Exploring ex vivo biofilm dynamics: consequences of low ampicillin concentrations on the human oral microbiome.

Prolonged exposure to antibiotics at low concentration can promote processes associated with bacterial biofilm formation, virulence and antibiotic resistance. This can be of high relevance in microbial communities like the oral microbiome, where commensals and pathogens share a common habitat and where the total abundance of antibiotic resistance genes surpasses the abundance in the gut. Here, we used an ex vivo model of human oral biofilms to investigate the impact of ampicillin on biofilm viability. The ecological impact on the microbiome and resistome was investigated using shotgun metagenomics. The results showed that low concentrations promoted significant shifts in microbial taxonomic profile and could enhance biofilm viability by up to 1 to 2-log. For the resistome, low concentrations had no significant impact on antibiotic resistance gene (ARG) diversity, while ARG abundance decreased by up to 84%. A positive correlation was observed between reduced microbial diversity and reduced ARG abundance. The WHO priority pathogens Streptococcus pneumoniae and Staphylococcus aureus were identified in some of the samples, but their abundance was not significantly altered by ampicillin. Most of the antibiotic resistance genes that increased in abundance in the ampicillin group were associated with streptococci, including Streptococcus mitis, a well-known potential donor of ARGs to S. pneumoniae. Overall, the results highlight the potential of using the model to further our understanding of ecological and evolutionary forces driving antimicrobial resistance in oral microbiomes.

Characterization of a Signaling System in Streptococcus mitis That Mediates Interspecies Communication with Streptococcus pneumoniae.

Streptococcus mitis is found in the oral cavity and nasopharynx and forms a significant portion of the human microbiome. In this study, in silico analyses indicated the presence of an Rgg regulator and short hydrophobic peptide (Rgg/SHP) cell-to-cell communication system in S. mitis Although Rgg presented greater similarity to a repressor in Streptococcus pyogenes, autoinducing assays and genetic mutation analysis revealed that in S. mitis Rgg acts as an activator. Transcriptome analysis showed that in addition to shp, the system regulates two other downstream genes, comprising a segment of a putative lantibiotic gene cluster that is in a conjugative element locus in different members of the mitis group. Close comparison to a similar lantibiotic gene cluster in Streptococcus pneumoniae indicated that S. mitis lacked the full set of genes. Despite the potential of SHP to trigger a futile cycle of autoinduction, growth was not significantly affected for the rgg mutant under normal or antibiotic stress conditions. The S. mitis SHP was, however, fully functional in promoting cross-species communication and increasing S. pneumoniae surface polysaccharide production, which in this species is regulated by Rgg/SHP. The activity of SHPs produced by both species was detected in cocultures using a S. mitis reporter strain. In competitive assays, a slight advantage was observed for the rgg mutants. We conclude that the Rgg/SHP system in S. mitis regulates the expression of its own shp and activates an Rgg/SHP system in S. pneumoniae that regulates surface polysaccharide synthesis. Fundamentally, cross-communication of such systems may have a role during multispecies interactions.IMPORTANCE Bacteria secrete signal molecules into the environment which are sensed by other cells when the density reaches a certain threshold. In this study, we describe a communication system in Streptococcus mitis, a commensal species from the oral cavity, which we also found in several species and strains of streptococci from the mitis group. Further, we show that this system can promote cross-communication with S. pneumoniae, a closely related major human pathogen. Importantly, we show that this cross-communication can take place during coculture. While the genes regulated in S. mitis are likely part of a futile cycle of activation, the target genes in S. pneumoniae are potentially involved in virulence. The understanding of such complex communication networks can provide important insights into the dynamics of bacterial communities.

High-resolution profiles of the Streptococcus mitis CSP signaling pathway reveal core and strain-specific regulated genes.

BACKGROUND: In streptococci of the mitis group, competence for natural transformation is a transient physiological state triggered by competence stimulating peptides (CSPs). Although low transformation yields and the absence of a widespread functional competence system have been reported for Streptococcus mitis, recent studies revealed that, at least for some strains, high efficiencies can be achieved following optimization protocols. To gain a deeper insight into competence in this species, we used RNA-seq, to map the global CSP response of two transformable strains: the type strain NCTC12261T and SK321. RESULTS: All known genes induced by ComE in Streptococcus pneumoniae, including sigX, were upregulated in the two strains. Likewise, all sets of streptococcal SigX core genes involved in extracellular DNA uptake, recombination, and fratricide were upregulated. No significant differences in the set of induced genes were observed when the type strain was grown in rich or semi-defined media. Five upregulated operons unique to S. mitis with a SigX-box in the promoter region were identified, including two specific to SK321, and one specific to NCTC12261T. Two of the strain-specific operons coded for different bacteriocins. Deletion of the unique S. mitis sigX regulated genes had no effect on transformation. CONCLUSIONS: Overall, comparison of the global transcriptome in response to CSP shows the conservation of the ComE and SigX-core regulons in competent S. mitis isolates, as well as species and strain-specific genes. Although some S. mitis exhibit truncations in key competence genes, this study shows that in transformable strains, competence seems to depend on the same core genes previously identified in S. pneumoniae.

Comprehensive Transcriptome Profiles of Streptococcus mutans UA159 Map Core Streptococcal Competence Genes.

In Streptococcus mutans, an oral colonizer associated with dental caries, development of competence for natural genetic transformation is triggered by either of two types of peptide pheromones, competence-stimulating peptides (CSPs) (18 amino acids [aa]) or SigX-inducing peptides (XIPs) (7 aa). Competence induced by CSP is a late response to the pheromone that requires the response regulator ComE and the XIP-encoding gene comS. XIP binds to ComR to allow expression of the alternative sigma factor SigX and the effector genes it controls. While these regulatory links are established, the precise set of effectors controlled by each regulator is poorly defined. To improve the definition of all three regulons, we used a high-resolution tiling array to map global changes in gene expression in the early and late phases of the CSP response. The early phase of the CSP response was limited to increased gene expression at four loci associated with bacteriocin production and immunity. In the late phase, upregulated regions expanded to a total of 29 loci, including comS and genes required for DNA uptake and recombination. The results indicate that the entire late response to CSP depends on the expression of comS and that the immediate transcriptional response to CSP, mediated by ComE, is restricted to just four bacteriocin-related loci. Comparison of the new data with published transcriptome data permitted the identification of all of the operons in each regulon: 4 for ComE, 2 for ComR, and 21 for SigX. Finally, a core set of 27 panstreptococcal competence genes was identified within the SigX regulon by comparison of transcriptome data from diverse streptococcal species. IMPORTANCES. mutans has the hard surfaces of the oral cavity as its natural habitat, where it depends on its ability to form biofilms in order to survive. The comprehensive identification of S. mutans regulons activated in response to peptide pheromones provides an important basis for understanding how S. mutans can transition from individual to social behavior. Our study placed 27 of the 29 transcripts activated during competence within three major regulons and revealed a core set of 27 panstreptococcal competence-activated genes within the SigX regulon.

Genome editing by natural genetic transformation in Streptococcus mutans.

Classical mutagenesis strategies using selective markers linked to designed mutations are powerful and widely applicable tools for targeted mutagenesis via natural genetic transformation in bacteria and archaea. However, the markers that confer power are also potentially problematic as they can be cumbersome, risk phenotypic effects of the inserted genes, and accumulate as unwanted genes during successive mutagenesis cycles. Alternative mutagenesis strategies use temporary plasmid or cassette insertions and can in principle achieve equally flexible mutation designs, but design of suitable counter-selected markers can be complex. All these drawbacks are eased by use of direct genome editing. Here we describe a strategy for directly editing the genome of S. mutans, which is applied to the widely studied reference strain UA159 (ATCC 700610) and has the advantage of extreme simplicity, requiring construction of only one synthetic donor amplicon and a single transformation step, followed by a simple PCR screen among a few dozen clones to identify the desired mutant. The donor amplicon carries the mutant sequence and extensive flanking segments of homology, which ensure efficient and precise integration by the recombination machinery specific to competent cells. The recipients are highly competent cells, in a state achieved by treatment with a synthetic competence pheromone.