RESEARCH REPORTS P. Lif Holgerson*, L. Harnevik', O. Hernell2 A.C.R. Tanner Mode of Birth Delivery Affects and l. Johansson Oral microbiota in Infants University, 901 87 Umea, Sweden: Department of Clinical Sciences, Pediatrics Section, Umea University, Sweden; and Department of Molecular Genetics, The Forsyth Institut Cambridge, MA, and Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Bosto MA, USA; *corresponding author, perillalif(@odont.um J Dent Res9o(10):11831188 ABSTRACT INTRODUCTION Establishment of the microbiota of the gut has been shown to differ between infants delivered by e first exposure to micro-organisms in vaginally delivered infants occurs Caesarian section(C-section) and those delivered during passage through the birth canal, whereas the first exposure to bacte vaginally. The aim of the present study was to ria in infants born by Caesarian section( C-section)is from the skin of parents and health providers, and medical equipment. Different modes of delivery compare the oral microbiota in infants delivered lead to differences in the intestinal microbiota in infants(Penders et al., 2006 by these different routes. The oral biofilm was Dominguez-Bello et al., 2010). Vaginally bom children have been reported to assayed by the Human Oral Microbe Identification have a more diverse gut microbiota, whereas children born by C-section had Microarray(HOMIM) in healthy three-month-old infants, 38 infants borm by C-section, and 25 nigher numbers of Clostridium difficile and delaved acquisition of bifidobac infants delivered vaginally. Among over 300 bac teria and Escherichia coli(ahrme et al, 2005; Penders et al., 2006). In the oral cavity, mutans streptococci were detected more frequently and at a younger terial taxa targeted by the HOMIM microarray, age in children delivered by c-section than in those delivered vaginally ( Slackia exigua was detected only in infants deliv- et al, 2005). These authors hypothesized that C-section, compared with vaginal ered by C-section. Further, significantly more bac- birth, lowered the exposure to commensal, protective bacteria from the mother terial taxa were detected in the infants delivered vaginally(79 specieslspecies clusters)compared during birth, reducing the natural barrier to colonization by oral pathogens Acquisition of oral bacteria in early childhood results mainly from transmis with infants delivered by C-section(54 species/ sion from the mother (Kononen, 2000; Tanner et al., 2002), but there is less species clusters). Multivariate modeling revealed a strong model that separated the microbiota of information about other factors influencing establishment of the microbiota in he oral cavity than reported for the gut. Establishment of the gut microbiota C-section and vaginally delivered infants into two was found not to be a predetermined species-by-species succession, but rather tudy indicated differences in the oral microbiota a coordinated interplay between extemal and intemal factors(Fanaro et al in infants due to mode of delivery, with vaginally environment during birth, the mother's microbiota, and infant feeding method delivered infants having a higher number of taxa (Fallani et al, 2010). Internal factors included the developmental stage of the detected by the HOMIM microarray gastrointestinal tract and host factors(Benson et al., 2010) The aim of the present study was to compare oral microbiota, seeking dif- KEY WORDS: newborn, Caesarian section, vag- ferences in colonization patterns in infants delivered vaginally or by C-section. inal delivery, bacterial taxa, HOMIM, Slackia The human Oral Microbe Identification Microarray was used to detect bacte rial taxa STUDY POPULATION METHODS DOI:10.11770022034511418973 Received February 16, 2011; Last revision July 6, 2011 All mothers living in a small inland town and a coastal university city in Northem Sweden who had delivered a healthy baby in the previous 3 mos were invited to consent for their infant to participate in the study. From 300 invited women, A supplemental appendix to this article is published elec- 207 accepted (69%), and all infants delivered by C-section(n=41)and 26 tronicallyonlyathttp://jdr.sagepub.com/supplemental. andomly selected vaginally delivered infants were selected for microbia O Intemational American Associations for Dental Research analyses. Phone interviews were conducted with the non-participants, and the
1183 RESEARCH REPORTS Clinical DOI: 10.1177/0022034511418973 Received February 16, 2011; Last revision July 6, 2011; Accepted July 9, 2011 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research P. Lif Holgerson1 *, L. Harnevik1 , O. Hernell2 , A.C.R. Tanner3 , and I. Johansson1 1 Department of Odontology, Cariology Section, Umeå University, 901 87 Umeå, Sweden; 2 Department of Clinical Sciences, Pediatrics Section, Umeå University, Sweden; and 3 Department of Molecular Genetics, The Forsyth Institute, Cambridge, MA, and Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA, USA; *corresponding author, pernilla.lif@odont.umu.se J Dent Res 90(10):1183-1188, 2011 Abstract Establishment of the microbiota of the gut has been shown to differ between infants delivered by Caesarian section (C-section) and those delivered vaginally. The aim of the present study was to compare the oral microbiota in infants delivered by these different routes. The oral biofilm was assayed by the Human Oral Microbe Identification Microarray (HOMIM) in healthy three-month-old infants, 38 infants born by C-section, and 25 infants delivered vaginally. Among over 300 bacterial taxa targeted by the HOMIM microarray, Slackia exigua was detected only in infants delivered by C-section. Further, significantly more bacterial taxa were detected in the infants delivered vaginally (79 species/species clusters) compared with infants delivered by C-section (54 species/ species clusters). Multivariate modeling revealed a strong model that separated the microbiota of C-section and vaginally delivered infants into two distinct colonization patterns. In conclusion, our study indicated differences in the oral microbiota in infants due to mode of delivery, with vaginally delivered infants having a higher number of taxa detected by the HOMIM microarray. KEY WORDS: newborn, Caesarian section, vaginal delivery, bacterial taxa, HOMIM, Slackia exigua. Introduction T he first exposure to micro-organisms in vaginally delivered infants occurs during passage through the birth canal, whereas the first exposure to bacteria in infants born by Caesarian section (C-section) is from the skin of parents and health providers, and medical equipment. Different modes of delivery lead to differences in the intestinal microbiota in infants (Penders et al., 2006; Dominguez-Bello et al., 2010). Vaginally born children have been reported to have a more diverse gut microbiota, whereas children born by C-section had higher numbers of Clostridium difficile and delayed acquisition of bifidobacteria and Escherichia coli (Ahrné et al., 2005; Penders et al., 2006). In the oral cavity, mutans streptococci were detected more frequently and at a younger age in children delivered by C-section than in those delivered vaginally (Li et al., 2005). These authors hypothesized that C-section, compared with vaginal birth, lowered the exposure to commensal, protective bacteria from the mother during birth, reducing the natural barrier to colonization by oral pathogens. Acquisition of oral bacteria in early childhood results mainly from transmission from the mother (Könönen, 2000; Tanner et al., 2002), but there is less information about other factors influencing establishment of the microbiota in the oral cavity than reported for the gut. Establishment of the gut microbiota was found not to be a predetermined species-by-species succession, but rather a coordinated interplay between external and internal factors (Fanaro et al., 2003; Penders et al., 2006). External factors for the gut microbiota included the environment during birth, the mother’s microbiota, and infant feeding method (Fallani et al., 2010). Internal factors included the developmental stage of the gastrointestinal tract and host factors (Benson et al., 2010). The aim of the present study was to compare oral microbiota, seeking differences in colonization patterns in infants delivered vaginally or by C-section. The human Oral Microbe Identification Microarray was used to detect bacterial taxa. Study Population & Methods Study Population All mothers living in a small inland town and a coastal university city in Northern Sweden who had delivered a healthy baby in the previous 3 mos were invited to consent for their infant to participate in the study. From 300 invited women, 207 accepted (69%), and all infants delivered by C-section (n = 41) and 26 randomly selected vaginally delivered infants were selected for microbial analyses. Phone interviews were conducted with the non-participants, and the Mode of Birth Delivery Affects Oral Microbiota in Infants
1184 Lif Helgerson et al. J Dent Res 90(10)2011 only reason given for non-participation was lack of time. The or later than gestational wk 37 only was also run. Variables were study was approved by The Regional Ethical Review Board, autoscaled to unit variance, and cross-validated prediction ofY Umea, Sweden, and participating mothers signed informed con- calculated(Wold, 1978). Cross-validation was done by a sys- sent at recruitment tematic prediction of 1/th of the data by the remaining 6/7th of Mode of delivery(C-section or vaginal), intravenous treat- the data. The importance of each x-variable in the model is ment with antibiotics during delivery, and body weight and given by a variable importance in projection(VIP)value. VIP> length were checked against medical records. The mothers com- 1.0 was considered influential and ViP> 1.5 highly influential leted a questionnaire on other possible confounders, such as ( Sjostrom et al, 1986). The R2-and Q -values give the capacity health issues(allergy, infections, stomach problems ) the infant's of the x-variables to explain(R)and predict (Q)the outcome use of antibiotics, feeding mode(breast-or bottle-fed), use of a pacifier, and the presence of teeth. RESULTS Microbiota by 16S rRNA Probes in HOMIM Microarray Cohort Descripti We collected oral biofilm samples by carefully swabbing the There were no differences by gender or by other characteristics, cheeks,tongue, and alveolar ridges. DNA was purified from including breast-feeding, between infants born vaginally and samples with the use of the Gen Elute Bacterial Genomic DNA those bom by C-section(Table 1). Two infants were born in ges- kit(Sigma Aldrich, St. Louis, MO, USA) to obtain 60-1220 ng tational wk 35(one delivered by C-section and one by the vaginal DNA, which exceeded the amount required for the microarray route), whereas all other infants were born in gestational wk or assay. Four samples were excluded because of low yield after later. All infants were healthy at birth and at 3 mos of age. None DNA extraction, leaving 38 and 25 of the samples from of the infants had ever received antibiotic treatment, and none was C-section and vaginal delivery groups, respectively ever given supplements containing probiotic bacteria. With the The purified DNA of samples was assayed with 422 oligo- exception of 15 mothers who received intravenous antibiotics in nucleotide probes to the 16S rRNa gene targeting more than association with a C-section because of an acute clinical compli 300bacterialtaxaintheHomiMmicroarray(http://mim.forcationnonehadantibioticsatdeliveryTherewerenosignificant syth. org/homim. html). Samples were analyzed at the homim differences between participating and non-participating infants in microarray facility at The Forsyth Institute, Cambridge, MA, length and weight at birth and at 3 mos of age, or in the socio- USA(Colombo et al, 2009). Hybridization signals were read on economic variables of their families(data not shown) a six-level scale(0-5), with a lower limit of detection of 10 cells Colombo et al., 2009) Bacteria Detected by HOMIM Microarray Statistical procedures There was reactivity to 85 of the 300 taxa in the HOMIM micro- array in oral biofilms of three-month-old infants(Appendix body length and weight at birth and at 3 mos were averaged among Table). Bacteria detected belonged to 6 phyla or divisions, and infants delivered by the two birth delivery modes. Differences approximately half of the taxa detected belonged in Firmicutes, Dichotomized scores from the HOMIM microarray analyses were infants(Table 2). Other genera detected in 80 to 99%of all used. Lack of signal was set to 0, and all signal levels I to children were Actinomyces. Gemella and Veillonella. a smaller Differences n prevalence distribution between groups were tested proportion of the infants (<15% of the combined groups) had th a Chi? test. The False Discovery Rate method was used to species in the genera Bacteroides, Selenomonas, Aggregatibacter identify a p-value with less than one false rejection of HO when true Kingella, Neisseria, and the TM7 division. (p <0.005). Thus, a p-value < 0.005 was considered statistically Species or species clusters detected in all infants were Streptococcus Cluster Il, Streptococcus Cluster Ill, Streptococcus Multivariate partial least-squares discriminant analysis anginosus/intermedius, Streptococcus oralis(Append (PLS-DA)modeling was performed (SIMCA P+, version 12.0, Table). Species detected in 2 80% of all children were Umetrics AB, Umea, Sweden)as described(Sjostrom et al, Streptococcus Cluster I, Streptococcus mitis biovar 2, 986: Bylesjo et al, 2006). In contrast to traditional regression Streptococcus australis, Streptococcus parasanguinis I and models, the PLS-DA technique, which defines the maximum Actinomyces gerensceriae, Gemella hemolysans, veillonella separation between class members(here mode of delivery) in atypical, and Veillonella parvula Species detected in only a few the data, is suitable for data where the number of observations is (<15%)infants included Streptococcus sanguinis and smaller than the number of variables, and where the independent Streptococcus mutans, species of Neisseria, Aggregatibacter, variables co-vary. Dichotomous HOMIM signals, and the and Kingella, and Actinomyces naeslundii genospecies and selected individual characteristics, gender, weight and length at (Actinomyces clusters I and Il, respectively)(Appendix Table) birth and 3 mos, gestational wks at delivery, treatment antibiotics during delivery, feeding mode(bottle- or breast-fed), use of pacifier, presence and number of teeth, and town of resi- Species Distribution by Mode of Delivery dence built the X-block, and mode of delivery the Y-block(out- There were higher numbers of taxa detected by the microarray come). An identical model including breast-fed infants born in in swabs from infants delivered vaginally (79 species/clusters)
1184 Lif Holgerson et al. J Dent Res 90(10) 2011 only reason given for non-participation was lack of time. The study was approved by The Regional Ethical Review Board, Umeå, Sweden, and participating mothers signed informed consent at recruitment. Mode of delivery (C-section or vaginal), intravenous treatment with antibiotics during delivery, and body weight and length were checked against medical records. The mothers completed a questionnaire on other possible confounders, such as health issues (allergy, infections, stomach problems), the infant’s use of antibiotics, feeding mode (breast- or bottle-fed), use of a pacifier, and the presence of teeth. Microbiota by 16S rRNA Probes in HOMIM Microarray We collected oral biofilm samples by carefully swabbing the cheeks, tongue, and alveolar ridges. DNA was purified from samples with the use of the Gen Elute Bacterial Genomic DNA kit (Sigma Aldrich, St. Louis, MO, USA) to obtain 60-1220 ng DNA, which exceeded the amount required for the microarray assay. Four samples were excluded because of low yield after DNA extraction, leaving 38 and 25 of the samples from C-section and vaginal delivery groups, respectively. The purified DNA of samples was assayed with 422 oligonucleotide probes to the 16S rRNA gene targeting more than 300 bacterial taxa in the HOMIM microarray (http://mim.forsyth.org/homim.html). Samples were analyzed at the HOMIM microarray facility at The Forsyth Institute, Cambridge, MA, USA (Colombo et al., 2009). Hybridization signals were read on a six-level scale (0-5), with a lower limit of detection of 104 cells (Colombo et al., 2009). Statistical Procedures Body length and weight at birth and at 3 mos were averaged among infants delivered by the two birth delivery modes. Differences between means were tested by two-sided, independent t tests. Dichotomized scores from the HOMIM microarray analyses were used. Lack of signal was set to 0, and all signal levels ≥ 1 to 1. Differences in prevalence distribution between groups were tested with a Chi2 test. The False Discovery Rate method was used to identify a p-value with less than one false rejection of H0 when true (p 1.0 was considered influential and VIP ≥ 1.5 highly influential (Sjöström et al., 1986). The R2 - and Q2 -values give the capacity of the x-variables to explain (R2 ) and predict (Q2 ) the outcome. Results Cohort Description There were no differences by gender or by other characteristics, including breast-feeding, between infants born vaginally and those born by C-section (Table 1). Two infants were born in gestational wk 35 (one delivered by C-section and one by the vaginal route), whereas all other infants were born in gestational wk 37 or later. All infants were healthy at birth and at 3 mos of age. None of the infants had ever received antibiotic treatment, and none was ever given supplements containing probiotic bacteria. With the exception of 15 mothers who received intravenous antibiotics in association with a C-section because of an acute clinical complication, none had antibiotics at delivery. There were no significant differences between participating and non-participating infants in length and weight at birth and at 3 mos of age, or in the socioeconomic variables of their families (data not shown). Bacteria Detected by HOMIM Microarray There was reactivity to 85 of the 300 taxa in the HOMIM microarray in oral biofilms of three-month-old infants (Appendix Table). Bacteria detected belonged to 6 phyla or divisions, and approximately half of the taxa detected belonged in Firmicutes, particularly Streptococcus species, which were detected in all infants (Table 2). Other genera detected in 80 to 99% of all children were Actinomyces, Gemella, and Veillonella. A smaller proportion of the infants (< 15% of the combined groups) had species in the genera Bacteroides, Selenomonas, Aggregatibacter, Kingella, Neisseria, and the TM7 division. Species or species clusters detected in all infants were Streptococcus Cluster II, Streptococcus Cluster III, Streptococcus anginosus/intermedius, and Streptococcus oralis (Appendix Table). Species detected in ≥ 80% of all children were Streptococcus Cluster I, Streptococcus mitis biovar 2, Streptococcus australis, Streptococcus parasanguinis I and II, Actinomyces gerensceriae, Gemella hemolysans, Veillonella atypical, and Veillonella parvula. Species detected in only a few (< 15%) infants included Streptococcus sanguinis and Streptococcus mutans, species of Neisseria, Aggregatibacter, and Kingella, and Actinomyces naeslundii genospecies1 and 2 (Actinomyces clusters I and II, respectively) (Appendix Table). Species Distribution by Mode of Delivery There were higher numbers of taxa detected by the microarray in swabs from infants delivered vaginally (79 species/clusters)
J Dent Res 90(10)2011 Mode of Delivery and Oral Microbiota 1185 Table 1. Gender Proportions, Body Weight and Length, and Feeding Method for Infants Delivered Vaginally or by Caesarian Section Caesarian Section n=38 Boys/Girls(numbers) 16/10 19/21 0.264 51.1(50.0-522) 49.2(484-50.0 61.3(60.4-624 60.2(592-60.8) 0.100 Weight (g) 3603{3394-3813) 3425{3190-3660 0.202 at 3 mos of ag 6364(5986-6742) 6069{5807-6331 0.22 Breast-fed at 3 mos of age(%)3 sively or partially 0.836 Not at all 8 le missing values. Oral from four infants, three born by C-section and one vaginally, were not analyzed by microarray, because of low DNA yield 2Mean(95%CI limits). Differences of means were tested with Student's independent t test after confirmation of a normal distribution Differences in proportions were tested with the Chi2-test Table 2. Proportions (%)of Three-month-old Infants with a Positive HOMIM Signal by Genus Phylogenetic Group and Ger Vaginal Delivery(%) Caesarian Section(%) Actinobacteria 92.0 81.6 65.8 Bacteroides 8.0 0.076 16.0 0.0 0.011 0.0 Prevotella Firmicutes Eubacterium 13.2 0.752 Gemella 960 100.0 0.214 granulicatella 36.0 18.4 12.0 Solobacterium 20.0 0.293 0.055 Fusobacterium 44.0 34.2 0.434 Leptotrichia 44.0 0.006 ggregatibacter 0.363 280 8.0 0.0 0.076 5.3 TMZ Division 12.0 0.0 0.029 than in infants delivered by C-section (54 species/clusters) detected more frequently from C-section compared with vagi (p=0.001) Of the species or clusters detected, 31 were detected nally born infants were Slackia exigua (p <0.001)and only in infants born vaginally, compared with 6 species or Lactobacillus Cluster I (p <0.001)(Fig ) Haemophilus parain clusters that were detected only in C-section infants(Appendix fluenzae(p=0.005) was detected more frequently from vagi 1) nally delivered compared with C-section infants (Fig) The detection frequencies of 22 species and 2 clusters dif- Additional species/clusters displayed marginal fered between modes of infant delivery. Species or clusters els(p-values between 0.02 and 0.006; Fig
J Dent Res 90(10) 2011 Mode of Delivery and Oral Microbiota 1185 than in infants delivered by C-section (54 species/clusters) (p = 0.001). Of the species or clusters detected, 31 were detected only in infants born vaginally, compared with 6 species or clusters that were detected only in C-section infants (Appendix Fig. 1). The detection frequencies of 22 species and 2 clusters differed between modes of infant delivery. Species or clusters detected more frequently from C-section compared with vaginally born infants were Slackia exigua (p < 0.001) and Lactobacillus Cluster I (p < 0.001) (Fig.). Haemophilus parainfluenzae (p = 0.005) was detected more frequently from vaginally delivered compared with C-section infants (Fig.). Additional species/clusters displayed marginal significance levels (p-values between 0.02 and 0.006; Fig.). Table 1. Gender Proportions, Body Weight and Length, and Feeding Method for Infants Delivered Vaginally or by Caesarian Section Vaginal Delivery n = 251 Caesarian Section n = 381 p-value Boys/Girls (numbers) 16/10 19/21 0.264 Length (cm)2 at birth 51.1 (50.0−52.2) 49.2 (48.4−50.0) 0.849 at 3 mos of age 61.3 (60.4−62.4) 60.2 (59.2−60.8) 0.100 Weight (g)2 at birth 3603 (3394−3813) 3425 (3190−3660) 0.202 at 3 mos of age 6364 (5986−6742) 6069 (5807−6331) 0.221 Breast-fed at 3 mos of age (%)3 Exclusively or partially 27 32 0.836 Not at all 73 68 1 Numbers vary slightly for various analyses due to single missing values. Oral samples from four infants, three born by C-section and one vaginally, were not analyzed by microarray, because of low DNA yield. 2 Mean (95% CI limits). Differences of means were tested with Student’s independent t test after confirmation of a normal distribution. 3 Differences in proportions were tested with the Chi2 -test. Table 2. Proportions (%) of Three-month-old Infants with a Positive HOMIM Signal by Genus Phylogenetic Group and Genus Vaginal Delivery(%) Caesarian Section(%) p-value Actinobacteria Actinomyces 92.0 81.6 0.247 Rothia 68.0 65.8 0.856 Bacteroidetes Bacteroides 8.0 0.0 0.076 Capnocytophaga 16.0 0.0 0.011 Porphyromonas 4.0 0.0 0.214 Prevotella 40.0 7.9 0.002 Firmicutes Eubacterium 16.0 13.2 0.752 Gemella 96.0 100.0 0.214 Granulicatella 36.0 60.5 0.057 Lactobacillus 16.0 63.2 0.000 Parvimonas 4.0 18.4 0.093 Selenomonas 12.0 10.5 0.856 Solobacterium 20.0 10.5 0.293 Streptococcus 100 100 1.000 Veillonella 84.0 97.4 0.055 Fusobacteria Fusobacterium 44.0 34.2 0.434 Leptotrichia 44.0 13.2 0.006 Proteobacteria Aggregatibacter 4.0 0.0 0.214 Campylobacter 40.0 28.9 0.363 Haemophilus 28.0 7.9 0.033 Kingella 8.0 0.0 0.076 Neisseria 8.0 5.3 0.663 TM7 Division 12.0 0.0 0.029
1186 Lif Helgerson et al J Dent Res 90(10)2011 saskia exigua was detected exclusively, and in LaciobacWus Custer i Streptococcus parasanguinis i and r prevalence, in infants delivered by C-section. These findings indicate that there were differences in the microbiota of the oral Bundobactenum animals ss. animals, Brdobactenwm laces delivery method, as has been reported for TM7]G-1)sp HoT 347 morea haemolysin the microbiota of the lower gastrointestinal tract(Penders et al, 2006; Domingue Bello et al., 2010). ahonen marty In the current study, 85 species or spe- Prewotewa loeschew, Prevotella sp HOT 472(clone GU027) cies clusters out of the approximately 300 taxa evaluated by the HOMIM Capnocytophaga granulosa, Cano aga sp, HOT 326 microarray were detected in the three- month-old infants. This is fewer bacteri taxa than reported for adults in whom Leptotmchia buccalis, L goodfellowi, Sneathia sanguinegens approximately 65 to 70% of the microar ray species were detected by the same assay( Colombo et al, 2009: Preza et al. 2009). While there are no direct com- Reactivity to 24 probes (out of 85 probe reactions) that differed significantly (p parisons between the numbers of taxa 0.005)or marginally (p the infant's saliva, mode of feeding, and microbial cross-talkIn 1.5)with being born by C-section were Slackia exigua the neonate, oral bacterial colonization starts with streptococci Lactobacillus Cluster I, veillonella sp. EF509966, Veillonella from the viridans group(Pearce et al., 1995; Kononen, 2000) atypical, and K parvula, and those associated with being vagi- whereas significant colonization of anaerobes was not detected ally delivered were S sanguinis, Streptococcus sp HOT 058, in infants before 2 mos of age(Kononen, 2000).While there are hominis (Table 3). The model rema strong(R=0.693, Q2=0.496), and the same taxa remained no comparable data with HOMIM in infants, the present fre- quent detection of species in Firmicutes, and particularly within rongly influential when the two pre-term (gestational wk 35) the genus Streptococcus, is consistent with previous reports of and all formula-fed infants were excluded oral colonization by streptococci in infants(Pearce et al., 1995 Kononen, 2005). It is unlikely that these species are transients, DISCUSSION considering the detection threshold of about 10 cells for the HOMIM microarray, indicating that species detection in this The present study investigated the oral microbiota in infants assay likely reflects colonization and growth (Paster and ferences associated by birth delivery mode. Higher numbers of mothers with antibiotics during delivery was not influent, the delivered vaginally or by C-section to evaluate if there were dif- Dewhirst, 2009; Olson et al., 2011). Notably, treatment of taxa were detected among infants delivered vaginally, compared the oral microbiota in three-month-old infants. with those delivered by C-section, with probes to the 16s rrna The present dataset was characterized by a larger number of of cultivated and uncultivated oral bacteria in a microarray variables than study participants, and by the presence of species format(HOMIM: Paster and Dewhirst, 2009 ). Further, the that might be interdependent based on shared environmental esults indicated differences in the microbiota depending on needs or inter-species co-aggregation. Under these conditions, delivery method, including a novel finding that Slackia exigua the multivariate PLS-DA method is suitable to search for subject
1186 Lif Holgerson et al. J Dent Res 90(10) 2011 PLS-DA Multivariate Modeling of HOMIM 16S rRNA-based Microarray Data A model with two significant components was obtained by PLS-DA modeling with mode of delivery as outcome (y-variable) and dichotomized HOMIM microarray signals as the x-block, including selected individual characteristics (see statistics) as potential confounders. This model virtually clustered infants delivered by C-section separately from those delivered vaginally (Appendix Fig. 2). The multivariate model, which had an explanatory and predictive capacity of 62% (R2 = 0.618) and 44% (Q2 = 0.0.457), respectively, confirmed associations found in the univariate analyses. Species strongly associated (VIP ≥ 1.5) with being born by C-section were Slackia exigua, Lactobacillus Cluster I, Veillonella sp. EF509966, Veillonella atypical, and V. parvula, and those associated with being vaginally delivered were S. sanguinis, Streptococcus sp. HOT 058, and Cardiobacterium hominis (Table 3). The model remained strong (R2 = 0.693, Q2 = 0.496), and the same taxa remained strongly influential when the two pre-term (gestational wk 35) and all formula-fed infants were excluded. Discussion The present study investigated the oral microbiota in infants delivered vaginally or by C-section to evaluate if there were differences associated by birth delivery mode. Higher numbers of taxa were detected among infants delivered vaginally, compared with those delivered by C-section, with probes to the 16S rRNA gene of cultivated and uncultivated oral bacteria in a microarray format (HOMIM; Paster and Dewhirst, 2009). Further, the results indicated differences in the microbiota depending on delivery method, including a novel finding that Slackia exigua was detected exclusively, and in high prevalence, in infants delivered by C-section. These findings indicate that there were differences in the microbiota of the oral cavity depending on birth delivery method, as has been reported for the microbiota of the lower gastrointestinal tract (Penders et al., 2006; DominguezBello et al., 2010). In the current study, 85 species or species clusters out of the approximately 300 taxa evaluated by the HOMIM microarray were detected in the threemonth-old infants. This is fewer bacterial taxa than reported for adults in whom approximately 65 to 70% of the microarray species were detected by the same assay (Colombo et al., 2009; Preza et al., 2009). While there are no direct comparisons between the numbers of taxa detected by HOMIM in infants and those in adults, a lower species diversity of infants compared with individuals in later ages is consistent with separate reports for infants and adults (Könönen, 2000; Hao and Lee, 2004; Morelli, 2008). The lower number of taxa detected in oral biofilms from C-section compared with vaginally delivered infants is in accord with the lower diversity reported for the gut in samples taken immediately after C-section birth (DominguezBello et al., 2010). After birth, bacterial colonization of the gastrointestinal tract, including the mouth, is influenced by the transmission of bacteria from the environment and by genetic factors (Mandar and Mikelsaar, 1996; Dominguez-Bello et al., 2010). In the first few months of life, the major influences on the oral microbial succession are person-to-person transmission, composition of the infant’s saliva, mode of feeding, and microbial cross-talk. In the neonate, oral bacterial colonization starts with streptococci from the viridans group (Pearce et al., 1995; Könönen, 2000), whereas significant colonization of anaerobes was not detected in infants before 2 mos of age (Könönen, 2000). While there are no comparable data with HOMIM in infants, the present frequent detection of species in Firmicutes, and particularly within the genus Streptococcus, is consistent with previous reports of oral colonization by streptococci in infants (Pearce et al., 1995; Könönen, 2005). It is unlikely that these species are transients, considering the detection threshold of about 104 cells for the HOMIM microarray, indicating that species detection in this assay likely reflects colonization and growth (Paster and Dewhirst, 2009; Olson et al., 2011). Notably, treatment of the mothers with antibiotics during delivery was not influential on the oral microbiota in three-month-old infants. The present dataset was characterized by a larger number of variables than study participants, and by the presence of species that might be interdependent based on shared environmental needs or inter-species co-aggregation. Under these conditions, the multivariate PLS-DA method is suitable to search for subject Figure. Reactivity to 24 probes (out of 85 probe reactions) that differed significantly (p < 0.005) or marginally (p < 0.01) in three-month-old infants born vaginally or by Caesarian section. ***p ≤ 0.005, **p < 0.01 tested by Chi-square. No indication for p-values between 0.02 and 0.01
J Dent Res 90(10)2011 Mode of Delivery and Oral Microbiota 1187 Table 3. Variable Importance (P) for Bacteria Associated with Mode of Delivery from PLS-DA Multivariate Modeling Associated with Vaginal Delivery Associated with Caesarian Section Delivery acterial Group Bacterial grou 1.50 Slackia 3.26 Cardiobacterium hominis 1.44 Veillonella sp. EF509966(intestinal isolate 1.54 Prevotella Cluster P 1.43 Veillonella atypica, Veillonella parvula 1.51 Prevotella loescheii, Prevotella sp HOT 472 1.43 Streptococcus parasanguinis I and ll 1.34 Eubacterium[11][G-7l yurii 1. 43 Veillonella parvula 1.23 Catonella morbi, Catonella sp. HOT 164 43 Gemella haemolysin IM7[G-1]sp HOT 347, TM7[G-2] sp. HOT 350 1.43 Gemella morbillorum 1.26 1.41 Streptococcus australis 1.20 Leptotrichia buccalis, Leptotrichia goodfellowi, Sneath Bifidobacterium animalis nimalis. Bifidobacterium 1.13 lactis Bacteroidetes phylum 1.28 Streptococcus cristatus Kingella oralis, Eikenella sp HOT 0O 1.26 Selenomonas noxia sp HOT 326 Haemophilus sp HOT 035, HOT 036 1.25 Selenomonas sputigena, Selemonas sp HOT 143 1.03 1. 18 Aggregatibacter segnis, Aggregatibacter sp HOT 512 1.0 Prevotella nigrescens 1.05 Campylobacter concisus .03 Corynebacterium matruchotii 05 Streptococcus sp HOT 070, 071 Neisseria elong granulicatella elegans Capnocytophaga sputig streptococcus mutans Prevotella clusterⅣ Prevotella melaninogenica, Prevotella histicola Results are shown for a basic model including all infants with HOMIM microarray data ( n= 63)and (in bold) a second model restricted to breas 0435), length at birth, gestational weeks, and town of residence were associated with mode of birth (P 2 1.0), whereas in the PLS.- A model tricked to breast fed infants and infants born in or after gestational week 3(n=42)(model R2=0.693, Q2=0.496), only length at 3 mos was influential in addition to bacteric 'Unnamed taxa are identified by their Human Oral Taxon(HoT) number from HOMD(Dewhirst et al., 2010) Lactobacillus cluster I (probe w94)targets L. casei, L. paracasei, and L rhamnosus clustering and for identifying the variables characterizing clus- delivery group. S. exigua is a Gram-positive, strictly anaerobic, ers. With x-variables that were scaled to unit variance, and saccharolytic species that has been isolated in root canal infec cross-validation of the explanatory capacity to account for over- tions, periodontitis, extra-oral surgical wounds, and intestinal interpretation, there was good power to discriminate between abscesses(Abiko et al., 2010: Kim et aL., 2010). While it is not modes of delivery. The results were also stable after the model known why S. exigua was detected in the C-section but not in as restricted to infants born in wk 37 or later and those being the vaginal delivery group, it seems possible that the more breast-fed. With PLS-DA, several bacterial species differed diverse microbial biofilm of vaginally delivered infants could between infants based on their delivery method. The biological suppress establishment of this periodontitis-associated species nce of the species evaluated, and dete The association between S. exigua and mode of delivery requires species in the mouth in early infancy of infants delivered by further investigation. C-section, compared with those delivered vaginally, however, is In conclusion, the present study indicates a different coloni- unknown and will require longitudinal evaluation. It is neverthe- zation pattern in the oral cavity between three-month-old infants less notable that a greater microbial diversity in the intestine and delivered vaginally and those delivered by Caesarian section in the mouth has been reported to be associated with health The reasons for the differences are unknown, as is whether (Marsh, 2006: Preza et al., 2008; Sjogren et al, 2009; Turnbaugh these differences have long-term impact on the oral or general et aL, 2009: Luoto et al., 2011) health of the child. Possible reasons for differences will likely a difference based on mode of delivery was detection of include the relative influence of host receptor and mucosal and Slackia exigua in over 76% of the C-section infants(all with saliva immune phenotypes, and interactions with environmental HOMIM scores 22)compared with non-detection in the vaginal exposures
J Dent Res 90(10) 2011 Mode of Delivery and Oral Microbiota 1187 clustering and for identifying the variables characterizing clusters. With x-variables that were scaled to unit variance, and cross-validation of the explanatory capacity to account for overinterpretation, there was good power to discriminate between modes of delivery. The results were also stable after the model was restricted to infants born in wk 37 or later and those being breast-fed. With PLS-DA, several bacterial species differed between infants based on their delivery method. The biological significance of the species evaluated, and detection of fewer species in the mouth in early infancy of infants delivered by C-section, compared with those delivered vaginally, however, is unknown and will require longitudinal evaluation. It is nevertheless notable that a greater microbial diversity in the intestine and in the mouth has been reported to be associated with health (Marsh, 2006; Preza et al., 2008; Sjögren et al., 2009; Turnbaugh et al., 2009; Luoto et al., 2011). A difference based on mode of delivery was detection of Slackia exigua in over 76% of the C-section infants (all with HOMIM scores ≥ 2) compared with non-detection in the vaginal delivery group. S. exigua is a Gram-positive, strictly anaerobic, asaccharolytic species that has been isolated in root canal infections, periodontitis, extra-oral surgical wounds, and intestinal abscesses (Abiko et al., 2010; Kim et al., 2010). While it is not known why S. exigua was detected in the C-section but not in the vaginal delivery group, it seems possible that the more diverse microbial biofilm of vaginally delivered infants could suppress establishment of this periodontitis-associated species. The association between S. exigua and mode of delivery requires further investigation. In conclusion, the present study indicates a different colonization pattern in the oral cavity between three-month-old infants delivered vaginally and those delivered by Caesarian section. The reasons for the differences are unknown, as is whether these differences have long-term impact on the oral or general health of the child. Possible reasons for differences will likely include the relative influence of host receptor and mucosal and saliva immune phenotypes, and interactions with environmental exposures. Table 3. Variable Importance (VIP) for Bacteria Associated with Mode of Delivery from PLS-DA Multivariate Modeling Associated with Vaginal Delivery Associated with Caesarian Section Delivery Bacterial Group1 VIP Bacterial Group1 VIP Streptococcus sanguinis 1.50 Slackia exigua 3.26 Cardiobacterium hominis 1.50 Lactobacillus Cluster I3 1.85 Streptococcus anginosus, Streptococcus gordonii 1.44 Veillonella sp. EF509966 (intestinal isolate) 1.54 Prevotella Cluster I2 1.43 Veillonella atypica, Veillonella parvula 1.51 Prevotella loescheii, Prevotella sp. HOT 472 1.43 Streptococcus parasanguinis I and II 1.34 Eubacterium [11] [G-7] yurii 1.43 Veillonella parvula 1.23 Catonella morbi, Catonella sp. HOT 164 1.43 Gemella haemolysans 1.32 TM7 [G-1] sp. HOT 347, TM7 [G-2] sp. HOT 350 1.43 Gemella morbillorum 1.26 Haemophilus parainfluenzae 1.41 Streptococcus australis 1.20 Leptotrichia buccalis, Leptotrichia goodfellowii, Sneathia sanguinegens 1.31 Bifidobacterium animalis ss. animalis, Bifidobacterium lactis 1.13 Bacteroidetes phylum 1.28 Streptococcus cristatus 1.08 Campylobacter gracilis 1.27 Kingella oralis, Eikenella sp. HOT 009 1.03 Capnocytophaga granulose, Capnocytophaga sp. HOT 326 1.26 Selenomonas noxia 1.03 Haemophilus sp. HOT 035, HOT 036 1.25 Selenomonas sputigena, Selemonas sp. HOT 143 1.03 Kingella oralis 1.18 Aggregatibacter segnis, Aggregatibacter sp. HOT 512 1.03 Prevotella nigrescens 1.05 Campylobacter concisus 1.03 Corynebacterium matruchotii 1.05 Streptococcus sp. HOT 070, 071 1.02 Neisseria elongata 1.05 Granulicatella elegans 1.02 Fusobacterium periodontium 1.05 Capnocytophaga sputigena 1.05 Streptococcus mutans 1.04 Prevotella Cluster IV 1.01 Prevotella melaninogenica, Prevotella histicola 1.01 Results are shown for a basic model including all infants with HOMIM microarray data (n = 63) and (in bold) a second model restricted to breastfed infants and those born in gestational week 37 or later (n = 42). In the PLS-DA model including all 63 infants (model R2 = 0.620, Q2 = 0435), length at birth, gestational weeks, and town of residence were associated with mode of birth (VIP ≥ 1.0), whereas in the PLS-DA model restricted to breast-fed infants and infants born in or after gestational week 3 (n = 42) (model R2 = 0.693, Q2 = 0.496), only length at 3 mos was influential in addition to bacteria. 1 Unnamed taxa are identified by their Human Oral Taxon (HOT) number from HOMD (Dewhirst et al., 2010). 2 Prevotella cluster I (probe Y65) targets P. loescheii, Prevotella spp clone GU027, Prevotella spp strain C3MKM081, and Prevotella spp strain TFI B31FD (HOT numbers 317, 472, 658). 3 Lactobacillus cluster I (probe W94) targets L. casei, L. paracasei, and L. rhamnosus
1188 Lif Helgerson et al J Dent Res 90(10)2011 ACKNOWLEDGMENTS Kononen E(2000). Development of oral bacterial flora in young children. Ann med32:107-112. The present study was supported by grants from Vasterbotten Kononen E(2005). Anaerobes in the upper respiratory tract in infancy. County Council(TUA/FoU) and The Swedish Patent Revenue Foundation, and by a Public Health Service Grant DE-015847 Li Y, Caufield PW, Dasanayake AP, Wiener HW, Vermund SH(2005). Mode of delivery and other maternal factors influence the acquisition of (AT)from the National Institute of Dental and Craniofacial Streptococcus mutans in infants. J Dent Res 84: 806-811 Research, USA, and by Henning and Johan Throne-Holst's Luoto R, Kalliomaki M, Laitinen K, Delzenne NM, Cani PD, Salminen S, Foundation(PL). Dr. Conny Wikstrom, Umetrics, Sweden, is et al. (2011). Initial dietary and microbiological environments deviate acknowledged for expertise support in PLS-DA. The authors in normal-weight compared to overweight children at 10 years of age. J Pediatr Gastroenterol Nutr 52- 90-95 clare no potential conflicts of interest with respect to the Mandar R. Mikelsaar M(1996). Transmission of mother 's microflora to the authorship and/or publication of this article newbom at birth Biol Neonate 69: 30-35 Marsh PD (2006). Dental plaque as a biofilm and a microbial community plications for health and disease. BMC Oral Health 6(Suppl REFERENCES Abiko Y, Sato T, Mayanagi G, Takahashi N(2010). Profiling of subgingival Morelli L(2008). Postnatal development of intestinal microflora as influ- plaque biofilm microflora from periodontally healthy subjects and from enced by infant nutrition. J Nutr 138: 1791S-1795S using quantitative real-time PCR. J Olson JC, Cuff CE. Lukomski S, Lukomska E, Canizales Y, Wu B. et al. Periodontal Res 45: 389-395 (2011). 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1188 Lif Holgerson et al. J Dent Res 90(10) 2011 Acknowledgments The present study was supported by grants from Västerbotten County Council (TUA/FoU) and The Swedish Patent Revenue Foundation, and by a Public Health Service Grant DE-015847 (AT) from the National Institute of Dental and Craniofacial Research, USA, and by Henning and Johan Throne-Holst’s Foundation (PL). Dr. Conny Wikström, Umetrics, Sweden, is acknowledged for expertise support in PLS-DA. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article. References Abiko Y, Sato T, Mayanagi G, Takahashi N (2010). Profiling of subgingival plaque biofilm microflora from periodontally healthy subjects and from subjects with periodontitis using quantitative real-time PCR. J Periodontal Res 45:389-395. Ahrné S, Lönnermark E, Wold AE, Aberg N, Hesselmar B, Saalman R, et al. (2005). Lactobacilli in the intestinal microbiota of Swedish infants. Microbes Infect 7:1256-1262. 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