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Oral biofilms: a reservoir of transferable, bacterial, antimicrobial resistance Expert Rev. Anti Infect. Ther. 8(12),

Oral biofilms: a reservoir of transferable, bacterial, antimicrobial resistance

Expert Rev. Anti Infect. Ther. 8(12), 1441–1450 (2010)

Adam P Roberts 1 and Peter Mullany 1

1 Department of Microbial Diseases, UCL Eastman Dental Institute, University College London, 256 Gray’s Inn Road, London, WC1X 8LD, UK Author for correspondence:

Tel.: +44 207 915 1044 Fax: +44 207 915 1127 adam.roberts@ucl.ac.uk

Oral microbes are responsible for dental caries and periodontal diseases and have also been implicated in a range of other diseases beyond the oral cavity. These bacteria live primarily as complex, polymicrobial biofilms commonly called dental plaque. Cells growing within a biofilm often exhibit altered phenotypes, such as increased antibiotic resistance. The stable structural properties and close proximity of the bacterial cells within the biofilm appears to be an excellent environment for horizontal gene transfer, which can lead to the spread of antibiotic resistance genes amongst the biofilm inhabitants. This article will present an overview of the different types and amount of resistance to antibiotics that have been found in the human oral microbiota and will discuss the oral inhabitants’ role as a reservoir of antimicrobial resistance genes. In addition, data on the genetic support for these resistance genes will be detailed and the evidence for horizontal gene transfer reviewed, demonstrating that the bacteria inhabiting the oral cavity are a reservoir of transferable antibiotic resistance

K eywords : antibiotic resistance • biofilms • caries • dental plaque • horizontal gene transfer • mobile genetic elements • oral biofilms • periodontitis • reservoir of resistance

Antibiotic resistance in pathogenic bacteria cur- rently represents one of the greatest challenges to modern medicine throughout the world. The increase in morbidity and mortality due to hos- pital-acquired ‘superbugs’ is annually increas- ing. In a recent EU report on the problem of antibiotic resistance, it was stated that each year approximately 25,000 patients die in the EU from an infection with one of eight different nosocomial, multidrug-resistant bacteria [101] . In addition, economic implications for health- care providers, in terms of increased patient stay in hospitals, isolation procedures and additional medical intervention are increasingly imping- ing on the available budgets. In the EU this has resulted in extra healthcare costs and productiv- ity losses of at least 1.5 billion each year [101] . Similar figures are available for the USA [1] . There are numerous ways that researchers and clinicians can tackle this problem, including the much needed discovery or generation of novel antimicrobials, highlighted by the recent global ‘10 × ‘20 initiative’, which aims to produce ten new antibiotics by 2020 [102] . Another, not mutually exclusive approach is to try and deter- mine where the resistance comes from. With the increasing use of antibiotics, it is important to

understand where the reservoirs of resistance are, what genes they contain and more importantly the possibilities for transfer of these resistances to pathogenic microbes. This knowledge will be of help in predicting emerging resistances among pathogens [2] . Most pathogens do not intrinsi- cally contain resistance genes, as has been dem- onstrated by the numerous completed genome sequences of pathogenic bacteria, and it is only the acquisition of such genes that leads them to become the multiple antibiotic-resistant bacte- ria that are the scourge of modern healthcare environments. These genes must be acquired from somewhere and recent investigations have identified the normal human microflora as one such potential reservoir for these antibiotic resis- tance genes. Of clinical importance is the fact that many of these antibiotic resistance genes have been found to be located on mobile genetic elements capable of broad host-range horizontal gene transfer among different bacteria (Box 1). This article will give an overview of our cur- rent understanding of the resistance found in bacteria inhabiting the oral cavity, usually within oral biofilms, and will highlight their potential to transfer these resistance genes to other oral and transient bacteria.

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Roberts & Mullany

Box 1. Mechanisms of gene transfer among bacteria.

DNA can transfer from one bacterial cell to another by one or more of three mechanisms; namely, transformation, conjugation and transduction.

Transformation is the process whereby bacteria take up free DNA from the environment and incorporate it into their genome. The source of the transforming DNA is usually lysed bacterial cells. Bacteria able to take up DNA by this process are termed competent.

Conjugation is the transfer of DNA (usually a conjugative/mobilizable plasmid or conjugative/mobilizable transposon) between two metabolically active bacterial cells and requires intimate cell to cell contact for transfer to occur.

Transduction is the process in which bacterial DNA gets erroneously packaged into the heads of bacteriophage, when the phage infects another bacterial cell the packaged DNA is incorporated into the new host’s genome.

An overview of these mechanisms is shown in Figure 1.

The oral cavity

The oral cavity is one of the most bacterially colonized envi- ronments in the human body. The microbial diversity present in the oral cavity is a result of the many different ecological niches present, ranging from the hard, nonshedding surfaces of the teeth, both above and below the gingival margin, and the mucosal surfaces of the various soft tissues, such as the tongue, cheek and the hard and soft palate. Each of these sur- faces presents a different environment to bacteria; therefore, the microbial populations found at each site are distinct [3] , particularly in the elderly [4] . In addition to the varied environ- ment, there is an enormous range of growth substrates that the resident bacteria encounter during ingestion of foods. It has been shown that increases in dietary sugar, for example, can push the population structure of the oral community to one with predominantly more cariogenic streptococci, which can subsequently lead to caries [5] . A recent study on the effect of different drinks has shown that the number of species is signifi- cantly lower between coffee and wine drinkers compared with the control group (water drinkers) [6] . The numbers of different types of bacteria present in the oral cavity are only recently beginning to be appreciated. Owing to the fact that less than half of the species present can currently be cul- tured in the laboratory the use of next-generation, massively paral- leled sequencing technologies are enabling researchers to gain a more accurate estimate of the true species diversity. A recurring theme among recent reports is that, whilst there is a core micro- biome emerging, for example, streptococci are the predominant species in a healthy mouth, there are significant interindividual differences in the microflora detected in each study [7–9] .

Oral biofilms

Some of the microbes present in oral biofilms are responsible for two of the most common diseases to affect humans, dental caries and inflammatory periodontal disease, which have been estimated to affect up to 90% of the planet’s population [10] . Development of oral biofilms begins when the newly exposed surface of the tooth (following eruption of a new tooth or fol- lowing mechanical removal of existing plaque by brushing or scaling) is covered by an acquired pellicle. This process is esti- mated to take minutes [11] . The pellicle is composed of different host-derived molecules, including mucins, proteins and agglu- tinins, which act as a source of receptors that are recognized by

various oral bacteria. In addition, there are other host proteins, for example, lysozyme and histatins, which are present in the pellicle and are hypothesized to protect from bacterial coloni- zation [12,13] . Bacteria that can recognize these receptors and bind to the pellicle-covered surface of the tooth are known as early colonizers and include Streptococcus spp., Actinomyces spp., Capnocytophaga spp., Eikenella spp., Haemophilus spp., Prevotella spp., Propionibacterium spp. and Veillonella spp. [14] . Subsequently the biofilm is colonized by the late colonizers, which include Actinobacillus spp., Prevotella spp., Eubacterium spp., Porphorymonas spp. and Treponema spp. Coaggregation between pairs of early colonizers is common, however it is relatively rare between late colonizers. Fusobacterium nucleatum however is able to coaggregate to all members of both the early and late colonizers so far examined and also binds many host-derived molecules found in the pellicle [15] . As the attached bacterial cells grow and divide, many will start to express a biofilm phenotype, which will include the secretion of extracellular polymeric substances (EPS),which include poly- peptides, carbohydrates and nucleic acids. These EPS will eventu- ally form the bulk of the mature biofilm structure [16] and afford the cells and microcolonies growing within this matrix physical stability from shear forces experienced within the oral cavity, such as those from the flow of saliva and the action of the tongue and other mucosal surfaces.

Oral diseases

The organisms present in oral biofilms are responsible for a number of different diseases. Acid-producing streptococci, such as Streptococcus mutans, are the primary cause of den- tal caries. In addition, oral biofilms contain other pathogens, such as Tannerella forsythensis, Porphyromonas gingivalis and Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, that have all been shown to be involved in the formation of peri- odontal disease [17] . Periodontitis is complex in that there are a number of different bacteria that are associated with the major- ity of (but not all) cases of the disease. A. actinomycetemcomitans is the bacteria that most closely fulfils Koch’s postulates as the etiological agent of periodontitis and is most often associated with the aggressive juvenile form of the disease [18] . The oral cav- ity provides an excellent portal to the rest of the body, either to the alimentary canal or through the gingival surfaces following cuts or abrasions sustained from brushing or eating, leading to

Oral biofilms: a reservoir of transferable, bacterial, antimicrobial resistance

Review

short-term bacteremia [19] . In addition, oral bacteria can often be isolated from the blood of dental patients following dental pro- cedures, for example, tooth extraction, and many of the bacteria isolated have been shown to cause endocarditis [20] . In recent years, research has demonstrated associations of oral bacteria with a range of other diseases and systemic conditions such as coronary heart disease and a higher risk of preterm, low- birthweight babies [21,22] . Similarly, recognition of the threats posed by periodontal diseases to individuals with other chronic diseases, such as diabetes [23] and hypertension [24] , are only just beginning to be understood. There is also an increasing amount of foreign implants, such as dentures and bridges, being used. These share a common prop- erty with the surface of the tooth in that they are nonshedding. Therefore this surface, once coated with the acquired pellicle, will be readily colonized by bacteria, leading to the formation of biofilms on these devices [25] that may compromise the comfort and treatment of the patient. The majority of bacteria living on the surfaces of the mouth do so in symbiosis with the host [10] . The normal oral biofilm at the gingival margin has beneficial effects as its constant pres- ence provides colonization resistance for incoming and possibly pathogenic microorganisms. This is illustrated by the fact that disease is usually accompanied by a shift in the populations of the predominant bacteria within oral biofilms when compared with a healthy state [26] .

The use of antibiotics to treat periodontal disease

The treatment of periodontitis is quite different from the treat- ment of the majority of other diseases caused by bacterial infec- tions due to the underlying polymicrobial nature of the disease. There has been a wide variety of different antibiotics used to treat periodontitis. These have been administered in different doses, in combinations with other antibiotics and used both alone and in combination with mechanical plaque removal [18] . Some of the most common antibiotics that have been shown to result in the required levels necessary to kill or inhibit bacterial growth in gingi- val crevicular fluid are amoxicillin and amoxicillin/clavulanic acid, the tetracyclines, clindamycin and metronidazole. Dentists (in the UK) prescribe amoxicillin, penicillin and metronidazole most often with other antibiotics (including cephalosporins, tetracyclines and macrolides) being prescribed less often [27] ; however, owing to the polymicrobial nature of oral biofilms there is likely to be certain species of bacteria present that are resistant to certain antibiotics. Because of this, the outcome of identical treatment regimes may be different for different patients and it is for this reason that combi- nation therapies, such as metronidazole and amoxicillin, are now becoming increasingly important [28,29] and are recommended to be adjunctive with mechanical debridement [30] .

Intrinsic biofilm resistance to antimicrobials

Bacteria growing within biofilms often exhibit differing pheno- types compared with planktonically grown counterparts. One of the most clinically important phenotypes is increased resistance to antimicrobial compounds.

As the biofilm matures, the EPS surrounding the bacterial cells will increase and can exclude antibiotics from the immedi-

ate vicinity of bacteria deep within the biofilm [31] . A similar effect may occur with compounds that are secreted by bacte- ria that confer antimicrobial resistance, such as b-lactamases.

A high concentration of these compounds will form near to the

cells producing them if diffusion through the biofilm structure

is impeded by the EPS, providing a local and transient area of

relatively high resistance. In addition, because of diffusion being hampered by the EPS, nutrients and chemicals other than antimicrobials or resistance proteins will also form gradients within the biofilms. Deep within the biofilm, cells are likely to be less metabolically active than those nearer the surface owing to the lack of nutrients reach- ing them. Many antibiotics act against metabolic targets such

as protein synthesis (e.g., tetracycline); therefore, if a cell is not

metabolically active it will be transiently resistant to the action

of these antibiotics. In addition, many antimicrobial compounds,

for example, mercury, are actively pumped into the bacterial cells and therefore if metabolic activity is low a lower amount of the antimicrobial will be pumped across the cell membrane, leading

to a reduced susceptibility to that agent. Another important difference between bacteria grown in bio- films compared with planktonically grown cells is altered gene expression. There is a multitude of different genes that are either

positively or negatively regulated when the bacteria are growing as

a biofilm compared with planktonically grown cultures [32] . The

complex regulatory networks controlling the expression of these genes and their response to biofilm formation, and in some cases the presence of antimicrobial compounds, are likely to control

apparatus such as efflux pumps, which will alter the sensitivity

to various

antimicrobials [32,33] .

Oral commensal bacteria as a reservoir of transferable resistance genes

There are many reports of individual bacterial isolates from the oral cavity becoming resistant to one or more antibiotics, for example, b-lactamases in Gram-negative respiratory and oral bac- teria (reviewed in [34] ), but these do not give us a comprehensive view of the resistance genes and any cognate mobile genetic ele- ments (Box 2) within this environment as a whole. Here we review the studies that have investigated the entire resistant cultivable microflora and the metagenome from the oral environment for resistance to specific antibiotics.

Studies on the cultivable oral flora

A study on children who had not received antibiotics in the pre-

ceding 3 months demonstrated that 15 out of 18 subjects had tetra- cycline-resistant bacteria in their oral cavity. The children were highly unlikely to have come into direct contact with tetracycline

as it is not prescribed for children owing to undesirable side effects, such as the discolouration of the teeth and nails [35] . The most common gene identified in this cohort was tet(M), which encodes

a ribosomal protection protein, contained on Tn916 /Tn1545 -like

conjugative transposons in Streptococcus, Granulicatella, Veillonella

Review

Roberts & Mullany

Box 2. Mobile genetic elements responsible for the interbacterial transfer of resistance genes.

Plasmids usually exist as independently replicating units; however, on some occasions they will integrate into the bacterial chromosome. They are grouped into incompatibility (Inc) groups based on their inability to coexist in the same cell. Plasmids from the same Inc group usually have identical or similar replication/partition systems. Only one plasmid from one Inc group can stably exist in a cell at one time, if another plasmid enters the cell belonging to the same Inc group, one plasmid will eventually be lost during cell division owing to mutual interference of the replication process by the other plasmid, leading to an unequal amount of the two plasmids in the dividing cell. Plasmids are found in both Gram-positive and Gram-negative organisms isolated from the oral cavity. Conjugative transposons, also known as Integrative Conjugative Elements, are mobile elements that integrate into their hosts’ genome. They encode all of the necessary information for intracellular transposition and intercellular conjugation. Some conjugative transposons have a very broad host range. They are commonly found in Gram-positive and Gram-negative bacteria. The Tn916 -like elements are very common in oral bacteria. Conjugative transposons can carry accessory genes, which often encode antibiotic resistance.

and Neisseria isolates [36] . Tn916 is a paradigm of a large family (the Tn916 /Tn1545 family) of related promiscuous conjugative transposons (Box 2) that have been found in or have been intro- duced into over 30 different genera of bacteria [37] , including many commonly found in the oral cavity. In another study it was found that 46 out of 47 children harbored tetracycline-resistant bacteria, and that 56% of these were resistant to an additional drug, usually erythromycin. Again the most common tetracy- cline resistance gene was tet(M), predominantly within the strep- tococci [38] . In a further study on the cultivable oral microflora of children it was shown that in addition to tetracycline, resistance to ampicillin, erythromycin and penicillin is also present [39] . All of the antibiotic-resistant bacteria were identified to at least genus level with the streptococci being the most resistant genus isolated (TaBle 1). This study also demonstrated that resistance to at least two different antibiotics was present in 111 (27.8%) of the 432 antibiotic-resistant organisms isolated from the 35 sub- jects [39] . Studies on the oral flora of healthy adults, who had not received antibiotics for the previous 3 months also demonstrated the presence of many different tetracycline resistance genes [40] , the most common being tet(M) followed by tet(W), another gene encoding a ribosomal protection protein originally dis- covered in the rumen anaerobic species Butyrivibrio fibrisolvens [41] . An endodontic study examining tetracycline-resistant root canal isolates demonstrated that tet(M) was the most common detectable gene and that it was also present on the Tn916 -like conjugative transposons. One Neisseria sp. isolate was capable of transferring this mobile element to Enterococcus faecalis – itself an endodontic pathogen [42] . Macrolide-resistant bacteria have also been characterized from the oral cavity [39,43,44] . Recently, however, the total cultivable flora was assayed for resistance to erythromycin and the mef and ermB genes were the most common. On average, 7% of the cultivable microflora were resistant to erythromycin and carried at least one erythromycin resistance gene [45] . The ermB genes in many oral isolates are present on Tn916 /Tn1545 -like conjugative transposons, which also contain tet(M) and aphA-3 conferring tetracycline and kanamycin resistance, respectively [45] . Similar results were found in other studies investigating the molecular basis of resistance to macrolides in commensal viridans strepto- cocci and Gemella spp. [46–48] . These studies also demonstrated that the majority of strains with an identifiable erythromycin resistance gene contained the mef gene. Recently the mef gene

has been found on a novel conjugative transposon Tn1207.3 in Streptococcus pyogenes [49] . Also the results from these studies show that ermB was again linked to Tn916 /Tn1545 -like conju- gative transposons [48] . The observation that both mef and ermB are contained within conjugative transposons that usually also contain genes encoding tetracycline resistance proteins may go some way to explain their broad distribution.

Metagenomic studies of the oral flora

Metagenomics is a powerful experimental approach that bypasses the need to culture any of the inhabitants of a par- ticular environment. Instead, the entire DNA from a particular niche, in this case the oral cavity, is isolated and analyzed. One such study, looking at various tetracycline and erythromycin resistance genes present in the oral and fecal communities from individuals from six different European countries showed that tet(M) predominated in the oral environment [50] , agreeing with cultivable studies described above. Conversely, for the erythromycin resistance genes no particular gene was predomi- nant in all locations. There was, however, a diverse range of resistance genes detected for both antibiotics [50] . This study also contained probes specific for the recombinase genes from various common conjugative and mobilizable transposons. The recombinase proteins are responsible for the excision and inser- tion of their cognate mobile elements [51] . Without exception the probe for the Tn916 integrase gene hybridized strongly to all oral samples screened. In five out of six samples the hybridization to the Tn916 integrase probe was in excess of that to tet (M) suggesting there is more Tn916 intTn present than tet (M); this could be due to some Tn916 -like elements in the oral flora not containing tet(M) as an accessory gene [50] . Functional studies aimed at isolating resistance genes from the human microflora have also been carried out using a phe- notypic screen of metagenomic libraries. DNA from the oral and fecal cavity was cloned into a vector to make the metage- nomic library. This was used to transform Escherichia coli and transformants selected for on the basis of resistance to one of 13 antimicrobials [52] . The insert DNA from the resistant clones were sequenced and this showed that the majority of the resistance genes found were new. The authors estimate that, based on the uniqueness (in the sequenced clones) of many of the novel antibiotic resistance genes, the full sequencing of the library would reveal hundreds more novel resistance genes from

Oral biofilms: a reservoir of transferable, bacterial, antimicrobial resistance

Review

Table 1. Number of isolates of different genera resistant to antibiotics isolated from children.

Bacterial genus

Amp R

Erm R

Pen R

Tc R

Actinobacillus spp.

1

3

2

0

Bacillus sp.

0

1

1

0

Bacteroides sp.

1

-

1

0

Capnocytophaga spp.

3

3

7

0

Eubacterium sp.

0

0

0

2

Fusobacterium spp.

0

-

2

0

Granulicatella sp.

0

0

0

1

Haemophilus spp.

37

-

-

7

Leptotrichia sp.

0

-

0

2

Moraxella sp.

0

1

0

0

Neisseria spp.

14

14

10

1

Patsurella sp.

1

-

1

0

Porphyromonas spp.

0

-

2

0

Prevotella spp.

4

-

5

0

Stomatococcus sp.

2

0

0

0

Streptococcus spp.

41

36

37

38

Veillonella spp.

43

-

82

8

-: Genera not included in antibioitc resistance guidelines; Amp R : Ampicillin resistance; Erm R : Erythromycin resistance; Pen R : Penicillin resistance; Tc R : Tetracycline resistance. Taken with permission from [36] .

these environments. The sequence of the inserts from this study also showed the presence of many transposase genes, indicat- ing that some of the resistance genes were likely to be present on mobile genetic elements [52] . Other previous metagenomic studies have also revealed novel antibiotic resistance genes by phenotypic screening of metagenomic libraries including the tetracycline resistance gene tet(37), the product of which enzy- matically breaks down tetracycline [53] and tet(32) encoding for a ribosomal protection protein [54] .

Evidence for horizontal gene transfer of antibiotic resistance in the oral cavity

For the oral microbiota to act as a reservoir for transferable anti- biotic resistance the resistance genes must either be contained on mobile genetic elements or be capable of being transferred into a new host by transformation. Whole-genome sequencing and the analysis of individual genes can determine the likelihood of par- ticular genes being acquired by horizontal transfer as genes that are more than 95% identical at the nucleotide level in diverse bac- teria, for example, tet(M), are highly likely to have been acquired in this way. There are many complete genome sequences [103,104] with many more nearing completion which reveal the presence of antibiotic resistance genes within putative mobile genetic ele- ments (reviewed in [55] ). However, functionality of the majority of putative mobile elements discovered during genome sequencing projects remains to be experimentally determined.

Evidence for transformation in the oral cavity

Successful transformation of a bacterial cell depends on physico- chemical factors of the DNA molecules, such as their size, con- formation and concentration of the transforming DNA and other environmental factors, including UV light, salt, pH, temperature and the presence of extracellular nucleases. In addition, there are genetic hurdles to overcome for successful transformation, such as the presence of restriction systems and the ability of the incom- ing DNA to either replicate autonomously or integrate into the recipient’s genome [56] . Transformation has no requirement for live donor cells as the DNA released upon cell death is the principle source of transform- ing DNA (Figure 1). In addition, some bacteria can actively release DNA into their environment [57] . Therefore, the rate-limiting steps for transformation of bacteria growing in an oral biofilm is the lon- gevity of DNA molecules in both the biofilm and the cytoplasm of the transformed cell. Researchers have investigated the persistence of Lactococcus lactis chromosomal and plasmid DNA (pVACMC1 and pIL253) in human saliva, and although partially degraded, the DNA was still visible on an agarose gel after 3.5 min incubation. Furthermore demonstration of the presence of a 520-bp fragment of the pVACMC1 DNA was demonstrated by PCR after 24 h of incubation in human saliva [58] . pVACMC1 could transform Streptococcus gordonii DL1 after being incubated in saliva but with a tenfold decrease in transformation efficiency after being incu- bated in saliva for 2 min compared with untreated DNA [58] . Later experiments demonstrated the transformation of S. gordonii by pVACMC1 in the oral cavity of a human volunteer [59] . Another study using the plasmid pUC18 demonstrated that it could trans- form E. coli to ampicillin resistance following 24 h incubation in clarified ovine saliva [60] . These studies show that DNA is able to survive in saliva for a long enough period of time to be able to transform competent bacteria. A recent development in the study of biofilm biology is the obser- vation that extracellular DNA is present within many of the biofilm matrices investigated and is actually required for the formation of some biofilms [61,62] . A more recent in vitro study has demonstrated that the genomic DNA of a Veillonella dispar strain carrying the con- jugative transposon Tn916 was able to transform Streptococcus mitis to tetracycline resistance within an oral biofilm grown in a fermen- tor [63] . It has also been demonstrated that some of the bacterial DNA that can be isolated from human saliva is from bacteria not normally considered to be members of the oral microflora, such as Phytoplasma sp. [50] , which is normally associated with plants. This observation demonstrates the presence of DNA from exogenous sources in the oral cavity. Virtually any DNA that is found free in the oral cavity, whether it is derived from oral bacteria or bacteria pres- ent on foodstuffs, is able to be a substrate for transformation. The successful acquisition of resistance genes from the transient micro- flora may be a mechanism that enables the normal flora to build up its reservoir. In addition, this reservoir of genes will be available in the form of free DNA from dead biofilm residents to transient, competent bacteria passing through the oral cavity. Therefore, it is possible that broad host range gene transfer within, out of and into oral biofilm inhabitants can occur by transformation.

Review

Roberts & Mullany

Additional evidence for the involve- ment of bacteriophage in the transfer of DNA among residents of oral biofilms comes from studies carried out in vitro. For example, tetracycline resistance pres- ent on Tn916 and chloramphenicol resis- tance present on plasmid pKT210 has been transferred between A. actinomycetemcomi- tans by the generalized transducing phage AaØ23 [68] . Additional evidence that A. actinomycetemcomitans bacteriophage may be transducing DNA between bac- teria in the oral environment comes from the direct isolation of bacteriophage par- ticles from subgingival plaque from peri- odontitis patients [68,69] . In a recent study investigating the differential gene expres- sion of Treponema denticola, a pathogenic oral spirochaete, growing either as part of a

biofilm or planktonically, researchers dem- onstrated the upregulation of various pro- phage genes and transposases in the bio- film samples [70] . In addition, functional bacteriophage were isolated from this bacterium and the authors hypothesized that there is a larger potential for genetic mobility when T. denticola is growing as part of a biofilm [70] .

Conjugation in the oral cavity

A B C D
A
B
C
D

Expert Rev. Anti Infect. Ther. © Future Science Group (2010)

Figure 1. Transfer of DNA between bacterial cells. The bacterial cells are

represented by the green and purple ovals. The DNA is represented by the helix. Transposons and plasmids are either blue bars or circles. The arrows show the direction of transfer of the DNA. (A) Transformation: the donor cell (top left) has lysed and the DNA released into the environment. This can be taken up by competent bacteria and incorporated into the recipient genome. (B) Conjugation of plasmids through a pilus. (C) Conjugation of conjugative transposons via a mating pore. (D) Transduction mediated by the injection of DNA by a bacteriophage.

Transduction in the oral cavity

One of the main barriers to the activity of bacteriophage in oral biofilms is the access to the cells within the EPS substances secreted by the biofilm residents themselves [64] . Little is known about the effect of phage and the extent to which transduction contributes to genetic exchange within oral biofilms, however, there are some studies that indicate trans- duction may by occurring within the oral cavity. In one study, three distinct bacteriophages were isolated from enriched human saliva, however, none of the bacteriophage could infect any of the oral pathogens tested but did infect Proteus mirabilis, also isolated from the saliva. The P. mirabilis cells were thought to have been transient in the samples tested, either introduced on food materials or transported manually to the mouth [65] . In a study investigating endodontic Enterococcus faecalis isolates, lysogeny was demonstrated and bacteriophage were isolated in four of the ten strains [66] . In another study, bacteriophage spe- cific for E. faecalis were isolated from the saliva of seven out of 31 volunteers [67] . If bacteriophage-mediated cell lysis of the P. mirabilis or the E. faecalis occurred within the oral cavity, this DNA released would be available for transforming other naturally competent bacteria.

Conjugative plasmids and conjugative transposons (particularly of the Tn916 -like

family) have been found in many differ- ent oral bacteria. Some of these mobile elements are transferable in vitro [55] . Both conjugative transposons and conjugative plas- mids have been transferred from oral donor bacteria to recipients of different species and genera [45,71,72] . To determine if transfer can occur in more complex environments that mimic the oral cavity, fermentor studies that allow growth of oral biofilms from an inoculum of saliva or a defined consortia of known and char- acterized oral species have been carried out. The constant depth film fermentor (CDFF) has been used to demonstrate that tran- sient bacteria may be able to act as a donor to oral bacteria in the short time that they are present in the oral cavity. Tn5397 (a conjugative transposon carrying tet[M]) in a B. subtilis donor can transfer to a streptococcal recipient growing as part of an artificial oral biofilm in the CDFF [73] . The B. subtilis donor could not be recovered from the biofilms 24 h after inoculation, showing that even though the donor bacteria are no longer present, the genetic information contained within them can persist. Further work has demonstrated transfer of native Tn916 -like elements between oral streptococci grown in the CDFF [74] . However, DNase was not included in the growth medium fed into the fermentor during these experiments and therefore it is not possible to distinguish between transformation and conjugation.

Oral biofilms: a reservoir of transferable, bacterial, antimicrobial resistance

Review

Tn916 -like conjugative transposons have been shown to be common in tetracycline-resistant Veillonella spp. and some of these were transferable within a mixed species consortia consisting of 21 tetracycline-sensitive members [75] . In vivo transfer of a Tn916 -like conjugative transposon, Tn6002, has been reported. The element was hypothesized to have been transferred from Streptococcus oralis to Streptococcus cristatus dur- ing systemic doxycycline treatment for periodontitis [76] . Another study has shown that the conjugative plasmid pAM81 was capable of transfer between Streptococcus gordonii and E. faecalis in the root canals of human teeth ex vivo [77] . The transfer of plasmids is often stimulated by pheromones, molecules that are produced by the bacteria which are intercellular signaling molecules. Often, when there is enough of the molecule present (such as in a biofilm where there is a high cell density), a signaling cascade will result in the transfer of plasmid or chromosomal elements [78] . Horizontal transfer of DNA, stimulated by intercellular signaling molecules, is one of many consequences of interbacterial communication, which also includes development of the biofilms themselves [14,15] . These results are highly indicative that gene transfer is occurring within the oral community, possible by all three mechanisms of horizontal gene transfer. In addition researchers and clinicians alike must remember that the individual mechanisms of horizontal gene transfer are not mutually exclusive and it is likely that the same region of DNA could be transferred by more than one mech- anism. The transfer of resistance to, from and within a human reservoir highlights the need for rigorous control of antibiotic use.

Expert commentary & five-year view

While there is an increasing wealth of evidence supporting the idea that oral bacteria are a reservoir of antibiotic resistance genes, how extensive this is will only be determined when complete oral meta- genomes have been sequenced. The ultimate proof of transferability, however, requires that gene transfer experiments are performed.

Acquired antibiotic resistance can persist in the environ- ment even in the absence of selective pressure (antibiotics) [79] . Presumably this is due to low fitness costs associated with acqui- sition of mobile elements carrying resistance genes. This has been demonstrated for both plasmids and transposons. A more thorough understanding of these metabolic burdens and the way host cells evolve to cope with the acquisition of foreign DNA is likely to be facilitated with the improved and increas- ingly cheap molecular techniques currently on, or coming to, the market. Recently various transcriptional and translational regulatory systems have been described for a variety of different mobile genetic elements that contain antibiotic resistance genes and a common theme emerging from the studies is that these elements are able to control their transcription and only express genes when they ‘sense’ certain, as yet ill-defined, environmental cues that can promote their transfer. The continued research in this area will allow an understanding of what triggers their transfer and give insights into how to control, or stop it occurring.

Financial & competing interests disclosure

The authors have received financial support from the Commission of the European Communities, specifically the Infectious Diseases research domain of the Health theme of the 7th Framework Programme, contract 241446, “The effects of antibiotic administration on the emergence and persistence of antibiotic-resistant bacteria in humans and on the composition of the indigenous microbiotas at various body sites”. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject mat- ter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Key issues

The oral microflora is extremely diverse and many inhabitants live within oral biofilms.

The normal oral flora contains many different antibiotic resistance genes.

Many of these antibiotic resistance genes are located on mobile genetic elements.

The biofilm environment is ideal for horizontal gene transfer.

Treatment with antibiotics selects for resistant strains and can also promote the mobility of mobile elements conferring this resistance.

Gene transfer in many different biofilm environments and of many different mobile genetic elements has been demonstrated.

The normal commensal oral flora acts as a reservoir of transferable antibiotic resistance genes, which are available to transient inhabitants.

Moreover, it is possible that these transient inhabitants act as donors of resistance genes to the normal oral flora.

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Websites

101 European Medicines Agency www.ema.europa.eu/pdfs/human/

antimicrobial_resistance/EMEA-576176–

2009.pdf

102 Infectious Disease Society of America

www.idsociety.org/10x20.htm

103 NCBI, Genomic BLAST www.ncbi.nlm.nih.gov/sutils/genom_table. cgi

104 Human Oral Microbiome Database www.homd.org