CONTINUING EDUCATION Advances in microbiology and bacteriology Dr. Navid Saberi presents an overview of microbiology and bacteriology in endodontic treatment t has been almost 340 years since the Ibirth of microbiology and the discovery Educational aims and objectives of bacteria by Robert Hooke and Antonie This clinical article aims to present an overview of the many advances in microbiology van Leeuwenhoek (Gest, 2004) and around and bacteriology, and which bacteria are safe to leave in the root canal after endodontic treatment. 150 years since the dawn of modern bacteriology by the concurrent and rivalry- Expected outcomes driven studies of Robert Koch and Louis Endodontic Practice US subscribers can answer the CE questions on page XX to Pasteur (Ullmann, 2007). earn 2 hours of CE from reading this article. Correctly answering the questions will In the field of endodontology, W.D. Miller demonstrate the reader can: was the first to associate bacteria with pulpal • Realize the many advances in microbiology and bacteriology. • Identify how these remaining bacteria can be detected. disease in 1894. However, even prior to • Recognize which bacteria are safe to remain in the root canals. Miller’s experiments, dental practitioners • Identify how many bacteria is it safe to leave in the root canal after endodontic treatment. were aware of the important role of disinfec- tion and antiseption in endodontic outcomes. In fact, in 1886, Dental Cosmos published therapy is therefore to achieve this goal by of bacterial detection have been associated a report titled “Disinfection and antiseption mechanical instrumentation and biochemical with some drawbacks. by heat.” According to the author, in this irrigation of the root canal systems, which can Culturing has been advocated as a rather procedure “a fine wire heated to redness and be referred to as biomechanical cleansing of simple means of bacterial identification. inserted into the canal to the apical foramen infected root canals. However, up until the late 1960s, culturing … the application repeated according to However, despite being desirable to methods were unable to propagate anaer- circumstances … is all that is necessary” and render the root canals sterile, it is logical and obic bacteria predictably. The routine use of “by his [Dr. G.O. Rogers’] method complete realistic to assume that achieving sterility may anaerobic glove box and sterilized anaerobic disinfection and antiseption are secured” not be feasible in every infected root canal. In culture media has significantly enhanced (Pomeroy, 1886). fact, sterility may not even be required for a accurate identification of these bacteria. In addition, devitalization by means of successful outcome. Actor (2012) reported Furthermore, standardized field decontami- arsenic trioxide and instrumentation with that there are 20 times more bacterial cells nation protocols in the late 1960s reduced modified watch springs were commonplace in human bodies than eukaryotic cells. the possibility of false positive results (Moore, (Grossman, 1982; 1987). These communities live in either a symbiotic 1966; Kantz and Henry, 1974; Ng, et al., It is now well established that bacteria or mutualistic manner and benefit from the 2003). are essential for the development of pulpal existence of one another without causing Although cultivability of all bacteria was and periradicular diseases (Kakehashi, et al., harm. Therefore, at least in theory, it can be first reported in the 1960s, it was not until 1965; Möller, et al., 1981), and the presence assumed that some bacteria in inaccessible the 1990s that scientists fully recognized of bacteria in the root canal space or peri- areas of the root canal systems may be left that not all bacteria were culturable or iden- apical tissue will undermine the success of behind without compromising the overall tifiable (Socransky, et al., 1963; Hugenholtz endodontic treatment (Sjögren, et al., 1997; outcome of the treatment. and Pace, 1996). This meant previous Byström, et al., 1987). In spite of many advances in micro- bacteriological studies based on culturing Therefore, for a successful treatment, all biology and bacteriology, there still remain alone probably discovered only half of the root canals should be identified, thoroughly many unanswered questions. How can we true taxa associated with endodontic infec- disinfected, and rendered bacteria-free prior detect these remaining bacteria? Which tions (Paster, et al., 2001). These uncultivable to obturation. The aim of modern endodontic bacteria are safe to remain in the root canals? bacteria could indeed be the more impor- And, more importantly, how many bacteria tant entities in the pathogenesis of periapical Navid Saberi, BDS, MFDSRCS, MSc, graduated from Manchester is it safe to leave in the root canal after disease. University and worked in the general, salaried, and hospital dental endodontic treatment? The development of polymerase chain services in Scotland for several years. He completed his MSc The aim of this article is to provide reaction (PCR) in the late 1980s and its use with distinction at Glasgow University and became a member of the faculty of dental surgery of the Royal College of Surgeons answers to the above questions. in endodontics by the early 1990s radi- of Edinburgh in 2010. He has limited his practice to endodontics cally changed the dogma that had been and works at several prestigious private practices in London, the Bacterial identification founded on a culture-based understanding South East of England, and the South East of Scotland, including Harley Street Dental Group in London, Elms Lea Dental Practice In order to determine the safe number of endodontic microbiology (Mullis and in Brighton, and Wessex Specialist Dental Centre in Hampshire, of bacteria that may possibly be left in root Faloona, 1987; Pollard, et al., 1989; Spratt, just to name a few. He co-founded the Scottish Endodontic Study canals after endodontic treatment, it is Weightman, and Wade, 1999). Group in 2010 and is an active member of the British Endodontic Society. imperative for researchers to identify these PCR, however, is not without pitfalls. microorganisms. However, most methods This highly sensitive method of nucleic acid 32 Endodontic practice Volume 8 Number 2 CONTINUING EDUCATION amplification requires a strict decontami- nation protocol if false positive results are to be avoided (Ng, et al., 2003). In addi- For a successful treatment, tion, Hayden and colleagues (1991) found that PCR amplification methods inherently all root canals should be identified, detect bacteria without specifying whether the microorganisms were dead or alive at thoroughly disinfected, and rendered bacteria-free the time of sampling. This predicament can be overcome by amplifying genomic ribo- prior to obturation. somal ribonucleic acid (RNA), which can only be detected in active and dividing cells. However, bacteriological results based on ribosomal ribonucleic acid (rRNA) amplifica- tion should also be scrutinized for method- of these bacteria could establish an infection Therefore, a certain combination of ology as a very short half-life of rRNA could (Sundqvist, 1994; Paster, et al., 2006). These bacteria, a favorable environment, nutrient potentially lead to false negative results if the bacteria live in symbiosis with the host within availability, and the host response must be procedure is not carried out swiftly. the oral cavity (Avila, Ojcius, and Yilmaz, the main determining factors in the patho- In spite of all major improvement in 2009). Nonetheless, they are opportunistic genesis of apical periodontitis (Siqueira and bacteriological identification methods, one microorganisms and may cause disease, Rocas 2009a,b; Paster, et al., 2006). major shortcoming still remains unsolved, particularly where the host loses the ability to The main inhabitants of primary and that is access to intraradicular micro- maintain the homeostasis in the ecosystem endodontic infection are gram negative organisms. Even though the main root canal (Actor, 2012). anaerobic rods with different pathogenicities. and associated lateral canals may be acces- As explained earlier, almost half of The most common bacterial species involved sible for direct or indirect sampling, most of disease-producing endodontic bacteria are in primary endodontic infections with their the root canal system, including canal walls, uncultivable (Hugenholtz and Pace, 1996). genera, phyla, and degree of pathogenicity dentinal tubules, isthmuses, fins, and webs, Although with the aid of PCR many uncul- are shown in Table 1 (Siqueira and Rocas may be untouched, inaccessible, or blocked tivable bacteria have been cloned, they are 2009b,c; Siqueira, et al., 2009; Ribeiro, 2011). by debris, hence making accurate sampling yet to be named and meticulously analyzed It is important to mention, however, that an impossible task. In addition, the discovery (Rolph, et al., 2001; Munson, et al., 2002, individual virulence of single species does of biofilms transformed our understanding Siqueira and Rocas, 2005). Therefore, a not directly translate to an overall degree of of bacterial ecology, according to Costerton thorough knowledge of the type, character- pathogenicity of a bacterial colony (Siqueira and colleagues (1994; 1999). Planktonic istics, metabolism, pathogenicity and partic- and Rocas, 2009a,c). Virulence is a direct root canal bacteria seldom sustain peri- ularly interactions of these microorganisms result of bacterial