The Bacterial Oral Microbiome in Children with Congenital Heart Disease: An Extensive Review

Children with congenital heart disease have poorer oral health compared with healthy children. Oral diseases, such as dental caries and gingivitis, are associated with the oral microbiome. The objective of this review was to find evidence of differences in the bacterial colonization of the oral cavity of children with congenital heart disease (CHD) versus healthy children. A literature review was conducted according to predetermined criteria, including the need for controlled clinical trials. Half of the 14 studies that met the inclusion criteria reported significant differences in bacterial colonization in children with congenital heart disease. A variety of influencing factors were discussed. There is some evidence for alterations in the oral microflora as a result of physiopathological and treatment-related factors in children with CHD, but additional research is required to validate these findings.


Introduction
Annually, 1.35 million infants are born with congenital heart disease (CHD) [1].Children with CHD endure invasive surgical procedures, physical limitations, and ambiguous prognoses [2].Moreover, CHD imposes a financial and emotional burden on affected families [3].
There are several studies that observed that children with CHD have poorer oral health, including more caries [4,5] and gingivitis [4], higher DMFT/DMFS values (higher number of teeth with caries history), a higher number of untreated teeth with dental caries [6][7][8][9], and higher plaque and gingival indices [10,11] compared with healthy control individuals of the same age.
Possible explanations for these associations between a congenital defect, such as CHD, and an acquired disease, such as caries, are that the focus of the affected families is often not primarily on oral health, the parents' lack of knowledge about adequate oral preventive measures or the disease burden of CHD [5,12].
In order to investigate whether other host factors, such as the individual bacterial colonization of the oral cavity, could be responsible for a higher caries risk in children with CHD, it is necessary to look at the factors of origin of dental caries.
It is known that dental caries are a multifactorial disease.Examples of causal factors include the amount and frequency of dietary intake (particularly of sugars), the individual qualitative and quantitative properties of saliva, oral hygiene practices, the local use of fluorides, and oral health awareness [13].
With respect to oral hygiene practices, tooth brushing [12,14] and the use of fluoridated toothpaste [14] occur less frequently in children with CHD than in healthy children of Pathogens 2023, 12, 1269 2 of 18 the same age, whereas the consumption of cariogenic foods [12] is substantially higher in children with CHD.Moreover, parents of children with CHD frequently have a very limited understanding of the significance of oral health [15].
Oral bacteria play a significant role as a cause of oral diseases such as dental caries and inflammatory diseases of the periodontium [13,16].
Concerning dental caries, the bacterial balance in the oral cavity is dominated by acidtolerant and acid-producing microorganisms, such as Streptococcus mutans and Lactobacillus spp.(species), which produce a low pH environment that can result in the demineralization of dental tissues [17,18].
Lactobacillus spp.are responsible for the progression of carious lesions [19], whereas Streptococcus mutans is one of the major causative agents of dental caries [20].Streptococcus mutans, a natural resident of the human oral cavity, can induce changes in the plaque microbiome by synthesizing large amounts of extracellular glucan polymers and acids.Dominant Lactobacillus spp. in carious lesions are, for example, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus pontis, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus oris and Lactobacillus vaginalis [19,21].Lactobacillus acidophilus, another representative of Lactobacillus spp., is a cariogenic bacterium [22] that has a sugar metabolism system functionally similar to that of Streptococcus mutans [23].
In gingivitis, a periodontal disease caused by plaque accumulation, the oral microflora in the biofilm favors Actinomyces spp.and capnophilic and obligate anaerobic bacteria such as Fusobacterium spp., Capnocytophaga spp., and Prevotella spp.[17].
A congenital heart defect can be a risk factor for the development of a brain abscess due to the dissemination of odontogenic bacteria into the bloodstream during dental procedures [24].In addition, many patients with CHD are susceptible to infective endocarditis (IE) due to oral infections [25] and dental procedures [26].IE, a disease of the heart tissue that can be predisposed because of CHD [27], shows a high mortality rate [28] and is one of the most severe complications caused by oral bacteria-induced bacteremia [27].
In order to prevent complications such as IE, antibiotic prophylaxis is recommended for patients at high risk, such as those with specific cardiac conditions [26,32].This could lead to the hypothesis that changes in the bacterial spectrum of the host organism can occur because of the frequent intake of antibiotics in children with cardiac conditions.
In addition, it is assumed that physiopathological processes associated with CHD may also result in alterations in bacterial colonisations within the human body.For children with CHD and heart failure, alterations in the intestinal bacterial spectrum due to heart failure have already been demonstrated [33].
For newborns with critical CHD, Huang et al. (2022) have already demonstrated a correlation between this congenital disease and an altered gut microbiome.According to the authors, this altered gut microbiome is associated with an imbalance of the immune system and unfavorable clinical outcomes [34].Since the oral cavity serves as the entry point of the intestinal tract, the question arises whether the oral microbiome is also determined by the congenital general disease and whether this predisposition is a potential cause for poorer oral health in children with CHD.So far, there are no published data on this.This article's primary objective is to explore whether there is evidence of differences in the bacterial colonization of the oral cavity between children with CHD and healthy children.

Materials and Methods
A literature review was carried out until August 2023 using the database "PubMed" (https://pubmed.ncbi.nlm.nih.gov/; last accessed on 1 August 2023).The research question Pathogens 2023, 12, 1269 3 of 18 "Is there any evidence of differences in the bacterial colonization of the oral cavity between children with CHD and healthy children?" was generated using the "PICO" scheme (Table 1).
Table 1.Items of the research question according to the PICO scheme (CHD = congenital heart disease).

Intervention
Gaining of samples of the oral cavity and investigation of these samples for bacteria.

Comparison
Healthy children (0-18 years of age) without CHD.

Outcome
Information on the composition of the bacterial colonization of the oral cavity.
Since the literature search with MeSH (Medical Subject Headings) terms analogous to the research question according to the PICO scheme did not yield any results, the search terms shown in Figure 1 were used.These search terms should represent all studies that examine children with congenital heart disease or congenital heart defects for bacterial colonization of the oral cavity.Furthermore, search items were added that referred to various inflammatory processes of the oral cavity and/or to the sample medium.
in the bacterial colonization of the oral cavity between children with CHD and healthy children.

Materials and Methods
A literature review was carried out until August 2023 using the database "PubMed" (https://pubmed.ncbi.nlm.nih.gov/; last accessed on 1 August 2023).The research question "Is there any evidence of differences in the bacterial colonization of the oral cavity between children with CHD and healthy children?" was generated using the "PICO" scheme (Table 1).
Table 1.Items of the research question according to the PICO scheme (CHD = congenital heart disease).

Intervention
Gaining of samples of the oral cavity and investigation of these samples for bacteria.

Comparison
Healthy children (0-18 years of age) without CHD.

Outcome
Information on the composition of the bacterial colonization of the oral cavity.
Since the literature search with MeSH (Medical Subject Headings) terms analogous to the research question according to the PICO scheme did not yield any results, the search terms shown in Figure 1 were used.These search terms should represent all studies that examine children with congenital heart disease or congenital heart defects for bacterial colonization of the oral cavity.Furthermore, search items were added that referred to various inflammatory processes of the oral cavity and/or to the sample medium.There were no limitations on the language or date of publication in the literature.Included were clinical trials that studied the oral bacteria of children with CHD.In addition, an active control population without any heart defects had to be part of the trials.Only studies reporting the procedure of sampling and detection of the examined bacteria were included.
Case reports, retrospective analyses, non-human trials, or studies with test subjects older than eighteen years of age were excluded.
The screening of the literature was carried out by one examiner and was repeated three times.
The taxonomy of the literature research process is depicted in Figure 2.
Included were clinical trials that studied the oral bacteria of children with CHD.In addition, an active control population without any heart defects had to be part of the trials.Only studies reporting the procedure of sampling and detection of the examined bacteria were included.
Case reports, retrospective analyses, non-human trials, or studies with test subjects older than eighteen years of age were excluded.
The screening of the literature was carried out by one examiner and was repeated three times.
The taxonomy of the literature research process is depicted in Figure 2.

Results
For this review article, fourteen studies spanning the years 1986 to 2021 were included.
Seven of these studies come from industrialized nations, such as the United States of America [35,36], the United Kingdom [6,37], Sweden [38,39] or Germany [40].Three of the included studies were conducted in emerging countries, such as Turkey [41,42] or Brazil [43].Four of the studies presented are from developing countries, such as Sudan [22], Iran [44,45] or India [11].
Eleven of the fourteen included studies [44,6,38,37,45,39,35,36,11,42,43] examined bacteria using bacterial culture on various specific agar plates in conjunction with the determination of the number of colony-forming units (CFUs).One of these eleven investigations described the use of additional Gram-staining to distinguish between Gram-positive and Gram-negative bacteria [37].A second study used species-specific 16S rRNA gene sequences for the identification of specific bacteria [42].A third study reported on a DNA probe test that followed the bacterial culture [36].

Results
For this review article, fourteen studies spanning the years 1986 to 2021 were included.Seven of these studies come from industrialized nations, such as the United States of America [35,36], the United Kingdom [6,37], Sweden [38,39] or Germany [40].Three of the included studies were conducted in emerging countries, such as Turkey [41,42] or Brazil [43].Four of the studies presented are from developing countries, such as Sudan [22], Iran [44,45] or India [11].
Eleven of the fourteen included studies [6,11,[35][36][37][38][39][42][43][44][45] examined bacteria using bacterial culture on various specific agar plates in conjunction with the determination of the number of colony-forming units (CFUs).One of these eleven investigations described the use of additional Gram-staining to distinguish between Gram-positive and Gram-negative bacteria [37].A second study used species-specific 16S rRNA gene sequences for the identification of specific bacteria [42].A third study reported on a DNA probe test that followed the bacterial culture [36].
A graphical representation of the media tested for bacteria and the differences in bacterial colonization between groups in relation to the number of studies found is given in Figure 3.
Only one study [40] did not focus solely on the identification of predefined bacterial species but instead examined the most comprehensive coverage of the microbiome possible.
Seven studies reported statistically significant differences in bacterial colonization between groups of children with CHD and groups of healthy children [44,38,22,45,39,40,43], three studies did not find any statistically significant differences [41,6,42], and four studies mention differences but do not report statistical significance for every variable investigated [37,35,36,11].

Saliva
Only one study [40] did not focus solely on the identification of predefined bacterial species but instead examined the most comprehensive coverage of the microbiome possible.
The characteristics of the studies are summarised in Table 2.

Distribution of Oral Bacteria in Children with CHD
The findings of this review suggest that the oral bacterial spectrum is altered in children with CHD compared with healthy controls.Suvarna et al. (2011), for instance, discovered a higher number of Streptococcus mutans CFUs in the saliva of infants with CHD compared with healthy controls [11].Hansson et al. (2012) were also able to demonstrate substantially higher Streptococcus mutans levels in children with CHD than in healthy controls at twelve months of age but not at six or nine months [38].The authors propose that factors such as increased frequency of meals, changes in salivary composition due to the use of diuretics, and increased use of antibiotics could explain the significant differences at 12 months of age [38].Regarding the fact that the first teeth erupt between six and twelve months of age [46] and that the awareness of the importance of good oral hygiene among parents of children with CHD is very low [15], another assumption could be the neglect of dental hygiene, especially considering that the majority of children with CHD in the study by Hansson et al. (2012) have already undergone corrective surgery within the first twelve months of life [38] and that this risky intervention, with all its implication for the child's potential health.Franco et al. (1996), in contrast, found no significant difference in the distribution of the number of CFUs of plaque or saliva Streptococcus mutans or Lactobacillus spp. between children with cardiovascular disease and healthy controls [6].As a possible explanation, the authors noted that the control group was comprised of individuals with more severe dental disease than the general population.As previously mentioned, children with CHD frequently have poorer oral health than healthy children [4,5,[7][8][9][10][11].

The Microbiome of Dental Caries and Its Implications in Children with CHD
Schulz-Weidner et al. (2021) [40] answered the question of whether the microbial composition of dental caries itself differs in this population with higher oral healthcare requirements.The authors were able to demonstrate statistically significant differences in the bacterial composition of carious dentinal samples between preschool children with CHD and healthy individuals [40].Fusobacterium spp., Prevotella spp., Capnocytophaga spp., and Oribacterium spp.were found to be substantially more abundant in the CHD group [40] at the level of operational taxonomic units (OTUs).
As previously described, Fusobacterium spp., Capnocytophaga spp., and Prevotella spp.are associated with gingivitis [17], so the findings of Schulz-Weidner et al. (2021) may account for the higher incidence of gingivitis in children with CHD [4].
This hypothesis is supported by the findings of Mohamed Ali et al. (2017), who observed a positive correlation between the gingival index and Tanerella forsythia, Camphylobacter rectus, and various Fusobacterium spp. in only children with CHD.In the study by Mohamed Ali et al. [22], children with CHD also had higher caries and gingivitis incidences.
In addition, Mohamed Ali et al. (2017) discovered a positive correlation between the amount of CFUs of Streptococcus mutans and the number of decayed teeth in both children with CHD and healthy controls [22].Pourmoghaddas et al. (2018) were unable to demonstrate a significant correlation between a higher amount of CFUs of bacteria (Streptococcus mutans or Lactobacillus spp.) and other factors, such as the number of decayed, missing, or filled teeth as a result of caries, or the gingival bleeding index [45].In this context, it is important to note that in the study by Pourmoghaddas et al. (2018), the healthy control group had a substantially higher carbohydrate intake than the examined children with CHD [45].
Mohamed Ali et al. (2017) [22] were the only authors reporting on the examination of one specific Lactobacillus bacterium: Lactobacillus acidophilus.L. acidophilus is a bacterium that is commonly found in saliva samples of subjects with dental caries [21].The fact that Mohamed Ali et al. (2017) examined dental plaque instead of saliva as substrate could explain the low L. acidophilus detection levels found during the study.

Infective Endocarditis (IE) and Oral Bacteria
Concerning the risk of IE in children with CHD, Topcuoglu et al. [42] examined saliva samples from children with CHD for the presence of Streptococcus mutans serotype k, a strain that was found in significantly greater amounts in heart valve specimens from patients with subacute IE [47].There was no significant difference in the composition of salivary cariogenic microorganisms between children with CHD and healthy controls, but Streptococcus mutans serotype k was only found in CHD cases [42].Due to the small sample sizes in both groups, further studies with larger numbers of cases are required to draw final conclusions about the prevalence of Streptococcus mutans serotype k in children with CHD.
Bozdogan et al. examined the presence of Aggregatibacter actinomycetemcomitans, a Gram-negative bacterium from the HACEK group, in samples of saliva and cardiac tissue in relation to the role of HACEK group bacteria as a cause of IE.There was no significant difference in the presence of this bacterium in saliva samples between children with CHD and healthy controls; however, children with CHD had a higher amount of colony-forming units despite having healthier gingiva [41].
If oral bacteria are suspected in heart tissue samples, it is reasonable to assume that these bacteria should also be detectable, at least temporarily, in the blood in the form of bacteremia.During the literature search for the present article, no studies could be found in which human infant blood was examined for oral bacteria.
At this point, however, it must be emphasized that a limitation of the results of the included studies on the topic of IE is that primarily young children with CHD were integrated into the studies, in whom the oral microbiome changes due to various external factors, such as the frequent use of antibiotics.
Steelman et al. published a study in 2000 containing evidence of a correlation between the severity of gingival inflammation and the presence of specific HACEK bacteria in both infants with CHD and healthy individuals [35].Steelman et al. (2003) found no correlation between the number of HACEK microbes and the degree of gingival inflammation in children with CHD, whose gingival inflammation was comparable with that of healthy controls [36].These findings may be explained by the fact that the HACEK group is part of the normal oral flora [29,30], highlighting the importance of using prophylactic antibiotics prior to invasive dental treatment in patients with a high risk of IE, even if there is no acute infection in the oral cavity.

Influence of Medications on the Oral Microflora
However, antibiotic resistance increases when they are used frequently [48].Koh et al. (1986) focused [37] on amoxicillin-resistant Streptococcus strains in the dental plaque of infants with CHD and adults with a history of rheumatic fever.Children and adults in good health served as control groups.Comparing the two test groups with the control groups, the results revealed that the percentage of antibiotic-resistant Streptococcus strains was elevated in the two test groups [37].
The proportion of Streptococcus strains resistant to 1 mg/mL amoxicillin was more than twice as high in adults with a history of rheumatic fever compared with healthy controls.The authors assume that this is owing to the routine prophylactic administration of antibiotics to the test group [37].
In addition, Folwaczny et al. (2019) demonstrated that adults with CHD who require antibiotic prophylaxes prior to specific dental treatments have higher DMFT values and greater radiographic bone loss despite lower incidences of caries and periodontitis in comparison with healthy adults [49].Torres et al. (2001) discovered a correlation between the frequent use of antibiotics and decreased Lactobacillus species counts, which occurred when antibiotics were regularly consumed.This correlation was not observed [43] for Streptococcus mutans.
These multiple findings lead to the conclusion that physicians should be aware of appropriate antibiotic use and should consider that in patients with CHD, a history of regular antibiotic use can potentially influence therapeutic outcomes and cause complications.Diuretics, which can affect the rate of salivation [50], are also frequently used as an additional medication in pediatric patients with cardiomyopathies [51].Rosén et al. (2010) did not find any significant differences in Streptococcus mutans or Lactobacillus spp.counts between children with CHD taking ACE inhibitors and/or diuretics and healthy children, but the total viable bacteria count was significantly higher in the healthy controls [39].This may indicate a shift in the balance of bacterial diversity in children with CHD, which may be caused by a reduced saliva flow [52], especially considering that 29% of the CHD group in the study by Rosén et al. had very low salivary secretions, whereas the control infants did not [39].

Subgroup Factors
Several studies conducted subgroup analysis or examined additional groups in addition to children with CHD and healthy controls.Steelman et al. (2000) and Steelman et al. (2003) distinguished cyanotic from acyanotic congenital heart disease infants.These subgroups did not differ significantly with regard to the presence of Aggregatibacter actinomycetemcomitans and Eikenella corrodens pathogens [35,36].Ajami et al. (2015) also investigated a group of children with acquired cardiac diseases, in addition to healthy controls and children with CHD.There was no correlation between the type of heart disease and the presence of Streptococcus mutans in salivary samples, but children with acquired heart diseases had substantially lower Streptococcus mutans counts and lower DMFT scores [44].
Concerning the question of whether dental preventive treatment can compensate for oral dysbiosis in children with CHD, Suvarna et al. (2011) were able to demonstrate a reduction in CFUs of Streptococcus mutans as well as improved oral health after a preventive treatment consisting of local antibacterial agents, pits and fissure sealants, topical fluoride application, and oral hygiene education for children with CHD and their parents [11].
Considering that younger patients with increased dental treatment needs frequently receive treatments under general anesthesia, which is a risky approach in children with CHD [53] and also includes inpatient admission and an economic burden [54], it is all the more crucial that preventive treatments and oral health education be utilized to avoid these highly invasive procedures.
In summing up the findings of the present review, some evidence for differences in the oral bacterial spectrum of individuals with CHD, as well as a large number of potential influencing factors, were identified.However, only one of the included studies [40] examined a broad spectrum of bacteria as opposed to focusing on a few specific bacteria.

Conclusions
There is some evidence for alterations in the oral microflora in children with CHD due to physiopathological and treatment-related factors, but additional research is required to validate these findings.The changes in oral flora as an indicator of a general infection could be helpful for the prevention of endocarditis, especially for this group of vulnerable patients, and thus also increase the quality of life of these patients.In addition, future research should investigate the influencing factors that may be present in children with CHD.

Figure 2 .
Figure 2. Taxonomy of the literature search.

Figure 2 .
Figure 2. Taxonomy of the literature search.