Detection of Enterococcus faecalis in subgingival biofilms of healthy, gingivitis, and chronic periodontitis subjects.

BACKGROUND: Enterococcus faecalis is a Gram-positive, facultative anaerobic coccus that can survive under harsh conditions. Studies have shown a positive relationship between biofilm formation and gelE gene expression in E. faecalis. The production of gelatinase (MMP 2) has been detected in 50% of E. faecalis isolates from endodontic and periodontal infections, which suggests its role in the pathogenesis of apical and marginal periodontitis. Although E. faecalis is not considered a periodontopathogen, this species has been more frequently detected in subgingival samples with periodontitis than from periodontally healthy subjects, suggesting that the local conditions in periodontitis may favor its colonization. Hence, the aim of the current study was to detect and compare the presence of E. faecalis in subgingival biofilms of healthy, gingivitis, and periodontitis subjects.
MATERIALS AND METHODS: A total of 100 subjects aged between 25 and 55 years, from the Outpatient Department of Periodontics were recruited for the study. All the subjects were screened for gingival and periodontal status using plaque index, gingival index, and clinical attachment loss. They were divided into three groups based on the clinical findings.• Group A: 18 healthy individuals (gingival index with score zero)• Group B: 34 gingivitis patients (gingival index with score >1)• Group C: 48 chronic periodontitis patients (clinical attachment loss >5 mm in >30% of sites).Subgingival plaque samples of all the enrolled subjects were collected using a sterile curette, later poured into a transport medium (Viability Medium Goteborg Agar III) and sent for microbial culturing within 2 h for detection of E. faecalis.
RESULTS: E. faecalis was detected in 26.8% of all samples evaluated. There was a significantly higher frequency of E. faecalis in subgingival biofilms of periodontitis group (41.7%), compared to gingivitis (5.9%) and healthy group (0%).
CONCLUSION: Enterococci may contribute to increased collagen and periodontal destruction and may further lead to disease progression in patients with chronic periodontitis.

INTRODUCTION

Enterococci are Gram-positive, facultative anaerobic cocci, which are resilient by nature and able to survive a wide array of hostile conditions.[1] They can persist in the environment for long periods and are known as commensals in the gastrointestinal tract. Enterococci have gained significance as they cause nosocomial infections and for their ability to resist the currently available antibiotics via horizontal gene transfer.

Enterococcus faecalis, a Gram-positive anaerobic coccus, has been isolated from patients with posttreatment apical periodontitis, refractory marginal periodontitis, and peri-implantitis. The ability of E. faecalis to cause infections has been linked to variable traits that enhance its virulence.[2]

Enterococci surface protein (ESP) was detected in most strains isolated from endodontic, periodontal, and oral infections. Since ESP has been associated with higher biofilm production of the strains, the high prevalence of ESP within oral isolates suggests that this surface protein may be a potential virulence trait that participates in the colonization of different niches of the oral cavity.[2]

Gel E (secretory metalloprotease gelatinase E), an extracellular metalloprotease, is a genetic factor of E. faecalis with ability to hydrolyze and degrade collagen, fibrinogen, and hemoglobin.[3] This suggests a role for this factor in the pathogenesis of apical and marginal periodontitis.

In light of this knowledge, the current study was designed to assess and compare the presence of E. faecalis in subgingival biofilms of healthy periodontium, gingivitis, and chronic periodontitis subjects.

MATERIALS AND METHODS

A total of 100 individuals including males and females who aged between 25 and 55 years with a minimum of 10 natural teeth and were systemically healthy with no history of antibiotics intake in the past 6 months were enrolled from the Outpatient Department of Periodontics during December 2017 to February 2018. Ethical committee clearance was obtained from the institution (SDC/SMG/2017/661-A). Consent was obtained from all the participants. Smokers, immunocompromised, pregnant, and lactating mothers, and patients with extensive dental caries and history of previous endodontic or periodontal treatments were excluded from the study.

Clinical parameters such as plaque index,[4] gingival index,[5] and clinical attachment loss using Williams graduated probe were assessed. Based on convenience sampling, patients with no bleeding on probing and a score zero of gingival index and no clinical attachment loss were categorized as healthy group (Group A). Patients with score one or above of gingival index were grouped as gingivitis (Group B) and those with generalized bleeding on probing and clinical attachment loss equal to or more than 5 mm, in more than 30% of sites were grouped as periodontitis (Group C). Total number of patients allotted for the study = 100 (n).

Group A: 18 healthy individuals

Group B: 34 gingivitis patients

Group C: 48 chronic periodontitis patients.

Assessment of bacteria

Subgingival plaque samples were collected from the enrolled patients using sterile Gracey curette and were pooled in fresh transport media “Viability Medium Goteborg Agar III” which was anaerobically prepared and sterilized. Samples were then sent to laboratory for microbial analysis, culturing and identification of E. faecalis within 2 h.

Microbial culturing

Samples were thoroughly shaken in a mixer for 60 s, and from each sample, 1 ml was collected for culturing. From serial dilutions, 0.1 ml was inoculated and plated on anaerobic blood agar plates. It was then incubated in an anaerobic chamber at 37°C for 2 days and the colonies of E. faecalis were identified. After completion of incubation, automation was done with Vitek® II system (manufactured by Biomerieux, USA). The purity of the cultures was confirmed by further biochemical tests on the basis of Gram staining, colony morphology, esculin hydrolysis, nitrate reduction, indole production, and oxidase and catalase activities.

Statistical analysis

Statistical evaluation was done using SPSS system version 16- manufactured by SPSS Inc., Chicago, USA. The Kolmogorov–Smirnov and the Shapiro–Wilk test and the test of normality indicated that the data were not normally distributed. Hence, Kruskal–Wallis one-way ANOVA was used to compare the plaque index, gingival index, and clinical attachment loss in three groups and also the presence of E. faecalis between three groups.

RESULTS

The study reported the results of 100 individuals. Table 1a and b reveals and compares the mean plaque index score, gingival index score, and clinical attachment loss for the three groups.

Table 1a

Comparison of the plaque index score, gingival index score and clinical attachment level for the three groups

	Group	Mean	SD	Minimum	Maximum	Quartiles				Test statistic	P
						Q1	Median	Q3	IQR
PI	Periodontitis	0.80	0.57	0.00	2.00	0.31	0.60	1.30	0.99	χ2 (2)=51.37	<0.001
	Gingivitis	0.31	0.25	0.00	1.00	0.12	0.26	0.40	0.28
	Healthy	0.017	0.038	0.00	0.10	0.00	0.00	0.00	0.00
GI	Periodontitis	2.18	2.44	1.00	17.00	1.60	1.70	1.98	0.38	χ2 (2)=47.76	<0.001
	Gingivitis	1.56	0.33	0.87	2.10	1.35	1.60	1.80	0.45
	Healthy	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
CAL	Periodontitis	1.67	0.43	0.50	2.37	1.30	1.70	2.08	0.78	χ2 (2)=86.32	<0.001
	Gingivitis	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	Healthy	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00

P value 0f 0.05 were considered significant. IQR – Interquartile range; SD – Standard deviation; PI – Plaque index; GI – Gingival index; CAL – Clinical attachment loss; P – Probability value; χ2 – Chi squared test statistics

Table 1b

Comparison of the mean plaque index score, gingival index score, and clinical attachment level for the three groups

	Group	n	Mean rank
PI	Periodontitis	48	68.56
	Gingivitis	34	45.21
	Healthy	18	12.33
GI	Periodontitis	48	64.31
	Gingivitis	34	52.71
	Healthy	18	9.50
CAL	Periodontitis	48	76.50
	Gingivitis	34	26.50
	Healthy	18	26.50

PI – Plaque index; GI – Gingival index; CAL – Clinical attachment loss; n – Number of subjects

Kruskal–Wallis H-test showed that overall, there was a statistically significant difference in plaque index between all the three different experimental groups, χ2 (2) = 51.37, P < 0.001 with a mean rank plaque index for Group C being 68.56, for Group B being 45.21, and for Group A being 12.33. There was a statistically significant difference in Gingival Index between all three experimental groups, χ2 (2) = 47.76, P < 0.001 with a mean rank gingival index for Group C being 64.31, for Group B being 52.71, and for Group A being 9.50. Kruskal–Wallis H-test also showed that there was again a statistically significant difference in clinical attachment loss between all the three experimental groups, χ2 (2) = 86.32, P < 0.001 with a mean rank clinical attachment loss for Group C being 76.50 and for Group B and Group A being 26.50.

The prevalence of E. faecalis is demonstrated in Table 2. Among the samples from 18 healthy periodontium, none of them showed the presence of E. faecalis, and of 34 gingivitis and 48 chronic periodontitis patients, one and eight samples were detected with the presence of E. faecalis, respectively. Hence, the present study demonstrated a statistically significant difference in the presence of E. faecalis in individuals with healthy periodontium (0%), gingivitis patients (5.9%), and chronic periodontitis patients (41.7%).

Table 2

Comparison of the presence of Enterococcus faecalis between periodontitis and gingivitis groups

	Frequency (%)
	Enterococcus faecalis		Total
	Present	Absent
Group
Periodontitis	20 (41.7)	28 (58.3)	48 (58.54)
Gingivitis	2 (5.9)	32 (94.1)	34 (41.46)
Total	22 (26.8)	60 (73.2)	82 (100.0)
χ2, P	12.98, <0.001

χ2 – Chi squared test statistics; P – Probability value

DISCUSSION

E. faecalis is not a normal commensal in the oral cavity; it can be conceived directly or indirectly based on the oral status. E. faecalis can be commonly retrieved from dorsum of the tongue (55%), gingival sulcus (22%), and oral rinse samples (29%).[6] It can get incorporated into oral cavity by food contaminants and also by nosocomial infections. Souto and Colombo in their study on the incidence of E. faecalis in the subgingival plaque samples of chronic periodontitis patients reported an increased incidence of E. faecalis in diseased compared to periodontally healthy subjects.[7] Similarly, the results of the present study revealed that chronic periodontitis subjects had 41.7% of E. faecalis in their subgingival plaque samples. It was also found that there was an absolute absence of E. faecalis in periodontally healthy subjects.

E. faecalis has the ability to induce antibiotic resistance through horizontal gene transfer.[8] It colonizes the oral cavity through ESP and ESP gene, a major virulence factor. Since it produces secretory metalloprotease gelatinase E gene, it is commonly found in endodontic infections. Gelatinases produced by E. faecalis can lyse the type I collagen fibers, suggesting its role in pathogenicity and initiation of periodontal destruction. Capsule produced by E. faecalis can aid in evading the host innate immunity and disease initiation.[9] This antiphagocytic capsule sustains during chronic inflammation without being targeted by host immune system. This could be the reason for elevated levels of E. faecalis in chronic periodontitis.

CONCLUSION

The current trends on periodontal therapy do not aim at certain nonoral anaerobes which seem to play a vital role in periodontal disease activity and progression. Enterococci may contribute to increased collagen and periodontal destruction and may further lead to disease progression in patients with chronic periodontitis. However, long-term, multicenter, interventional trials are required to evaluate the pathogenicity of E. faecalis in periodontal disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.