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Mother-to-child transmission of mutans streptococci
Jinthana Lapirattanakul*,1 & Kazuhiko Nakano2
Abstract: Mutans streptococci (MS) are the major group of pathogens implicated in dental caries. Like other infectious diseases, transmission of the causative microorganisms is the initial and essential step that should be understood relative to disease control and prevention. This review summarizes current knowledge regarding MS transmission, especially from mothers to their children. Included are methods used to study transmission, sources of MS, initial acquisition, factors concerning transmission and prevention of transmission. Information accumulated over many decades showed the involvement of MS transmission in the pathogenesis of caries, hence several preventive measurements have been proposed. Nevertheless, some essential aspects remain to be elucidated for more benefits of practical application. Dental caries is one of the most prevalent diseases in the field of dentistry, in which transmission of mutans streptococci (MS), the major bacterial group involved in caries initiation, is one of the essential factors in its onset. MS consist of seven closely related species, among which Streptococcus mutans and Streptococcus sobrinus are the two major species isolated from human oral cavities [1] . Their pathogenic properties related to dental caries include their ability to colonize efficiently on tooth surfaces as well as their acidogenic and aciduric potentials [2] . MS utilize two distinct mechanisms for tooth adherence. The initial step is a sucrose-independent adhesion, which initiates the primary attachment [3] . The following step involves sucrose-dependent adhesion that helps enhance the firm and irreversible tooth adherence involving glucosyltransferases (Gtfs) and glucan-binding proteins (Gbps) [4] . As for the acidogenic and aciduric properties that provide the ecological advantage for MS over other oral bacteria, these characteristics are mainly based on the high efficiency of sugar transport and metabolism as well as the function of ATPase-driven proton pumps [2] . Moreover, most MS strains are also able to synthesize and store intracellular polysaccharides, which serve as reserve nutrients during prolonged starvation, thereby maintaining an acidic pH environment. Based on the cariogenic properties of MS, the acquisition of these bacteria in the oral cavities of infants is one of the important research subjects for paediatric dentistry. This review aims to summarize current knowledge regarding MS transmission, especially from mothers to their children. In addition, future research important for this field are also pointed out.
Keywords
• factors • initial acquisition • mother-to-child transmission • mutans streptococci • prevention • sources • typing methods
Methods used to study transmission Since evidence for transmission was mainly dependent on the detection of strains with identical characteristics, various typing methods were used in the study of MS transmission. The methods based on characteristics expressed by bacteria are called phenotyping, while the techniques relying Department of Oral Microbiology, Faculty of Dentistry, Mahidol University, Bangkok, 10400, Thailand Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, Osaka, 565-0871, Japan *Author for correspondence: Tel.: +66 2200 7804; Fax: +66 2200 7804; [email protected]
1 2
10.2217/FMB.14.37 © 2014 Future Medicine Ltd
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Review Lapirattanakul & Nakano on genetic properties are termed genotyping. For the transmission of MS, both phenotypic and genotypic methods were utilized to discriminate MS isolates from children, and their suspected sources (Figure 1) . ●●Serotyping
Serotyping is based on the structure of the carbohydrate antigen presented on the bacterial cell wall. A total of nine serotypes, a to h and k, were designated in MS [5,6] . Moreover, a recent report concerning a new serotype (serotype p) of MS from the pig oral cavity is now available [7] . As for the serotypes of the two MS species most frequently detected in the oral cavities of humans, S. mutans could be classified into four serotypes, c, e, f and k, while S. sobrinus has two serotypes, d and g [1,6] . For S. mutans, serotype c is the most common serotype in the oral cavity with a prevalence of approximately 70–80%, followed by serotype e (∼20%) [8] . On the other hand, the distribution frequency of serotypes f and k in the oral cavity is quite low with a prevalence of less than 5%. By contrast, definite information regarding the prevalence of each serotype of S. sobrinus is still limited, although it is widely accepted that the prevalence of S. sobrinus is not as high as that of S. mutans [9] . Since the MS harbored in the oral cavity of humans predominantly belong to serotype c, the discriminatory power of their serotyping is quite low, and the use of only serotyping is not sufficient to define the transmission in subjects harboring serotype c strains. However, in the families that possess the rare serotypes, data based on serotyping have been used as a tentative clue for the transmission of such strains [9,10] . Moreover, some studies also used serotyping with other typing methods in order to gain more precise information regarding MS transmission [11] . ●●Biotyping
This phenotypic method for MS was initially introduced based on biochemical properties, including the fermentation of mannitol (with and without bacitracin), sorbitol, raffinose, melibiose, and the production of ammonia from arginine [12] . According to this method, MS could be divided in to five biotypes (I–V), which the authors could correlate with serotypes a through g. In addition, variation and modification of some biochemical reactions for typing were also observed in some studies [5,13] . In the early period of MS transmission studies,
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biotyping was used together with other typing tools in order to evaluate the similarity of MS strains among intrafamily members [13] . In those studies, the harboring of multiple types of MS in individual mouths as well as the intrafamily sharing of MS types, primarily between mothers and their children, were observed. Nevertheless, since the number of biotypes is limited and the isolated strains were mainly classified in biotype I [12] , the lack of discriminatory power appeared to impede the use of only the biotyping method in epidemiological research, including those related to MS transmission. ●●Bacteriocin typing
The early evidence for MS transmission was strongly influenced by bacteriocin typing studies, which showed identical bacteriocin profiles of MS strains from mothers and their children [13,14] . Bacteriocins are peptide antibiotics produced by bacteria to interfere with the growth of their closely related strains. Biogenesis of bacteriocins is believed to modulate the growth of competitor microorganisms occupying the same ecological niche [15] . Bacteriocins synthesized by MS were named mutacins, and the synthesis of these bacteriocins has been shown to be relatively common [16] . In addition, the possibility of randomly sharing identical bacteriocin profiles appears to be low, since there was no similar profiles reported in MS strains from nonrelated subjects [14,16–17] . Therefore, bacteriocin typing appears to be appropriate as a typing tool for MS. To determine the bacteriocin profiles of MS isolates, both bacteriocin production and sensitivity could be used [13,14] . For bacteriocin production profiles, bacteriocin activity produced by MS is tested against other oral indicator bacteria. Initially, stab cultures of tested MS are grown on solid media, and then covered by a soft agar overlay containing the indicator strains. Bacteriocin production profiles are determined by the zones of inhibition around the stab MS strains. By contrast, bacteriocin sensitivity profiles are derived from the growth inhibition zones of MS around the stab inocula of known bacteriocinogenic strains. Although bacteriocin typing was successfully employed in various MS transmission studies, there were variations in both numbers and kinds of the indicator strains [18,19] . In addition, inconsistency was also noticed in the criteria used to interpret the inhibition zones to generate the profiles [16–18] .
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Mother-to-child transmission of mutans streptococci
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1970s–1980s
Serotyping
1980s–1990s
1
Biotyping
Bacteriocin typing
1
1
2
2 1
2
RFLP
Plasmid analysis
2
AP-PCR
1
2000s–2010s
1
2
2
1 2 1
2
PCR-RFLP
1
PFGE
MLST
2
REP-PCR
Figure 1. Transitional development of the tools for mutans streptococci transmission study. Bacteriocin typing, RFLP and AP-PCR are the standard methods commonly used by various researchers. AP-PCR: Arbitrarily-primed PCR; MLST: Multilocus sequence typing; PFGE: Pulsed field gel electrophoresis; REP-PCR: Repetitive-element PCR; RFLP: Restriction fragment length polymorphism. ●●Plasmid analysis
In 1982, the first typing method based on genetic components was utilized to show the possibility of MS transmission among familial members [20] . A cryptic plasmid, an extrachromosomal DNA element encoding for unknown function, was used as the epidemiological marker to compare MS strains from familial members. This 5.6-kb cryptic plasmid was isolated from clinical populations at a low prevalence of 5–13% [20,21] . Based on the presence of this rarely found plasmid, Caufield and coworkers reported that the relatives of plasmidpositive children were 4.5-times more likely to harbor plasmid-positive strains than the control child group [20] . A few years later, procedures for
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further characterization of MS strains harboring such a plasmid were proposed [22] . These included the bacteriocin profiles of the strains as well as the restriction endonuclease (RE) patterns of the plasmids. Based on these procedures, the plasmid-containing strains could be divided into two groups with different bacteriocin profiles and RE patterns. The study of MS transmission using plasmid analysis procedures could confirm the fidelity of MS transmission in either familial or racial lines [23] . ●●Restriction fragment length
polymorphism
To perform genotyping of MS strains by chromosomal DNA fingerprinting, a technique
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Review Lapirattanakul & Nakano termed restriction fragment length polymorphism (RFLP) is generally used. RFLP measures the sizes of DNA fragments digested by frequentcutter RE and separated by conventional gel electrophoresis. Thus, the obtained band patterns reflect the DNA polymorphism at RE recognition sites found in the genomes of each bacterial strain [24] . RFLP used to study the transmission of MS could be mainly divided into two approaches, with or without hybridization probes. RFLP without hybridization is also known as RE analysis (REA). REs often utilized for cleaving MS genomic DNA were HaeIII [25–29] , HindIII [25,30–31] and EcoRI [16,25,29] , and the fragment products were separated on 0.55–0.7% (w/v) agarose gels using low-voltage electrophoresis. A large number of MS transmission publications relied on the fidelity of this traditional technique [11,25,28,32] . However, since the digestion of MS chromosomal DNA with frequent-cutter RE in RFLP without hybridization could yield hundreds of short restriction fragments, the interpretation of DNA band patterns might be ambiguous. The utilization of Southern blot hybridization in RFLP with hybridization probes helped to simplify this problem [24] . Conserved regions of 16S or 23S rRNA genes were commonly utilized to probe the different RFLP band patterns in MS genotyping [30,31] . Therefore, the term ‘ribotyping’ often refers to this technique. Research based on ribotyping have primarily provided information such as the sources and persistence of MS colonization [30–31,33–34] . ●●Pulsed field gel electrophoresis
Pulsed field gel electrophoresis (PFGE) is a variation of gel electrophoresis used for the separation of large DNA molecules [24] . In PFGE, genomic DNA is cleaved by rare-cutter REs, yielding large DNA fragmental products. Since DNA fragments larger than 20 kb show the same mobility in a size-dependent manner, they could not be separated with conventional electrophoresis. However, the separation of such fragments could be attained by application of an electric field that periodically changes direction in PFGE. Thus, variation among bacterial strains could be detected as the distinct band patterns on a flat agarose gel. PFGE is considered to be the gold standard of molecular typing methods and was extensively applied with many kinds of bacterial species [35] . For MS, PFGE was initially employed to study the size and organization of the S. mutans
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chromosome in 1990 [36,37] . However, the first usage of PFGE as a typing tool was reported in 2004 [38] . This study showed that PFGE following BssHII digestion was useful for the characterization of species and serotypes of the MS group. In addition, PFGE was also utilized for epidemiological purposes, such as to examine the emergence of new S. mutans strains during orthodontic treatment [39] , and to compare S. mutans strains from caries-active and caries-free children [40] . As for the use of this technique for analyzing MS transmission, one study utilized PFGE to demonstrate that MS genotypes that did not match maternal strains were the majority strains in children with severe early childhood caries [41] . Moreover, PFGE was also compared with recently developed genotyping method for the investigation of S. mutans diversity and transmission [42] . The authors indicated the comparable results between this method and PFGE. ●●PCR-RFLP
One study for MS transmission used PCR-RFLP to indicate the diversity of S. mutans strains from mothers and their children [43] . Unlike RFLP for chromosomal DNA fingerprinting in which whole genomic DNA is digested with RE and then separated for strain profiles, PCR-RFLP involves only the digestion and separation of PCR products from a specific locus amplified with gene-specific primers [44] . In this study, the specific locus used to classify S. mutans strains was the gtfB gene encoding a GtfB, an enzyme involved in extracellular polysaccharide synthesis [43] , since such enzymes play crucial roles in the development of virulent dental plaque [45] . PCR-RFLP investigation of the gtfB gene digested with HaeIII yielded diverse band patterns for discriminating the evaluated strains and could also show the identical band profiles between S. mutans isolates from mother–child pairs, although the percentages of such matching pairs were only 13 and 44% in caries-free and caries-active groups, respectively. ●●Arbitrarily-primed PCR
Arbitrarily-primed PCR (AP-PCR), which is often referred to as random amplification of polymorphic DNA (RAPD), was first reported in 1990 [46,47] . At low, nonstringent annealing temperatures, amplicons throughout the genome are targeted and amplified using arbitrary sequence primers of approximately ten-base lengths. Since primers for AP-PCR
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Mother-to-child transmission of mutans streptococci are not specifically designed based on any target DNA information, the primers may or may not produce the amplicons, depending on the positions that are complementary to the primer sequences. Given that the number and location of such complementary positions vary for different strains of bacterial species, the separation of these AP-PCR products on agarose gels could provide distinct patterns for particular strains with different genotypes (Figure 2) . In order to generate the best DNA patterns for strain differentiation, a screening process for appropriate primers is necessary. AP-PCR is one of the most widely used genotyping methods for MS [48–52] , due to lower costs, and less time and labor consumption as compared with several other genotyping techniques [44] . However, the reproducibility for both intra- and inter-laboratory results remains a challenge, as the amplification process is extremely sensitive and the changes in annealing temperatures, reagents and instrumentation tend to influence the results. Many short primers were widely used to characterize MS isolates by AP-PCR, for example, OPA-02 (5´-TGCCGAGCTG-3´) [48,52–53] , OPA-05 (5´-AGGGGTCTTG-3´) [49,54–55] , OPA-13 (5´-CAGCACCCAC-3´) [54,55] . Moreover, a commercial kit providing consistent conditions for amplification was also available and successfully utilized for MS strains [50,51] . For MS transmission studies, the results from AP-PCR methods clearly presented evidence for transmission of MS strains from mothers and the other close sources to children [32,48,52,56] . ●●Multilocus sequence typing
Multilocus sequence typing (MLST) is one of the molecular epidemiological methods based on nucleotide sequence comparison of multiple housekeeping genes [57] . As for MS bacterial species, a MLST scheme was initially developed for S. mutans in 2007 [58] . One year later, this highly discriminating method was applied to confirm the importance of maternal S. mutans in children [59] . The main advantages of MLST over the other typing methods are the reproducibility and the convenience of data comparison between laboratories [57] . Thus, the problems usually associated with conventional gel-based approaches could be generally resolved. With MLST, S. mutans strains are classified based on eight internal fragments of housekeeping genes with sequence lengths ranging from 387 to 462 base pairs (Figure 3) . After PCR amplification
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and sequence determination of the eight target genes, each distinct nucleotide sequence for each gene is assigned with a different allele number. Allele numbers for eight different housekeeping genes define an allelic profile for each isolated strain and consequently enable a sequence type (ST) to be assigned to each strain. Moreover, if a research problem is concerned with the possible relationship between the analyzed strains, the dendrogram constructed from the matrix of pairwise differences between allelic profiles could also be performed. Besides the MLST scheme mentioned above, one additional MLST approach for S. mutans using different gene loci was also published [60] . This scheme also included the nucleotide sequences of two putative virulence genes in order to increase the discriminatory power of the method. Nevertheless, this latter approach has not yet been applied to study MS transmission. The public database collecting MLST data for S. mutans using both approaches is also available [61] . However, to date, no MLST scheme has been developed for S. sobrinus and other MS species. ●●Repetitive-element PCR
Repetitive-element PCR (REP-PCR) is another genotyping strategy based on genomic fingerprint patterns. This genotyping method uses primers that are complementary to unknownfunction, noncoding intergenic repetitive sequences dispersed throughout the genome [24] . The amplification of DNA between adjacent repetitive elements produces multiple amplicons, depending on the distribution of these repetitive elements across the genome. The amplicon sizes could be detected by either agarose gel electrophoresis or capillary electrophoresis, and band patterns are compared in order to determine the genetic relatedness between the analyzed strains (Figure 4) . Three main families of repetitive DNA elements used for typing are: the 35–40-bp repetitive extragenic palindromic (REP), the 124–127-bp enterobacterial repetitive intergenic consensus (ERIC) and 154-bp BOX sequences. REP-PCR has been used to study the transmission of S. mutans strains since 2010 [42] . These studies used REP as the repetitive DNA elements for analysis. The DiversiLab system (bioMerieux, NC, USA), a semiautomated method for performing REP-PCR, was also used for the resolution and reproducibility of MS typing with this technique [42,62] , since this standardized commercial approach was
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B
Figure 2. Representative arbitrarily-primed PCR results of Streptococcus mutans strains with OPA-13. The strains were isolated from two mother–child pairs. (A) Strains PM2ma through PM2mc were isolated from a mother, while strains PM2a through PM2c were from her child. The patterns of PCR products were different, indicating no transmission of these strains between them. (B) The patterns of strains PF3ma through PF3mc from a mother and those of PF3a through PF3c from her child is consistent, suggesting that these strains were transmitted between them.
successfully used to evaluate several pathogenic organisms [63] . In addition, REP-PCR was assessed by comparing its reproducibility, discriminatory power and concordance with those of PFGE and MLST techniques [42,62] . These results also indicated mother-to-child transmission occurrence as well as the genetic diversity of the strains analyzed. Sources of MS Knowledge involving the sources of MS transmission is considered important, since inhibition of the transmission process is a crucial strategy for caries prevention. Accumulated evidence suggests that children could receive MS from both mothers and other sources, although several investigators reported the fidelity of vertical transmission from mothers to children [13,19,26,31,59] . The term ‘vertical transmission of MS’ was generally utilized in research works
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concerning this field in order to indicate the detection of similar MS strains between mothers and their children [64,65] . Saliva is considered as the major vehicle of transmission by physical contact. From some studies indicating mothers as the main source of MS infection, MS levels in maternal saliva were also examined [30–31,56] and shown to be a possible influencing factor in transmission, since mothers with high MS levels tended to be the sources of the MS in their children. Such findings appeared to be logical, since mothers generally have intimate contact with their own children via maternal saliva [66–68] . Besides the main transmission route from mothers to their children, the sharing of identical MS strains was also reported in other family members such as fathers and siblings [11,27,29–30] . Thus, family members, such as fathers and siblings were also suspected as alternative MS sources of children. However, the percentages of children harboring the same strains with fathers were not as high as those of children sharing the strains with their mothers. Moreover, since the results based on intrafamilial subjects could not exclude the effect of strain transferring between mothers and their children as well as between spouses (mothers and fathers), the interpretation of such results should be done with caution. Recently, the review regarding intrafamilial sources of MS in children was extensively summarized by Douglass and colleagues [69] . As for the acquisition of nonfamilial MS sources in children, although MS strains showing different phenotypes and genotypes from the strains of their mothers and other family members have been repeatedly demonstrated [11,13,27–28,30] , the information concerning these extrafamilial sources is still limited compared with the large amounts of intrafamilial evidence. Most of the researchers were interested in studying the similarity of MS isolates from children within the same classes as well as the nonfamilial caretakers in nurseries or schools [29,32,53,55,70] . The results suggested the occurrence of horizontal transmission among the children in these same classes or schools even though such transmission was not a major event. However, no evidence of transmission was seen between the children and their nonfamilial caretakers [29,70] , despite the high oral levels of MS indicated in some of these caretakers [70] . Interestingly, some reports indicated that the maternal MS strains were observed less frequently in the nursery school children than in
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Mother-to-child transmission of mutans streptococci
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Chromosomal DNA of S. mutans Gene 1
Gene 2
Gene 3
Gene 4
Gene 5
Gene 6
Gene 7
Gene 8
PCR amplification of eight target housekeeping genes
Determination of nucleotide sequence
Sequence comparison of each gene fragment with the known sequence data to assign allele number
Eight allele numbers define allelic profile and ST of each strain Gene 1 Gene 2 Gene 3 Gene 4 Gene 5 Gene 6 Gene 7 Gene 8 A
ST1
1
1
11
14
3
4
2
1
B
ST2
3
1
15
1
6
4
5
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C
ST3
7
2
11
1
5
7
5
1
D
ST4
7
2
11
1
5
7
5
1
Construction of dendrogram based on allelic profiles of isolates ST1 A ST2 B ST3 C ST4 D
Figure 3. Multilocus sequence typing procedure for Streptococcus mutans. Internal fragments of housekeeping genes are amplified and characterized by nucleotide sequencing. The results are then compared with accumulated sequences in the MLST database to assign allele numbers, allelic profiles and STs. The relationship among S. mutans isolates could be also demonstrated by the dendrogram based on allelic profiles of the isolates. MLST: Multilocus sequence typing; ST: Sequence type.
the children who stay at home [29,70] . Thus, the horizontal transmission of MS might become gradually important nowadays given current socioeconomic changes.
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While the persistence of MS, especially the early acquired strains from mothers, was reported in the human oral cavities [25,28,30,34] , the gain and loss of strains were also observed [28,30–31,34] .
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Bacterial genome with multiple sites of repetitive elements
Strain 1
Strain 2
PCR amplification with primers complementary to the repetitive elements
Strain 1
Strain 2
Separation of amplicons by electrophoresis for strain differentiation 1 2
Figure 4. Repetitive-element PCR procedure. By using primers that are complementary to the noncoding repetitive regions throughout bacterial genomes, multiple amplicons could be produced. After electrophoresis, band patterns of the amplicons are compared to determine the genetic relatedness between the analyzed strains.
Several studies showed that the number of MS types per individual were higher in adults than that found in children [16–17,26,31] . Therefore, the receipt of additional strains from new sources with increasing social exposure is also an interesting possibility. One support for this hypothesis was the observation that the frequency of matching genotypes between mother–child pairs decreased as the age of the child increased [26] . In fact, the gain of strains from new sources could have occurred even in adults with established oral microbiota, since a homology of genotypes was also observed in the MS strains isolated from spouses [33] . Nevertheless, the longitudinal persistence of such strains is still unknown. Initial acquisition Based upon pioneering MS transmission research, the initial acquisition of MS was proposed to be after tooth eruption [10,71] . Since the principal ecological niche for MS has been shown to be on solid surfaces, such as teeth and prosthetic devices, this supports the notion that nonshedding tissue surfaces are essential for
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MS colonization. Many researchers have shown that there was no detection of MS in predentate infants, while the establishment of such organisms occurred soon after the eruption of teeth in the oral cavities [10,71–73] . Nevertheless, evidence for the existence of MS in predentate infants has gradually accumulated [66,74–77] . Hence, the definite time period for initial MS acquisition has yet to be firmly established. The inconsistency of such information could be due to inherent differences among the analyzed populations or simply reflect the variations in the methodologies used. Both cross-sectional and longitudinal experimental designs were applied to study the initial acquisition of MS. As shown in Table 1, variations found in MS acquisition periods among different populations and study designs could be observed. Longitudinal studies were considered to be more appropriate for ensuring persistent colonization, and could help rule out the transient contamination of MS that might be detected by cross-sectional study. As for the detection of MS in predentate children, it is an interesting aspect. However, since there is still
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Mother-to-child transmission of mutans streptococci inconsistency about the initial acquisition period for MS, such MS detection in predentate infants could be either transient existence or persistent colonization. Thus, this aspect would be of interest for future research. Even though knowledge concerning the initial acquisition of MS is still ambiguous, various studies suggested that the MS infection rates were elevated when the ages of children and the number of erupted teeth also increased [67,72–73,76] . Moreover, the increases in both surface areas as well as retention sites of the primary molars were considered to be important
Review
for MS colonization [72,78–79] . Caufield and colleagues introduced the term of ‘window of infectivity’, a period between 19 and 31 months of age, as the critical period for MS acquisition [79] . The significance of such a period was suggested to be due to the eruption of primary molars. However, some studies of children, particularly from lower socioeconomic populations with high risk of caries showed the possibility of early MS acquisition [73,76,80,82] . In addition, some studies also reported late MS transmission in children [31,81] . For example, there has been evidence for MS acquisition in children
Table 1. Representative studies about mutans streptococci acquisition in children. Study (year)
Country (n†)
Age of children
Study design
Earliest period for MS detection
Remark
Berkowitz et al. (1975)
USA (141)
3 weeks to 14 months old
Cross-sectional study
In predentate cleft palate children with obturator
[10]
Carlsson et al. (1975)
Sweden (25)
Followed-up children from 0 to 5 years old
Soon after 1 year
Catalanotto et al. (1975)
USA (92)
0–5 years old
Longitudinal study during a 5-year period Cross-sectional study
MS was not detected in predentate children without cleft palate, but detection was observed at 10 weeks after the eruption of the first incisor No detection of MS before tooth eruption No detection of MS before tooth eruption
[78]
Fujiwara et al. (1991)
Japan (356)
0–2 years old
Cross-sectional study
No detection of MS before tooth eruption
[72]
Caufield et al. (1993)
USA (46)
Followed-up children from 0 to 5 years old
This study introduced the term ‘window of infectivity’. No detection of MS before tooth eruption
[79]
Karn et al. (1998) Mohan et al. (1998) Straetemans et al. (1998)
USA (149)
8–15 months old 6–24 months old
At the age of 10 months At the age of 6–9 months At the age of 11 years
Wan et al. (2001)
Australia (172)
MS detection in predentate children was not performed No detection of MS before tooth eruption Study was performed to detect MS acquisition in children free from MS until 5 years old –
[80]
USA (118)
Longitudinal study for the presence of MS at 3-month interval from the age of 0–5 years old Cross-sectional study Cross-sectional study Cross-sectional study
In the group with primary incisors and primary first molar At the age of 0.5–1 years with 1–4 erupted teeth At the age of 9 months
[66]
Flório et al. (2004)
Brazil (33)
–
[76]
The Netherlands 11 years old (30)
Followed-up children Longitudinal from 6 to 9 months old study during a period of 3 months Followed-up children Longitudinal with mean age 5.9 ± study for the 1.5 (standard deviation) presence of MS at months for 24-month 2-month intervals period during a period of 24 months
In predentate children at the age of 6 months In predentate children at the age of 6 months
Ref.
[71]
[73] [81]
Total number of children in the study. MS: Mutans streptococci.
†
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Review Lapirattanakul & Nakano who were free from MS until aged 5 years [81] . Such acquisition is postulated to be due to the new environment created by the eruption of permanent first molars [79] . Factors concerning transmission Various factors influencing MS transmission have been reported. Both parents and dentists should be aware of these factors in order to prevent or delay MS transmission. ●●Maternal factors
Mothers are generally the major sources of MS as well as the major caretakers of their own children, thus maternal factors should have a great impact on MS acquisition in children. Successful MS colonization in children was proposed to be due to both the magnitude and frequency of inoculation [83] . Accordingly, the magnitude of MS inoculum could be related to the MS numbers in maternal saliva and the importance of the frequency of inoculation could be associated with the behaviors promoting mother–child salivary contacts. Many researchers have shown the influence of maternal MS levels on MS transmission to their children [67,84–85] . Mothers with high numbers of MS tended to easily transfer their MS strains to their infants and have children with early MS colonization [56,77,86–87] . Most studies demonstrated that having mothers with MS levels greater than 105 to 106 CFUs/ml is one of the risk factors for MS infection in children. In addition, some studies also could show a statistical association between the salivary MS levels of mothers and those of their children [75,85,88] . Nevertheless, other studies did not find such a relationship [59,76] . The difference in these findings might be the consequence of subject selection, since the mothers used as the subjects in these latter studies harbored either high or low levels of MS in their saliva. As for the behaviors promoting mother–child salivary contacts, maternal behaviors such as sharing utensils and pretasting food for children are possibly involved in MS transmission [66–68,84] . Since a large number of vital MS cells could be detected on metal spoons contaminated with saliva [84] , a high risk for repeated exposures to maternal MS by such behaviors could be expected. Nevertheless, a negative association between the frequency of mother–child salivary contacts and the number of salivary MS in children was also reported [89] .
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An explanation for these apparently contradictory results was hypothesized to be the involvement of the children’s protective immunity gained by the exposure to antigens of maternal MS before tooth eruption. Besides the magnitude and frequency of MS infections from maternal saliva, some investigators also indicated the importance of some maternal factors concerning oral health in MS transmission. Mothers with poor oral hygiene, high caries experience, high numbers of unrestored dental cavities, poor periodontal health and high frequency of snacking tended to serve as potential reservoirs of MS for their own children [56,66,77] . Moreover, the additional maternal factors such as low socioeconomic status and education were also pointed out as the risk factors for transferring MS to children [56,66,68] . ●●MS virulence factors
Among the virulence factors of MS, the production of mutacins was suggested to be an important facilitator of colonization and establishment of MS [90] . As for the role of mutacins in transmission, this hypothesis was initially presented by Grönroos and colleagues in 1998 [16] . This research group observed that the transmitted MS strains showed a broader spectrum of mutacin activity than the nontransmitted strains. Moreover, the sizes of the inhibition zones against all types of indicator bacteria were also larger for the transmitted strains than the nontransmitted strains. Thus, it was believed that the MS strains producing high levels of mutacin activity were more easily transmitted than the strains with low activity. A similar observation regarding the association between mutacins and MS transmission has also been reported [91] . In that study, the transmitted MS strains produced more mutacin against S. sobrinus strain 6715 than the nontransmitted strains, whereas there was no difference in biofilm formation between such transmitted and nontransmitted strains. Moreover, there has been research suggesting the possible involvement of some mutacin structural genes in MS transmission [52] . Although mutacin activity seemed to provide an ecological advantage in colonization for the producing strains, a later study could not show an influence of mutacin activity on MS transmission in children acquiring the MS strains after the age of 5 years [17] . Therefore, additional research in this aspect of MS virulence for transmission is still required.
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Mother-to-child transmission of mutans streptococci ●●Dietary factors
A role for dietary carbohydrates such as sucrose in the establishment and cariogenic property of MS is generally accepted. High-sugar diets dramatically increased the prevalence of dental caries in various societies around the world. As for MS transmission, sugar consumption, breastfeeding and using nursing bottles were three major dietary factors typically studied for their influences on transmission [73,92] . Children who frequently consume sugar-containing snacks and drinks were demonstrated to be at risk for early MS colonization as well as high levels of MS [66–68,73,85] . Also, unfavorable dietary habits such as exposure to sugar more than three-times a day, intake of sweets between meals and consumption of sugar-containing beverages at night were stated as the significant predictors for early MS acquisition in various studies [67,82,85] . One additional dietary factor concerns a child’s breastfeeding experience. The association between breastfeeding and MS transmission is controversial. It was shown that breastfeeding, especially feeding on demand at night, may predispose infants to early colonization of MS [66,82] . Additionally, Li and colleagues used the similarity of the chromosomal DNA fingerprints of MS strains to demonstrate the high fidelity of mother-to-child transmission in children who were breastfed [92] . However, some studies showed that breastfeeding did not affect the MS levels of the children [93,94] , and there was no significant difference in breastfeeding duration between children with and without MS transmission [56,59] . In fact, inconclusive results were not only found for the relationship between breastfeeding and MS transmission but also for the correlation between breastfeeding and caries [95] . Thus, recent recommendations tend to support breastfeeding for health, developmental and psychosocial advantages of the infants, together with intensive oral hygiene care for the prevention of bacterial colonization and the risk of caries. As for the usage of nursing bottles, evidence has accumulated demonstrating the influence of prolonged bottle feeding upon early MS acquisition [68,80] . The strong involvement of this factor could be clearly observed when the nursing bottles contained cariogenic components and misuse of the bottles were also taken into consideration [73,77,85] . For example, it was shown that children who consumed sweetened beverages in their nursing bottles had a fourfold increase in
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Review
the odds of colonization by MS relative to children who consumed milk. Furthermore, the use of nursing bottles at bedtime was also pointed out as the potential risk factor for MS infection. ●●Host factors in children
Various characteristics of children were also raised as potential host factors related to MS transmission. For example, oral hygiene appeared to be important for initial MS acquisition. Poor oral hygiene, indicated by visible plaque, lack of parental assistance with toothbrushing and commencement of toothbrushing after age 12 months was significant factor in MS infection [96] . By contrast, oral hygiene practices, such as toothbrushing, were associated with noncolonization by MS [67,68] . Apart from oral hygiene, there were some investigations showing the possible influence of some tooth defects, such as enamel hypoplasia, on the acquisition of MS [94,96] . In addition, the presence of oral developmental nodules (Bohn’s nodules) and oral cleft defects was also reported to promote early MS infection in predentate infants [10,75] . The increase in retention sites on the irregular surfaces of teeth and the mucosa appear to enhance bacterial colonization. As for demographic characteristics, ethnic grouping or the races of the subjects were also proposed to influence the differences in MS acquisition [26,82,97] . However, some studies suggested that such observations might be partially the consequence of differences in the socioeconomic status or dietary habits of the subjects [82,97] . Moreover, the gender of children may also impact the MS transmission rate, since mothers transmitted the MS strains to female children with significantly greater fidelity than to male children [26–27,59,91] . Such gender-specific conservation was reported in American, Chinese and Japanese families. Nevertheless, the scientific explanation for this aspect has not been established. ●●Other factors
Apart from the aforementioned factors, certain additional influences such as antibiotic usage and mode of infant delivery may be involved in MS colonization. For instance, antibiotic usage has been reported to help decrease MS levels and caries [98–100] . Some researchers also discussed the decline in MS-associated caries as a consequence of the medical usage of antibiotics [99] , since only one single exposure to a short-term antibiotic
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Review Lapirattanakul & Nakano regimen could reduce the levels of salivary MS for a period of at least 3 months in children [100] . As for long-term utilization, long-term antibiotic therapy or the usage of multiple courses of antibiotics in children with some diseases have been reported to delay the acquisition of MS [66–67,101] . However, such benefits were proven to occur only during the active administration of the drugs [101] . On the other hand, contradictory results were also observed, that is, there was no significant difference in antibiotic utilization recorded from birth to 3 years of age between groups of children with and without MS colonization [79] . Actually, one study showed that children administered antibiotics during early childhood exhibited a higher MS prevalence than those who were untreated [102] . Regarding the mode of delivery, infants born either by vaginal delivery or Caesarean section (C-section) were compared for MS infection. However, inconsistency in the results from each study was observed. Li and coworkers initially introduced the idea that children born by C-section acquired MS significantly earlier and at higher levels than vaginally delivered children [97] . When the number of subjects increased, this group of researchers could also confirm such results as well as demonstrate the high fidelity of mother-to-child transmission in children who were born by C-section, since all MS genotypes colonized in these C-section babies were all found in their mothers [56] . However, some studies indicated no difference in MS infection rates between vaginally delivered children and Caesarean-born children [68,75] . Moreover, there is a recent work indicating that vaginally born children were 1.8-times more likely to have high MS scores than the C-section group [88] . Prevention of transmission It was scientifically proven that early infection by MS could lead to a high prevalence of dental caries in children [103] . Hence, it seems logical to assume that the strategies for reduction of maternal MS levels in order to impede MS transfer from mothers to children should be valuable. The first report in this regard was published in the early 1980s, in which an achievement in reduction of maternal MS levels from ≥106 to
Mother-to-child transmission of mutans streptococci
Jinthana Lapirattanakul*,1 & Kazuhiko Nakano2
Abstract: Mutans streptococci (MS) are the major group of pathogens implicated in dental caries. Like other infectious diseases, transmission of the causative microorganisms is the initial and essential step that should be understood relative to disease control and prevention. This review summarizes current knowledge regarding MS transmission, especially from mothers to their children. Included are methods used to study transmission, sources of MS, initial acquisition, factors concerning transmission and prevention of transmission. Information accumulated over many decades showed the involvement of MS transmission in the pathogenesis of caries, hence several preventive measurements have been proposed. Nevertheless, some essential aspects remain to be elucidated for more benefits of practical application. Dental caries is one of the most prevalent diseases in the field of dentistry, in which transmission of mutans streptococci (MS), the major bacterial group involved in caries initiation, is one of the essential factors in its onset. MS consist of seven closely related species, among which Streptococcus mutans and Streptococcus sobrinus are the two major species isolated from human oral cavities [1] . Their pathogenic properties related to dental caries include their ability to colonize efficiently on tooth surfaces as well as their acidogenic and aciduric potentials [2] . MS utilize two distinct mechanisms for tooth adherence. The initial step is a sucrose-independent adhesion, which initiates the primary attachment [3] . The following step involves sucrose-dependent adhesion that helps enhance the firm and irreversible tooth adherence involving glucosyltransferases (Gtfs) and glucan-binding proteins (Gbps) [4] . As for the acidogenic and aciduric properties that provide the ecological advantage for MS over other oral bacteria, these characteristics are mainly based on the high efficiency of sugar transport and metabolism as well as the function of ATPase-driven proton pumps [2] . Moreover, most MS strains are also able to synthesize and store intracellular polysaccharides, which serve as reserve nutrients during prolonged starvation, thereby maintaining an acidic pH environment. Based on the cariogenic properties of MS, the acquisition of these bacteria in the oral cavities of infants is one of the important research subjects for paediatric dentistry. This review aims to summarize current knowledge regarding MS transmission, especially from mothers to their children. In addition, future research important for this field are also pointed out.
Keywords
• factors • initial acquisition • mother-to-child transmission • mutans streptococci • prevention • sources • typing methods
Methods used to study transmission Since evidence for transmission was mainly dependent on the detection of strains with identical characteristics, various typing methods were used in the study of MS transmission. The methods based on characteristics expressed by bacteria are called phenotyping, while the techniques relying Department of Oral Microbiology, Faculty of Dentistry, Mahidol University, Bangkok, 10400, Thailand Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, Osaka, 565-0871, Japan *Author for correspondence: Tel.: +66 2200 7804; Fax: +66 2200 7804; [email protected]
1 2
10.2217/FMB.14.37 © 2014 Future Medicine Ltd
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Review Lapirattanakul & Nakano on genetic properties are termed genotyping. For the transmission of MS, both phenotypic and genotypic methods were utilized to discriminate MS isolates from children, and their suspected sources (Figure 1) . ●●Serotyping
Serotyping is based on the structure of the carbohydrate antigen presented on the bacterial cell wall. A total of nine serotypes, a to h and k, were designated in MS [5,6] . Moreover, a recent report concerning a new serotype (serotype p) of MS from the pig oral cavity is now available [7] . As for the serotypes of the two MS species most frequently detected in the oral cavities of humans, S. mutans could be classified into four serotypes, c, e, f and k, while S. sobrinus has two serotypes, d and g [1,6] . For S. mutans, serotype c is the most common serotype in the oral cavity with a prevalence of approximately 70–80%, followed by serotype e (∼20%) [8] . On the other hand, the distribution frequency of serotypes f and k in the oral cavity is quite low with a prevalence of less than 5%. By contrast, definite information regarding the prevalence of each serotype of S. sobrinus is still limited, although it is widely accepted that the prevalence of S. sobrinus is not as high as that of S. mutans [9] . Since the MS harbored in the oral cavity of humans predominantly belong to serotype c, the discriminatory power of their serotyping is quite low, and the use of only serotyping is not sufficient to define the transmission in subjects harboring serotype c strains. However, in the families that possess the rare serotypes, data based on serotyping have been used as a tentative clue for the transmission of such strains [9,10] . Moreover, some studies also used serotyping with other typing methods in order to gain more precise information regarding MS transmission [11] . ●●Biotyping
This phenotypic method for MS was initially introduced based on biochemical properties, including the fermentation of mannitol (with and without bacitracin), sorbitol, raffinose, melibiose, and the production of ammonia from arginine [12] . According to this method, MS could be divided in to five biotypes (I–V), which the authors could correlate with serotypes a through g. In addition, variation and modification of some biochemical reactions for typing were also observed in some studies [5,13] . In the early period of MS transmission studies,
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biotyping was used together with other typing tools in order to evaluate the similarity of MS strains among intrafamily members [13] . In those studies, the harboring of multiple types of MS in individual mouths as well as the intrafamily sharing of MS types, primarily between mothers and their children, were observed. Nevertheless, since the number of biotypes is limited and the isolated strains were mainly classified in biotype I [12] , the lack of discriminatory power appeared to impede the use of only the biotyping method in epidemiological research, including those related to MS transmission. ●●Bacteriocin typing
The early evidence for MS transmission was strongly influenced by bacteriocin typing studies, which showed identical bacteriocin profiles of MS strains from mothers and their children [13,14] . Bacteriocins are peptide antibiotics produced by bacteria to interfere with the growth of their closely related strains. Biogenesis of bacteriocins is believed to modulate the growth of competitor microorganisms occupying the same ecological niche [15] . Bacteriocins synthesized by MS were named mutacins, and the synthesis of these bacteriocins has been shown to be relatively common [16] . In addition, the possibility of randomly sharing identical bacteriocin profiles appears to be low, since there was no similar profiles reported in MS strains from nonrelated subjects [14,16–17] . Therefore, bacteriocin typing appears to be appropriate as a typing tool for MS. To determine the bacteriocin profiles of MS isolates, both bacteriocin production and sensitivity could be used [13,14] . For bacteriocin production profiles, bacteriocin activity produced by MS is tested against other oral indicator bacteria. Initially, stab cultures of tested MS are grown on solid media, and then covered by a soft agar overlay containing the indicator strains. Bacteriocin production profiles are determined by the zones of inhibition around the stab MS strains. By contrast, bacteriocin sensitivity profiles are derived from the growth inhibition zones of MS around the stab inocula of known bacteriocinogenic strains. Although bacteriocin typing was successfully employed in various MS transmission studies, there were variations in both numbers and kinds of the indicator strains [18,19] . In addition, inconsistency was also noticed in the criteria used to interpret the inhibition zones to generate the profiles [16–18] .
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Mother-to-child transmission of mutans streptococci
Review
1970s–1980s
Serotyping
1980s–1990s
1
Biotyping
Bacteriocin typing
1
1
2
2 1
2
RFLP
Plasmid analysis
2
AP-PCR
1
2000s–2010s
1
2
2
1 2 1
2
PCR-RFLP
1
PFGE
MLST
2
REP-PCR
Figure 1. Transitional development of the tools for mutans streptococci transmission study. Bacteriocin typing, RFLP and AP-PCR are the standard methods commonly used by various researchers. AP-PCR: Arbitrarily-primed PCR; MLST: Multilocus sequence typing; PFGE: Pulsed field gel electrophoresis; REP-PCR: Repetitive-element PCR; RFLP: Restriction fragment length polymorphism. ●●Plasmid analysis
In 1982, the first typing method based on genetic components was utilized to show the possibility of MS transmission among familial members [20] . A cryptic plasmid, an extrachromosomal DNA element encoding for unknown function, was used as the epidemiological marker to compare MS strains from familial members. This 5.6-kb cryptic plasmid was isolated from clinical populations at a low prevalence of 5–13% [20,21] . Based on the presence of this rarely found plasmid, Caufield and coworkers reported that the relatives of plasmidpositive children were 4.5-times more likely to harbor plasmid-positive strains than the control child group [20] . A few years later, procedures for
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further characterization of MS strains harboring such a plasmid were proposed [22] . These included the bacteriocin profiles of the strains as well as the restriction endonuclease (RE) patterns of the plasmids. Based on these procedures, the plasmid-containing strains could be divided into two groups with different bacteriocin profiles and RE patterns. The study of MS transmission using plasmid analysis procedures could confirm the fidelity of MS transmission in either familial or racial lines [23] . ●●Restriction fragment length
polymorphism
To perform genotyping of MS strains by chromosomal DNA fingerprinting, a technique
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Review Lapirattanakul & Nakano termed restriction fragment length polymorphism (RFLP) is generally used. RFLP measures the sizes of DNA fragments digested by frequentcutter RE and separated by conventional gel electrophoresis. Thus, the obtained band patterns reflect the DNA polymorphism at RE recognition sites found in the genomes of each bacterial strain [24] . RFLP used to study the transmission of MS could be mainly divided into two approaches, with or without hybridization probes. RFLP without hybridization is also known as RE analysis (REA). REs often utilized for cleaving MS genomic DNA were HaeIII [25–29] , HindIII [25,30–31] and EcoRI [16,25,29] , and the fragment products were separated on 0.55–0.7% (w/v) agarose gels using low-voltage electrophoresis. A large number of MS transmission publications relied on the fidelity of this traditional technique [11,25,28,32] . However, since the digestion of MS chromosomal DNA with frequent-cutter RE in RFLP without hybridization could yield hundreds of short restriction fragments, the interpretation of DNA band patterns might be ambiguous. The utilization of Southern blot hybridization in RFLP with hybridization probes helped to simplify this problem [24] . Conserved regions of 16S or 23S rRNA genes were commonly utilized to probe the different RFLP band patterns in MS genotyping [30,31] . Therefore, the term ‘ribotyping’ often refers to this technique. Research based on ribotyping have primarily provided information such as the sources and persistence of MS colonization [30–31,33–34] . ●●Pulsed field gel electrophoresis
Pulsed field gel electrophoresis (PFGE) is a variation of gel electrophoresis used for the separation of large DNA molecules [24] . In PFGE, genomic DNA is cleaved by rare-cutter REs, yielding large DNA fragmental products. Since DNA fragments larger than 20 kb show the same mobility in a size-dependent manner, they could not be separated with conventional electrophoresis. However, the separation of such fragments could be attained by application of an electric field that periodically changes direction in PFGE. Thus, variation among bacterial strains could be detected as the distinct band patterns on a flat agarose gel. PFGE is considered to be the gold standard of molecular typing methods and was extensively applied with many kinds of bacterial species [35] . For MS, PFGE was initially employed to study the size and organization of the S. mutans
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chromosome in 1990 [36,37] . However, the first usage of PFGE as a typing tool was reported in 2004 [38] . This study showed that PFGE following BssHII digestion was useful for the characterization of species and serotypes of the MS group. In addition, PFGE was also utilized for epidemiological purposes, such as to examine the emergence of new S. mutans strains during orthodontic treatment [39] , and to compare S. mutans strains from caries-active and caries-free children [40] . As for the use of this technique for analyzing MS transmission, one study utilized PFGE to demonstrate that MS genotypes that did not match maternal strains were the majority strains in children with severe early childhood caries [41] . Moreover, PFGE was also compared with recently developed genotyping method for the investigation of S. mutans diversity and transmission [42] . The authors indicated the comparable results between this method and PFGE. ●●PCR-RFLP
One study for MS transmission used PCR-RFLP to indicate the diversity of S. mutans strains from mothers and their children [43] . Unlike RFLP for chromosomal DNA fingerprinting in which whole genomic DNA is digested with RE and then separated for strain profiles, PCR-RFLP involves only the digestion and separation of PCR products from a specific locus amplified with gene-specific primers [44] . In this study, the specific locus used to classify S. mutans strains was the gtfB gene encoding a GtfB, an enzyme involved in extracellular polysaccharide synthesis [43] , since such enzymes play crucial roles in the development of virulent dental plaque [45] . PCR-RFLP investigation of the gtfB gene digested with HaeIII yielded diverse band patterns for discriminating the evaluated strains and could also show the identical band profiles between S. mutans isolates from mother–child pairs, although the percentages of such matching pairs were only 13 and 44% in caries-free and caries-active groups, respectively. ●●Arbitrarily-primed PCR
Arbitrarily-primed PCR (AP-PCR), which is often referred to as random amplification of polymorphic DNA (RAPD), was first reported in 1990 [46,47] . At low, nonstringent annealing temperatures, amplicons throughout the genome are targeted and amplified using arbitrary sequence primers of approximately ten-base lengths. Since primers for AP-PCR
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Mother-to-child transmission of mutans streptococci are not specifically designed based on any target DNA information, the primers may or may not produce the amplicons, depending on the positions that are complementary to the primer sequences. Given that the number and location of such complementary positions vary for different strains of bacterial species, the separation of these AP-PCR products on agarose gels could provide distinct patterns for particular strains with different genotypes (Figure 2) . In order to generate the best DNA patterns for strain differentiation, a screening process for appropriate primers is necessary. AP-PCR is one of the most widely used genotyping methods for MS [48–52] , due to lower costs, and less time and labor consumption as compared with several other genotyping techniques [44] . However, the reproducibility for both intra- and inter-laboratory results remains a challenge, as the amplification process is extremely sensitive and the changes in annealing temperatures, reagents and instrumentation tend to influence the results. Many short primers were widely used to characterize MS isolates by AP-PCR, for example, OPA-02 (5´-TGCCGAGCTG-3´) [48,52–53] , OPA-05 (5´-AGGGGTCTTG-3´) [49,54–55] , OPA-13 (5´-CAGCACCCAC-3´) [54,55] . Moreover, a commercial kit providing consistent conditions for amplification was also available and successfully utilized for MS strains [50,51] . For MS transmission studies, the results from AP-PCR methods clearly presented evidence for transmission of MS strains from mothers and the other close sources to children [32,48,52,56] . ●●Multilocus sequence typing
Multilocus sequence typing (MLST) is one of the molecular epidemiological methods based on nucleotide sequence comparison of multiple housekeeping genes [57] . As for MS bacterial species, a MLST scheme was initially developed for S. mutans in 2007 [58] . One year later, this highly discriminating method was applied to confirm the importance of maternal S. mutans in children [59] . The main advantages of MLST over the other typing methods are the reproducibility and the convenience of data comparison between laboratories [57] . Thus, the problems usually associated with conventional gel-based approaches could be generally resolved. With MLST, S. mutans strains are classified based on eight internal fragments of housekeeping genes with sequence lengths ranging from 387 to 462 base pairs (Figure 3) . After PCR amplification
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and sequence determination of the eight target genes, each distinct nucleotide sequence for each gene is assigned with a different allele number. Allele numbers for eight different housekeeping genes define an allelic profile for each isolated strain and consequently enable a sequence type (ST) to be assigned to each strain. Moreover, if a research problem is concerned with the possible relationship between the analyzed strains, the dendrogram constructed from the matrix of pairwise differences between allelic profiles could also be performed. Besides the MLST scheme mentioned above, one additional MLST approach for S. mutans using different gene loci was also published [60] . This scheme also included the nucleotide sequences of two putative virulence genes in order to increase the discriminatory power of the method. Nevertheless, this latter approach has not yet been applied to study MS transmission. The public database collecting MLST data for S. mutans using both approaches is also available [61] . However, to date, no MLST scheme has been developed for S. sobrinus and other MS species. ●●Repetitive-element PCR
Repetitive-element PCR (REP-PCR) is another genotyping strategy based on genomic fingerprint patterns. This genotyping method uses primers that are complementary to unknownfunction, noncoding intergenic repetitive sequences dispersed throughout the genome [24] . The amplification of DNA between adjacent repetitive elements produces multiple amplicons, depending on the distribution of these repetitive elements across the genome. The amplicon sizes could be detected by either agarose gel electrophoresis or capillary electrophoresis, and band patterns are compared in order to determine the genetic relatedness between the analyzed strains (Figure 4) . Three main families of repetitive DNA elements used for typing are: the 35–40-bp repetitive extragenic palindromic (REP), the 124–127-bp enterobacterial repetitive intergenic consensus (ERIC) and 154-bp BOX sequences. REP-PCR has been used to study the transmission of S. mutans strains since 2010 [42] . These studies used REP as the repetitive DNA elements for analysis. The DiversiLab system (bioMerieux, NC, USA), a semiautomated method for performing REP-PCR, was also used for the resolution and reproducibility of MS typing with this technique [42,62] , since this standardized commercial approach was
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B
Figure 2. Representative arbitrarily-primed PCR results of Streptococcus mutans strains with OPA-13. The strains were isolated from two mother–child pairs. (A) Strains PM2ma through PM2mc were isolated from a mother, while strains PM2a through PM2c were from her child. The patterns of PCR products were different, indicating no transmission of these strains between them. (B) The patterns of strains PF3ma through PF3mc from a mother and those of PF3a through PF3c from her child is consistent, suggesting that these strains were transmitted between them.
successfully used to evaluate several pathogenic organisms [63] . In addition, REP-PCR was assessed by comparing its reproducibility, discriminatory power and concordance with those of PFGE and MLST techniques [42,62] . These results also indicated mother-to-child transmission occurrence as well as the genetic diversity of the strains analyzed. Sources of MS Knowledge involving the sources of MS transmission is considered important, since inhibition of the transmission process is a crucial strategy for caries prevention. Accumulated evidence suggests that children could receive MS from both mothers and other sources, although several investigators reported the fidelity of vertical transmission from mothers to children [13,19,26,31,59] . The term ‘vertical transmission of MS’ was generally utilized in research works
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concerning this field in order to indicate the detection of similar MS strains between mothers and their children [64,65] . Saliva is considered as the major vehicle of transmission by physical contact. From some studies indicating mothers as the main source of MS infection, MS levels in maternal saliva were also examined [30–31,56] and shown to be a possible influencing factor in transmission, since mothers with high MS levels tended to be the sources of the MS in their children. Such findings appeared to be logical, since mothers generally have intimate contact with their own children via maternal saliva [66–68] . Besides the main transmission route from mothers to their children, the sharing of identical MS strains was also reported in other family members such as fathers and siblings [11,27,29–30] . Thus, family members, such as fathers and siblings were also suspected as alternative MS sources of children. However, the percentages of children harboring the same strains with fathers were not as high as those of children sharing the strains with their mothers. Moreover, since the results based on intrafamilial subjects could not exclude the effect of strain transferring between mothers and their children as well as between spouses (mothers and fathers), the interpretation of such results should be done with caution. Recently, the review regarding intrafamilial sources of MS in children was extensively summarized by Douglass and colleagues [69] . As for the acquisition of nonfamilial MS sources in children, although MS strains showing different phenotypes and genotypes from the strains of their mothers and other family members have been repeatedly demonstrated [11,13,27–28,30] , the information concerning these extrafamilial sources is still limited compared with the large amounts of intrafamilial evidence. Most of the researchers were interested in studying the similarity of MS isolates from children within the same classes as well as the nonfamilial caretakers in nurseries or schools [29,32,53,55,70] . The results suggested the occurrence of horizontal transmission among the children in these same classes or schools even though such transmission was not a major event. However, no evidence of transmission was seen between the children and their nonfamilial caretakers [29,70] , despite the high oral levels of MS indicated in some of these caretakers [70] . Interestingly, some reports indicated that the maternal MS strains were observed less frequently in the nursery school children than in
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Mother-to-child transmission of mutans streptococci
Review
Chromosomal DNA of S. mutans Gene 1
Gene 2
Gene 3
Gene 4
Gene 5
Gene 6
Gene 7
Gene 8
PCR amplification of eight target housekeeping genes
Determination of nucleotide sequence
Sequence comparison of each gene fragment with the known sequence data to assign allele number
Eight allele numbers define allelic profile and ST of each strain Gene 1 Gene 2 Gene 3 Gene 4 Gene 5 Gene 6 Gene 7 Gene 8 A
ST1
1
1
11
14
3
4
2
1
B
ST2
3
1
15
1
6
4
5
1
C
ST3
7
2
11
1
5
7
5
1
D
ST4
7
2
11
1
5
7
5
1
Construction of dendrogram based on allelic profiles of isolates ST1 A ST2 B ST3 C ST4 D
Figure 3. Multilocus sequence typing procedure for Streptococcus mutans. Internal fragments of housekeeping genes are amplified and characterized by nucleotide sequencing. The results are then compared with accumulated sequences in the MLST database to assign allele numbers, allelic profiles and STs. The relationship among S. mutans isolates could be also demonstrated by the dendrogram based on allelic profiles of the isolates. MLST: Multilocus sequence typing; ST: Sequence type.
the children who stay at home [29,70] . Thus, the horizontal transmission of MS might become gradually important nowadays given current socioeconomic changes.
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While the persistence of MS, especially the early acquired strains from mothers, was reported in the human oral cavities [25,28,30,34] , the gain and loss of strains were also observed [28,30–31,34] .
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Bacterial genome with multiple sites of repetitive elements
Strain 1
Strain 2
PCR amplification with primers complementary to the repetitive elements
Strain 1
Strain 2
Separation of amplicons by electrophoresis for strain differentiation 1 2
Figure 4. Repetitive-element PCR procedure. By using primers that are complementary to the noncoding repetitive regions throughout bacterial genomes, multiple amplicons could be produced. After electrophoresis, band patterns of the amplicons are compared to determine the genetic relatedness between the analyzed strains.
Several studies showed that the number of MS types per individual were higher in adults than that found in children [16–17,26,31] . Therefore, the receipt of additional strains from new sources with increasing social exposure is also an interesting possibility. One support for this hypothesis was the observation that the frequency of matching genotypes between mother–child pairs decreased as the age of the child increased [26] . In fact, the gain of strains from new sources could have occurred even in adults with established oral microbiota, since a homology of genotypes was also observed in the MS strains isolated from spouses [33] . Nevertheless, the longitudinal persistence of such strains is still unknown. Initial acquisition Based upon pioneering MS transmission research, the initial acquisition of MS was proposed to be after tooth eruption [10,71] . Since the principal ecological niche for MS has been shown to be on solid surfaces, such as teeth and prosthetic devices, this supports the notion that nonshedding tissue surfaces are essential for
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MS colonization. Many researchers have shown that there was no detection of MS in predentate infants, while the establishment of such organisms occurred soon after the eruption of teeth in the oral cavities [10,71–73] . Nevertheless, evidence for the existence of MS in predentate infants has gradually accumulated [66,74–77] . Hence, the definite time period for initial MS acquisition has yet to be firmly established. The inconsistency of such information could be due to inherent differences among the analyzed populations or simply reflect the variations in the methodologies used. Both cross-sectional and longitudinal experimental designs were applied to study the initial acquisition of MS. As shown in Table 1, variations found in MS acquisition periods among different populations and study designs could be observed. Longitudinal studies were considered to be more appropriate for ensuring persistent colonization, and could help rule out the transient contamination of MS that might be detected by cross-sectional study. As for the detection of MS in predentate children, it is an interesting aspect. However, since there is still
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Mother-to-child transmission of mutans streptococci inconsistency about the initial acquisition period for MS, such MS detection in predentate infants could be either transient existence or persistent colonization. Thus, this aspect would be of interest for future research. Even though knowledge concerning the initial acquisition of MS is still ambiguous, various studies suggested that the MS infection rates were elevated when the ages of children and the number of erupted teeth also increased [67,72–73,76] . Moreover, the increases in both surface areas as well as retention sites of the primary molars were considered to be important
Review
for MS colonization [72,78–79] . Caufield and colleagues introduced the term of ‘window of infectivity’, a period between 19 and 31 months of age, as the critical period for MS acquisition [79] . The significance of such a period was suggested to be due to the eruption of primary molars. However, some studies of children, particularly from lower socioeconomic populations with high risk of caries showed the possibility of early MS acquisition [73,76,80,82] . In addition, some studies also reported late MS transmission in children [31,81] . For example, there has been evidence for MS acquisition in children
Table 1. Representative studies about mutans streptococci acquisition in children. Study (year)
Country (n†)
Age of children
Study design
Earliest period for MS detection
Remark
Berkowitz et al. (1975)
USA (141)
3 weeks to 14 months old
Cross-sectional study
In predentate cleft palate children with obturator
[10]
Carlsson et al. (1975)
Sweden (25)
Followed-up children from 0 to 5 years old
Soon after 1 year
Catalanotto et al. (1975)
USA (92)
0–5 years old
Longitudinal study during a 5-year period Cross-sectional study
MS was not detected in predentate children without cleft palate, but detection was observed at 10 weeks after the eruption of the first incisor No detection of MS before tooth eruption No detection of MS before tooth eruption
[78]
Fujiwara et al. (1991)
Japan (356)
0–2 years old
Cross-sectional study
No detection of MS before tooth eruption
[72]
Caufield et al. (1993)
USA (46)
Followed-up children from 0 to 5 years old
This study introduced the term ‘window of infectivity’. No detection of MS before tooth eruption
[79]
Karn et al. (1998) Mohan et al. (1998) Straetemans et al. (1998)
USA (149)
8–15 months old 6–24 months old
At the age of 10 months At the age of 6–9 months At the age of 11 years
Wan et al. (2001)
Australia (172)
MS detection in predentate children was not performed No detection of MS before tooth eruption Study was performed to detect MS acquisition in children free from MS until 5 years old –
[80]
USA (118)
Longitudinal study for the presence of MS at 3-month interval from the age of 0–5 years old Cross-sectional study Cross-sectional study Cross-sectional study
In the group with primary incisors and primary first molar At the age of 0.5–1 years with 1–4 erupted teeth At the age of 9 months
[66]
Flório et al. (2004)
Brazil (33)
–
[76]
The Netherlands 11 years old (30)
Followed-up children Longitudinal from 6 to 9 months old study during a period of 3 months Followed-up children Longitudinal with mean age 5.9 ± study for the 1.5 (standard deviation) presence of MS at months for 24-month 2-month intervals period during a period of 24 months
In predentate children at the age of 6 months In predentate children at the age of 6 months
Ref.
[71]
[73] [81]
Total number of children in the study. MS: Mutans streptococci.
†
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Review Lapirattanakul & Nakano who were free from MS until aged 5 years [81] . Such acquisition is postulated to be due to the new environment created by the eruption of permanent first molars [79] . Factors concerning transmission Various factors influencing MS transmission have been reported. Both parents and dentists should be aware of these factors in order to prevent or delay MS transmission. ●●Maternal factors
Mothers are generally the major sources of MS as well as the major caretakers of their own children, thus maternal factors should have a great impact on MS acquisition in children. Successful MS colonization in children was proposed to be due to both the magnitude and frequency of inoculation [83] . Accordingly, the magnitude of MS inoculum could be related to the MS numbers in maternal saliva and the importance of the frequency of inoculation could be associated with the behaviors promoting mother–child salivary contacts. Many researchers have shown the influence of maternal MS levels on MS transmission to their children [67,84–85] . Mothers with high numbers of MS tended to easily transfer their MS strains to their infants and have children with early MS colonization [56,77,86–87] . Most studies demonstrated that having mothers with MS levels greater than 105 to 106 CFUs/ml is one of the risk factors for MS infection in children. In addition, some studies also could show a statistical association between the salivary MS levels of mothers and those of their children [75,85,88] . Nevertheless, other studies did not find such a relationship [59,76] . The difference in these findings might be the consequence of subject selection, since the mothers used as the subjects in these latter studies harbored either high or low levels of MS in their saliva. As for the behaviors promoting mother–child salivary contacts, maternal behaviors such as sharing utensils and pretasting food for children are possibly involved in MS transmission [66–68,84] . Since a large number of vital MS cells could be detected on metal spoons contaminated with saliva [84] , a high risk for repeated exposures to maternal MS by such behaviors could be expected. Nevertheless, a negative association between the frequency of mother–child salivary contacts and the number of salivary MS in children was also reported [89] .
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An explanation for these apparently contradictory results was hypothesized to be the involvement of the children’s protective immunity gained by the exposure to antigens of maternal MS before tooth eruption. Besides the magnitude and frequency of MS infections from maternal saliva, some investigators also indicated the importance of some maternal factors concerning oral health in MS transmission. Mothers with poor oral hygiene, high caries experience, high numbers of unrestored dental cavities, poor periodontal health and high frequency of snacking tended to serve as potential reservoirs of MS for their own children [56,66,77] . Moreover, the additional maternal factors such as low socioeconomic status and education were also pointed out as the risk factors for transferring MS to children [56,66,68] . ●●MS virulence factors
Among the virulence factors of MS, the production of mutacins was suggested to be an important facilitator of colonization and establishment of MS [90] . As for the role of mutacins in transmission, this hypothesis was initially presented by Grönroos and colleagues in 1998 [16] . This research group observed that the transmitted MS strains showed a broader spectrum of mutacin activity than the nontransmitted strains. Moreover, the sizes of the inhibition zones against all types of indicator bacteria were also larger for the transmitted strains than the nontransmitted strains. Thus, it was believed that the MS strains producing high levels of mutacin activity were more easily transmitted than the strains with low activity. A similar observation regarding the association between mutacins and MS transmission has also been reported [91] . In that study, the transmitted MS strains produced more mutacin against S. sobrinus strain 6715 than the nontransmitted strains, whereas there was no difference in biofilm formation between such transmitted and nontransmitted strains. Moreover, there has been research suggesting the possible involvement of some mutacin structural genes in MS transmission [52] . Although mutacin activity seemed to provide an ecological advantage in colonization for the producing strains, a later study could not show an influence of mutacin activity on MS transmission in children acquiring the MS strains after the age of 5 years [17] . Therefore, additional research in this aspect of MS virulence for transmission is still required.
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Mother-to-child transmission of mutans streptococci ●●Dietary factors
A role for dietary carbohydrates such as sucrose in the establishment and cariogenic property of MS is generally accepted. High-sugar diets dramatically increased the prevalence of dental caries in various societies around the world. As for MS transmission, sugar consumption, breastfeeding and using nursing bottles were three major dietary factors typically studied for their influences on transmission [73,92] . Children who frequently consume sugar-containing snacks and drinks were demonstrated to be at risk for early MS colonization as well as high levels of MS [66–68,73,85] . Also, unfavorable dietary habits such as exposure to sugar more than three-times a day, intake of sweets between meals and consumption of sugar-containing beverages at night were stated as the significant predictors for early MS acquisition in various studies [67,82,85] . One additional dietary factor concerns a child’s breastfeeding experience. The association between breastfeeding and MS transmission is controversial. It was shown that breastfeeding, especially feeding on demand at night, may predispose infants to early colonization of MS [66,82] . Additionally, Li and colleagues used the similarity of the chromosomal DNA fingerprints of MS strains to demonstrate the high fidelity of mother-to-child transmission in children who were breastfed [92] . However, some studies showed that breastfeeding did not affect the MS levels of the children [93,94] , and there was no significant difference in breastfeeding duration between children with and without MS transmission [56,59] . In fact, inconclusive results were not only found for the relationship between breastfeeding and MS transmission but also for the correlation between breastfeeding and caries [95] . Thus, recent recommendations tend to support breastfeeding for health, developmental and psychosocial advantages of the infants, together with intensive oral hygiene care for the prevention of bacterial colonization and the risk of caries. As for the usage of nursing bottles, evidence has accumulated demonstrating the influence of prolonged bottle feeding upon early MS acquisition [68,80] . The strong involvement of this factor could be clearly observed when the nursing bottles contained cariogenic components and misuse of the bottles were also taken into consideration [73,77,85] . For example, it was shown that children who consumed sweetened beverages in their nursing bottles had a fourfold increase in
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the odds of colonization by MS relative to children who consumed milk. Furthermore, the use of nursing bottles at bedtime was also pointed out as the potential risk factor for MS infection. ●●Host factors in children
Various characteristics of children were also raised as potential host factors related to MS transmission. For example, oral hygiene appeared to be important for initial MS acquisition. Poor oral hygiene, indicated by visible plaque, lack of parental assistance with toothbrushing and commencement of toothbrushing after age 12 months was significant factor in MS infection [96] . By contrast, oral hygiene practices, such as toothbrushing, were associated with noncolonization by MS [67,68] . Apart from oral hygiene, there were some investigations showing the possible influence of some tooth defects, such as enamel hypoplasia, on the acquisition of MS [94,96] . In addition, the presence of oral developmental nodules (Bohn’s nodules) and oral cleft defects was also reported to promote early MS infection in predentate infants [10,75] . The increase in retention sites on the irregular surfaces of teeth and the mucosa appear to enhance bacterial colonization. As for demographic characteristics, ethnic grouping or the races of the subjects were also proposed to influence the differences in MS acquisition [26,82,97] . However, some studies suggested that such observations might be partially the consequence of differences in the socioeconomic status or dietary habits of the subjects [82,97] . Moreover, the gender of children may also impact the MS transmission rate, since mothers transmitted the MS strains to female children with significantly greater fidelity than to male children [26–27,59,91] . Such gender-specific conservation was reported in American, Chinese and Japanese families. Nevertheless, the scientific explanation for this aspect has not been established. ●●Other factors
Apart from the aforementioned factors, certain additional influences such as antibiotic usage and mode of infant delivery may be involved in MS colonization. For instance, antibiotic usage has been reported to help decrease MS levels and caries [98–100] . Some researchers also discussed the decline in MS-associated caries as a consequence of the medical usage of antibiotics [99] , since only one single exposure to a short-term antibiotic
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Review Lapirattanakul & Nakano regimen could reduce the levels of salivary MS for a period of at least 3 months in children [100] . As for long-term utilization, long-term antibiotic therapy or the usage of multiple courses of antibiotics in children with some diseases have been reported to delay the acquisition of MS [66–67,101] . However, such benefits were proven to occur only during the active administration of the drugs [101] . On the other hand, contradictory results were also observed, that is, there was no significant difference in antibiotic utilization recorded from birth to 3 years of age between groups of children with and without MS colonization [79] . Actually, one study showed that children administered antibiotics during early childhood exhibited a higher MS prevalence than those who were untreated [102] . Regarding the mode of delivery, infants born either by vaginal delivery or Caesarean section (C-section) were compared for MS infection. However, inconsistency in the results from each study was observed. Li and coworkers initially introduced the idea that children born by C-section acquired MS significantly earlier and at higher levels than vaginally delivered children [97] . When the number of subjects increased, this group of researchers could also confirm such results as well as demonstrate the high fidelity of mother-to-child transmission in children who were born by C-section, since all MS genotypes colonized in these C-section babies were all found in their mothers [56] . However, some studies indicated no difference in MS infection rates between vaginally delivered children and Caesarean-born children [68,75] . Moreover, there is a recent work indicating that vaginally born children were 1.8-times more likely to have high MS scores than the C-section group [88] . Prevention of transmission It was scientifically proven that early infection by MS could lead to a high prevalence of dental caries in children [103] . Hence, it seems logical to assume that the strategies for reduction of maternal MS levels in order to impede MS transfer from mothers to children should be valuable. The first report in this regard was published in the early 1980s, in which an achievement in reduction of maternal MS levels from ≥106 to
