Investigating oral microbiome profiles in patients with cleft lip and palate compared with the healthy control.

BACKGROUND: Patients with cleft lip and palate (CLP) have an oronasal communication differed from the closed state in healthy individuals, leading to a unique oral microbiome. This study aimed to determine if variances in the oral microbiota persist among CLP patients who have received treatments for the closure of these fistulas compared to the microbiota of healthy individuals. METHODS: Saliva samples were collected from a cohort comprising 28 CLP patients (CLP group) and 30 healthy controls (HC group). Utilizing 16S rRNA sequencing on the Illumina NovaSeq platform, we conducted a comprehensive analysis of the diversity and composition of the oral microbiota. RESULTS: The analysis of the microbiota in the saliva samples revealed a total of 23 microbial phyla, 38 classes, 111 orders, 184 families, 327 genera and 612 species. The alpha diversity with microbial abundance and evenness indicated the significant difference between the CLP and HC groups. Principal coordinate analysis (PCoA) and the ADONIS test further supported the presence of distinct microorganisms between the two groups. The CLP group displayed elevated abundances of Neisseria, Haemophilus, Porphyromonas, and Granulicatella, as indicated by LefSe analysis. Conversely, Rothia, Veillonella, and Pauljensenia exhibited significant reductions in abundance in the CLP group. The results of the PICRUSt analysis indicated significant differences in the relative abundance of 25 KEGG pathways within the CLP group. Through Spearman correlation analysis, strong associations between Rothia, Veillonella, and Pauljensenia and 25 functional pathways linked to CLP were identified. CONCLUSION: Findings of this study offer a thorough comprehension of the microbiome profiles of CLP patients after the restoration of oronasal structure and are anticipated to present innovative concepts for the treatment of CLP.

Characterization and functional prediction of the dental plaque microbiome in patients with alveolar clefts.

Introduction: Alveolar cleft (AC) is a common congenital defect in people with cleft lip and palate (CLP). Alveolar bone grafting (ABG) is typically performed during adolescence, resulting in the fissure remaining in the mouth for a longer length of time. Patients with AC have a greater rate of oral diseases such as dental caries than the normal population, and the precise characteristics of the bacterial alterations caused by AC are unknown. Methods: We recruited a total of 87 subjects and collected dental plaque samples from AC adolescents (AAP), post-operative ABG adolescents (PAP), healthy control adolescents (CAP), AC young adults (AYP), post-operative ABG young adults (PYP), and healthy control young adults (CYP). The sequencing of 16S rRNA genes was performed. Results: The microbial composition of plaque from alveolar cleft patients differed significantly from age-matched healthy controls. Linear discriminant analysis effect size (LEfSe) analysis revealed that AAP was enriched for Neisseria, Haemophilus, Fusobacterium, Rhodococcus, Aggregatibacter, Gemella, and Porphyromonas, whereas AYP was enriched for Capnocytophaga, Rhodococcus, and Actinomyces-f0332. There were phenotypic differences in facultatively anaerobic, Gram-negative, Gram-positive, and oxidative stress tolerance between the AYP group with longer alveolar cleft and the healthy control group according to Bugbase phenotypic predictions. Alveolar bone grafting did not alter the functional phenotype of alveolar cleft patients but reduced the number of differential genera between alveolar cleft patients and healthy controls at both ages. Conclusions: Our study systematically characterized the supragingival plaque microbiota of alveolar cleft patients, post-alveolar bone grafting patients, and matched healthy controls in two ages to gain a better understanding of plaque ecology and microbiology associated with alveolar clefts.

A novel delivery system of probiotic drop and its effect on dental caries risk factors in cleft lip/palate children.

OBJECTIVE: The aim of the present study was to investigate the effect of the probiotic bacterium Lactobacillus reuteri on the levels of salivary mutans streptococci and lactobacilli in children with cleft lip/palate who used the novel drop containing L. reuteri. MATERIAL AND METHODS: The study group consisted of 19 operated cleft lip/palate children aged 4 to 12 years. The study had a double-blind, randomized crossover design, and the experimental period consisted of four consecutive time periods. During periods 2 and 4, consisting of 25 days each, parents were instructed that their children should consume 5 drops per day (0.15 to 0.20 g) of probiotic or placebo drops produced by the same manufacturer. The probiotic drop, BioGaia Reuteri drops, contained L. reuteri DSM 17938 and L. reuteri ATCC PTA 5289 (≥1 × 10(8) CFU/5 drops). The counts of salivary mutans streptococci and lactobacilli were evaluated using the CRT tests. The data were processed with NCSS 2007 software using chi-square and McNemar tests. RESULTS: There was no statistically significant (p > .05) reduction of salivary mutans streptococci and lactobacilli after 25 days of consumption of both drops. CONCLUSIONS: The novel drop containing L. reuteri may not reduce the levels of salivary mutans streptococci and lactobacilli in cleft lip/palate children.

Oral candidal colonization in cleft patients as a function of age, gender, surgery, type of cleft, and oral health.

PURPOSE: To assess the colonization rate of oral Candida species and the influence of age, gender, oral health status, number of surgeries, and type of cleft. PATIENTS AND METHODS: A prospective study of 60 patients with cleft and 60 control subjects was carried out at the Cleft Centre at King Abdullah University Hospital and the Maxillofacial Unit at Jordan University of Science and Technology between October 2007 and June 2008. Oral health was assessed using the Gingival, Plaque, and Decayed, Missing, and Filled (DMFT/dmft) indexes using World Health Organization criteria. A culture swab was obtained from the tongue and buccal and palatal mucosae. Candida albicans and other Candida species were identified using the germ tube test and the automated biochemical test panel VITEK. RESULTS: The colonization rate of Candida in patients with cleft (63.3%) was significantly higher than in healthy control subjects (18.3%). The colonization rate of Candida and the distribution of C albicans varied with age but were not significantly associated with gender in patients with cleft and healthy controls. The candidal colonization rate was highest in patients with cleft who had at least 3 surgeries (78.2%) and in patients with bilateral clefts (77.7%). Patients with cleft had a significantly poorer health status than healthy controls; however, this was not influenced by the type of the cleft or the number of surgeries. CONCLUSION: Patients with cleft had a significantly higher rate of oral candidal colonization compared with control subjects, which varied with age, type of cleft, and the number of surgical interventions. Oral health status was significantly poorer in patients with cleft.

Bacteria and salivary profile of adolescents with and without cleft lip and/or palate undergoing orthodontic treatment.

BACKGROUND: Patients with cleft lip and/or palate (CL&/P) experience a higher caries prevalence. This study aimed to determine if patients with CL&/P, undergoing and not undergoing orthodontic treatment, have a different salivary biochemical profile and different salivary levels of Mutans Streptococci (MS) and Lactobacilli (LB) compared to patients undergoing and not undergoing orthodontic treatment without CL&/P. METHODS: One hundred and ten subjects aged between 12 and 17 years were recruited into one of four different groups comprising two control groups and two treatment groups. The control groups comprised of subjects with and without CL&/P who were not undergoing orthodontic treatment. The treatment groups comprised of subjects with and without CL&/P undergoing orthodontic treatment. Regular reinforcement of oral hygiene instructions, dietary counselling and debridement, when necessary, were offered to subjects in the treatment groups following their orthodontic adjustment appointments. The salivary secretion time, pH of resting and stimulated saliva, salivary flow rate, buffering capacity, quantity of salivary MS and LB were measured. RESULTS: Subjects with CL&/P undergoing orthodontic treatment at the Children's Oral Health Service tended to present with microbiological and salivary profiles that were less favourable for caries development. There was a significant difference in the percentage of subjects with > or = 10(5) colony forming units (CFU)/mL of MS between the cleft treatment and non-cleft treatment groups. Subjects in the non-cleft treatment group had the highest percentage of subjects (86.7 per cent) with > or = 10(5) CFU/mL of MS whereas subjects in the cleft treatment group had the lowest percentage of subjects (60 per cent) with > or = 10(5) CFU/mL of MS. For LB, there were significantly higher percentages of subjects with > or =10(5) CFU/mL of LB in the non-cleft treatment (76.7 per cent) and cleft treatment (73.3 per cent) groups compared to the non-cleft control (46.7 per cent) and cleft control (40.0 per cent) groups. CONCLUSIONS: Regular oral hygiene reinforcement and dental health education appears to have a positive effect in reducing the percentage of subjects with > or = 10(5) CFU/mL of MS.

Investigating Oral Microbiome Profiles in Children with Cleft Lip and Palate for Prognosis of Alveolar Bone Grafting.

In this study, we sought to investigate the oral microbiota structure of children with cleft lip and palate (CLP) and explore the pre-operative oral bacterial composition related to the prognosis of alveolar bone grafting. In total, 28 patients (19 boys, 9 girls) with CLP who were scheduled to undergo alveolar bone grafting for the first time were recruited. According to the clinical examination of operative sites at the third month after the operation, the individuals were divided into a non-inflammation group (n = 15) and an inflammation group (n = 13). In all, 56 unstimulated saliva samples were collected before and after the operation. The v3-v4 hypervariable regions of the 16S rRNA gene were sequenced using an Illumina MiSeq sequencing platform. Based on the beta diversity of the operational taxonomic units (OTUs) in the inflammation and non-inflammation samples, the microbial variation in the oral cavity differed significantly between the two groups before and after the operation (P < 0.05). Analysis of the relative abundances of pre-operative OTUs revealed 26 OTUs with a relative abundance higher than 0.01%, reflecting a significant difference of the relative abundance between groups (P < 0.05). According to a principal component analysis of the pre-operative samples, the inflammation-related OTUs included Tannerella sp., Porphyromonas sp., Gemella sp., Moraxella sp., Prevotella nigrescens, and Prevotella intermedia, most of which were enriched in the inflammation group and showed a significant positive correlation. A cross-validated random forest model based on the 26 different OTUs before the operation was able to fit the post-operative status of grafted sites and yielded a good classification result. The sensitivity and specificity of this classified model were 76.9% and 86.7%, respectively. These findings show that the oral microbiota profile before alveolar bone grafting may be related to the risk of post-operative inflammation at grafted sites.

Dental caries, oral hygiene, and oral clearance in children with craniofacial disorders.

The reason that children with cleft palates tend to have a greater prevalence of tooth decay than normal children is unclear. We hypothesized that children with cleft palates would have increased oral clearance times for foods and, consequently, higher levels of caries and caries-associated micro-organisms than control children. Children aged 6-16 yrs, with (n = 81) or without (n = 61) cleft palates, were studied. Children with cleft palates had DMFT and dmft scores greater (p < 0.01) than those of the control group. The number of caries-associated organisms was greater in the saliva of the cleft palate children (all p < 0.001). The oral hygiene, plaque and gingival index scores were greater (p < 0.0001), oral clearance was longer (p < 0.01), and levels of sucrose and starch-derived saccharides higher (p < 0.01) in the cleft palate group. However, salivary concentrations of organic acids were lower in the children with craniofacial disorders, probably reflecting the altered physiology of the more mature dental biofilm. The longer oral clearance times of foods and the consequent generation of fermentable sugars from starches may contribute to the higher caries prevalence observed in children with cleft palates.

Candida albicans in patients with oronasal communication and obturator prostheses.

Patients using obturator prostheses often present denture-induced stomatitis. In order to detect the presence of oral Candida albicans in patients with oronasal communications and to evaluate the effectiveness of a topical antifungal treatment, cytological smears obtained from the buccal and palatal mucosa of 10 adult patients, and from the nasal acrylic surface of their obturator prostheses were examined. A therapeutic protocol comprising the use of oral nystatin (Mycostatin) and prosthesis disinfection with sodium hypochlorite was prescribed for all patients. Seven patients were positive for C. albicans in the mucosa, with 1 negative result for the prosthetic surface in this group of patients. Post-treatment evaluation revealed the absence of C. albicans on prosthesis surface and on the oral mucosa of all patients. The severity of the candidal infection was significantly higher in the palatal mucosa than in the buccal mucosa, but similar in the palatal mucosa and prosthesis surface, indicating that the mucosa underlying the prosthesis is more susceptible to infection. The therapeutic protocol was effective in all cases, which emphasizes the need for denture disinfection in order to avoid reinfection of the mucosa.

Staphylococcus aureus transmission through oronasal fistula in children with cleft lip and palate.

OBJECTIVE: To determine the presence of Staphylococcus aureus in a nasal flora and oral environment, the correlation between frequency of transmission of S. aureus and oronasal fistula size, and the pattern of methicillin resistance on S. aureus strains in children with cleft lip and palate (CLP). DESIGN: Thirty-two CLP children with and without oronasal fistulas, ranging in age from 5 to 13 years were examined for oronasal fistula presence and size. Stimulated saliva samples and nasal swab samples were taken and investigated for S. aureus presence. S. aureus presence and counts were correlated with fistula presence and size. RESULTS: Saliva samples showed statistical differences between the groups with and without oronasal fistulas with an area ranging from 0.80 to 28.26 mm2. The S. aureus counts were significantly higher (r = .535, p = .002) in saliva samples from children with larger oronasal fistula. The S. aureus count was not significantly different (r = -.013, p = .942) in nasal samples compared with oronasal fistula size. Methicillin resistance with disk-diffusion method was recorded as sensitive (> or =13 mm) in all S. aureus strains. CONCLUSIONS: The results of this study indicate a positive correlation between fistula size and S. aureus transmission to one oral environment through oronasal fistulae, and a positive correlation between frequency of S. aureus transmission and fistula size. All S. aureus strains were sensitive to methicillin. These results may have implications for preventive treatment of CLP children.

The bacteriology of children before primary cleft lip and palate surgery.

OBJECTIVE: Bacterial infections can complicate any surgery. Knowledge of potentially pathogenic bacterial flora in children with cleft lip and palate allows appropriate risk management, including the need for prophylactic antibiotics. This project reviewed the bacteriology of children before primary cleft lip and palate surgery. DESIGN: A retrospective study of the results of nose, throat, and ear microbiological swabs taken from children, aged 1 to 26 months, before repair of primary cleft lip, cleft palate, or both was carried out. Swabs with Staphylococcus aureus and beta-hemolytic streptococcus were considered positive. RESULTS: From October 1987 to May 2002, 321 primary cleft lip or palate operations were performed in 250 patients. Results from 326 sets of preoperative swabs were available, including five repeat sets from patients whose operations were postponed. There were 235 (72.1%) negative sets and 91 (27.9%) positive sets. Of the positive swabs, 86 sets grew S. aureus, and 10 sets grew beta-hemolytic streptococcus. CONCLUSIONS: Children with unrepaired cleft lip and palate have a significant risk of carrying S. aureus and a small risk of carrying beta-hemolytic streptococci. These risks need to be considered when deciding on protocols for preoperative bacteriology tests and prophylactic antibiotics.

Study of viridans streptococci and Staphylococcus species in cleft lip and palate patients before and after surgery.

OBJECTIVE: To determine the effect of surgery on types and colony count of Streptococcus and Staphylococcus species in cleft lip and palate (CLP) patients. DESIGN: Saliva samples were collected after the morning meal by placing a sterile cotton swab in the vestibule of the oral cavity from cleft lip and palate patients immediately preoperative and 12 weeks postoperative. Normal children were examined as a control group. Samples were cultured; Staphylococcus and Streptococcus isolates were identified and quantified. PATIENTS: Fifteen cleft lip and palate patients and 22 normal children, aged 3 to 39 months were examined. RESULTS: Streptococcus mitis biovar 1, Streptococcus salivarius and Streptococcus oralis of the viridans group of streptococci were the most commonly found in normal children, as well as in cleft lip and palate children. In the cleft lip and palate group, mean streptococcal count was 32.41 (29.80) and 46.46 (42.80) in the pre- and postoperative periods, respectively; in the normal group, the count was 20.93 (27.93) and 49.92 (34.72) at 0 week and 12 weeks, respectively. Staphylococcus aureus was the most common Staphylococcus species found in CLP patients, representing 47.4% postoperatively. In the cleft lip and palate children, mean staphylococcal count was 5.34 (8.13) and 0.56 (0.92) in the pre- and postoperative periods, respectively; in normal children, the count was 0.82 (1.98) and 0.60 (2.55) at 0 and 12 weeks, respectively. The differences were statistically significant only for the staphylococcal count between pre- and postoperative periods in children with cleft lip and palate as tested by analysis of variance (p < .05). CONCLUSIONS: Cleft lip and palate patients had more colonization by S. aureus compared with normal children, and the colony count decreased significantly following surgical repair of the cleft lip and palate.

Microbiota associated with residual clefts and neighboring teeth in patients with cleft lip, alveolus, and palate.

Twenty patients with residual clefts or pronounced soft tissue grooves, treated for uni- or bilateral cleft lip, alveolus, and palate were included in this study. Ten patients were recalled for dental prophylaxis at regular intervals, 10 patients were not. One microbiologic sample was obtained from the cleft area and two samples from a tooth adjacent to the cleft (sites adjacent and distant to the cleft). Between the recall and the nonrecall group there were notable differences in the presence of anaerobic Gram-negative organisms. Fusobacterium spp., Prevotella melaninogenica, and P. intermedia were more often found in nonrecall patients. While rarely seen in recall patients, spirochetes and motile rods were a common feature of nonrecall patients. The putative periodontal pathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis were not detected in either group. The differences between the recall and the nonrecall groups were more pronounced when the respective samples from teeth were related to each other than when the samples obtained from the clefts were compared. The cleft flora was less complex irrespective of how good maintenance was and resembled the flora of teeth of well-maintained patients. Samples from clefts were never Wolinella positive, and harbored significantly less Capnocytophaga and Actinomyces viscosus than samples from dental sites.

Prevalence of Streptococcus mutans and lactobacilli in 18-month-old children with cleft lip and/or palate.

The prevalence of Streptococcus mutans and lactobacilli was determined in 62 18-month-old Dutch children with a cleft lip and/or palate. Plaque and saliva samples were collected, a dental examination was performed, and the parents were interviewed with a structured questionnaire regarding general health, dietary habits, fluoride exposure, and socioeconomic class. Appropriate dilutions of the plaque and saliva samples were cultured on selective media to count all viable bacteria, S. mutans and lactobacilli. S. mutans was detected in the saliva of 45% of the children, and lactobacilli was detected in 16%. Also, S. mutans was detected in 48% of the plaque samples and lactobacilli in 8%. Of all of the variables examined, consumption of more than three snacks and beverages between main meals was significantly associated with presence of S. mutans in saliva. Preoperative infant orthopedic treatment (i.e., wearing an acrylic plate from shortly after birth) was significantly associated with presence of lactobacilli in saliva. The presence of S. mutans in the plaque samples was also significantly associated with presence of lactobacilli in saliva. These results indicate that children with oral cleft are at an increased risk of being infected by S. mutans and lactobacilli at a very early age. Such early colonization indicates a high risk for caries in the primary dentition.

Transmission of mutans streptococci between mothers and children with cleft lip and/or palate.

OBJECTIVE: The aim of this study was to investigate the transmission of Streptococcus mutans between children with cleft lip and/or palate and their mothers. DESIGN: Saliva samples of 21 mother-child pairs were collected and cultured on plates containing a selective growth medium for mutans streptocci. At least five separate colonies of each colony morphotype were isolated. A polymerase chain reaction (PCR) with randomly chosen primers was used to type the isolates. RESULTS: The number of morphotypes and PCR types was significantly lower in the children than in the mothers. Significant correlations were found between the number of morphotypes and PCR types, in the children as well as in the mothers. In only 38% of the mother-child pairs were the same PCR types found in mother and child. CONCLUSIONS: This suggests that S. mutans had been transmitted from mother to child in one-third of the population studied. No correlations were found among the number of colony-forming units, the number of colony-colony-morphotypes, and the number of PCR types of the mothers and transmission. Similar PCR types in mother and child were found significantly more often in children who had more than one PCR type. The results indicate that transmission of S. mutans from mother to child is not frequent in children with oral cleft. This may have consequences for preventive treatment of cleft lip and/or palate children and their mothers.

Clinical and microbiological evaluation of the periodontal status of children with unilateral complete cleft lip and palate.

OBJECTIVE: To compare periodontal conditions in children with and without cleft. DESIGN: Clinical examinations and microbiological analysis of 57 selected children, including 30 with unilateral complete cleft lip and palate (experimental group) and 27 without clefts (control group). SETTING: Hospital of Rehabilitation of Craniofacial Anomalies (HRCA) in Bauru, Sao Paulo, Brazil. PATIENTS, PARTICIPANTS: All children examined were healthy and between the ages of 5 and 6 years. RESULTS: The mean plaque index (PI) in the experimental group was higher (1.82 +/- 0.3) than in the control group (1.63 +/- 0.38), although this difference was not statistically significant. The mean gingival index (GI) in the experimental group (1.82 +/- 0.38) was found to be significantly higher (p <.05) than that of the control group (0.79 +/- 0.33). The cleft area in the experimental group, with a mean PI of 2.04 +/- 0.58 and mean GI of 1.11 +/- 0.26, compared with the posterior area, with a mean PI of 1.74 +/- 0.37 and mean GI of 1.04 +/- 0.26, showed a statistically significant difference only in the PI. Most of the children in both experimental and control groups presented a moderate PI degree (73.33% and 81.48%, respectively) and a high prevalence of mild gingivitis (53.33% and 70.37%, respectively). Analysis of the organisms showed that Prevotella nigrescens was detected in 16.67% of the experimental group and 11.11% of the control, whereas Porphyromonas gingivalis and Treponema denticola were not detected. CONCLUSION: Children with clefts showed greater gingival inflammation, despite the same amount of plaque and prevalence of microorganisms.

Candida albicans in patients with oronasal communication and obturator prostheses

Patients using obturator prostheses often present denture-induced stomatitis. In order to detect the presence of oral Candida albicans in patients with oronasal communications and to evaluate the effectiveness of a topical antifungal treatment, cytological smears obtained from the buccal and palatal mucosa of 10 adult patients, and from the nasal acrylic surface of their obturator prostheses were examined. A therapeutic protocol comprising the use of oral nystatin (Mycostatin®) and prosthesis disinfection with sodium hypochlorite was prescribed for all patients. Seven patients were positive for C. albicans in the mucosa, with 1 negative result for the prosthetic surface in this group of patients. Post-treatment evaluation revealed the absence of C. albicans on prosthesis surface and on the oral mucosa of all patients. The severity of the candidal infection was significantly higher in the palatal mucosa than in the buccal mucosa, but similar in the palatal mucosa and prosthesis surface, indicating that the mucosa underlying the prosthesis is more susceptible to infection. The therapeutic protocol was effective in all cases, which emphasizes the need for denture disinfection in order to avoid reinfection of the mucosa.

Os pacientes portadores de prótese obturadora freqüentemente apresentam estomatite protética. Com o objetivo de detectar a presença de Candida albicans oral em pacientes com comunicação oronasal e avaliar a eficácia de um tratamento tópico antifúngico foi realizada citologia esfoliativa da mucosa palatina e jugal e da superfície acrílica nasal da prótese obturadora. O protocolo terapêutico consistiu de nistatina (Mycostatin®) para tratamento da mucosa oral e uma solução de hipoclorito de sódio para desinfecção da prótese. Sete pacientes (70 por cento) apresentaram resultado positivo para C. albicans na mucosa, com um resultado negativo para a superfície protética neste grupo. A avaliação após o tratamento revelou ausência de C. albicans na mucosa oral de todos os pacientes, bem como na superfície protética. A infecção por C. albicans das mucosas jugal e palatina diferiram significantemente, enquanto que a mucosa palatina e a superfície protética apresentaram valores semelhantes. O grau de infecção da mucosa palatina foi significantemente maior quando comparado àquele da mucosa jugal e semelhante ao apresentado pela prótese, sugerindo que a mucosa subjacente à prótese é mais susceptível à infecção. O protocolo terapêutico foi efetivo em todos os casos, o que enfatiza a necessidade da desinfecção protética para se evitar a reinfecção da mucosa oral.