As one of the most essential trace elements for the growth of virtually all organisms, iron plays an important role in determining the bio-abundance and bio-diversity within a variety of different ecosystems (32
). However, only a limited number of studies have evaluated the effect of iron availability on the bacterial composition of human-associated microbiota (37
). In this study, using in vitro
human salivary microbial communities, we investigated whether the accessibility of iron plays a role in modulating the microbial populations within the oral microbiota.
We generated culturing conditions with differential free iron availability by adding different concentrations of Bipy, a ferrous iron (Fe2+) chelator to SHI medium containing sheep blood. Our data revealed that titration of free Fe2+ from SHI medium resulted in a drastic shift in the bacterial composition of an in vitro salivary microbial community, with Streptococcus spp, Gemella spp. and Granulicatella spp. becoming the most dominant bacterial species (). The Bipy-induced shift in microbial composition was observed both in pooled and individual saliva-derived microbial communities (, ). The similarity in the resultant bacterial profiles among different communities and the seemingly abrupt shift occurring at 0.3μM Bipy suggested that, the addition of 0.3μM Bipy could reduce the concentration of free iron in the medium below a threshold that might be required for the optimal growth of oral community. Once the free iron level is below the threshold, it could shift the microbial population toward bacterial species that might be more capable of utilizing alternative iron sources and/or more resistant to the iron starvation conditions.
Interestingly, the identified major bacterial species growing under iron-limited condition are phylogenetically related to different branches of Streptococci
spp. was previously classified into the genus Streptococcus
), and reclassified as genus Gemella
later based on the nucleotide sequence of the 16S rRNA(41
). While for Granulicatella
spp. and Abiotrophia
spp. (identified from subject 3), they were originally described as “nutritionally variant streptococci (NVS)”, a type of streptococci exhibiting satellitism around colonies of other bacteria (42
). These bacterial species share many physiological and biochemical properties with the viridans group streptococci, including the range of infections that they cause (43
). One of the common characteristics among these bacterial species is their α-hemolytic activity (44
), which was further confirmed by our hemolysis assay results (). The SHI medium used in this study contains 5% sheep blood (24
), the abundant iron-containing heme within sheep red blood cells could potentially serve as an iron source for oral bacteria under free iron-limited condition. The α-hemolytic activity would allow bacteria to partially lyse the red blood cells to release iron-complexes, e.g. heme and hemoglobin. Although the mechanisms regarding heme uptake in Gemella
spp., is currently unknown, many streptococci
have been shown to employ high-affinity iron uptake systems to obtain heme (20
Intriguingly, some of the selected Streptococcus
strains, including Streptococcus
spp. oral clone C3AKM006 and G. haemolysans
isolated from both Bipy-supplemented ASS and SHI medium displayed enhanced α-hemolytic activity under iron-limited condition (). For many host-associated bacterial species, iron starvation could act as an important environmental cue and induce specific adapted response in bacteria, including the induction of siderophore-mediated iron uptake systems and other potentially pathogenic features (1
). Certain Streptococcus
spp., including S. pyogenes
displayed increased hemolytic activity due to high hemolysin production when encountering iron-limited condition (49
). Our data clearly suggested that, during iron starvation, many oral bacterial species, such as Gemella haemolysans
, Granulicatella adiacens,
and certain Streptococci, including S. cristatus
might be able to sustain their growth by retrieving host iron-complexes via hemolytic activity.
Furthermore, when using chemically defined ASS medium without the inclusion of sheep blood, the addition of Bipy resulted in a community with a profile that was very similar microbial to the one obtained with Bipy-supplemented SHI medium (). Even in the presence of Bipy at a concentration that would chelate most of the free Fe2+
iron within the medium, several Streptococci,
species could still be isolated from the culture. 16s rDNA sequence analysis revealed that their identities matched a subset of Streptococcus
strains (including S. parasanguinis
and G. haemolysans)
isolated from Bipy-treated SHI medium (data not shown) which had been show to possess hemolytic activity. These results indicated that besides being capable of acquiring host iron-complexes, certain oral strains are also highly resistant to iron starvation conditions. Although iron is required for the growth of the virtually all microbes, different bacteria might have differential iron requirement for sustaining their normal growth and possess different ability to persist under iron deficient condition (1
). Furthermore, certain bacterial species, including oral Lactobacilli
) adapt their metabolism towards an absolute manganese requirement instead of absolute iron requirement as a possible defense mechanism against endogenous superoxide during aerobic metabolism (51
). However, we did not isolate any Lactobacilli
strains from salivary communities cultivated using either Bipy-supplemented SHI medium or ASS medium. This could be due to the fact that although SHI medium has been shown to sustain the growth of a variety of oral microbes, it does not seem to favor the growth of Lactobacilli
The varying capability of oral bacterial species in exploring iron sources and acquiring iron from their environments, as well as their differential resistance to iron starvation, are likely to make iron, this scarce and growth-limiting trace element, one of the modulating factors in shaping the host associated microbial community. This is corroborated by our observation that, for a mixed community composed of populations obtained from both iron-limiting and iron-containing medium, iron supplementation can restore the original bacterial profile; while the addition of an iron chelator drove the mixed culture towards a community that had been shown to be specifically selected under iron-deficient condition ().
The most intriguing finding of this study was that the majority of the oral bacterial species isolated under iron deficient condition have previously been implicated in infectious endocarditis (IE)(52
). IE is an infection of the lining of the heart chambers and heart valves that is caused by bacteria or other infectious substances. Since the description of the systemic significance of oral infection in late 19th
), every major body system has been identified as a potential targeting site for bacterial metastasis of oral origin (58
). The involvement of oral bacteria in the pathogenesis of endocarditis had been previously established (52
). It has been estimated that 65% of IE clinical cases were due to infection of α-hemolytic streptococci and majority of them were of oral origin. With the improvement of culturing technique and non-culture based detection methods, more and more fastidious bacteria have been identified to be involved in IE, including Gemella
), as well as Abiotrophia spp
). Normal oral functional activities like chewing, as well as most dental procedures, such as periodontal cleaning, tooth extraction and endodontic procedures could cause tissue surface trauma and transient bacteremia (62
). In individuals with heart malformations or the presence of vascular prostheses, the transient bacteremia could potentially lead to attachment of bacteria and result in IE. It is intriguing to speculate that, for an individual with an oral microbial community containing more IE-related bacterial species, such as α-hemolytic bacteria as a result of particular oral or systemic conditions, there could be a potentially higher risk of endocarditis. Further investigation is needed to determine whether there is a possible association between the saliva iron content and individual’s risk of infectious endocarditis.
The human oral cavity represents one of the most complex host-associated microbial ecosystems ever identified. Like gut-associated microbial flora, the microbial composition within the oral habitat could be a complex polygenic trait shaped and modulated by multiple host and environmental factors. A healthy and balanced oral commensal flora with stable microbial composition could play an important role in maintaining the community stability and preventing foreign/pathogenic colonization; while unbalanced population structure due to local or systemic conditions could lead to the deterioration of oral ecological conditions and induce adverse effect on the host. Our data suggested that, as one of the most essential trace elements, the accessibility of free iron could play an important role in modulating the population structure of the commensal oral microbiaota and have potential clinical relevance in inducing certain disease conditions.