Study of Viridans Streptococci and Staphylococcus Species in Cleft Lip and Palate Patients Before and After Surgery ERRY MOCHAMAD ARIEF, D.D.S., M.SC. (PERIODONTICS) ZEEHAIDA MOHAMED, M.D., M.PATH. FAUZIAH MOHAMAD IDRIS, M.D., M.PATH. 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 preand 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. KEY WORDS: cleft lip and palate, Staphylococcus, viridans streptococci
Cleft lip and palate (CLP) is a congenital malformation that occurs in the embryonic and early fetal stages and represents the most common congenital deformity of the head and neck (Medeiros et al., 2000). The cleft lip and palate creates communication between the nasopharyngeal space and the oral cavity, which may predispose to alteration of normal flora at both sites. Viridans streptococci were the first persistent colonizer of the human mouth (Pearce et al., 1995; Kononen et al., 1999), and Streptococcus, Staphylococcus, Veilonella, and Neisseria species were con-
sistently found toward the end of the first year of life (Schuster, 1999). Pearce et al. (1995) found that Streptococcus mitis biovar 1 and Streptococcus oralis composed 55.0% of the pioneer Streptococcus isolated from neonates. Streptococcus salivarius constituted 25.3% of the isolates, whereas Streptococcus angiosus, Streptococcus mitis biovar 2, Streptococcus sanguis and Streptococcus gordonii accounted collectively for 11.4%. Dental caries is known to be prevalent in CLP patients. Bokhout et al., (1997) determined the incidence of dental caries in primary dentition of these children. Dental caries occurred in 30.9% of children with oral cleft and 6.5% in the control group. The overall incidence of manifest caries was significantly higher for the oral cleft children, with a crude incidence rate ratio of 9.25 in oral cleft children compared with the control group. The development of dental caries involves multiple factors, and oral streptococci play a significant role in the initiation process (Samarayanake, 2002). The aim of this study is to determine the effect of surgery (palatoplasty and lip repair) on the types and the colony count of Streptococcus and Staphylococcus species in cleft lip and cleft palate patients.
Dr. Arief is Lecturer, School of Dental Sciences; Dr. Zeehaida is Lecturer, Microbiology Department, School of Medical Sciences; and Dr. Fauziah is Lecturer and Head of Microbiology Department, School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu, Kelantan, Malaysia. This paper was presented orally at the 7th National Conference on Medical Sciences, 17–18 May 2002, Kota Bharu, Malaysia. This study was supported by IRPA short term, USM, Grant 6131144. Submitted June 2004; Accepted September 2004. Address correspondence to: Dr. Erry Mochamad Arief, D.D.S., M.Sc. (Periodontics), School of Dental Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian Kota Bharu, Kelantan, Malaysia. E-mail
[email protected]. 277
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TABLE 1 The isolation frequency of gram-positive cocci oral flora in pre- and postoperative cleft lip and palate (CLP) patients, and in normal children at 0 and 12 weeks Normal Children Organism
S. aureus S. mutans S. mitis S. mitis biovar 1 S. mitis biovar 2 S. salivarius S. oralis S. intermedius S. acidominimus A. viridans E. faecium S. bovis biovar 2 S. morbillorum S. hemolysian S. sanguis
0 week (%)
12 weeks (%)
7.7 3.3 3.3 20.3 3.3 23.3 16.7 3.3 3.3 13.3 0.0 3.3 0.0 3.3 3.3
0.0 6.3 0.0 18.7 6.3 24.9 37.5 0.0 0.0 0.0 0.0 0.0 6.3 0.0 0.0
CLP Patients Preoperative Postoperative 0 week (%) 12 weeks (%)
47.4 3.0 6.0 25.0 0.0 21.0 21.0 3.0 6.0 9.0 3.0 3.0 0.0 0.0 0.0
0.0 4.7 0.0 28.7 4.7 23.8 28.7 0.0 4.7 0.0 0.0 0.0 0.0 4.7 0.0
METHOD All CLP patients who were admitted to a tertiary teaching center for palatoplasty and lip repair were selected for the study, except for those who were diagnosed with isolated cleft lip, those who wore acrylic plates, those who were on antibiotics the preceding 5 weeks, and those with associated congenital abnormalities. In this prospective study, 17 CLP patients of both sexes aged 3 to 39 months were examined immediately preoperative and at 12 weeks postoperative. Twentytwo normal children, matched for sexes and ages, were examined as a control group. Saliva samples were collected after morning meals by placing a sterile cotton swab in the vestibule of the mouth. The samples were inoculated on blood agar and incubated at 378C for 48 hours. Staphylococcal and streptococcal isolates were identified to the species level. Catalase-negative and optochin-resistant Streptococcus colonies were further identified by API 20 Strept (Bio-Merieux, Durham, NC). Coagulase production and sensitivity to novobiocin were used to speciate the Staphylococcus colonies. The samples were serially diluted for colony counting. Method for bacterial count was modified from a surface viable count by spreading method (Brown et al., 1989). Data entry and data analysis were done using Statistical Package for Social Science (SPSS; SPSS, Inc., Chicago, IL) software version 10. The descriptive statistic was used for sex, age, diagnosis of CLP, and mean bacterial counts. Stata version
7 (StataCorp. LP, College Station, TX) was used to test the significant difference in the bacterial counts and changes of the bacterial species after operation in CLP patients. The outcome variables measured were the bacterial counts (colonyforming unit per mL) and the types of Streptococcus and Staphylococcus species. Univariate analysis using t test for the outcome variables was done in the pre- and postoperative periods. Repeated analysis of variance (ANOVA) measured the effect of surgery on the outcome variables, taking into account the time factor, age, and sex. A nonparametric test was carried out to support the parametric test finding. For all tests, a significant level is p , .05. RESULTS Of 17 CLP patients, 7 (41.2%) were girls and 10 (58.8%) were boys. The ages ranged from 3 to 39 months (average, 11.8 months). Nine (52.9%) were diagnosed to have bilateral CLP, six (35.3%) had unilateral CLP, and two (11.8%) had cleft palate. Two CLP patients defaulted follow-up and were excluded from the study, thus the final sample was 15. The control group comprised 22 children, 8 (36.4%) girls and 14 (63.6%) boys, ages ranging from 6 to 25 months (average, 12.5 months). Streptococcus mitis biovar 1, Streptococcus salivarius and Streptococcus oralis were the more frequently found Streptococcus species in normal children, as well as in CLP patients. Staphylococcus aureus was the most common Staphylococcus species found in CLP patients, comprising 47.4% detection preoperatively, is shown in Table 1. The comparison of the bacterial counts in the 15 preoperative and postoperative CLP patients observed is shown in Table 2. DISCUSSION In this study, the mean Streptococcus count for the CLP patients was noted to increase following closure of the CLP. Even though the finding was not statistically significant, studies in normal infants had shown that the majority of the normal oral floras are of the Streptococcus species (Kononen et al., 1999). Surgical closure of the cleft lip and palate would make the oral cavity similar to that of a normal infant. The comparatively reduced amount of oxygen in the oral cavity following the closure of the cleft palate would favor the growth of facultative oral streptococci. Anaerobic streptococci, mainly peptostreptococci, would also be expected to increase, as was ob-
TABLE 2 Comparison of bacterial count in pre- and postoperative cleft lip and palate (CLP) patients, and in normal children at 0 and 12 weeks (mean, SD) CLP Patients Bacterial Count
Preoperative (3 103 CFU/mL)
Postoperative (3 103 CFU/mL)
Streptococcus Species Staphylococcus Species
32.41 (7.69) 5.34 (2.10)
46.46 (11.01) 0.56 (0.24)
* Sig. 5 Significance; S 5 significant, p , .05; NS 5 not significant, p . .05.
Normal Children Sig.*
0 week (3 103 CFU/mL)
12 weeks (3 103 CFU/mL)
Sig.
NS S
20.93 (27.93) 0.82 (1.98)
49.92 (34.72) 0.60 (2.55)
NS NS
Arief et al., GRAM POSITIVE FLORA IN CLP PATIENTS AFTER SURGERY
served in normal infants (Sarkonen et al., 2000). However, no studies had been done regarding the anaerobic streptococci of the oral cavity in CLP patients. Reduction in the oral pH environment following food intake could alter the oral ecosystem. Increasing the amount of sugar intake would encourage growth of certain streptococci that are able to tolerate a lower pH environment (De Soet et al., 2000). Older children, with more teeth than their younger counterparts, would eat a greater variety of foods and thus would have more Streptococcus species in their oral cavity. This study did not attempt to differentiate between dentate and nondentate subjects, because the sample population was too small for such analysis. Therefore, the increase in the Streptococcus colony count could not be attributed to the operation alone. Moreover, the CLP patients might have taken a greater variety of foods postoperatively than before the closure of the cleft lip and palate, which could explain the increase in the Streptococcus count. The above factors could also contribute to the large variation seen in the frequency and colony count of Streptococcus species in normal children. The mean Staphylococcus count of the CLP patients during the preoperative period showed a significantly higher count of (5.34 3 103 CFU/mL) as compared to 12 weeks postoperative period (0.56 3 103 CFU/mL; p , .01). This is in agreement with the fact that the majority of the Staphylococcus aureus inhabit the nose and nasopharyngeal space, and oral colonization by the bacteria would occur in CLP patients. The postoperative mean Staphylococcus counts for the postoperative CLP patients were comparable to those in normal children, indicating that the oral environment of CLP patients was normalized following the reconstructive surgery. S. aureus, which represented 47.4% of the staphylococcal species identified preoperatively, was reduced to zero postoperatively (p , .05). Streptococcus mutans had been identified as predominant organisms in dental caries (Roberson et al., 2002). However, our study did not find any significant difference between preand postoperative colonization of S. mutans. Dental caries in CLP patients were attributed mainly to poor oral hygiene associated with malocclusion and other anatomical anomalies (Roberson et al., 2002). S. mutans and other bacteria of plaque metabolize refined carbohydrates, such as sucrose, to produce organic acids. These acids may cause a carious lesion by demineralizing the enamel structure (Roberson et al., 2002). The low pH also promotes the growth of these plaque-forming organisms and therefore more demineralization occurs than remineralization. The imbalance between those processes results in dental caries. With our findings of predominant Staphylococcus aureus in preoperative, as opposed to postoperative, patients, we postulate that the organism interacts with Streptococcus mutans to somehow increase the demineralization process or the progression of cavitated lesions. The latter is known to be associated with lactobacilli, whereas early stages of caries formation are strongly associated with S. mutans (Roberson et al., 2002). Staphylococcus aureus is a known pathogen of skin and soft tissue infection, which includes su-
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perficial infection, such as abscesses and angular cheilitis, and deep infection, such as osteomyelitis, endocarditis, and septicemia (Samaranayake et al., 2002). The virulence factors of S. aureus include the production of proteolytic and lipolytic enzymes. Abscess formation in S. aureus infection is characterized by the disruption of protein and lipid constituents (Waldvogel, 2000). The saliva sampling method in this study is by oral swabbing and this method may cause inconsistency in sample volume. However, a similar method was used by Wan et al. (2001) in quantifying the oral microflora of 6-month-old predentate infants. Saliva sampling by pipetting is a more reliable method in standardizing the sample volume (Kononen et al., 1999). Standardization of sample collection time also is important in minimizing variation in the content of oral microflora. CONCLUSION CLP patients had more colonization by S. aureus than normal children had, and the colony count decreased significantly following surgical repair of the CLP. Acknowledgments. Thanks are due to Dr. Than Winn from the Department of Community Medicine for statistical analysis and to Mrs. Rosliza Abdul Rahman from the Department of Medical Microbiology and Parasitology for laboratory assistance; both are from the School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu, Kelantan, Malaysia.
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