamada. Masuda and Kotani. 1980; Masuda pf ul.. 19801. Colonization of Strep. 777ut~777~in man is favoured by a large inoculum (van Houte and Green 1974; Duchin and van Houte, 197X: Svanberg and Locsche. 1977. 1978) and repeated inoculation (Krasse et d.. 1967. Jordan et ctl.. 1972). These factors might explain why the mother is a probable reservoir for the primary infection of her infant by Srrcp. 117ut~117s. Earlier, it was observed that mothers highly infected with Strep, n71m777~ tended to have children who also were highly infected. whereas mothers with low salivary concentrations tended to have noninfected children (Kb;hler and Bratthall. 1978). This observation was confirmed in later studies (Berkowitz et rrl., 1980, 1981; can Houte. Yanover and Brecher, 1981). It therefore seemed of interest to determine whether reduction of the number of Strep. 177utu77.s in highly infected mothers could prevent the initial oral establishment of these microorganisms in their infants.
MATERIALS
AKD METHODS
All mothers attending the child health centre in Oskarshamn. Swec.en, with their first-born babies (3-8 months old) were sampled for salivary concen-
trations of Srrrp. 177uru/7s and lactobacilli. Sampling was conducted by dentists and specially trained dental nurses employed at the public dental office in Oskarshamn. Over a 2; year period. 249 mothers were sampled. Mothers harbouring 10b or more Strep. 7777rrrtr7.s colony-forming-units (c.f.u.) per ml saliva and their infants were selected. These 87 mothers were assigned alternately to experimental and control groups (Kiihler et al.. 1982). Due to various circumstances, such as moving from the district (2 experimental and 2 control mothers) and refusal to particisubjects), 6 subjects were expate (2 experimental cluded. The mean. median and range of salivary Strep. mttms and lactobacilli in the initial sample of the control group and the experimental group are given in Table I. In the control group. 23 (57.5 per cent) of the infants were males; in the experimental group. 26 (63.4 per cent) were males.
In a letter to the mothers in the experimental group. the purpose and design of the study were explained. To reduce the salivary concentration of Strep. r77utan.s below the pre-selected threshold value of 3 x IO5 c.f.u. per ml saliva. the experimental mothers were given a basic preventive programme. This programme consisted of: (1) information about the purpose of the study, (2) dietary counselling, (3) professional tooth-cleaning and oral hygiene instructions, (4) fluoride treatment. (5) excavation of large cavities. The programme was carried out in the public dental clinic in Oskarshamn by three dentists in cooperation with specially-trained preventive nurses. The effect of the treatment was controlled by microbiological analysis. Mothers harbouring t3 x 10’ c.f.u. per ml saliva after the initial treatment were recalled after 4 months for a new saliva sample. Subjects having 23 x 10’ Strep. 777utan~ per ml saliva after the basic preventive programme were prescribed chlorhexidine-
226
Birgitta KShler. D. Bratthall and B. Krasse
Table 1. Salivary numbers
of Strrp.
mtms
and lactobacilli in control the study
and experimental
c.f.u. Strep. mi4tutls per ml saliva x lOh
t1* Control Experimental
40 41
Mean
Median
3.9 2.1:
1.9 1.5:
group at the start of
c.f.u. lactobacilli per ml saliva x IO” Range
t1
Mean
Median
Range
0.9-26.0 0%9.4$
39t 41
364 291
62 46
&3600 @I920
* Number of subjects. t One sample lost. $ Two of the experimental mothers with very high salivary Strep. lower mean, median and range values in this &ou,.
containing gel in individually designed applicators according to the method of Emilson (198 I ). The gel, which contained 1.0 per cent chlorhexidine digluconate (Hibitane. ICI Ltd. England). was used once a day. for 5 min. for 2 weeks. One week after the termination of the chlorhexidine treatment, saliva samples were collected to determine the effect of the gel application on the number of Strep. mutans. The mothers treated with chlorhexidine were recalled after 2 3 months to check the salivary level of Strep. mutans. Details of the preventive programme and the short-term effect after the first treatment period on Strep. mutans and lactobacilli have been described by Kiihler et (11.(1982). In mothers having 23 x IO’ Strep. ~nutm.s per ml saliva at the subsequent appointments. the basic preventive programme a&or the chlorhexidine treatment was repeated. In some cases. short-term chlorhexidine treatment (Maltr. Zickert and Krasse, 1981) was used as an alternative to the chlorhexidine treatment. Mothers who were pregnant during the course of the study did not use chlorhexidine during their pregnancies. instead. topical application with 8 per cent stannous-lluoridc was sometimes used (Keene. Shklair and Hoerman, 1976). The effect of each of the treatments used was again controlled by microbiological analysis. The preventive measures were carried out throughout the study unless the infant acquired detectable levels of Strep. mutuns on successive samplings, if the mother did not comply. or if we were unsuccessful in keeping her salivary level low (< 3 x 10’ c.f.u. per ml). These mothers. however. agreed to be recalled with their infants every 4 months for sampling. The infants in the experimental and control groups were sampled for the presence of Stwp. tmftun.7 at 4-month intervals starting at the age of I5 months. The mothers were also sampled at this time. Due to circumstances such as illness, moving from town and other problems, subjects occasionally failed to leave samples as planned. In addition, 4 mothers in the test group elected during the course of the study not to participate further in the preventive programme, although their infants were not infected at that time. The number of subjects examined is indicated in each table. At the age of 36 months, the examination of the child was extended to include collection of paraffinstimulated saliva and of 21 pooled plaque sample.
mutuns
levels
were lost, which resulted
in
Mothrrs. One millilitre of parafin-stimulated saliva was collected. transferred to 5.7 ml VMGII transport medium (Miiller. 1966) and mailed to the Department of Cariology of the Dental School in Giiteborg. On arrival. usually the following day. the samples were cultured on mitis salivarius bacitracin (MSB) agar selective for Strep. ttxums (Gold, Jordan and van Houte. 1973). using the micropipette method described by Westergren and Krasse (1978). The number of Strep. muru/ls c.f.u. per ml saliva was estimated. Childrrtl. The presence of Strrp. mututts in saliva was determined by a sampling technique reported by Kiihler and Bratthall (1979). A wlooden spatula was rotated in the mouth of the infant to wet it with saliva. Each side of the spatula was then pressed directly against an agar plate containing MSB agar medium. The plates were incubated in exhaled air at 37 C for 2 days. The incubation was done at the clinic in Oskarshamn and the plates were then mailed to Giiteborg for identification and enumeration. The mean number of Strep. tmltcmS colonies recovered on a predetermined area for the two sides pressed against the MSB-agar was calculated. The infants are described as infected when Strep. rmrtutn was detected by the spatula method and was also present in any of the subsequent samples. On a few occasions, the spatula sampling was not performed due to unwillingness of the child to cooperate. The result of a plaque sample was then used instead. Colonies with both typical and atypical morphology were isolated from all mothers and their infants for identification and serotyping by immunofluorescent identification (Bratthall. 1972). At 36months examination. paraffin-stimulated saliva was also collected and transferred to I ml of reduced transport fluid (RTF) (Syed and Loesche, 1972) and mailed to our laboratory. According to the child’s ability to cooperate. 0.05%1.0 ml saliva was collected. 0.025 ml were taken both directly and from dilutions IO- ’ and 10~’ and cultured on MSB agar. The number of Strep. muturn c.f.u. per ml saliva was estimated by the micropipette method described by Westcrgren and Krasse (I 978). To estimate the frequency of Srrrp. rmrrctm infection in infants of mothers with a low sahvary level ot Strep. mutons, all infants of mothers with <3 x IO5 Strep. m~tu~s per ml saliva at selection were recalled
Prevention
of Srrrp.
tnutum
0
Control group
m m
Test group Low gro”p
infection
in infants
227
AI
f '5s rl
15
23
36
33
15
23
86
33
Months Fig. I. Percentage of infants infected with Strep. wuuus at the age of 15, 23 and 26 months. The figures in the colurlns represent the number of infected infants out of the number of examined infants in the group. The differences between the control group and the test group were statistically significant at *p < 0.02 ald **p < 0.01. (AI) All subjects; (AH) infants in the control group and infants in the test group of mothers with a salivary level of Srrrp. IHU~UIIS below the threshold value (~3 x IO’ c.f.u. per ml); (BI) infants of mothers with a low salivary level of Strep. tnutwn (< 3 x IO’ per ml) at the selection; (BII) infants of mothers with a low number of Srrep. I~IU~L~~IS both at selection and at sampling when their infants were approximately 33 months of age. The salivary level is expressed at I5 months of age as the highest recovery in the mother since the initial preventive measures and expressed at 23 and 36 months of age as the median of all samples collected after the initial sample until the infant was 23 and 36 months old.
for sampling if the!/ had reached the approximate age of 36 months during the winter of 1981-82. Thus. 24 infants, 7 boys and 17 girls with an average age of 33.1 months (range 25-38 months) were examined. The infants were :sampled by the spatula technique described above. A paraffin-stimulated saliva sample was collected from the mothers to estimate their present salivary level of Stuq~. mut~m. Statistical significance was determined using the chi-square test with Yates’ correction. A difference at the level of p < O.Cl2was regarded significant. RESLLTS
On all sampling ages. fewer infants were infected with Strrp. 1777m~7.~ in the test group than in the control group (Fig. I). In both groups. the percentage of
Table 2. Number
infected infants increased with increasing age of the infants. Once Strep. 177utu17.~ was detected in the infant, the organism was usually also present in the subsequent samples and usually in increasing numbers (Table 2). Only in 4 infants (2 in the test group and 2 in the control group), was Srrep. 777utunstransiently present on one sampling occasion. Out of the 40 infants in the control group. a total of 26 infants (65 per cent) became infected during the study. In the test group. on the other hand, 13 out of 37 infants were infected (35 per cent). The difference between the two was statistically groups significant (p = 5.73, p < 0.02). Because the effect of the preventive measures on the salivary level of Srrrp. 17717r~177s in the mothers varied. the number of infected infants at various ages will be
cf infants with various levels of Srrep. 171um.s infection at the ages of 15. 23 and 36 months. The number of Strep. r77ut~177.s c.f.u. is determined with the spatula technique Control group Level of infection with Strep. 177utu7s*
Age
(months)
No. of infected No. of examined
I5 23
9 38 17,‘38
36
17 27
* Mean
number
c.f.u.
(a) 5 2
Strep.
(b)
(c)
(d)
I 4
2 5
3
II 4
Test group Level of infection with Srrrp. r~nrttrr7s* (e) I
n71mr7.srecovered
I 4
No. of infected No. of examined
4/‘40 6,3X 8;21
from a predetermined
(b)+ = l-19; ‘c)+ -- = 20. 49; ‘d’+ + + = 50-99; ‘e’+ + + + = 2100.
(a)
(b)
(c)
I
2 4 I
1 1
(d)
(c)
4
I I 2
area on the MSB agar. ‘“‘f = < I;
?‘Y __<
Birgitta
K6hler.
D. Bratthall
and B. Krasse
Table 3. The effect of preventive measures on Strep. rnutc~ts in the mothers and their I5 months old infants. The number of infected infants is related to the salivary Strep. rn~ctms infection in the mothers Strep. WU~UHS c.f.u. per ml saliva (mothers)* <3 x 105
No. of mothers Control Test
3X 40
No. of infected infants total
23
IO’ < IO”
x
2106
No. of infected infants No. of examined infants
9 (23.7)t 4(10.0)
l,,9(ll.l) l,‘9(l 1.1)
0.!4 (0) 0.:14(O)
* The highest Strep, IHU~UIUrecovery old. t Per cent infected infants.
after the initial preventive
described relative to the salivary level of in their mothers (Tables 336).
Strep.
~~~utum
At this age Srrep. mutum was detected in 9 out of 3X infants in the control group (24 per cent) and in 4 out of 40 infants in the test group (IO per cent) (Table 3). The difference between the groups was not significant (p = 1.73. p > 0.1). The mothers of 8 of the 9 infected infants in the control group harboured 2106 Strep. J~ut~~m per ml saliva. Only I4 mothers in the test group kept their salivary Strep. rmtum level below 3 x 10’ c.f.u. per ml saliva, through the preventive measures up to this time. None of their infants harboured detectable levels of Strep. J?JJJfUfLS: 3 infants were infected of the I7 test mothers who had shown high levels of Strep. JHLJTUJS ( 2 IOh c.f.u. per ml) during this period. The mothers of the 4 infected infants in the test group had all received chlorhexidine treatment.
At this age. I7 out of 3X infants in the control group (45 per cent) and 6 out of 3X infants in the test group (I6 per cent) harboured Strep. I~JUINJJS (Table 4); the difference between the two groups was significant (p = 6.23. p < 0.02). In the control group I2 infants of the 25 mothers with a median value of > lOh were
measures
8 ‘25 (27.5) 3 17(17.6)
until the infants were I5 months
infected (4X per cent) as compared with 3 infants of the 2X test mothers with a median value <3 x IO’ c.f.u. per ml saliva (1 I per cent). The difference between the number of infected infants in this group (3/2X) and the number of infected infants m the whole control group (17/38) was significant (I, = 7.30, p < 0.01). Of the 2X mothers in the test group with a low median value. only 6 mothers had shown salivary levels below the threshold value of 3 x IO’ c.f.u. on all sampling occasions after the initial preventive measures and none of their infants harboured Strep. J~~utun.s. In I5 mothers of the test group. all saliva samples collected after the initial sample exhibited < IOh Strep. JIJII~CJJI.S per ml saliva. None of their infants harboured Strep. rmrtcm. In contrast, I5 mothers in the control group showed > lOh Strep. J~~~JIIS per ml saliva in all samples collected up to this time and 9 of their infants were infected (60 per cent).
Tvventy-seven infants in the control group and 21 infants in the test group (excluding the 3 infants of test mothers who had withdrawn from the preventive programme) reached the age of 36 months. Seventeen infants in the control group (63 per cent) and X infants in the test group (3X per cent) harboured Strep. mrtml.s at this age (Table 5) (p = 2.02. p > 0.1).
Table 4. The effect of preventive measures on Strep. ~m~t~ms in the mothers and their 23-months old infants. The number of infected infants is related to the salivary infection of Strrp. M~LJJJS in the mothers
(Sfwp.
<3
No. of mothers Control Test
3X 3X
x
IO5
No. of infected infants total I7 (45): 6(16)*
I;‘1 (100) 3.128(I I)**
mutms
Median? c.f.u. per ml saliva)
23 x lOi < lOh No. of infected infants No. of examined infants 4/12 (33) 3 ‘9 (33)
2106
12.25 (4X) o:I (0)
* Difference between control group and test group significant at *p < 0.02 and **p < 0.01 (chi-square test with Yates’ correction). t Median of the results of all samples collected from the mother after the initial sample until the infant was 23 months old. : Per cent infected infants.
229
Prevention of Strep. tnnutansinfection in infants
Table 5. The effect of preventive measures on Strep. mutuns in the mothers and their 36 months old infants. The number of infected infants is related to the salivary infection of Strep. mutans in the mothers Median? (Strep. mutans c.f.u. per ml saliva) <3 x 105 No. of infected infants total
No. of mothers Control Test
21 21
17 (63)f 8 (38)
O/O 3/16 (19)*
23 x 105 < lo6 No. of infected infants No. of examined infants 419 (44) 313 (100)
2106
13/18 (72) 212 (100)
* Difference between control group and test group significant at *p < 0.02 (chi-square test with Yates’ correction). t Median of the results of all samples collected from the mother after the initial sample until the time when the infant was 36 months old. : Per cent infecl.ed infants.
Table 5 shows the number of Strep. mutuns-infected infants in relationship to the median value of the Strep. I~U~WSinfections in their mothers. All mothers in the control grcup had a median value 23 x lo5 Strep. IIIUIUMSper ml saliva and 63 per cent of their infants were infected. In the test group, only 16 mothers showed a median value of Strep. mutans ~3 x 10’ c.f.u. per ml and only 3 of their infants were infected (19 per cent). There was a significant difference between this group of infants and the number of infected infants in the control group (p = 6.22. p < 0.02). Twenty-one infants in the control group and 29 infants in the test group who were not infected at the age of 23 months. were sampled between 23 and 36 months of age. At this time, 4 subjects in the test group had elected not to participate further. Nine of the infants in the control group and 7 in the test group became infected during this period. The detection of Strep. muturls in 5 of the infants in the test group coincided uith a high number of Strep. mutun.s in their mothers. Table 6 shows the sampling results of the infants of mothers who had, at the initial selection, few Strep. mutuns and thus had not been included in the original control and test groups. Six out of 24 infants were infected as compared with 17 out of 27 infants in the
Table 6. Number of infected infants of mothers with <3 x lo5 Strep. mufuns per ml saliva at selection. related to the salivary level of their mothers when the infants were sampled at the approximate age of 33 months Strep.
mutans
c.f.u. per ml saliva (mothers)
No. of mothers
No. of infected infant:, total
<3 x 10’ 23 x lo5 < lOh 2106 No. of infected infants No. of examined infants
24
6 (25)*
3116 (19)
* Per cent infec-:ed infants.
215 (40)
l/3 (33)
control group at the age of 36 months (Fig. 1, AI and BI); the difference was significant (p = 5.94, p < 0.02). The percentage of infected infants to mothers with a low level of Strep. mutans on both sampling occasions was exactly the same as that of the infected infants of the test mothers, who had achieved a satisfactory reduction of Strep. mutans (Fig. 1. AI1 and BII).
DISCL’SSION
Our study demonstrates that a reduction of the salivary level of Strep. mutans in highly infected mothers can inhibit or delay the establishment of this microorganism in their infants. The results are consistent with observations in cross-sectional studies which show that infants of mothers with few salivary Strep. mutans are seldom infected (KGhler and Bratthall, 1978; Berkowitz et al., 1980, 1981; van Houte er ul., 1,981). The low prevalence of Strep. mutans infections in such cases is generally ascribed to the few cells available for transfer. There are, however, other explanations. A low number of Strep. mutuns has been associated with a low sucrose intake (Hoover et al., 1980), so mothers with low Strep. mutuns levels may provide their infants with a low sucrose diet, i.e. a diet not conducive to implantation of Strep. mutuns (Krasse, 1965; Krasse et ul., 1967; reviewed by van Houte 1976). This suggestion is supported by our finding that at 23 months only 1 out of 14 infants (7 per cent) became infected when their mothers had achieved a reduction of the Strep. mutans level by adopting the basic preventive programme. On the other hand, 5 out of 24 infants (21 per cent) of mothers, in whom this programme proved insufficient and had to be supplemented with chlorhexidine were infected. The fewness of cells available for transfer in the mother does not preclude the risk of Strep. rnutarls infection. In our study, as in others, infected infants were found whose mothers showed low levels of Strep. mutuns. It is conceivable that the father or someone else in close contact with the infant could also have served as a source of infection. Most of the fathers had their saliva sampled once or twice during the course of the study (data not included). The 3 infants
Birgitta
230
who
were
infected
at
23
months,
KGhler, D. Bratthall
although
the
showed a low level, all had fathers with a salivary level exceeding 3 x lo5 c.Eu.; 2 of them harboured > lo6 c.f.u. Strep. mutans per ml. However, 10 non-infected infants of mothers with a low median value, also had fathers who harboured > 10” c.f.u. Another possibility is that the selected threshold value of 3 x IO5 was not always low enough to eliminate the risk of spreading the infection. The Strep. mutans strain harboured by these mothers could be highly communicable. Furthermore, in some mother and child pairs, salivary factors could favour the initial adherence (Kiihler, Krasse and Carl&, 1981) whereas in other combinations they might inhibit the initial adherence (Kiihler et ul., 198 1; Gahnberg rt al., 1982). This could then explain why some infants of highly infected mothers never became infected. Serotype c predominated in both the mothers and their infants. When colonies with atypical morphologies were found, however, a striking resemblance in colonies from mother and her infant was observed. Also when several serotypes were found in the infants, the same serotypes occurred in their mothers; Dr A. H. Rogers, Adelaide, Australia, has kindly carried out bacteriocin typing on strains from such motherinfant pairs. He found two bacteriocin types in common; one serotype c and one serotype d/g. These observations further support the concept of maternal transfer. One important question is whether some samples are false negatives, i.e. Strep. mutans colonizes the teeth but is not detectable. The spatula sampling technique has a certain margin of error (KBhler and Bratthall, 1979), especially at low numbers of Strep. mutans in saliva. In the present study, however, plaque samples were also obtained when the infants were 19 or 23 and 36 months old. When Strep. mutans was detected with one method it was, with a few exceptions, also detected with the other. Furthermore, once Strep. mutans had been detected, it was usually present in the subsequent samples and usually in increasing numbers. The total number of infected infants also increased with increasing age. Between 23 and 36 months, a large number of infants in both groups became infected. The explanation for this might be increased exposure to new sources of infection and changes in oral conditions such as an increased number of retentive sites and changes in dietary habits, e.g. increased sucrose consumption. Changes in dietary habits and an increased number of retentive sites ought to favour colonization and growth of attached Strep. mutuns, resulting in a detectable number of organisms in the sample. A difference between the groups remained, however; more children became infected with Strep. mutans in the control group than in the test group. During this period, several mothers in the experimental group were pregnant. This occurred in the mothers of 5 out of 7 infants who became infected. In 4 of these mothers, a high number of Strep. mutnns was also observed in saliva. Of the original 41 mothers in the test group, 4 broke the preventive programme and the infants of 3 of them later became infected with Strep. mutans. In addition, during the study, the salivary level of Strep. mutuns in several mothers could not be effectively mothers
and B. Krasse
controlled due to pregnancy, illness and social problems. This resulted in poor cooperation, often with concomitant Strep. muruns infection in their infants. However, when the preventive measures gave a considerable reduction of Strep. mut~ms in the mothers for a prolonged period of time, their infants rarely became infected. These facts illustrate both the difficulties and the value of the preventive approach. The value is further emphasized by the development of dental caries in all infants who at the age of 36 months had > lo6 Strep. muruns per ml saliva. The increased risk of infection of the infant with Strep. mutuns when the mother carries this microorganism in high numbers implies that caries-preventive measures ought to be concentrated on mothers with high counts of Strep. mutuns. In our study, only the mother was subject to the preventive measures but the programme ought to be extended to the whole family. The approach could also be applied to other social groupings. Moreover. the results suggest, in accordance with observations from other infectious diseases, that reduction or elimination of a highly cariogenic microflora in the population could be an important approach to the prevention of caries. Acknowlrllyer,lrrlfs~Thanks are due to Drs Ingrid Andrken, Elna Hultqvist and Berit Jonsson and their dental nurses Anita Aldebert, Lilian Axelsson. Ann Gustavsson, Ann-Charlotte Karlsson and Katarina Skoog for assistance with the clinical work. The skilful technical assistance of Mrs Ann-Britt Lundberg. MS Ann-Charlotte BGrjesson, Ingela Carlsson and Ann-Christine Reinhold is gratefully acknowledged. This study was supported by the Swedish Medical Research Council (Project No. 4548). REFERENCES Berkowitz R. J.. Jordan H. V. and White G. 1975. The early establishment of Srruptoc’cjcc~~.~ wu~m1.s in the mouths of infants. Arch., owl Biol. 20, 171& 174. Berkowitz R. J. and Jordan H. V. 1975. Similarity of bacteriocins of Streptococcus luuturl.\ from mother and infant. Arc/Is ortrl Biol. 20, 725 730. Berkowitz R. J.. Turner J. and Green P. 1980. Primary oral infection of Infants with Streptoc’oc’cl,\ tw~w~. Arc,h orrrl Biol. 25, 221-224. Berkowitz R. J.. Turner J. and Green P. 1981. Maternal levels of Strrptococ,cus nwtom and primary oral infection of infants. Arc/Is orul Biol. 26. 147-149. Bratthall D. 1972. Immunofluoresccnt identification of Strrptococcus w~tcms. Odotlr. Rc2r.y 23. I 16. Carlsson J., Grahnkn H. and Jonsson G. 1975. Lactobacilh and streptococci in the mouth of children. Crrricjs Rrs. 9. 333-339. Catalanotto F. A., Shklair 1. L. and Keene H. J. 1975. Prevalence and localization of Sfrrprococc~rra mutull.s in infants and children. J. AUI. drut. Ass.91. 606-m609. Duchin S. and van Houte J. 197X. Colonization of teeth in humans by Strepfococcus mrttrm as related to its concentration in saliva and host age. Irlfwr. Imr~uo~. 20. 12@125. Emilson C. G. 1981. Effect of chlorhexidine gel treatment on Streptococcus mutws population in human saliva and dental plaque. Stand. /. dwt. Rrs. 89, 239 246. Gahnberg L., Olsson J.. Krasse B. and Carl& A. 1982. Interference of salivary immunoglobulin A antibodies and other salivary fractions with adherence of Sweprococcus mutans to hydroxyapatite. Infect. IIW~~II. 37. 401-406. Gibbons R. J. and van Houte J. 1975. Dental caries. ,.I. Reo. Med. 26. 121&136.
Prevention
of
Strep.tmttm.sinfection in infants
Gold 0. G., Jordan H. V. and van Houte J. 1973. A selective medium for Strrptococcus mutms. Archs ortrl Biol. 18, 1357-1364. Hamada S. and Slade H. D. 1980. Biology, immunology. and cariogenicity of Strrptococcusmutam Microhiol. Rw. 44, 331-384. Hamada S.. Masuda N. and Kotani S. 1980. Isolation and serotyping of Srrrptocomrs mutms from teeth and feces in children. J. c/ill. Microhiol. 11, 314-318. Hoover C. I.. Newbrun E.. Mettraux G. and Graf H. 1980. Microflora and chemical composition of dental plaque from subjects with hereditary fructose intolerance. Iufh. f/~trmr~. 28, 853-1859. Jordan H. V.. En&lander H. R.. Engler W. 0. and Kulczyk D. 1972. Observ.ztions on the implantation and transmission of Strrptwoc’c’us vmtm.~ in humans. J. dent. Rex 51.515~5lX. Keene H. J., Shklair 1. L. and Hoerman K. C. 1976. Partial elimination of Streptococ.c~us mfum from selected tooth surfaces after re:,toration of carious lesions and SnF, prophylaxis. J. Am. derlr. Ass. 93, 328-333. Krasse B. 1965. Tht effect of caries inducing streptococci in hamsters fed die,s with sucrose or glucose. Archs oru/ Biol. IO, 223-226 Krasse B.. Edwardsson S.. Svensson I. and Trell L. 1967. Implantation of caries-inducing streptococci in the human oral cavity. Arch oral Biol. 12, 331-236. Kohler B. and Br;.tthall D. 1978. Intrafamiliar levels of STrrp. rmmws ansd some aspects of the bacterial transmission. SU&. J. drrlr. Res. 86, 35-42. Kahler B. and Bratthall D. 1979. Practical method to facilitate estimat on of Srreptococws mutom levels in saliva, J. c,/itl. Microhiol. 9. 5X4-588. Klihler B.. Krasse B. and Carl& A. 1981. Adherence and Strepfouxcus muIms infection: III vitro study with saliva from noninfectecj and infected pre-school children. I@cr. Irnrmr,~. 34. 633. 636. Kiihler B.. Andrier I., Jonsson B. and Hultqvist E. 1982. Effect of caries preventive measures on Strrptocowus mutms and lactobacilli in selected mothers. Sctrt~1. J. drrit.
Res, 90,
Maltz M.. Zickert
102~10%
I and Krasse
B. 1981. Effect of intensive
231
chlorhexidine on number of Streptoin saliva. Scund. J. dwt. Rex 89, 445~-449. Masuda N., Shimamoto T., Sobue S. and Hamada S. 1980. Transmission of Streptococcus tnutum in some selected families. J. dwr. Rrs. 59. 463 (Abstr. 784). MKller A. J. R. 1966. Microbiological examination of root canals and periapical tissues of human teeth. Thesis. Odorlt. Tidskr. 74, Special issue. Stiles H. M.. Meyers R., Brunelle J. A. and Wittig A. B. 1976. Occurrence of Strrptococcrrs mutam and StruptoW~CLI.S .smyuis m the oral cavity and feces of young children. In: Proc. Muohitrl Aspects of’ Lkwttrl Caries (Edited by Stiles H. M.. Loesche W. J. and O’Brien T. C.) Information Retrieval Inc.. Washington D.C.. Special Suppl. Microbial. Abstr. 1, 187-199. Svanberg M. and Loesche W. J. 1977. The salivary concentration of Streptococci rnutms and Streprococci saryuis and their colonization of artificial tooth fissures in man. Arc/n owl Biol. 22, 441-447. Svanberg M. and Loesche W. J. 1978. Implantation of .Swptoc.oc~cu.s mutms on tooth surfaces in man. Arch.7 orcrl Biol. 23, 55 l-556. Sved S. A. and Loesche W. J. 1972. Survival of human -dental plaque flora m various transport media. Appl. treatment coccus
with
mu~am
Microhiol.
24, 638-644.
van Houte J. and Green D. B. 1974. Relationship between concentration of bacteria in saliva and the colonization of teeth in humans. I,!frct. Inlrtlu~. 9. 624-630. van Houte J. 1976. Oral bacterial colonization: mechanisms and implications. In: Proc. :lfic~rohitr/ Asp~cfs qf Drnttrl Curia (Edited by Stiles H. M.. Loesche W. J. and O’Brien T. C.) Information Retrieval Inc., Washington D. C., Special Suppl. Microbial. Abstr. 1, 3-32. van Houtc J. 1980. Bacterial specificity in the etiology of dental caries. Iur. drrlt. J. 30, 305-326. van Houte J., Yanover L. and Brecher S. 1981. Relationship of levels of the bacterium Strrp~ococcus mutms in saliva of children and their parents. Awhs ortrl BIO/. 26, 38 l-386. Westergren G. and Krasse B. 1978. Evaluation of a micromethod for determination of Streptococcus rmrtcws and lactobacillus infection. J. clir~. Microhiol. 7, 82 -83.
OUII
Bid. Vol 1x,
PrInted it1C;re:ltBritam
vo. 3. pp.
0003-9961) 83 030225-07iK3.00,0
225 131. IYX.:
CopyrIght
All rlghrs reserved
PREVENTIVE
MEASURES
IN MOTHERS
ESTABLISHMENT STREPTOCOCCUS
OF
THE
MUTANS
c
I%?
INFLUENCE
Pergamon
Press Ltd
THE
BACTERIUM
IN THEIR
INFANTS
BIRC~ITTAKijHLER. D. BRATTHALLand B. KRAN Department
of Cariology.
Faculty
of Odontology.
University
of GGteborg.
S-400 33 GBteborg.
Sweden
Summary~First-time mothers who had a high salivary number of Strep. ~7t~f~f7.~ [ 2 1Oh colonyforming-unils (c.f.u.) per ml] were selected. Every second mother was given a special preventive programme to reduce her salivary level below 3 x IO5 c.f.u. per ml. Where a reduction of Strep. 177ufu17swas achieved in the mother. the establishment of Strep. 117ut~17sin her infant was prevented or delayed. Thus. 28 mothers were successfully treated until their infants were 23 months old ,.md only 3 of their infants (I I per cent) were infected with Strep. 117ut~t7.s, compared with I7 out of 38 infants in the control group (45 per cent). In both groups, the percentage of infected infants increased with increasing age, although at all ages fewer infants were infected with Strep. ,77utu77s in the test group than in the control group. Sixteen infants of successfully treated mothers had reached the age of 36 months. Three were infected (19 per cent) compared with I7 out of 27 in the control group (63 per cent). These findings show that the spread of Strep. rutrtar1.s can be delayed or prevented by measures directed against the main source of infection. an approach which is successful in the prevention of other infectious diseases.
INTRODlJCTIOiY
Strrptoumus mutuc.s is an important pathogen in the development of dental caries (reviewed by Gibbons and van Houte. 1975; Hamada and Slade, 1980: van Houte, 1980). This microorganism is either not detected at all or is only transiently present in the mouths of infants before emergence of teeth (Berkowitz, Jordan and W:lite, 1975; Carlsson. Grahnkn and Jonsson. 1975; Catalanotta, Shklair and Keene. 1975; Stiles et ~11.. 1976; Berkowitz. Turner and Green, 1980). The mother seems to be the major source of infection (Berkowitz and Jordan, 1975; Berkowitz et (I/.. 1975; Hamada. Masuda and Kotani. 1980; Masuda pf ul.. 19801. Colonization of Strep. 777ut~777~in man is favoured by a large inoculum (van Houte and Green 1974; Duchin and van Houte, 197X: Svanberg and Locsche. 1977. 1978) and repeated inoculation (Krasse et d.. 1967. Jordan et ctl.. 1972). These factors might explain why the mother is a probable reservoir for the primary infection of her infant by Srrcp. 117ut~117s. Earlier, it was observed that mothers highly infected with Strep, n71m777~ tended to have children who also were highly infected. whereas mothers with low salivary concentrations tended to have noninfected children (Kb;hler and Bratthall. 1978). This observation was confirmed in later studies (Berkowitz et rrl., 1980, 1981; can Houte. Yanover and Brecher, 1981). It therefore seemed of interest to determine whether reduction of the number of Strep. 177utu77.s in highly infected mothers could prevent the initial oral establishment of these microorganisms in their infants.
MATERIALS
AKD METHODS
All mothers attending the child health centre in Oskarshamn. Swec.en, with their first-born babies (3-8 months old) were sampled for salivary concen-
trations of Srrrp. 177uru/7s and lactobacilli. Sampling was conducted by dentists and specially trained dental nurses employed at the public dental office in Oskarshamn. Over a 2; year period. 249 mothers were sampled. Mothers harbouring 10b or more Strep. 7777rrrtr7.s colony-forming-units (c.f.u.) per ml saliva and their infants were selected. These 87 mothers were assigned alternately to experimental and control groups (Kiihler et al.. 1982). Due to various circumstances, such as moving from the district (2 experimental and 2 control mothers) and refusal to particisubjects), 6 subjects were expate (2 experimental cluded. The mean. median and range of salivary Strep. mttms and lactobacilli in the initial sample of the control group and the experimental group are given in Table I. In the control group. 23 (57.5 per cent) of the infants were males; in the experimental group. 26 (63.4 per cent) were males.
In a letter to the mothers in the experimental group. the purpose and design of the study were explained. To reduce the salivary concentration of Strep. r77utan.s below the pre-selected threshold value of 3 x IO5 c.f.u. per ml saliva. the experimental mothers were given a basic preventive programme. This programme consisted of: (1) information about the purpose of the study, (2) dietary counselling, (3) professional tooth-cleaning and oral hygiene instructions, (4) fluoride treatment. (5) excavation of large cavities. The programme was carried out in the public dental clinic in Oskarshamn by three dentists in cooperation with specially-trained preventive nurses. The effect of the treatment was controlled by microbiological analysis. Mothers harbouring t3 x 10’ c.f.u. per ml saliva after the initial treatment were recalled after 4 months for a new saliva sample. Subjects having 23 x 10’ Strep. 777utan~ per ml saliva after the basic preventive programme were prescribed chlorhexidine-
226
Birgitta KShler. D. Bratthall and B. Krasse
Table 1. Salivary numbers
of Strrp.
mtms
and lactobacilli in control the study
and experimental
c.f.u. Strep. mi4tutls per ml saliva x lOh
t1* Control Experimental
40 41
Mean
Median
3.9 2.1:
1.9 1.5:
group at the start of
c.f.u. lactobacilli per ml saliva x IO” Range
t1
Mean
Median
Range
0.9-26.0 0%9.4$
39t 41
364 291
62 46
&3600 @I920
* Number of subjects. t One sample lost. $ Two of the experimental mothers with very high salivary Strep. lower mean, median and range values in this &ou,.
containing gel in individually designed applicators according to the method of Emilson (198 I ). The gel, which contained 1.0 per cent chlorhexidine digluconate (Hibitane. ICI Ltd. England). was used once a day. for 5 min. for 2 weeks. One week after the termination of the chlorhexidine treatment, saliva samples were collected to determine the effect of the gel application on the number of Strep. mutans. The mothers treated with chlorhexidine were recalled after 2 3 months to check the salivary level of Strep. mutans. Details of the preventive programme and the short-term effect after the first treatment period on Strep. mutans and lactobacilli have been described by Kiihler et (11.(1982). In mothers having 23 x IO’ Strep. ~nutm.s per ml saliva at the subsequent appointments. the basic preventive programme a&or the chlorhexidine treatment was repeated. In some cases. short-term chlorhexidine treatment (Maltr. Zickert and Krasse, 1981) was used as an alternative to the chlorhexidine treatment. Mothers who were pregnant during the course of the study did not use chlorhexidine during their pregnancies. instead. topical application with 8 per cent stannous-lluoridc was sometimes used (Keene. Shklair and Hoerman, 1976). The effect of each of the treatments used was again controlled by microbiological analysis. The preventive measures were carried out throughout the study unless the infant acquired detectable levels of Strep. mutuns on successive samplings, if the mother did not comply. or if we were unsuccessful in keeping her salivary level low (< 3 x 10’ c.f.u. per ml). These mothers. however. agreed to be recalled with their infants every 4 months for sampling. The infants in the experimental and control groups were sampled for the presence of Stwp. tmftun.7 at 4-month intervals starting at the age of I5 months. The mothers were also sampled at this time. Due to circumstances such as illness, moving from town and other problems, subjects occasionally failed to leave samples as planned. In addition, 4 mothers in the test group elected during the course of the study not to participate further in the preventive programme, although their infants were not infected at that time. The number of subjects examined is indicated in each table. At the age of 36 months, the examination of the child was extended to include collection of paraffinstimulated saliva and of 21 pooled plaque sample.
mutuns
levels
were lost, which resulted
in
Mothrrs. One millilitre of parafin-stimulated saliva was collected. transferred to 5.7 ml VMGII transport medium (Miiller. 1966) and mailed to the Department of Cariology of the Dental School in Giiteborg. On arrival. usually the following day. the samples were cultured on mitis salivarius bacitracin (MSB) agar selective for Strep. ttxums (Gold, Jordan and van Houte. 1973). using the micropipette method described by Westergren and Krasse (1978). The number of Strep. muru/ls c.f.u. per ml saliva was estimated. Childrrtl. The presence of Strrp. mututts in saliva was determined by a sampling technique reported by Kiihler and Bratthall (1979). A wlooden spatula was rotated in the mouth of the infant to wet it with saliva. Each side of the spatula was then pressed directly against an agar plate containing MSB agar medium. The plates were incubated in exhaled air at 37 C for 2 days. The incubation was done at the clinic in Oskarshamn and the plates were then mailed to Giiteborg for identification and enumeration. The mean number of Strep. tmltcmS colonies recovered on a predetermined area for the two sides pressed against the MSB-agar was calculated. The infants are described as infected when Strep. rmrtutn was detected by the spatula method and was also present in any of the subsequent samples. On a few occasions, the spatula sampling was not performed due to unwillingness of the child to cooperate. The result of a plaque sample was then used instead. Colonies with both typical and atypical morphology were isolated from all mothers and their infants for identification and serotyping by immunofluorescent identification (Bratthall. 1972). At 36months examination. paraffin-stimulated saliva was also collected and transferred to I ml of reduced transport fluid (RTF) (Syed and Loesche, 1972) and mailed to our laboratory. According to the child’s ability to cooperate. 0.05%1.0 ml saliva was collected. 0.025 ml were taken both directly and from dilutions IO- ’ and 10~’ and cultured on MSB agar. The number of Strep. muturn c.f.u. per ml saliva was estimated by the micropipette method described by Westcrgren and Krasse (I 978). To estimate the frequency of Srrrp. rmrrctm infection in infants of mothers with a low sahvary level ot Strep. mutons, all infants of mothers with <3 x IO5 Strep. m~tu~s per ml saliva at selection were recalled
Prevention
of Srrrp.
tnutum
0
Control group
m m
Test group Low gro”p
infection
in infants
227
AI
f '5s rl
15
23
36
33
15
23
86
33
Months Fig. I. Percentage of infants infected with Strep. wuuus at the age of 15, 23 and 26 months. The figures in the colurlns represent the number of infected infants out of the number of examined infants in the group. The differences between the control group and the test group were statistically significant at *p < 0.02 ald **p < 0.01. (AI) All subjects; (AH) infants in the control group and infants in the test group of mothers with a salivary level of Srrrp. IHU~UIIS below the threshold value (~3 x IO’ c.f.u. per ml); (BI) infants of mothers with a low salivary level of Strep. tnutwn (< 3 x IO’ per ml) at the selection; (BII) infants of mothers with a low number of Srrep. I~IU~L~~IS both at selection and at sampling when their infants were approximately 33 months of age. The salivary level is expressed at I5 months of age as the highest recovery in the mother since the initial preventive measures and expressed at 23 and 36 months of age as the median of all samples collected after the initial sample until the infant was 23 and 36 months old.
for sampling if the!/ had reached the approximate age of 36 months during the winter of 1981-82. Thus. 24 infants, 7 boys and 17 girls with an average age of 33.1 months (range 25-38 months) were examined. The infants were :sampled by the spatula technique described above. A paraffin-stimulated saliva sample was collected from the mothers to estimate their present salivary level of Stuq~. mut~m. Statistical significance was determined using the chi-square test with Yates’ correction. A difference at the level of p < O.Cl2was regarded significant. RESLLTS
On all sampling ages. fewer infants were infected with Strrp. 1777m~7.~ in the test group than in the control group (Fig. I). In both groups. the percentage of
Table 2. Number
infected infants increased with increasing age of the infants. Once Strep. 177utu17.~ was detected in the infant, the organism was usually also present in the subsequent samples and usually in increasing numbers (Table 2). Only in 4 infants (2 in the test group and 2 in the control group), was Srrep. 777utunstransiently present on one sampling occasion. Out of the 40 infants in the control group. a total of 26 infants (65 per cent) became infected during the study. In the test group. on the other hand, 13 out of 37 infants were infected (35 per cent). The difference between the two was statistically groups significant (p = 5.73, p < 0.02). Because the effect of the preventive measures on the salivary level of Srrrp. 17717r~177s in the mothers varied. the number of infected infants at various ages will be
cf infants with various levels of Srrep. 171um.s infection at the ages of 15. 23 and 36 months. The number of Strep. r77ut~177.s c.f.u. is determined with the spatula technique Control group Level of infection with Strep. 177utu7s*
Age
(months)
No. of infected No. of examined
I5 23
9 38 17,‘38
36
17 27
* Mean
number
c.f.u.
(a) 5 2
Strep.
(b)
(c)
(d)
I 4
2 5
3
II 4
Test group Level of infection with Srrrp. r~nrttrr7s* (e) I
n71mr7.srecovered
I 4
No. of infected No. of examined
4/‘40 6,3X 8;21
from a predetermined
(b)+ = l-19; ‘c)+ -- = 20. 49; ‘d’+ + + = 50-99; ‘e’+ + + + = 2100.
(a)
(b)
(c)
I
2 4 I
1 1
(d)
(c)
4
I I 2
area on the MSB agar. ‘“‘f = < I;
?‘Y __<
Birgitta
K6hler.
D. Bratthall
and B. Krasse
Table 3. The effect of preventive measures on Strep. rnutc~ts in the mothers and their I5 months old infants. The number of infected infants is related to the salivary Strep. rn~ctms infection in the mothers Strep. WU~UHS c.f.u. per ml saliva (mothers)* <3 x 105
No. of mothers Control Test
3X 40
No. of infected infants total
23
IO’ < IO”
x
2106
No. of infected infants No. of examined infants
9 (23.7)t 4(10.0)
l,,9(ll.l) l,‘9(l 1.1)
0.!4 (0) 0.:14(O)
* The highest Strep, IHU~UIUrecovery old. t Per cent infected infants.
after the initial preventive
described relative to the salivary level of in their mothers (Tables 336).
Strep.
~~~utum
At this age Srrep. mutum was detected in 9 out of 3X infants in the control group (24 per cent) and in 4 out of 40 infants in the test group (IO per cent) (Table 3). The difference between the groups was not significant (p = 1.73. p > 0.1). The mothers of 8 of the 9 infected infants in the control group harboured 2106 Strep. J~ut~~m per ml saliva. Only I4 mothers in the test group kept their salivary Strep. rmtum level below 3 x 10’ c.f.u. per ml saliva, through the preventive measures up to this time. None of their infants harboured detectable levels of Strep. J?JJJfUfLS: 3 infants were infected of the I7 test mothers who had shown high levels of Strep. JHLJTUJS ( 2 IOh c.f.u. per ml) during this period. The mothers of the 4 infected infants in the test group had all received chlorhexidine treatment.
At this age. I7 out of 3X infants in the control group (45 per cent) and 6 out of 3X infants in the test group (I6 per cent) harboured Strep. I~JUINJJS (Table 4); the difference between the two groups was significant (p = 6.23. p < 0.02). In the control group I2 infants of the 25 mothers with a median value of > lOh were
measures
8 ‘25 (27.5) 3 17(17.6)
until the infants were I5 months
infected (4X per cent) as compared with 3 infants of the 2X test mothers with a median value <3 x IO’ c.f.u. per ml saliva (1 I per cent). The difference between the number of infected infants in this group (3/2X) and the number of infected infants m the whole control group (17/38) was significant (I, = 7.30, p < 0.01). Of the 2X mothers in the test group with a low median value. only 6 mothers had shown salivary levels below the threshold value of 3 x IO’ c.f.u. on all sampling occasions after the initial preventive measures and none of their infants harboured Strep. J~~utun.s. In I5 mothers of the test group. all saliva samples collected after the initial sample exhibited < IOh Strep. JIJII~CJJI.S per ml saliva. None of their infants harboured Strep. rmrtcm. In contrast, I5 mothers in the control group showed > lOh Strep. J~~~JIIS per ml saliva in all samples collected up to this time and 9 of their infants were infected (60 per cent).
Tvventy-seven infants in the control group and 21 infants in the test group (excluding the 3 infants of test mothers who had withdrawn from the preventive programme) reached the age of 36 months. Seventeen infants in the control group (63 per cent) and X infants in the test group (3X per cent) harboured Strep. mrtml.s at this age (Table 5) (p = 2.02. p > 0.1).
Table 4. The effect of preventive measures on Strep. ~m~t~ms in the mothers and their 23-months old infants. The number of infected infants is related to the salivary infection of Strrp. M~LJJJS in the mothers
(Sfwp.
<3
No. of mothers Control Test
3X 3X
x
IO5
No. of infected infants total I7 (45): 6(16)*
I;‘1 (100) 3.128(I I)**
mutms
Median? c.f.u. per ml saliva)
23 x lOi < lOh No. of infected infants No. of examined infants 4/12 (33) 3 ‘9 (33)
2106
12.25 (4X) o:I (0)
* Difference between control group and test group significant at *p < 0.02 and **p < 0.01 (chi-square test with Yates’ correction). t Median of the results of all samples collected from the mother after the initial sample until the infant was 23 months old. : Per cent infected infants.
229
Prevention of Strep. tnnutansinfection in infants
Table 5. The effect of preventive measures on Strep. mutuns in the mothers and their 36 months old infants. The number of infected infants is related to the salivary infection of Strep. mutans in the mothers Median? (Strep. mutans c.f.u. per ml saliva) <3 x 105 No. of infected infants total
No. of mothers Control Test
21 21
17 (63)f 8 (38)
O/O 3/16 (19)*
23 x 105 < lo6 No. of infected infants No. of examined infants 419 (44) 313 (100)
2106
13/18 (72) 212 (100)
* Difference between control group and test group significant at *p < 0.02 (chi-square test with Yates’ correction). t Median of the results of all samples collected from the mother after the initial sample until the time when the infant was 36 months old. : Per cent infecl.ed infants.
Table 5 shows the number of Strep. mutuns-infected infants in relationship to the median value of the Strep. I~U~WSinfections in their mothers. All mothers in the control grcup had a median value 23 x lo5 Strep. IIIUIUMSper ml saliva and 63 per cent of their infants were infected. In the test group, only 16 mothers showed a median value of Strep. mutans ~3 x 10’ c.f.u. per ml and only 3 of their infants were infected (19 per cent). There was a significant difference between this group of infants and the number of infected infants in the control group (p = 6.22. p < 0.02). Twenty-one infants in the control group and 29 infants in the test group who were not infected at the age of 23 months. were sampled between 23 and 36 months of age. At this time, 4 subjects in the test group had elected not to participate further. Nine of the infants in the control group and 7 in the test group became infected during this period. The detection of Strep. muturls in 5 of the infants in the test group coincided uith a high number of Strep. mutun.s in their mothers. Table 6 shows the sampling results of the infants of mothers who had, at the initial selection, few Strep. mutuns and thus had not been included in the original control and test groups. Six out of 24 infants were infected as compared with 17 out of 27 infants in the
Table 6. Number of infected infants of mothers with <3 x lo5 Strep. mufuns per ml saliva at selection. related to the salivary level of their mothers when the infants were sampled at the approximate age of 33 months Strep.
mutans
c.f.u. per ml saliva (mothers)
No. of mothers
No. of infected infant:, total
<3 x 10’ 23 x lo5 < lOh 2106 No. of infected infants No. of examined infants
24
6 (25)*
3116 (19)
* Per cent infec-:ed infants.
215 (40)
l/3 (33)
control group at the age of 36 months (Fig. 1, AI and BI); the difference was significant (p = 5.94, p < 0.02). The percentage of infected infants to mothers with a low level of Strep. mutans on both sampling occasions was exactly the same as that of the infected infants of the test mothers, who had achieved a satisfactory reduction of Strep. mutans (Fig. 1. AI1 and BII).
DISCL’SSION
Our study demonstrates that a reduction of the salivary level of Strep. mutans in highly infected mothers can inhibit or delay the establishment of this microorganism in their infants. The results are consistent with observations in cross-sectional studies which show that infants of mothers with few salivary Strep. mutans are seldom infected (KGhler and Bratthall, 1978; Berkowitz et al., 1980, 1981; van Houte er ul., 1,981). The low prevalence of Strep. mutans infections in such cases is generally ascribed to the few cells available for transfer. There are, however, other explanations. A low number of Strep. mutuns has been associated with a low sucrose intake (Hoover et al., 1980), so mothers with low Strep. mutuns levels may provide their infants with a low sucrose diet, i.e. a diet not conducive to implantation of Strep. mutuns (Krasse, 1965; Krasse et ul., 1967; reviewed by van Houte 1976). This suggestion is supported by our finding that at 23 months only 1 out of 14 infants (7 per cent) became infected when their mothers had achieved a reduction of the Strep. mutans level by adopting the basic preventive programme. On the other hand, 5 out of 24 infants (21 per cent) of mothers, in whom this programme proved insufficient and had to be supplemented with chlorhexidine were infected. The fewness of cells available for transfer in the mother does not preclude the risk of Strep. rnutarls infection. In our study, as in others, infected infants were found whose mothers showed low levels of Strep. mutuns. It is conceivable that the father or someone else in close contact with the infant could also have served as a source of infection. Most of the fathers had their saliva sampled once or twice during the course of the study (data not included). The 3 infants
Birgitta
230
who
were
infected
at
23
months,
KGhler, D. Bratthall
although
the
showed a low level, all had fathers with a salivary level exceeding 3 x lo5 c.Eu.; 2 of them harboured > lo6 c.f.u. Strep. mutans per ml. However, 10 non-infected infants of mothers with a low median value, also had fathers who harboured > 10” c.f.u. Another possibility is that the selected threshold value of 3 x IO5 was not always low enough to eliminate the risk of spreading the infection. The Strep. mutans strain harboured by these mothers could be highly communicable. Furthermore, in some mother and child pairs, salivary factors could favour the initial adherence (Kiihler, Krasse and Carl&, 1981) whereas in other combinations they might inhibit the initial adherence (Kiihler et ul., 198 1; Gahnberg rt al., 1982). This could then explain why some infants of highly infected mothers never became infected. Serotype c predominated in both the mothers and their infants. When colonies with atypical morphologies were found, however, a striking resemblance in colonies from mother and her infant was observed. Also when several serotypes were found in the infants, the same serotypes occurred in their mothers; Dr A. H. Rogers, Adelaide, Australia, has kindly carried out bacteriocin typing on strains from such motherinfant pairs. He found two bacteriocin types in common; one serotype c and one serotype d/g. These observations further support the concept of maternal transfer. One important question is whether some samples are false negatives, i.e. Strep. mutans colonizes the teeth but is not detectable. The spatula sampling technique has a certain margin of error (KBhler and Bratthall, 1979), especially at low numbers of Strep. mutans in saliva. In the present study, however, plaque samples were also obtained when the infants were 19 or 23 and 36 months old. When Strep. mutans was detected with one method it was, with a few exceptions, also detected with the other. Furthermore, once Strep. mutans had been detected, it was usually present in the subsequent samples and usually in increasing numbers. The total number of infected infants also increased with increasing age. Between 23 and 36 months, a large number of infants in both groups became infected. The explanation for this might be increased exposure to new sources of infection and changes in oral conditions such as an increased number of retentive sites and changes in dietary habits, e.g. increased sucrose consumption. Changes in dietary habits and an increased number of retentive sites ought to favour colonization and growth of attached Strep. mutuns, resulting in a detectable number of organisms in the sample. A difference between the groups remained, however; more children became infected with Strep. mutans in the control group than in the test group. During this period, several mothers in the experimental group were pregnant. This occurred in the mothers of 5 out of 7 infants who became infected. In 4 of these mothers, a high number of Strep. mutnns was also observed in saliva. Of the original 41 mothers in the test group, 4 broke the preventive programme and the infants of 3 of them later became infected with Strep. mutans. In addition, during the study, the salivary level of Strep. mutuns in several mothers could not be effectively mothers
and B. Krasse
controlled due to pregnancy, illness and social problems. This resulted in poor cooperation, often with concomitant Strep. muruns infection in their infants. However, when the preventive measures gave a considerable reduction of Strep. mut~ms in the mothers for a prolonged period of time, their infants rarely became infected. These facts illustrate both the difficulties and the value of the preventive approach. The value is further emphasized by the development of dental caries in all infants who at the age of 36 months had > lo6 Strep. muruns per ml saliva. The increased risk of infection of the infant with Strep. mutuns when the mother carries this microorganism in high numbers implies that caries-preventive measures ought to be concentrated on mothers with high counts of Strep. mutuns. In our study, only the mother was subject to the preventive measures but the programme ought to be extended to the whole family. The approach could also be applied to other social groupings. Moreover. the results suggest, in accordance with observations from other infectious diseases, that reduction or elimination of a highly cariogenic microflora in the population could be an important approach to the prevention of caries. Acknowlrllyer,lrrlfs~Thanks are due to Drs Ingrid Andrken, Elna Hultqvist and Berit Jonsson and their dental nurses Anita Aldebert, Lilian Axelsson. Ann Gustavsson, Ann-Charlotte Karlsson and Katarina Skoog for assistance with the clinical work. The skilful technical assistance of Mrs Ann-Britt Lundberg. MS Ann-Charlotte BGrjesson, Ingela Carlsson and Ann-Christine Reinhold is gratefully acknowledged. This study was supported by the Swedish Medical Research Council (Project No. 4548). REFERENCES Berkowitz R. J.. Jordan H. V. and White G. 1975. The early establishment of Srruptoc’cjcc~~.~ wu~m1.s in the mouths of infants. Arch., owl Biol. 20, 171& 174. Berkowitz R. J. and Jordan H. V. 1975. Similarity of bacteriocins of Streptococcus luuturl.\ from mother and infant. Arc/Is ortrl Biol. 20, 725 730. Berkowitz R. J.. Turner J. and Green P. 1980. Primary oral infection of Infants with Streptoc’oc’cl,\ tw~w~. Arc,h orrrl Biol. 25, 221-224. Berkowitz R. J.. Turner J. and Green P. 1981. Maternal levels of Strrptococ,cus nwtom and primary oral infection of infants. Arc/Is orul Biol. 26. 147-149. Bratthall D. 1972. Immunofluoresccnt identification of Strrptococcus w~tcms. Odotlr. Rc2r.y 23. I 16. Carlsson J., Grahnkn H. and Jonsson G. 1975. Lactobacilh and streptococci in the mouth of children. Crrricjs Rrs. 9. 333-339. Catalanotto F. A., Shklair 1. L. and Keene H. J. 1975. Prevalence and localization of Sfrrprococc~rra mutull.s in infants and children. J. AUI. drut. Ass.91. 606-m609. Duchin S. and van Houte J. 197X. Colonization of teeth in humans by Strepfococcus mrttrm as related to its concentration in saliva and host age. Irlfwr. Imr~uo~. 20. 12@125. Emilson C. G. 1981. Effect of chlorhexidine gel treatment on Streptococcus mutws population in human saliva and dental plaque. Stand. /. dwt. Rrs. 89, 239 246. Gahnberg L., Olsson J.. Krasse B. and Carl& A. 1982. Interference of salivary immunoglobulin A antibodies and other salivary fractions with adherence of Sweprococcus mutans to hydroxyapatite. Infect. IIW~~II. 37. 401-406. Gibbons R. J. and van Houte J. 1975. Dental caries. ,.I. Reo. Med. 26. 121&136.
Prevention
of
Strep.tmttm.sinfection in infants
Gold 0. G., Jordan H. V. and van Houte J. 1973. A selective medium for Strrptococcus mutms. Archs ortrl Biol. 18, 1357-1364. Hamada S. and Slade H. D. 1980. Biology, immunology. and cariogenicity of Strrptococcusmutam Microhiol. Rw. 44, 331-384. Hamada S.. Masuda N. and Kotani S. 1980. Isolation and serotyping of Srrrptocomrs mutms from teeth and feces in children. J. c/ill. Microhiol. 11, 314-318. Hoover C. I.. Newbrun E.. Mettraux G. and Graf H. 1980. Microflora and chemical composition of dental plaque from subjects with hereditary fructose intolerance. Iufh. f/~trmr~. 28, 853-1859. Jordan H. V.. En&lander H. R.. Engler W. 0. and Kulczyk D. 1972. Observ.ztions on the implantation and transmission of Strrptwoc’c’us vmtm.~ in humans. J. dent. Rex 51.515~5lX. Keene H. J., Shklair 1. L. and Hoerman K. C. 1976. Partial elimination of Streptococ.c~us mfum from selected tooth surfaces after re:,toration of carious lesions and SnF, prophylaxis. J. Am. derlr. Ass. 93, 328-333. Krasse B. 1965. Tht effect of caries inducing streptococci in hamsters fed die,s with sucrose or glucose. Archs oru/ Biol. IO, 223-226 Krasse B.. Edwardsson S.. Svensson I. and Trell L. 1967. Implantation of caries-inducing streptococci in the human oral cavity. Arch oral Biol. 12, 331-236. Kohler B. and Br;.tthall D. 1978. Intrafamiliar levels of STrrp. rmmws ansd some aspects of the bacterial transmission. SU&. J. drrlr. Res. 86, 35-42. Kahler B. and Bratthall D. 1979. Practical method to facilitate estimat on of Srreptococws mutom levels in saliva, J. c,/itl. Microhiol. 9. 5X4-588. Klihler B.. Krasse B. and Carl& A. 1981. Adherence and Strepfouxcus muIms infection: III vitro study with saliva from noninfectecj and infected pre-school children. I@cr. Irnrmr,~. 34. 633. 636. Kiihler B.. Andrier I., Jonsson B. and Hultqvist E. 1982. Effect of caries preventive measures on Strrptocowus mutms and lactobacilli in selected mothers. Sctrt~1. J. drrit.
Res, 90,
Maltz M.. Zickert
102~10%
I and Krasse
B. 1981. Effect of intensive
231
chlorhexidine on number of Streptoin saliva. Scund. J. dwt. Rex 89, 445~-449. Masuda N., Shimamoto T., Sobue S. and Hamada S. 1980. Transmission of Streptococcus tnutum in some selected families. J. dwr. Rrs. 59. 463 (Abstr. 784). MKller A. J. R. 1966. Microbiological examination of root canals and periapical tissues of human teeth. Thesis. Odorlt. Tidskr. 74, Special issue. Stiles H. M.. Meyers R., Brunelle J. A. and Wittig A. B. 1976. Occurrence of Strrptococcrrs mutam and StruptoW~CLI.S .smyuis m the oral cavity and feces of young children. In: Proc. Muohitrl Aspects of’ Lkwttrl Caries (Edited by Stiles H. M.. Loesche W. J. and O’Brien T. C.) Information Retrieval Inc.. Washington D.C.. Special Suppl. Microbial. Abstr. 1, 187-199. Svanberg M. and Loesche W. J. 1977. The salivary concentration of Streptococci rnutms and Streprococci saryuis and their colonization of artificial tooth fissures in man. Arc/n owl Biol. 22, 441-447. Svanberg M. and Loesche W. J. 1978. Implantation of .Swptoc.oc~cu.s mutms on tooth surfaces in man. Arch.7 orcrl Biol. 23, 55 l-556. Sved S. A. and Loesche W. J. 1972. Survival of human -dental plaque flora m various transport media. Appl. treatment coccus
with
mu~am
Microhiol.
24, 638-644.
van Houte J. and Green D. B. 1974. Relationship between concentration of bacteria in saliva and the colonization of teeth in humans. I,!frct. Inlrtlu~. 9. 624-630. van Houte J. 1976. Oral bacterial colonization: mechanisms and implications. In: Proc. :lfic~rohitr/ Asp~cfs qf Drnttrl Curia (Edited by Stiles H. M.. Loesche W. J. and O’Brien T. C.) Information Retrieval Inc., Washington D. C., Special Suppl. Microbial. Abstr. 1, 3-32. van Houtc J. 1980. Bacterial specificity in the etiology of dental caries. Iur. drrlt. J. 30, 305-326. van Houte J., Yanover L. and Brecher S. 1981. Relationship of levels of the bacterium Strrp~ococcus mutms in saliva of children and their parents. Awhs ortrl BIO/. 26, 38 l-386. Westergren G. and Krasse B. 1978. Evaluation of a micromethod for determination of Streptococcus rmrtcws and lactobacillus infection. J. clir~. Microhiol. 7, 82 -83.