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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 16  |  Issue : 5  |  Page : 333-337

Isolation of Enterococcus faecalis in the saliva samples of patient candidates for liver transplantation


1 Department of Oral and Maxillofacial Medicine, School of Dentistry, Shiraz University of Medical Sciences, Shiraz, Iran
2 Department of Microbiology, Burn and Wound Healing Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
3 Department of Oral and Maxillofacial Medicine, Oral and Dental Disease Research Center, School of Dentistry, Shiraz University of Medical Sciences Arak, Iran
4 Department of Oral and Maxillofacial Medicine, School of Dentistry, Arak University of Medical Science, Arak, Iran
5 Burn and Wound Healing Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Date of Web Publication5-Sep-2019

Correspondence Address:
Dr. Fahimeh Rezazadeh
Department of Oral and Maxillofacial Medicine, Oral and Dental Disease Research Center, School of Dentistry, Shiraz University of Medical Sciences, Shiraz
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1735-3327.266091

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  Abstract 


Background: Liver transplantation has turn into a standard management for chronic liver failure (CLF), and the number of recipients increased during the last few years. Enterococci are progressively related to nosocomial and opportunistic infections. Oral cavity may act as a reservoir for this species, especially in cases with oral infection. Immunocompromised patients are prone to serious enterococci-related disease. This study investigated the prevalence of Enterococcus faecalis in the saliva samples of patient candidates for liver transplantation.
Materials and Methods: In this cross-sectional study the saliva samples of 100 patient candidates for liver transplantation and 100 age- and sex-matched healthy control group were collected. Bacterial DNA was extracted from the samples and E. faecalis was detected using polymerase chain reaction test. Fisher's exact test and Mann–Whitney test were used to correlate the positive and negative cases with the disease. Statistically, a significant difference was considered when P < 0.05.
Results: There was no significant difference between both groups for the presence of E. faecalis. This bacterium isolated from the saliva of two cases in the study group and only one healthy control. The higher rate of carious teeth were detected in the oral cavity of CLF cases than control group (P = 0.001).
Conclusion: Patients with chronic liver diseases assessed in this research showed a higher incidence of poor oral health and caries compared with the healthy controls, but there was no statistical difference in the presence of E. faecalis in saliva samples of each group. Complete oral examination, dental treatment, and oral hygiene instruction are necessary for all these cases before liver transplantation.

Keywords: Enterococcus faecalis, liver transplantation, saliva


How to cite this article:
Ghapanchi J, Emami A, Rezazadeh F, Shakibasefat H, Pirbonyeh N. Isolation of Enterococcus faecalis in the saliva samples of patient candidates for liver transplantation. Dent Res J 2019;16:333-7

How to cite this URL:
Ghapanchi J, Emami A, Rezazadeh F, Shakibasefat H, Pirbonyeh N. Isolation of Enterococcus faecalis in the saliva samples of patient candidates for liver transplantation. Dent Res J [serial online] 2019 [cited 2019 Sep 20];16:333-7. Available from: http://www.drjjournal.net/text.asp?2019/16/5/333/266091




  Introduction Top


Several liver diseases may cause chronic or ongoing liver inflammation, and the most usual etiologies of end-stage liver failure are chronic viral hepatitis, alcoholic liver cirrhosis, autoimmune liver disease, primary sclerosing cholangitis, primary biliary cirrhosis, steatohepatitis, inherited liver disorders, and drug-induced hepatic failure.[1] The number of recipients of liver transplants has increased quickly in the recent few years and is expected to remain to do so in the next. The dentist should be ready to see participants who are planning or have had liver transplants. The main aim of dental intervention, before and after liver transplantation, is the inhibition of bacteremia originates from oral cavity that may lead to general infection. Furthermore, pre- and posttransplant medical problems that should be mentioned are inadequate drug metabolism, bleeding tendency, inadequate wound healing, immunosuppressive drugs, and higher rate of infections.[2],[3]

Infective disorders are the main factors that can cause morbidity and mortality in end-stage liver damage and transplant recipient.[4] Oral cavity infection is estimated as a major source for general infection in a large group of liver transplant candidates and recipients.[4]

The salivary bacteria play a major role in producing oral disorders and have an interaction with other microbiota of the human body, especially the intestinal tract, but there is little information's about the similarity of them.[5],[6] Relatively, most of studies focused on oral pathogenic bacteria that cause periodontal problems and caries, and human salivary microbiota did not consider perfectly.[7],[8]

In the past Enterococcus faecalis classified as a portion of D Streptococcus. It is a Gram-positive, commensal bacterium of humans' gastrointestinal tracts. As well as other types in the genus Enterococcus, E. faecalis isolated from healthy participants but can initiate serious infections in individuals, particularly in the nosocomial environment.[9]

A study showed that E. faecalis was more frequently detected in the subgingival microbiota of HIV-positive than HIV-negative participants with periodontal disease,[10] and another documents established that E. faecalis is commonly accompanying with necrotizing gingival disorders in the HIV-positive cases.[11] In another study, 60% of diabetic patients showed oral E. faecalis and Enterococcus faecium in the mouth.[12] To the best of our knowledge, there are no available data about the presence of this bacterium in the saliva of Iranian population, especially end-stage liver disease patients. Owing to the reservoir activity of the oral cavity for several pathogens related to systemic infections, this study was conducted to determine the prevalence of E. faecalis as noncommensal pathogenic bacteria in the saliva samples of patient candidates for liver transplantation.


  Materials and Methods Top


Ethical statement

This cross-sectional study was carried out in accordance with the guidelines of the Declaration of Helsinki as revised in Edinburgh (1975). The study protocol was approved by the Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran. The written informed consents were obtained from participants for sample collection, and in unable cases, verbal consent was obtained. Patients were informed about the nature of the study.

Participants

In a cross-sectional study, 100 dentate patient candidates for liver transplantation who referred to the Clinic of Emam Reza (Medical Clinic in Shiraz, Iran) for dental examination between December 2017 and April 2018 were collected. The study group comprised 63 males and 37 females. A control group consists of 100 age- and sex-matched participants that referred for routine checkups between the same times. The exclusion criteria were the patients with diabetes, pregnancy, smoking, using antibiotics within the past 2 months, and candidate for other organ transplantation. All patients in both groups were examined radiographically (panoramic X-ray) and clinically by oral medicine specialist as an examiner. The number of decayed teeth and gingival status of patients was recorded according to the Modified Gingival Index also by examiner [Table 1].
Table 1: Modified gingival index criteria

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Saliva sampling

Participants were prohibited from eating and washing their mouth 1 h before sampling. Each individual will be sampled using the oral rinse technique [13] with 10 mL of 0.9% sterile saline for 60 s, and the mouthwashes will be collected in sterile 50-mL polypropylene falcons. Samples freeze (−20°C) until polymerase chain reaction (PCR) amplification is performed.

DNA extraction

Bacterial DNA was extracted from all saliva samples, using the GeneAll DNA extraction mini-kit (GeneAll, Seoul, Korea), in accordance with the manufacturer's instructions. DNA quality and quantity were checked by a spectrophotometric method (BioPhotometer, Eppendorf, England). The extracted DNA was stored at −20°C for further use.

Molecular detection of Enterococcus faecalis

Molecular diagnosis of E. faecalis was performed by gene amplification of primers ddl. The primers listed in [Table 2] were used to amplify the genes applied for detection E. faecalis. As a first step, PCR was performed with a standard protocol. Each step was carried out using a 25-μL mixture containing 12.5 μL of ×10 buffer (supplied with Taq polymerase), 10 pmol of each primer, and 70 ng of genomic DNA. The PCR was performed with Bio-Rad T100 thermal cycler (USA) with microtubes under the following conditions: denaturation for 5 min at 95°C; 35 cycles of 45s at 95°C, 45s at 48°C, and 1 min at 72°C; and a final extension step of 10 min at 72°C.
Table 2: List of oligonucleotide primer used for detection bacteria by polymerase chain reaction

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Statistical analysis

Fisher's exact test and Mann–Whitney test with odds ratio (95% confidence interval) were used to correlate the positive and negative cases with the disease. Statistically, a significant difference was considered when P < 0.05.


  Results Top


The age in case group ranged from 10 to 67 years (mean age: 40.84 years) and in control group from 10 to 77 years (mean age: 39.14 years).

There was no significant difference between both groups for the presence of E. faecalis (P > 0.005). This bacterium isolated from the saliva of two cases and only one control [Figure 1]. The higher rate of carious teeth was detected in the oral cavity of chronic liver failure (CLF) cases (3.23) than control group (1.84) (P = 0.001) [Table 3].
Figure 1: Agarose gel electrophoresis for ddl gene of Enterococcus faecalis

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Table 3: Demographic and clinical parameters of participants

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A 45-year-old male with 14 carious teeth and moderate gingivitis and a 26-year-old female with good oral hygiene demonstrated E. faecalis in the saliva. In the healthy controls, a saliva sample of a 77-year-old male with severe gingival index was positive for bacterial DNA. About 10% of study group patients were positive for HBV and 1% was positive for HCV, and four patients had Wilson's disease.


  Discussion Top


In the past few years, many researches focused on the interaction between serious systemic diseases and oral microflora.[14] On the other hand, numerous studies showed that normal oral microflora may be changed due to systemic diseases or oral condition alteration. In the present study, the difference in E. faecalis in the saliva of both CLF and healthy participants has been compared. Although CLF cases were unable to adequately maintain the oral health, the presence of this bacterium did not differ significantly. For proper bacterial pathogenicity, the microorganism should be able to adhere to, grow on, and invade the host. Furthermore, it should be capable to overcome the host defense mechanisms.[15]

The previous study reported that improper oral hygiene, dry mouth, limited jaw movement, hospitalization, and immune system suppression can improve noncommensally bacterial colonization.[14] One of the major side effects of liver transplant cases is infections.[16] An increase in susceptibility to rejection resulted from oral infection was seen in numerous transplant recipients.[17]

Animal-based research showed that a different liver disturbance such as fatty liver disease, cirrhosis, and hepatocellular carcinoma may develop in cases with periodontitis; liver transplantation also affected by such problem.[18] Bajaj et al.[19] reported that there is an interdependence between the saliva and stool microbiota of cirrhotic patients. Inflammatory process, inadequate immunologic response, and hospitalization may accelerate this phenomenon.[14]

In cirrhotic cases, especially in those with encephalopathy, a massive circulatory inflammation associated with Th1 and Th17 is seen.[20] The saliva of CLF cases showed an increased level of interleukin (IL)-1β and IL-6 concentration and a prominent rise in secretory IgA. This procedure resulted in diminished innate local defenses and decreased histatins 1 and 5 and lysozyme. Higher levels of fecal secretory IgA in the saliva were also seen. This phenomenon may explain the entire initiation of systemic inflammation, possibly through interdependence between the intestine and the oral cavity.[19]

E. faecalis is a pathogenic bacteria that can own the fundamentals to establish an oral infection and preserve an inflammatory response that can be harmful to the host.[15]

To the best of our knowledge, there are no available data regarding the presence of E. faecalis in the saliva of patient candidates for liver transplant, so comparing the current research with other investigations is limited. A study demonstrated a meaningfully higher occurrence of E. faecalis in the saliva (40.5%) and subgingival biofilm (47.8%) of samples obtained from cases with periodontitis in comparison to healthy controls.[14] On opposite, Rams et al.[21] identified E. faecalis in only 1% of initial onset periodontitis and 5.1% of long-lasting periodontitis cases. This finding is similar to the current study result that showed only two positive samples for E. faecalis in participants with moderate-to-severe gingival index.

Definitely, oral enterococci yield virulence elements of potential importance to the pathogenesis of periodontitis, as well as aggregation materials, superficial adhesins, lipoteichoic acid, extracellular superoxide products, lytic enzymes such as gelatinase, hyaluronidase, and elastase, the toxin cytolysin, and hemolysins able to cause neutrophil impairment.[15]

In addition, E. faecalis may improve pathogenicity in complex infections with anaerobic microorganisms and is able to make experimental apical periodontitis after combination with other mouth microflora. Enterococci have long term concerned in chronic root canal infections and are the main type in failed endodontic-treated teeth. The origin of the bacteria is still unclear, as enterococci do not relate to the common oral microflora.[22]

Assessment of oral rinse of 100 dentistry students and 100 cases that had root canal-treated tooth revealed that 1% of the students and 11% of the second group showed E. faecalis in the samples. Genetic examination displayed that the isolates from the treated teeth were not linked to those from the usual gastrointestinal microbiota. The saliva samples of none of these cases exhibit enterococci.[23]

It seems that dissimilarities in isolating of E. faecalis can resulted from variations in the study groups, cultural and religious factors, nutrition, oral or systemic condition of the participants, count of the samples, approaches of the study geographical variations, and personal hygiene maintenance. Besides, saliva is extensively open and complex environment; a microflora in the saliva varied at all periods.

Moreover, it should be recognized that cross-sectional studies have a restricted time which can single observed the oral microbiota. The transitory oral microflora existing in a complicated active environment might arise in another time.[24]


  Conclusion Top


Patients with chronic liver diseases assessed in this research showed a higher incidence of poor oral health and caries compared with the healthy controls, but there was no statistical difference in the presence of E. faecalis in both groups. Complete oral examination, dental treatment, and oral hygiene instruction are necessary for all these cases before liver transplantation.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.



 
  References Top

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Silva Santos PS, Fernandes KS, Gallottini MH. Assessment and management of oral health in liver transplant candidates. J Appl Oral Sci 2012;20:241-5.  Back to cited text no. 1
    
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Guggenheimer J, Eghtesad B, Stock DJ. Dental management of the (solid) organ transplant patient. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:383-9.  Back to cited text no. 2
    
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Ghapanchi J, Haghnegahdar AA, Faghih M, Rezazadeh F, Derafshi R, Farzin M, et al. Evaluation of mandibular inferior cortex changes in patients candidate for liver and kidney transplantation using panoramic view. J Nephropathol 2017;6:317-23.  Back to cited text no. 3
    
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Lins L, Bittencourt PL, Evangelista MA, Lins R, Codes L, Cavalcanti AR, et al. Oral health profile of cirrhotic patients awaiting liver transplantation in the Brazilian Northeast. Transplant Proc 2011;43:1319-21.  Back to cited text no. 4
    
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Nasidze I, Li J, Quinque D, Tang K, Stoneking M. Global diversity in the human salivary microbiome. Genome Res 2009;19:636-43.  Back to cited text no. 5
    
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Rezazadeh F, Shahbazi F, Andisheh-Tadbir A. Evaluation of salivary level of IL-10 in patients with oral lichen planus, a preliminary investigation. Comp Clin Pathol 2017;26:531-4.  Back to cited text no. 6
    
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Kumar PS, Griffen AL, Barton JA, Paster BJ, Moeschberger ML, Leys EJ. New bacterial species associated with chronic periodontitis. J Dent Res 2003;82:338-44.  Back to cited text no. 7
    
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Faveri M, Mayer MP, Feres M, de Figueiredo LC, Dewhirst FE, Paster BJ, et al. Microbiological diversity of generalized aggressive periodontitis by 16S rRNA clonal analysis. Oral Microbiol Immunol 2008;23:112-8.  Back to cited text no. 8
    
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Ryan KJ, Ray CG. (Eds). Sherris medical microbiology. 4th Edition, McGraw Hill, New York. 2004. p. 551-2.  Back to cited text no. 9
    
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Gonçalves LS, Soares Ferreira SM, Souza CO, Souto R, Colombo AP. Clinical and microbiological profiles of human immunodeficiency virus (HIV)-seropositive Brazilians undergoing highly active antiretroviral therapy and HIV-seronegative Brazilians with chronic periodontitis. J Periodontol 2007;78:87-96.  Back to cited text no. 10
    
11.
Ramos MP, Ferreira SM, Silva-Boghossian CM, Souto R, Colombo AP, Noce CW, et al. Necrotizing periodontal diseases in HIV-infected Brazilian patients: A clinical and microbiologic descriptive study. Quintessence Int 2012;43:71-82.  Back to cited text no. 11
    
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Chomicz L, Szubińska D, Piekarczyk J, Wojtowicz A, Piekarczyk B, Starościak B, et al. Occurrence of oral subclinical infections in insulin treated diabetics. Wiad Parazytol 2004;50:177-80.  Back to cited text no. 12
    
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Paula SB, Morey AT, Santos JP, Santos PM, Gameiro DG, Kerbauy G, et al. Oral candida colonization in HIV-infected patients in londrina-PR, Brazil: Antifungal susceptibility and virulence factors. J Infect Dev Ctries 2015;9:1350-9.  Back to cited text no. 13
    
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Souto R, Colombo AP. Prevalence of Enterococcus faecalis in subgingival biofilm and saliva of subjects with chronic periodontal infection. Arch Oral Biol 2008;53:155-60.  Back to cited text no. 14
    
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Kayaoglu G, Árstavik D. Virulence factors of Enterococcus faecalis: Relationship to endodontic disease. Crit Rev Oral Biol Med 2004;15:308-20.  Back to cited text no. 15
    
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Grønkjær LL, Holmstrup P, Schou S, Schwartz K, Kongstad J, Jepsen P, et al. Presence and consequence of tooth periapical radiolucency in patients with cirrhosis. Hepat Med 2016;8:97-103.  Back to cited text no. 16
    
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Bissig KD, Zimmermann A, Bernasch D, Furrer H, Dufour JF. Herpes simplex virus hepatitis 4 years after liver transplantation. J Gastroenterol 2003;38:1005-8.  Back to cited text no. 17
    
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Han P, Sun D, Yang J. Interaction between periodontitis and liver diseases. Biomed Rep 2016;5:267-76.  Back to cited text no. 18
    
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Bajaj JS, Betrapally NS, Hylemon PB, Heuman DM, Daita K, White MB, et al. Salivary microbiota reflects changes in gut microbiota in cirrhosis with hepatic encephalopathy. Hepatology 2015;62:1260-71.  Back to cited text no. 19
    
20.
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21.
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Oktyabrskii O, Smirnova G. Redox potential changes in bacterial cultures under stress conditions. Microbiology 2012;81:131-42.  Back to cited text no. 22
    
23.
Sedgley CM, Lennan SL, Clewell DB. Prevalence, phenotype and genotype of oral enterococci. Oral Microbiol Immunol 2004;19:95-101.  Back to cited text no. 23
    
24.
Wang QQ, Zhang CF, Chu CH, Zhu XF. Prevalence of Enterococcus faecalis in saliva and filled root canals of teeth associated with apical periodontitis. Int J Oral Sci 2012;4:19-23.  Back to cited text no. 24
    


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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