The John Tagg Story

Professor Tagg’s first significant encounter with Streptococcus pyogenes – the bacteria that causes a strep sore throat – was as a 12-year old living in Melbourne. A series of sore throats culminated in him developing rheumatic fever. He then had to consume penicillin tablets daily over the following decade to help prevent any follow-up attacks of the disease. Fortunately, due to this antibiotic regime, he did not suffer any of the residual heart damage that can occur from recurrent episodes of rheumatic fever.

Prof. John Tagg

Nevertheless, it just seemed to the young John Tagg that there must be a better way to defend against streptococcal sore throats.

Following high school, John studied at Melbourne University, and in the third year of his Microbiology degree, he became influenced by the teachings of Dr Rose Mushin concerning the potential applications of bacterial interference as a targeted and natural means of infection prevention. Dr Mushin had become a devotee of an old-world strategy for infectious disease control, the origins of which pre-date the discovery of antibiotics and indeed can be traced back to the studies of Louis Pasteur.

Dr Mushin managed to convince the entire microbiology class to consume milk that had been seeded with so-called “friendly” Escherichia coli. These bacteria were equipped with a bacteriocin armament that would enable them, from their proposed site of lodgement in the intestinal tract, to kill any vulnerable salmonellae that happened to pass close by. Dr Mushin’s proposal sounded logical and feasible. Bacteriocins, she explained, were proteinaceous antibiotics produced by bacteria which had a bacteriocidal mode of action against various other relatively closely-related bacteria that were potentially capable of competing with them for occupancy of the same ecological niche (i.e. they were anti-competitor molecules).

As the young John Tagg listened to Dr Mushin it occurred to him that perhaps a similar strategy could be applied in the human oral cavity to gain some relatively-specific protection against S. pyogenes infections.

That insight provided him with an irresistible challenge – he now knew what he wanted to do. First John needed to familiarise himself with the idiosyncrasies of streptococcal behaviour and his conviction was that, in order to prevent

First John needed to familiarise himself with the idiosyncrasies of streptococcal behaviour and his conviction was that, in order to prevent S. pyogenes from assaulting its human host, it would be important to try to strengthen the territorial defensive capabilities of the non-virulent streptococcal component of our indigenous microbiota. It occurred to him that over the course of their lengthy co-evolution with humans it was the indigenous oral streptococcal populations that would surely have developed the most effective and specifically-targeted bacteriocin weaponry to counter competition for their space by rapidly-multiplying virulent streptococci.

John Tagg next undertook PhD research at Monash University – the theme of those studies being an exploration of the relationship between S. pyogenes infections and the induction of the autoimmune manifestations of rheumatic fever [2]. He set about screening many hundreds of oral streptococci for their bacteriocin-producing capability (i.e. bacteriocinogenicity) and included amongst these streptococci was a series of S. pyogenes isolates from the Fairfield Infectious Diseases Hospital in Melbourne. On September 1, 1969, John discovered that number 22 in this series produced bacteriocin-like inhibitory activity against some other S. pyogenes strains when tested in a deferred antagonism assay [3]. The inhibitory agent, later given the name streptococcin A-FF22 [4], was the first of the streptococcal bacteriocins to be isolated and characterisedNext it was time for John to undertake a post-doctoral apprenticeship, and from his reading of the current scientific literature, he knew where he wanted to continue his studies.

Prof. John Tagg

Next, it was time for John to undertake a post-doctoral apprenticeship, and from his reading of the current scientific literature, he knew where he wanted to continue his studies. Dr Lewis Wannamaker at the University of Minnesota was an influential leader in the field of streptococcal research and had played a major role in developing the original guidelines for the use of penicillin prophylaxis as a preventative against rheumatic fever recurrences [5]. In 1972 Dr Wannamaker hosted a workshop on ‘Streptococci and Streptococcal Diseases’ at the University of Minnesota which was attended by many of the leading S. pyogenes researchers of that era. In his workshop summary, Dr Wannamaker remarked that “working with the streptococcus is like a love affair, which I guess explains why so many of us find it difficult to give up” [6]. John Tagg could relate to this sentiment and wrote to him asking if they could work together, seeking a bacterial interference-based alternative to the use of penicillin for rheumatic fever prophylaxis

Three years of total streptococcal research immersion followed. Dr Wannamaker was generous in his support for John Tagg’s endeavours to find a harmless oral streptococcus capable of effectively engaging in bacteriocin-mediated warfare against S. pyogenes, while at the same time maintaining the view that what S. pyogenes really needed was greater scientific understanding – not extermination.

Before leaving Minnesota John Tagg wrote the first major review of the bacteriocins of gram-positive bacteria [7] and upon purifying streptococcin A-FF22 demonstrated that it was closely similar to nisin, the best known and still the most widely applied of all the bacteriocins of gram-positive bacteria [8].

Rheumatic fever is a major public health concern in New Zealand, with a particularly high occurrence in the native Maori and Pacific Islander populations. In view of this John Tagg was excited in 1975 to obtain an academic position in the Microbiology Department at the University of Otago in Dunedin. His research agenda in New Zealand became firmly focused on finding a harmless oral streptococcal antagonist of S. pyogenes. He developed a procedure for the bacteriocin ‘fingerprinting’ of streptococci based on the deferred antagonism test in which a set of nine standard indicator bacteria are evaluated for their relative sensitivity to inhibitory substances released into an agar medium during the growth of a diametric streak culture of the test bacterium [9]. It soon became clear that most (if not all) streptococci were probably capable of producing some sort of bacteriocin-like inhibitory activity. John Tagg then proposed the term BLIS as a ‘catchy’ preliminary descriptor for the molecular agents of these inter-bacterial inhibitory effects prior to the completion of more exhaustive tests designed to establish whether they were indeed bona fide bacteriocins [10]. The pattern of inhibition of the nine standard BLIS indicator bacteria when converted to code format is referred to as the BLIS Production (P) type of the test bacterium [9]. Over the next several decades John Tagg’s laboratory screened many thousands of streptococcal isolates for their BLIS activities and subsequently documented a remarkably heterogeneous array of proteinaceous inhibitory agents, ranging from post-translationally modified nisin-like peptides of the lantibiotic family to non-modified small peptides and relatively large proteins, some muralytic in their activity and others having an unusual circular conformation [11, 12]. Streptococci, especially those of the species uberis, mutans and salivarius appear to have been particularly inventive and acquisitive of unusual bacteriocin loci.

John Tagg’s second approach to identifying a naturally-occurring antagonist of S. pyogenes came in the form of a prospective study of 100 five-year-old Dunedin schoolchildren. The objective was to document changes occurring in the composition of the children’s oral microflora over the next six years and in particular to record if and when each child acquired S. pyogenes. Despite frequent outbreaks of S. pyogenes pharyngitis occurring in the school classrooms, not all similarly-exposed children experienced streptococcal infections. A particularly interesting observation was that many of the children who seldom acquired S. pyogenes had large populations of strong BLIS-producing S. salivarius on their tongues (i.e., the BLIS producers comprised at least 5% of the child’s total S. salivarius population) [13].

This led to the hypothesis that the presence in the oral cavity of certain BLIS-producing S. salivarius may afford some protection against S. pyogenes infection. A follow-up study of 780 Dunedin school children identified two major types of BLIS-producing S. salivarius, the corresponding P-type patterns being 226 (11% of children positive) and 677 (9% positive) [14]. A further 20% of the children had S. salivarius of various other P-type designations, including some (approximately 1% of all tested subjects) having particularly strong (P-type 777) BLIS activity. The children harbouring populations of either P-type 677 or P-type 777 S. salivarius had a significantly reduced rate of acquisition of S. pyogenes during the 10-month study period [14].  Strains of P-type 677 S. salivarius typically produce a 2315 Da bacteriocin of the lantibiotic class named salivaricin A (SalA) [15].  SalA differs from most other lantibiotics in that its inhibitory activity against S. pyogenes is bacteriostatic, rather than bacteriocidal [16]. SalA inhibits the in vitro growth of all tested S. pyogenes, although the extent of inhibition of strains of serotype M4 (themselves producers of a SalA variant lantibiotic) is relatively reduced [15]. SalA and its variants (SalA1, SalA2, SalA3 etc) function as cross-reactive signal peptides, capable of specifically up-regulating the production of all SalA lantibiotics [17, 18]. Perhaps even more significantly, this signal can effect inter-species communication, up-regulating production of SalA-like loci in strains of S. pyogenes, S. agalactiae and S. zooepidemicus [17]. Specific SalA auto-inducing activity has been detected in the saliva of subjects following their colonisation with SalA-producing S. salivarius, showing that this lantibiotic activity is produced and is biologically active in vivo   [19]. In other studies, it has been demonstrated that anti-S. pyogenes inhibitory activity is also produced when SalA-positive S. salivarius are grown in saliva in an in vitro test system [19]. Also, the dosing of children with SalA-producing S. salivarius stimulated clonal expansion of their pre-existing indigenous populations of SalA-producing S. salivarius [20]. Moreover, when a SalA-producing S. salivarius population is present on the tongue, some other bacterial species on the tongue show increased levels of specific resistance to this bacteriocin [21].

The commercial outcome of these laboratory discoveries was the launching of the Dunedin-based company Blis Technologies Ltd in August 2000 and two years later the first oral probiotic product, BLIS K12 ThroatGuard, appeared on the shelves of New Zealand pharmacies. A wide variety of BLIS K12 products in powder, lozenge, chewing gum and ice cream formulations have subsequently been developed and many are now marketed internationally. A more recently released S. salivarius oral probiotic (strain M18) expresses the megaplasmid-encoded bacteriocins salivaricins MPS, A2 and 9 as well as the chromosomally-encoded bacteriocin salivaricin M [23]. Some inhibitory spectrum differences are evident for strain M18, including increased inhibitory activity against the dental caries-associated species Streptococcus mutans [24].

Case For BLIS-Producing S. Salivarius As Preferred Probiotics For The Oral Cavity

The Case For BLIS-Producing S. Salivarius As Preferred Probiotics For The Oral Cavity

Within hours of our birth the species S. salivarius has achieved numerical prominence within our oral microbiota and large populations continue to flourish throughout our lives on the healthy oral mucosae, especially the tongue surfaces [25].  When S. salivarius numbers are depleted, unbalanced overgrowth of relatively ‘malevolent’ microbes such as some of the Candida spp. and black-pigmented anaerobes can sometimes occur, resulting respectively in the unpleasant clinical conditions of oral thrush [26] and halitosis [27]. It seems that due to its strategic lingual location, versatile anti-competitor weaponry and considerable numbers, S. salivarius is particularly well suited to execute a population surveillance and management or ‘sentinel’ role within the oral microbiota [28].

A high proportion of S. salivarius are BLIS producers and upon closer examination, it is common to find that two or more bacteriocins contribute to the total BLIS activity of individual strains [17]. Interestingly, most BLIS-producing S. salivarius appear to utilise very large (>100 kb) megaplasmids as the genetic receptacles for their bacteriocin loci [30]. This mega-plasmid DNA (as yet unreported in any other oral bacteria) appears to be transmissible in the human oral cavity between different S. salivarius [30]. Sequencing of pSsalK12, the megaplasmid of S. salivarius strain K12, has disclosed an abundance of mobile genetic elements (putative insertion sequences), especially flanking the loci for salivaricins A2 and B, indicating that these loci may have been acquired from other bacterial hosts via transposition events [22]. More recently, S. salivarius strain JH has been shown to produce at least four bacteriocins – salivaricin A3, streptin, streptococcin SA-FF22 and salivaricin E (Walker, unpublished).

Since bacteriocin production is energetically demanding and also requires the coordinated action of multiple gene products, it seems that it must surely confer a significant benefit upon the host bacterium. Furthermore, the ubiquity of bacteriocinogenicity in the bacterial world infers that bacteriocin production and/or the corresponding specific bacteriocin immunity phenotype must have a major role in the survival of individual bacterial clones in nature. The mode of action of type-A lantibiotic bacteriocins such as those produced by strain K12 involves pore formation in the cytoplasmic membranes of target bacteria and the process of insertion only occurs if there is a sufficiently high potential difference across the target cell membrane. More specifically, these lantibiotics may modulate the composition of the oral microbiota by killing relatively rapidly multiplying competitor bacteria – such as for example pathogenic S. pyogenes during the acute stage of a streptococcal throat infection.  On the other hand, when small populations of S. pyogenes are sequestered within the oral microbiota, or even intracellularly, as occurs when they are in the relatively quiescent carriage state, they may not then be so vulnerable to lantibiotic attack. The hypothesis underlying the probiotic application of BLIS K12 is that the colonisation of the oral cavity with lantibiotic-producing S. salivarius may help limit infection by newly acquired virulent S. pyogenes or shifts of indigenous S. pyogenes from innocuous carriage to active infection status.

Laboratory studies have shown that bacteriocin production is strongly influenced by both genetic and environmental factors [30]. In order to conserve energy, bacteriocin expression can temporarily be turned off. Alternatively, bacteriocin genetic loci may be either inactivated (via mutation) or entirely jettisoned (as in the course of megaplasmid elimination) should the production of that bacteriocin fail to confer a survival advantage to the cell as it multiplies within that particular ecosystem. Interestingly, the loci encoding homologs of some of the lantibiotics (i.e. SalA, SalB, streptin and streptococcin A-FF22) that have been identified in S. salivarius mega-plasmids have also been detected (albeit in a chromosomal-location) in various other oral streptococcal species, including Streptococcus mitis, S. pyogenes, Streptococcus equisimilis and S. mutans [28]. These observations are consistent with S. salivarius megaplasmids serving as flexible genetic receptacles for the acquisition and expression of bacteriocin loci that have putatively been gathered from (and then potentially donated to) a variety of other oral streptococci.

In previous research, John Tagg and his colleagues have been particularly interested in factors that may influence the population balance between commensal and potentially pathogenic streptococci in the human oral cavity. S. salivarius is principally located on the tongue surface [25]. Carriage of S. pyogenes (in the pharynx) is common, especially in school-aged children. When present in small numbers S. pyogenes causes no pathology, presumably any excessive proliferation being held in check either by competition from other members of the oral microbiota or by the host’s immune defences. However, failure of these controls can result in development of streptococcal pharyngitis. We have speculated that due to its strategic location and dominant numbers S. salivarius is well placed to help modulate the oral cavity population levels of S. pyogenes and various other streptococcal species [28].

Although principally located on the human tongue, S. salivarius can also be found in large numbers within the pharyngeal microbiota [25]. Here, bacteriocin-producing strains can more directly interact with and interfere with the proliferation of S. pyogenes.  Moreover, the bacteriocin products of S. salivarius will readily saturate the saliva, which is constantly flowing over all oral surfaces and it can be anticipated that the bacteriocin concentration and thus its killing action will be especially high during sleeping hours when the body’s natural circadian rhythm slows the rate of saliva formation [19]. S. salivarius has also been reported to sometimes represent substantial proportions of the microbial populations present in the intestine [31, 32], human breast milk [33] and even the lungs [34].

A number of the beneficial outcomes from the ingestion of probiotics can be traced to their immunomodulatory activity. Probiotic cell interaction with immune cells in the mucosal environment can influence various processes dependent on the mucosa-associated lymphoid tissue (MALT) such as tolerance induction, modulation of cytokine/chemokine release and general regulation of mucosal immune responses. Since this activity is often strain-specific, it is necessary for each candidate probiotic strain to be specifically assessed for its immunomodulatory activity. Effects of probiotics such as BLIS K12 include (a) maintenance of epithelial cell homeostasis, including the down-regulation of the pro-inflammatory response elicited by pathogens [35, 36], and (b) up-regulation of the unified interferon signaling pathway which is responsible for the expression of genes involved in innate immunity against viral infection, antitumor activity, priming of the lipopolysaccharide response and anti-inflammatory effects [37].

The species S. salivarius has a number of innate characteristics favouring its candidature as a probiotic [38].  Of all the bacterial species known to populate the human oral and nasopharyngeal mucosa in large numbers, S. salivarius is perhaps the most innocuous.  BLIS K12™ has a very well-documented safety record [39] and has now been granted self-affirmed GRAS (Generally Regarded As Safe) status for unrestricted food additive applications in the USA. S. salivarius is an early coloniser of the human oral cavity, with infants usually acquiring the mother’s predominant strain of S. salivarius within hours of birth [40, 41]S. salivarius typically then remains a numerically-prominent member of the oral cavity throughout the life of the host. It has been found to be present at levels of up to 1x107 colony forming units (cfu) per millilitre of saliva and based on the daily estimated average consumption of saliva in adults (up to 1.5 litres), large numbers (estimates of 1x1010 cfu) are ingested daily. S. salivarius can also be isolated from breast milk and this could be an important source of the bacterium for infants in the early months of life [42].

In other studies, it has been shown that BLIS K12™ (a) binds with strong avidity to human epithelial cells in vitro and prevents attachment of S. pyogenes [43] (b) persists in the human oral cavity following ingestion [44].

Actual And Potential Oral Probiotic Applications Of BLIS-Producing S. Salivarius

With the discovery of penicillin and the subsequent flourishing of the antibiotic era, the further development of probiotics and of bacterial interference as strategies for infection control was largely overlooked. More recently, however, as the efficacy and safety of many antibiotics have diminished, together with the associated resurgence of many infectious diseases and increased numbers of immunologically compromised and aged hosts, probiotics are once again being promoted either as an alternative or as a supplement to existing therapies. Intestinal commensals, especially lactobacilli and bifidobacteria, have long been consumed by humans in a variety of dairy product formulations either to nurture gut health or to treat a wide variety of ailments of the gastrointestinal tract and vagina. With increasing interest now in probiotic interventions for the modulation of microbial diseases and disequilibria in other body tissues, attention is logically being focused upon the predominant microbial indigenes of these tissues as a prime source of novel probiotics that are best equipped to colonise and help stabilise the populations of microbes incumbent to these tissues.

The term probiotic (literally meaning ‘for life’) is a general term now widely used to refer to ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’ [45]. More specifically, ‘oral probiotics’ can be considered to be probiotics, the persisting action and beneficial outcomes of which are a direct outcome of their interaction with microbial and/or host cellular components of the oral cavity. Thus, bona fide oral probiotics differ from the traditional orally-administered intestinal probiotics (i.e. yoghurts or other products comprising lactobacilli and bifidobacteria of intestinal origin). The latter do not primarily or persistently localise within the oral microbiota – even though some short-term beneficial outcomes may manifest within the oral cavity following their ingestion.

Every human harbours a personalised oral cavity microbiome, the presence of which is essential to heath maintenance, but also has a dark side capable of causing harm to host tissues, both oral and systemic. The biofilms coating every surface of the oral cavity comprise complex ecosystems that help to maintain our health when they are in equilibrium. Ecological shifts within the microbiome can sometimes allow pathogens that are either intrinsic to the biofilm (in a carriage state) or newly introduced to the oral cavity to become manifest and induce disease. The intentional seeding of the oral microbiome with harmless probiotics equipped with the capability of exerting targeted bacterial population control (i.e. bacterial interference or ‘germ warfare’) provides an approach to infection prevention that has as its basis the augmentation of naturally occurring processes. Within the oral cavity, the principal bacterial infections that have to date been identified for probiotic intervention have been streptococcal pharyngitis, otitis media, halitosis and dental caries.

Successful implantation of the oral microbiota with BLIS K12™ provides a potential means of preventing streptococcal pharyngitis and also an alternative to antibiotic prophylaxis for the prevention of rheumatic fever recurrences. Acute S. pyogenes infections and their non-suppurative sequelae continue to exact a severe toll on susceptible populations. Prior to the development of BLIS K12™ the only available strategy has been treatment of the acute streptococcal infections as they occur by administration of therapeutic doses of a broad-spectrum antibiotic such as penicillin. Studies by di Pierro and colleagues [ref] in Italy have shown that the taking of lozenges containing BLIS K12™ by children [46] and adults [47] can effect a reduction in the occurrence of streptococcal pharyngitis. Otitis media-prone infants given a paediatric formulation of strain K12 for two weeks prior to ventilation tube placement displayed colonisation of the adenoid tissue [48] and more recently the taking of BLIS K12™ was found to reduce the occurrence of secretory otitis media in children [49]. A relative deficiency of S. salivarius in the oral cavity has been linked to the overgrowth of malodorous bacteria on the tongue and the development of bad breath (halitosis) [27]. Administration of BLIS K12™ can help alleviate the symptoms [50]. The more recently developed probiotic S. salivarius strain M18 (BLIS M18 initially referred to as strain Mia) differs from BLIS K12™ in having additional BLIS activity against the mutans streptococci, bacteria that are commonly implicated in the development of dental caries. Strain M18 also produces strong dextranase activity. Successful colonisation of young schoolchildren with strain M18 has been shown to reduce both the levels of mutans streptococci and the plaque index [24]. Oral persistence of BLIS M18™ was shown to be dose dependent [51].

Oral cavity microbial disequilibria can lead not only to development of dental caries, periodontal disease and candidosis, but also can facilitate initiation of the transition between carriage state and active infection by bacteria such as S. pyogenes as well as possibly influencing the onset of preterm labour and a wide variety of non-transmissible diseases ranging from cardiovascular disease and diabetes to obesity. In other studies S. salivarius K12 has been shown to outcompete Candida albicans and Streptococcus agalactiae respectively in the rat oral cavity [52] and the mouse vagina [53] opening up prospects for future applications to the control of candida in the elderly and S. agalactiae infections in the newborn.

One practical issue concerns how best to facilitate probiotic colonisation of the host tissues. Clearly the attachment and flourishing of a newly introduced probiotic bacterium will be enhanced when the native microbiota of the host is relatively sparse, as occurs in the first days of life or during the course of antibiotic treatment. Indeed, in order to increase the prospects for probiotic implantation it can be recommended that any course of prescription antibiotics should be linked to a follow-up regimen of probiotics, with probiotic dosing beginning on the last day of antibiotic administration.

The Probiotics For All Ages

The time now appears opportune for the accelerated development and application of BLIS-producing S. salivarius probiotics to the control of a wide variety of specific bacterial infections or of other diseases of more complex etiologies that reflect undesirable population structures within the indigenous microbiota. The BLIS approach to infection control is cost-effective, easily implemented and does not present many of the complications associated with immunisation (hypersensitivity or antigenic cross-reactivity) or chemotherapy (resistance development and direct toxicity). The considerable scope for their potential applicability throughout the life of their natural human host encourages consideration of BLIS K12™ and BLIS M18™ as the probiotics for all ages.

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