Microbiology

Question 1

Q38 : Is there any association between presence of intra-radicular bacterial biofilms & periapical cysts?

Answer 1

According to Ricucci & Siqueira (JOE - 2010), intra-radicular biofilms were significantly associated with epithelialized lesions (cysts and epithelialized granulomas or abscesses)

Question 2

Q39 : Describe the bacterial community in primary endodontic infection and how it is different from secondary infection?

Answer 2

According to Siqueira & Roças (Journal of Oral Microbiology-2009)
Primary infection:
* Mixed bacterial community
* Predominated by anaerobic bacteria
* Diverse number of species in larger numbers are present
* The size of apical periodontitis lesion is proportional to the number of bacteria in the root canal
* Common bacteria in primary infections are Porphyromonas spp, Prevotella spp. Treponema spp. Diolestor spp. T forsythia
Secondary infection:
* Primarily, Gram ve facultative anaerobes
* Less diversity in species and fewer numbers compared to primary endodontic infection
* The number of bacteria in a failed case will vary according to the quality of the treatment (worse treatment more bacteria)
* -E faecalis is predominant in failed cases

Question 3

Q40 : Give examples of methods used to detect microbial species in the root canal system?

Answer 3

• Microscopy
• Immunological methods
• Culturing
• Pyrosequencing analysis.
• Molecular biology techniques
  a) PCR
  b) RT-PCR (Qualitative)
  c) qPCR (Quantitative)
  d) DGGE

Question 4

Q41 : What are the causes of failure or persistence of an apical infection following endodontic treatment?

Answer 4

According to Sundqvist & Figdor (Endo Topics-2003) The contributing factors to persistent infection can be divided in to
1- Intra-radicular infection. Such as missed canal, poor root canal treatment, coronal leakage, isthmus or lateral canals. This is the primary cause of failure in most of the root canal failures. It has been shown by Ricucci & Siqueira (JOE 2010) that intra radicular biofilm is present in 77% of cases with radiographic evidence of apical periodontitis. This percentage further increases in cases with symptoms, sinus tracts, and epithelialized lesions

2- Extra radicular infection. Can be either
a) Extra-radicular biofilm on the external root surface. It has been shown by Ricucci & Siqueira (JOE 2010) that the incidence of extra-radicular biofilm is as low as 6%. This incidence can increase up to 70% in cases with sinus tracts (Ricucci et al 2018)
b) Apical Actinomycosis which can only be successfully treated by apical surgery & curettage of the apical inflammatory lesion (Siqueira, Endo Topics-2003). It has been shown to occur between 2-10% of cases treated through endodontic surgery** (Borssén & Sundqvist, Oral Surg- 1981, Hirshberg et al, OOOE 2003,)**
3- Cystic lesion. It has been hypothesized by** Nair (IEJ-1998)** that true cysts may not resolve following endodontic treatment and surgical intervention is required.
4-Foreign body reaction. Presence of entrapped foreign body material from endodontic treatment such as gutta percha or sealer, or food particles may cause persistent of periapical disease. Clinical studies with long-term follow ups have showed that gutta purcha may retard the healing process but will not cause true failure per say (Molven et al, IEJ-2002, Fristad et al. 2004 Azim et al, IEJ - 2016)
5- Fibrous scar tissue healing. While scare tissue is not a true disease, it can mimic periapical disease radiographically (Nair, IEJ 2006).

Question 5

Q42 : Endotoxins level in primary endodontic infection is higher/lower than or similar to than that of secondary endodontic infection ?

Answer 5

Higher
Endotoxins are released by gram -ve bacteria. Gram -ve obligate anaerobes dominate the root canal space in primary infections (Baumgartner & Falker, JOE-1991) compared to secondary/persistent infections which are dominated by gram +ve bacteria (Chavez de Paz et al. IEJ-2003, Chavez de Paz et al, IEJ - 2004)
Gomes et al (JOE-2012) investigated the level of endotoxins in primary and secondary endodontic infections. They found that teeth with primary endodontic infections had higher levels of endotoxin and a more complex community of gram-negative bacteria than teeth with secondary infections Endotoxin levels were related to the severity of bone destruction in periapical tissues as well as the development of clinical symptoms in teeth with primary infections.

Question 6

Q43 : What is the prevalence of intra-radicular bacterial biofilms in teeth with apical periodontitis?

Answer 6

Intra-radicular biofilms were observed in the apical segment of 77% of the root canals. There is also a higher incidence of biofilms in cases with large periapical lesions. (Ricucci & Siqueira, JOE 2010)

Question 7

Q44 : Which of the following procedures will result in the highest incidence of bacteremia? EXTRACTION / Elevation flap / root canal therapy

Answer 7

All of the above procedures may cause bacteremia. Baumgartner et al. (JOE - 1976) showed that bacteremia is less likely to happen during non-surgical RCT unless teeth were over instrumented.
Debelian et al (Endod Dent Traumatol-1995) even showed that over instrumentation during RCT does not appear to influence the occurrence of bacteremia.
Bacteremia is more likely to occur during surgical endodontic treatment and surgical extraction (Baumgartner et al, JOE - 1977)

Question 8

Q45 : What are the “zones of fish”?

Answer 8

Zones of Fish were the early attempt to disprove focal infection theory by showing that bone infection can be contained and will not spread to distant organs. (Fish, JADA- 1939)
Zones of fish can be divided into 4 zones:
1) Zone of infection
2) Zone of contamination
3) Zone of irritation
4) Zone of stimulation

Question 9

Q46 : What are the dominating bacterial species in teeth with necrotic pulp/apical periodontitis?

Answer 9

Sunqvist (1976) showed that anaerobic bacteria accounted for more than 90% of the isolates bacteria in cases with necrotic pulp.
Fabricus et al. (Scan J Dent Res - 1982) showed that after 7 days of infection: proportion of facultative anaerobic greatly higher than strict anaerobic bacteria. After 90 days of infection, 85% of the bacterial cells from the apical region were anaerobic. After 180 and 1060 days of infection, 95-98% are anaerobic.
Baumgartner & Falkler (JOE-1991) showed that 68% of bacteria isolated from the apical 5mm were strict anaerobes.
Siqueria et al. (JOE-2011) investigated the diversity of the apical endodontic microbiota 187 bacterial species-level with 84 genera, and 10 phyla were identified using pyrosequencing analysis. The most abundant and prevalent phyla were :
Proteobacteria (43%) Firmicutes (25%) Bacteroidetes (9%) Fusobacteria (15%) Actinobacteria (5%)

Question 10

Q47 : Why it is difficult to remove a bacterial biofilm from the root canal space?

Answer 10

Explanation:
1) Bacterial biofilms can exhibit metabolic cross feeding (community can exist for longer time)
2) Resistant to intra-canal medication such as Ca(OH)2 (Distel et al, JOE - 2002)
3) Resistance to antibiotics (Stewart & Costerton, Lancet 2001)
4) Can survive deep into the dentinal tubules (Love, JOE 2001)
5) Endodontic instruments are unable to mechanically touch all canal walls, and thus won’t be able to mechanically disrupt biofilms along the entire canal wall (Peters et al, JOE 2001)

Question 11

Q48 : E faecalis is commonly detected in failed endodontic cases. Describe how E faecalis can resist the various disinfection procedures?

Answer 11

a) E. faecalis is able to penetrate dentinal tubules to a deep extent, protects it from the action of instruments and irrigant (Haapasalo & Ørstavik, JDR-1987)
b) Its Proton pump enable E Faecalis to resist High PH (Evans et al, IEJ-2002) as a result it can resist Ca(OH)2 and grow in its presence (Distel et al, JOE-2002)
c) It has the ability to invade tubules and binds with collagen (Love, JOE-2001)
d) Environmental signals can regulate the genetic expression of E faecalis (Jett et al. Clin Microbiol Rev-1994)

Question 12

Q49 : What is the primary cause of pulp and periapical disease?

Answer 12

Bacteria
Kakehashi et al. (000- 1965) showed that periapical disease did not develop in germ-free rates following pulp exposure compared to conventional rats. This study demonstrated that lesions of endodontic origin were a result of bacterial infection.
Sunqvist (1976), Moller et al. (Scan J Dent Res 1981) and Lin et al: (JOE 2006) showed that apical periodontitis was detected in teeth with bacteria in the root canal systems and uninfected necrotic tissue did not produce an apical inflammatory reaction.

Question 13

Q50 : What is the incidence of extra-radicular biofilm in endodontic infections?

Answer 13

The incidence is very low ranging from 4- 6% (Siqueira & Lopez, IEJ - 2001, Ricucci & Siqueira, JOE 2010). The incidence of extra radicular biofilm can increase to 70% in cases diagnosed with chronic apical abscess (Riccuci et al, JOE - 2018)

Question 14

Q51 : Which species are considered “black pigmented bacteria”?

Answer 14

There was a nomenclature change in the 1990’s which affected the taxonomic classification of certain species. Older papers referred to a broader group of ‘Bacteroides’ or ‘black-pigmented’ bacteria. These bacteria are gram negative obligate anaerobes. They were divided into two groups according to their ability to ferment carbohydrates. Prevotella (saccharolytic) and Porphyromonas (asaccharolytic) (Robertson & Smith, JMM - 2009)

Question 15

Q52 : What is the prevalence of Candida species in root canal infections?

Answer 15

In a recent systematic review and Meta Analysis of literature by Mergoni et al (JOE - 2018), the cumulative prevalence of Candida spp was 8.2%, which is quite a low value and places some doubt as to the real role of yeasts as pathogens in the majority of endodontic infections. They suggested that Candida may play a role in root canal infections although the body of evidence is not strong.

Question 16

Q53 : What is “Anachoresis”? And can anachoresis cause pulp disease?

Answer 16

Anachoresis is the localization of blood-borne bacteria during bacteremia to a site of inflammation. The hypothesis is that bacterial in the blood stream can seed directly into the inflamed pulp via the pulpal blood supply during bacteremia,

Delivanis et al (000-1981) conducted a study on a stagnant fluid in instrumented but not filled root canals of five cats. The fluid was analyzed for possible localization and growth of IV injected bacteria 48 hrs. after injection, all sample obtained from the canals were negative for the tested organism, Delivanis & Fan (JOE-1984) repeated the same study but they also over instrumented the canals to cause bleeding. They found that canals can get infected following over instrumentation, indicating that anachorasis can occur.

It was thought that anachoresis can cause pulp disease due to presence of bacteria in the blood stream in the inflamed pulp (Tziatas, JOE-1989) These studies, however, do not prove that the pulp subsequently become necrotic as a direct result of the bacteremia Lin et al (JOE-2006), however, showed that teeth with periapical lesions on one side of the jaw did not serve as foci of infection nor caused infection of teeth with devitalized pulps on the other side of the jaw.
Another argument against anachoresis as a route of pulpal infection comes from the study by Moller et al (Scan Dent Res-1981) were cases of aseptic necrosis remained bacteria-free after 6 to 7 months of observation.
It appears that anachoresis can occur, but it does not cause pulp/periapical disease. It can, however, aggravate a periapical condition making it more symptomatic Sabeti et al (JOE-2003) showed that Cytomegalovirus (HCMV), Epstein-Barr Virus (EBV) participates in the pathogenesis of symptomatic periapical lesions which can impairs local defenses, thereby inducing overgrowth of endodontopathic bacteria

Question 17

Q54 : Does LPS play a role in pulp and periapical disease? Explain

Answer 17

YES
Schein & Schilder (JOE-1975) investigated the levels of endotoxin in the fluid aspirated from the root canals of teeth that were opened for RCT. They found that necrotic teeth contained greater conc. of endotoxin than those with vital pulps, symptomatic teeth also contained more endotoxin than asymptomatic teeth. There were also more endotoxins in teeth with PAR compared to teeth without PAR. These results suggested a possible role of endotoxins in pulpal and periapical diseases.
Pitts et al (JOE - 1982), later evaluate the effect of endotoxin on periapical tissue by introducing endotoxin into the root canals of dogs. They found that root exposed to endotoxins tended to show periapical radiographic changes sooner and to a more severe degree. Histologically, they also showed larger bone defects with more intense degree of inflammation. The results of this study indicate that endotoxins may have a role in periapical inflammation and bone destruction.
Khabbaz et al (OOOE-2001) found that endotoxins were detected in all pulpal tissues of symptomatic & asymptomatic carious teeth. The conc., however, of endotoxins in the pulpal tissue of symptomatic teeth was significantly higher suggesting a possible association of endotoxins levels with severity of pulpal pain.

Question 18

Q55 : Can bacteria survive in the periapical area?

Answer 18

Normally bacteria cannot survive planktonically in the apical area. With exception to few species such as actinomyces (Bystrom et al, Endod Dent Traumatol - 1987)
They can only survive in a biofilm (Plaque) structure on the external root surface (Tronstad et al, Endod Dent Traumatol - 1990). The incidence, however, is very low (4-6%) (Siqueira & Lopez, IEJ-2001), (Ricucci & Siqueira, JOE - 2010)
Several earlier studies have shown the ability of bacteria to survive in the apical area such as Tronstad et al. (Endod Dent Traumatol, 1987), Waymen et al. (JOE-1992) and Iwu et al (000-1990). Since all these were clinical studies, it is possible that these samples were contaminated during sampling despite their careful measurements, since maintaining a surgical sterile environment in the oral cavity is not really possible.
Walton & Ardjman (JOE-1992) have shown in a monkey study that when a blocks sections (bone and lesion) were harvested from teeth where apical periodontitis was induced, there were no bacteria found in all samples.

Question 19

Q56 : What are the most commonly detected bacteria in teeth with acute apical abscess?

Answer 19

Sakamoto et al. (oral Micro & Immun- 2006) performed a molecular analysis on teeth with asymptomatic apical periodontitis and teeth with acute apical abscess. The species that most commonly isolated in teeth with acute apical abscess were
1- F nucleatum T-RF type II
2- P Intermedia
3- Dialister pneumosintes
Gomes et al (JOE 2006) showed that Filifactor alocis, Tannerella forsythia, and Treponema denticola were associated with acute apical abscess as well.

Question 20

Q57 : What is a bacterial biofilm?

Answer 20

Microorganisms attached to a surface and form 3-dimensional structure held together by extra cellular polymeric substance that can cope with environmental stresses
Holds different types of bacteria
Connected via water channels
Comunicate via quorum sensing** (Scoransky & Haffajee, Periodontology 2000-2002)**
Exchange gene coding thereby increasing their microbial resistance.

Question 21

Q58 : Describe the pathogenic role of E. faecalis?

Answer 21

• According to Rocas et al (JOE 2004), E. faecalis possesses certain virulence factors including lytic enzymes, cytolysin, aggregation substance, pheromones, and lipoteichoic acid. It has been shown to adhere to host cells, express proteins that allow it to compete with other bacterial cells, and alter host responses.
• E. faecalis is able to suppress the action of lymphocytes, potentially contributing to endodontic failure (Lee et al, JOE 2004)
  -E. faecalis is not limited to its possession of various virulence factors. It is also able to share these virulence traits among species, further contributing to its survival and ability to cause disease
  E. faecalis possesses a proton pump that allows it to adapt to harsh environments (Evans et al., IEJ 2002). This proton pump is theorized to contribute to E. faecalis unique resistance to CaOH2
  Check the review by Stuart et al (JOE-2006) and on Enterococcus faecalis and Its role in root canal treatment failure.

Question 22

Q59 : Describe the differences between endotoxins and Lipoteichoic acid?

Answer 22

Endotoxins lipopolysaccharides (LPS) are released by gram-negative bacteria and they are recognized mostly by toll-like receptor 4 (TLR-4) on antigen presenting cells (APC) or macrophage resulting in inflammatory cytokine production and activation of innate immune system.
Lipoteichoic acids (LTAs), on the other hand, are released by gram positive bacteria and they are recognized by TLR 2. They can also induce inflammation, but they are not as virulent as LPS.

Question 23

Q60 : How can an extra-radicular biofilm develop?

Answer 23

Extra-radicular biofilm can develop either through periodontal infection or endodontic infection spreading through the dentinal tubules and reaching the external root surface.
Recently, Ricucci et al. (JOE-2018) showed that extra-radicular biofilms were more prevalent in cases with chronic apical abscess suggesting that saliva may gain access to the apical root segment through sinus tracts and form bacterial biofilm/calculus on the external root surface

Question 24

Q61 : Is there any correlation between the bacterial load inside the root canal space and the lesion size?

Answer 24

Yes
Ricucci & Siqueira (JOE -2010) showed a higher incidence of bacterial biofilms in cases with large periapical lesions.

Question 25

Q62 : Other than bacteria, what other species can play a role in persistent apical periodontitis?

Answer 25

Candida species:
Candida species are normally present in saliva. Baumgartner et al (JOE-2000) evaluated the contents of infected root canals and aspirates of cellulitis/abscesses of endodontic origin. They found that C. albicans may be involved in root canal infections. Siqueira et al. (JOE-2002) showed that C albicans has the ability to colonize dentin while other types of fungal species were unable to, which helped explaining why C. albicans is the fungal species most often found in endodontic infections.
Viruses:
Sabeti et al (Oral Microbiol Immunol-2003) stated that Human Cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) active infections are associated with acute exacerbation of apical periodontitis. They hypothesize that periapical active virus infection can impairs local defenses, thereby inducing overgrowth of endodontopathic bacteria and the clinical flare-up of inflammation Sabeti et al. (JOE-2003). Later, Slots et al. (IEJ-2004) suggested adding HCMV and probably EBV to the list of putative pathogenic agents in symptomatic periapical disease. More recently, Jakovljevic & Andric (JOE-2014) conducted a systematic review to analyze the evidence indicating that HCMV and EBV can actually contribute to the pathogenesis of periapical lesions. The results of this study failed to establish a statistically significant relationship between HCMV and EBV detection and the clinical features of periapical lesions and the mechanisms of HCMV and EBV reactivation and participation in periapical disease progression remain to be revealed.

Question 26

Q63 : What type of bacteria appears to survive commonly after chemo-mechanical root canal treatment in teeth with apical periodontitis?

Answer 26

Chavez de Paz et al (IEJ-2003) showed that the most commonly isolated species after chemo-mechanical root canal treatment were:
Gram +Ve (85%)
Lactobacillus spp (22%)
nonmutants streptococci (18%)
Enterococcus spp. (12%)