Modulation Of Virulence Research Articles

Analysis of the Bacterial Diversity in Liver Abscess: Differences Between Pyogenic and Amebic Abscesses.

Several recent studies have demonstrated that virulence in Entamoeba histolytica is triggered in the presence of both pathogenic and nonpathogenic bacteria species using in vitro and in vivo experimental animal models. In this study, we examined samples aspirated from abscess material obtained from patients who were clinically diagnosed with amebic liver abscess (ALA) or pyogenic liver abscess (PLA). To determine the diversity of bacterial species in the abscesses, we performed partial 16S rRNA gene sequencing. In addition, the E. histolytica and Entamoeba dispar species were genotyped using tRNA-linked short tandem repeats as specific molecular markers. The association between clinical data and bacterial and parasite genotypes were examined through a correspondence analysis. The results showed the presence of numerous bacterial groups. These taxonomic groups constitute common members of the gut microbiota, although all of the detected bacterial species have a close phylogenetic relationship with bacterial pathogens. Furthermore, some patients clinically diagnosed with PLA and ALA were coinfected with E. dispar or E. histolytica, which suggests that the virulence of these parasites increased in the presence of bacteria. However, no specific bacterial groups were associated with this effect. Together, our results suggest a nonspecific mechanism of virulence modulation by bacteria in Entamoeba.

Exposure to sub-inhibitory concentrations of cefotaxime enhances the systemic colonization of Salmonella Typhimurium in BALB/c mice.

It has been proposed that sub-inhibitory concentrations of antibiotics play a role in virulence modulation. In this study, we evaluated the ability of Salmonella enterica serovar Typhimurium (hereafter S. Typhimurium) to colonize systemically BALB/c mice after exposure to a sub-inhibitory concentration of cefotaxime (CTX). In vivo competition assays showed a fivefold increase in systemic colonization of CTX-exposed bacteria when compared to untreated bacteria. To identify the molecular mechanisms involved in this phenomenon, we carried out a high-throughput genetic screen. A transposon library of S. Typhimurium mutants was subjected to negative selection in the presence of a sub-inhibitory concentration of CTX and genes related to anaerobic metabolism, biosynthesis of purines, pyrimidines, amino acids and other metabolites were identified as needed to survive in this condition. In addition, an impaired ability for oxygen consumption was observed when bacteria were cultured in the presence of a sub-inhibitory concentration of CTX. Altogether, our data indicate that exposure to sub-lethal concentrations of CTX increases the systemic colonization of S. Typhimurium in BALB/c mice in part by the establishment of a fitness alteration conducive to anaerobic metabolism.

Regulation of Indole Signalling during the Transition of E. coli from Exponential to Stationary Phase.

During the transition from exponential to stationary phase E. coli produces a substantial quantity of the small, aromatic signalling molecule indole. In LB medium the supernatant indole concentration reaches a maximum of 0.5–1 mM. At this concentration indole has been implicated in many processes inducing acid resistance and the modulation of virulence. It has recently been shown that cell-associated indole transiently reaches a very high concentration (approx. 60 mM) during stationary phase entry, presumably because indole is being produced more rapidly than it can leave the cell. It is proposed that this indole pulse inhibits growth and cell division, causing the culture to enter stationary phase before nutrients are completely exhausted, with benefits for survival in long-term stationary phase. This study asks how E. coli cells rapidly upregulate indole production during stationary phase entry and why the indole pulse has a duration of only 10–15 min. We find that at the start of the pulse tryptophanase synthesis is triggered by glucose depletion and that this is correlates with the up-regulation of indole synthesis. The magnitude and duration of the resulting indole pulse are dependent upon the availability of exogenous tryptophan. Indole production stops when all the available tryptophan is depleted and the cell-associated indole equilibrates with the supernatant.

Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants.

The tomato (Solanum lycopersicum) callose synthase genes CalS11-like and CalS12-like encode proteins that are essential for the formation of callose, a major component of pollen mother cell walls; these enzymes also function in callose formation during pathogen infection. This article describes the targeting of these callose synthase mRNAs by a small RNA derived from the virulence modulating region of two Potato spindle tuber viroid variants. More specifically, viroid infection of tomato plants resulted in the suppression of the target mRNAs up to 1.5-fold, depending on the viroid variant used and the gene targeted. The targeting of these mRNAs by RNA silencing was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Viroid mutants incapable of targeting callose synthase mRNAs failed to induce typical infection phenotypes, whereas a chimeric viroid obtained by swapping the virulence modulating regions of a mild and a severe variant of Potato spindle tuber viroid greatly affected the accumulation of viroids and the severity of disease symptoms. These data provide evidence of the silencing of multiple genes by a single small RNA derived from a viroid.

The Global Transcription Factor Lrp Controls Virulence Modulation in Xenorhabdus nematophila.

The bacterium Xenorhabdus nematophila engages in phenotypic variation with respect to pathogenicity against insect larvae, yielding both virulent and attenuated subpopulations of cells from an isogenic culture. The global regulatory protein Lrp is necessary for X. nematophila virulence and immunosuppression in insects, as well as colonization of the mutualistic host nematode Steinernema carpocapsae, and mediates expression of numerous genes implicated in each of these phenotypes. Given the central role of Lrp in X. nematophila host associations, as well as its involvement in regulating phenotypic variation pathways in other bacteria, we assessed its function in virulence modulation. We discovered that expression of lrp varies within an isogenic population, in a manner that correlates with modulation of virulence. Unexpectedly, although Lrp is necessary for optimal virulence and immunosuppression, cells expressing high levels of lrp were attenuated in these processes relative to those with low to intermediate lrp expression. Furthermore, fixed expression of lrp at high and low levels resulted in attenuated and normal virulence and immunosuppression, respectively, and eliminated population variability of these phenotypes. These data suggest that fluctuating lrp expression levels are sufficient to drive phenotypic variation in X. nematophila. Many bacteria use cell-to-cell phenotypic variation, characterized by distinct phenotypic subpopulations within an isogenic population, to cope with environmental change. Pathogenic bacteria utilize this strategy to vary antigen or virulence factor expression. Our work establishes that the global transcription factor Lrp regulates phenotypic variation in the insect pathogen Xenorhabdus nematophila, leading to attenuation of virulence and immunosuppression in insect hosts. Unexpectedly, we found an inverse correlation between Lrp expression levels and virulence: high levels of expression of Lrp-dependent putative virulence genes are detrimental for virulence but may have an adaptive advantage in other aspects of the life cycle. Investigation of X. nematophila phenotypic variation facilitates dissection of this phenomenon in the context of a naturally occurring symbiosis.

Evolutionary trajectories and diagnostic challenges of potentially zoonotic avian influenza viruses H5N1 and H9N2 co-circulating in Egypt

In Egypt, since 2006, descendants of the highly pathogenic avian influenza virus (HP AIV) H5N1 of clade 2.2 continue to cause sharp losses in poultry production and seriously threaten public health. Potentially zoonotic H9N2 viruses established an endemic status in poultry in Egypt as well and co-circulate with HP AIV H5N1 rising concerns of reassortments between H9N2 and H5N1 viruses along with an increase of mixed infections of poultry.Nucleotide sequences of whole genomes of 15 different isolates (H5N1: 7; H9N2: 8), and of the hemagglutinin (HA) and neuraminidase (NA) encoding segments of nine further clinical samples (H5N1: 2; H9N2: 7) from 2013 and 2014 were generated and analysed. The HA of H5N1 viruses clustered with clade 2.2.1 while the H9 HA formed three distinguishable subgroups within cluster B viruses. BEAST analysis revealed that H9N2 viruses are likely present in Egypt since 2009. Several previously undescribed substituting mutations putatively associated with host tropism and virulence modulation were detected in different proteins of the analysed H9N2 and H5N1 viruses.Reassortment between HP AIV H5N1 and H9N2 is anticipated in Egypt, and timely detection of such events is of public health concern. As a rapid tool for detection of such reassortants discriminative SYBR-Green reverse transcription real-time PCR assays (SG-RT-qPCR), targeting the internal genes of the Egyptian H5N1 and H9N2 viruses were developed for the rapid screening of viral RNAs from both virus isolates and clinical samples. However, in accordance to Sanger sequencing, no reassortants were found by SG-RT-qPCR.Nevertheless, the complex epidemiology of avian influenza in poultry in Egypt will require sustained close observation. Further development and continuing adaptation of rapid and cost-effective screening assays such as the SG-RT-qPCR protocol developed here are at the basis of efforts for improvement the currently critical situation.

Subinhibitory antibiotic therapy alters recurrent urinary tract infection pathogenesis through modulation of bacterial virulence and host immunity.

ABSTRACTThe capacity of subinhibitory levels of antibiotics to modulate bacterial virulence in vitro has recently been brought to light, raising concerns over the appropriateness of low-dose therapies, including antibiotic prophylaxis for recurrent urinary tract infection management. However, the mechanisms involved and their relevance in influencing pathogenesis have not been investigated. We characterized the ability of antibiotics to modulate virulence in the uropathogens Staphylococcus saprophyticus and Escherichia coli. Several antibiotics were able to induce the expression of adhesins critical to urothelial colonization, resulting in increased biofilm formation, colonization of murine bladders and kidneys, and promotion of intracellular niche formation. Mice receiving subinhibitory ciprofloxacin treatment were also more susceptible to severe infections and frequent recurrences. A ciprofloxacin prophylaxis model revealed this strategy to be ineffective in reducing recurrences and worsened infection by creating larger intracellular reservoirs at higher frequencies. Our study indicates that certain agents used for antibiotic prophylaxis have the potential to complicate infections.

An essential role for phosphatidylinositol 3-kinase in the inhibition of phagosomal maturation, intracellular survival and virulence in Candida glabrata.

The yeast class III phosphoinositide 3-kinase (PI3K) that catalyses production of the lipid signalling molecule, phosphatidylinositol-3-phosphate, is primarily implicated in vesicle-mediated transport and autophagy. In this study, we identified, through a genetic screen, the Candida glabrata CgVPS15 gene, an orthologue of the Saccharomyces cerevisiae PI3K regulatory subunit-encoding open reading frame (ORF) to be required for impairment of phagosomal maturation in human macrophages. We also disrupted catalytic subunit of the C. glabrata PI3K complex, CgVps34, and found it to be pivotal to arrest mature phagolysosome biogenesis. Further, deletion of either CgVPS15 or CgVPS34 rendered C. glabrata cells hyperadherent to epithelial cells and susceptible to the antimicrobial arsenal of primary murine and cultured human macrophages and diverse stresses. Despite no growth retardation at 37°C, Cgvps15Δ and Cgvps34Δ mutants were severely virulence attenuated in mice. We demonstrate that trafficking and/or processing of the vacuolar lumenal hydrolase, carboxypeptidase Y, and the major adhesin, Epa1, rely on PI3K regulatory mechanisms in C. glabrata. By disrupting autophagy-related PI3K complex genes, we show that C. glabrata PI3K-impeded phagolysosomal acidification is primarily owing to its role in cellular trafficking events. Altogether, our findings underscore the essentiality of PI3K signalling in modulation of host immune response, intracellular survival and virulence in C. glabrata.

Iron and copper as virulence modulators in human fungal pathogens.

Fungal pathogens have evolved sophisticated machinery to precisely balance the fine line between acquiring essential metals and defending against metal toxicity. Iron and copper are essential metals for many processes in both fungal pathogens and their mammalian hosts, but reduce viability when present in excess. However, during infection, the host uses these two metals differently. Fe has a long-standing history of influencing virulence in pathogenic fungi, mostly in regards to Fe acquisition. Numerous studies demonstrate the requirement of the Fe acquisition pathway of Candida, Cryptococcus and Aspergillus for successful systemic infection. Fe is not free in the host, but is associated with Fe-binding proteins, leading fungi to develop mechanisms to interact with and to acquire Fe from these Fe-bound proteins. Cu is also essential for cell growth and development. Essential Cu-binding proteins include Fe transporters, superoxide dismutase (SOD) and cytochrome c oxidase. Although Cu acquisition plays critical roles in fungal survival in the host, recent work has revealed that Cu detoxification is extremely important. Here, we review fungal responses to altered metal conditions presented by the host, contrast the roles of Fe and Cu during infection, and outline the critical roles of fungal metal homeostasis machinery at the host-pathogen axis.

Impact of Brief and Sequential Exposure to Nystatin on the Germ Tube Formation and Cell Surface Hydrophobicity of Oral Candida Albicans Isolates from Human Immunodeficiency Virus-Infected Patients

Objective: To evaluate the impact of brief and sequential exposure to nystatin on the germ tube formation and cell surface hydrophobicity of oral isolates of Candida albicans obtained from patients infected with human immunodeficiency virus (HIV). Materials and Methods: After determining the minimum inhibitory concentration of nystatin, 10 oral isolates of C. albicans from 10 different HIV-infected patients were briefly (1 h) and sequentially (10 days) exposed to subtherapeutic concentrations of nystatin. Following a subsequent drug removal, the germ tube formation and cell surface hydrophobicity of these isolates were determined via a germ tube induction assay and an aqueous hydrocarbon assay, respectively. The data obtained from these assays for the control (unexposed to nystatin) and nystatin-exposed isolates were analyzed using Student's t tests. Results: The mean percentage reduction in the germ tube formation and cell surface hydrophobicity of the nystatin-exposed isolates compared to the controls was 30.12 ± 1.99 (p < 0.001) and 29.65 ± 2.33 (p < 0.001), respectively. Conclusion: These data elucidate the possible pharmacodynamic mechanisms by which nystatin might operate in vivo in the modulation of candidal virulence.

MgtE is a dual-function protein in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections, including chronic biofilm infections in the lungs of individuals with cystic fibrosis. We previously found that the inner-membrane protein MgtE can function both as a magnesium transporter and a virulence modulator, although the exact mechanism governing these activities is unclear. To address this issue, we carried out an experimental characterization of P. aeruginosa MgtE and generated a computer-rendered model. Our in silico analysis demonstrated the structural similarity of P. aeruginosa MgtE to that of the crystal structure of MgtE in Thermus thermophilus. Experimentally, we verified that MgtE is not essential for growth and found that it may not be involved directly in biofilm formation, even under low-magnesium conditions. We demonstrated both magnesium transport and cytotoxicity-regulating functions, and showed that magnesium-binding sites in the connecting helix region of MgtE are vital in coupling these two functions. Furthermore, limiting magnesium environments stimulated mgtE transcriptional responses. Our results suggested that MgtE might play an important role in linking magnesium availability to P. aeruginosa pathogenesis.

The Indole Pulse: A New Perspective on Indole Signalling in Escherichia coli

Indole has diverse signalling roles, including modulation of biofilm formation, virulence and stress responses. Changes are induced by indole concentrations of 0.5–1.0 mM, similar to those found in the supernatant of Escherichia coli stationary phase culture. Here we describe an alternative mode of indole signalling that promotes the survival of E. coli cells during long-term stationary phase. A mutant that has lost the ability to produce indole demonstrates reduced survival under these conditions. Significantly, the addition of 1 mM indole to the culture supernatant is insufficient to restore long-term survival to the mutant. We provide evidence that the pertinent signal in this case is not 1 mM indole in the culture supernatant but a transient pulse of intra-cellular indole at the transition from exponential growth to stationary phase. During this pulse the cell-associated indole reaches a maximum of approximately 60 mM. We argue that this is sufficient to inhibit growth and division by an ionophore-based mechanism and causes the cells to enter stationary phase before resources are exhausted. The unused resources are used to repair and maintain cells during the extended period of starvation.

Effects of 6‐hydroxynicotinate 3‐monooxygenase gene knockout (∆nicC) on nicotinate degradation and virulence modulation in Bordetella pertussis (LB266)

Due to increased incidence of whooping cough, further investigation of the pathogenic mechanisms of its causative agent, Bordetella pertussis, is of medical importance. Previous in vitro assays indicate a correlation between elevated nicotinate concentrations and the down‐regulation of virulent toxins (e.g. pertussis toxin and adenylate cyclase) in B. pertussis. Furthermore, recent genomic analyses reveal a unique nicotinate degradation pathway in B. pertussis. This study analyzes the effects of disrupting the gene that codes for 6‐hydroxynicotinate 3‐monooxygenase (NicC), the enzyme predicted to catalyze the committed step in this pathway. A nicC knockout strain (ΔnicC) of B. pertussis Tohama I was created by insertion of a chloramphenicol‐resistance (CmR) gene via allelic exchange. Analyses by HPLC assays and RT‐PCR of nicotinate degradation and the resulting effects on virulence modulation, respectively, in ΔnicC and the wildtype Tohama I strains of B. pertussis are ongoing.

YmoA negatively controls the expression of insecticidal genes in Yersinia enterocolitica

Yersinia enterocolitica is toxic towards invertebrates due to the presence of the toxin complex (tc) genes that are activated by the thermolabile regulator TcaR2. In the search for further regulatory factors involved in insecticidal gene expression, the modulator of yersinial virulence, YmoA, was identified to silence all tc genes of the Y. enterocolitica strain W22703 (biovar 2, serovar O:9). Using promoter fusions with the luciferase reporter, we found that the deletion of ymoA results in elevated transcription of tcaR1, tcaR2, tcaA, tcaB, tcaC, tccC1 and tccC2 at both 15 °C and 37 °C. Complementation by episomal ymoA significantly reduced tc gene expression, thus validating the inhibitory activity of YmoA on the production of insecticidal proteins. YmoA contributes to the binding properties of H-NS to the tc promoters by forming a complex with this nucleoid-associated protein, and this complex not only binds to the upstream regions of all tc genes, but also to intragenic sites of tcaA and tcaB that play an important role in controlling the expression of both genes. At low temperature, the intracellular amount of thermostable YmoA is not reduced, but the repressor is less functional. These data point to H-NS/YmoA as an antagonist of the inducer TcaR2.

An ethA-ethR -Deficient Mycobacterium bovis BCG Mutant Displays Increased Adherence to Mammalian Cells and Greater Persistence In Vivo , Which Correlate with Altered Mycolic Acid Composition

Tuberculosis remains a major worldwide epidemic because of its sole etiological agent, Mycobacterium tuberculosis. Ethionamide (ETH) is one of the major antitubercular drugs used to treat infections with multidrug-resistant M. tuberculosis strains. ETH is a prodrug that requires activation within the mycobacterial cell; its bioactivation involves the ethA-ethR locus, which encodes the monooxygenase EthA, while EthR is a transcriptional regulator that binds to the intergenic promoter region of the ethA-ethR locus. While most studies have focused on the role of EthA-EthR in ETH bioactivation, its physiological role in mycobacteria has remained elusive, although a role in bacterial cell detoxification has been proposed. Moreover, the importance of EthA-EthR in vivo has never been reported on. Here we constructed and characterized an EthA-EthR-deficient mutant of Mycobacterium bovis BCG. Our results indicate that absence of the ethA-ethR locus led to greater persistence of M. bovis BCG in the mouse model of mycobacterial infection, which correlated with greater adherence to mammalian cells. Furthermore, analysis of cell wall lipid composition by thin-layer chromatography and mass spectrometry revealed differences between the ethA-ethR KO mutant and the parental strain in the relative amounts of α- and keto-mycolates. Therefore, we propose here that M. bovis BCG ethA-ethR is involved in the cell wall-bound mycolate profile, which impacts mycobacterial adherence properties and in vivo persistence. This study thus provides some experimental clues to the possible physiological role of ethA-ethR and proposes that this locus is a novel factor involved in the modulation of mycobacterial virulence.

In Nicotiana species, an artificial microRNA corresponding to the virulence modulating region of Potato spindle tuber viroid directs RNA silencing of a soluble inorganic pyrophosphatase gene and the development of abnormal phenotypes

Potato spindle tuber viroid (PSTVd) is a small non-protein-coding RNA pathogen that can induce disease symptoms in a variety of plant species. How PSTVd induces disease symptoms is a long standing question. It has been suggested that PSTVd-derived small RNAs (sRNAs) could direct RNA silencing of a targeted host gene(s) resulting in symptom development. To test this, we expressed PSTVd sequences as artificial microRNAs (amiRNAs) in Nicotiana tabacum and Nicotiana benthamiana. One amiRNA, amiR46 that corresponds to sequences within the PSTVd virulence modulating region (VMR), induced abnormal phenotypes in both Nicotiana species that closely resemble those displayed by PSTVd infected plants. In N. tabacum amiR46 plants, phenotype severity correlated with amiR46 accumulation and expression down-regulation of the bioinformatically-identified target gene, a Nicotiana soluble inorganic pyrophosphatase (siPPase). Taken together, our phenotypic and molecular analyses suggest that disease symptom development in Nicotiana species following PSTVd infection results from sRNA-directed RNA silencing of the host gene, siPPase.

Antibiotic treatment of Pseudomonas aeruginosa biofilms stimulates expression of the magnesium transporter gene mgtE

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with the capacity to cause serious disease, including chronic biofilm infections in the lungs of cystic fibrosis (CF) patients. These infections are treated with high concentrations of antibiotics. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified, including the magnesium transporter protein MgtE. We have recently found that isogenic deletion of mgtE leads to increased cytotoxicity through effects on the type III secretion system. To explore the role of the CF lung environment in MgtE activity, we investigated mgtE transcriptional regulation following antibiotic treatment. Utilizing quantitative real-time-PCR, we have demonstrated an increase in mgtE transcript levels following antibiotic treatment with most of the 12 antibiotics tested. To begin to determine the regulatory network governing mgtE expression, we screened a transposon-mutant library of P. aeruginosa to look for mutants with potentially altered mgtE activity, using cytotoxicity as a readout. In this screen, we observed that AlgR, which regulates production of the biofilm polysaccharide alginate, alters MgtE-mediated cytotoxicity. This cross-talk between MgtE and AlgR suggests that AlgR is involved in linking external inducing signals (e.g. antibiotics) to mgtE transcription and downstream virulence and biofilm activities. Analysing such interactions may lead to a better understanding of how the CF lung environment shapes P. aeruginosa biofilm infections.

Pro-inflammatory cytokines can act as intracellular modulators of commensal bacterial virulence

Interactions between commensal pathogens and hosts are critical for disease development but the underlying mechanisms for switching between the commensal and virulent states are unknown. We show that the human pathogen Neisseria meningitidis, the leading cause of pyogenic meningitis, can modulate gene expression via uptake of host pro-inflammatory cytokines leading to increased virulence. This uptake is mediated by type IV pili (Tfp) and reliant on the PilT ATPase activity. Two Tfp subunits, PilE and PilQ, are identified as the ligands for TNF-α and IL-8 in a glycan-dependent manner, and their deletion results in decreased virulence and increased survival in a mouse model. We propose a novel mechanism by which pathogens use the twitching motility mode of the Tfp machinery for sensing and importing host elicitors, aligning with the inflamed environment and switching to the virulent state.

Cell-free preparations of Lactobacillus acidophilus strain La-5 and Bifidobacterium longum strain NCC2705 affect virulence gene expression in Campylobacter jejuni.

Campylobacter spp. are among the most commonly reported bacterial causes of acute diarrheal disease in humans worldwide. Potential virulence factors include motility, chemotaxis, colonization ability, adhesion to intestinal cells, invasion and epithelial translocation, intracellular survival, and formation of toxins. Probiotic Lactobacillus and Bifidobacterium strains are known to have an inhibitory effect against the growth of various foodborne pathogens. The objective of this study was to investigate the effect of Lactobacillus acidophilus strain La-5 and Bifidobacterium longum strain NCC2705 cell-free spent media (CFSM) on the expression of virulence genes (cadF, cdtB, flaA, and ciaB) of Campylobacter jejuni strain 81-176 and a luxS mutant, using real-time PCR. Our results demonstrated that the CFSM of both probiotic strains were able to down-regulate the expression of ciaB (ratio of -2.80 and -5.51, respectively) and flaA (ratio of -7.00 and -5.13, respectively) in the wild-type Campylobacter strain. In the luxS mutant, where the activated methyl cycle is disrupted, only the ciaB gene (ratio -7.21) was repressed in the presence of La-5 CFSM. A supplementation of homocysteine to restore the disrupted cycle was able to partially reestablish the probiotic effect of both strains. luxS and the activated methyl cycle might play an active role in the modulation of virulence of C. jejuni by probiotic extracts.

Virulence modulation of Streptococcus mutans biofilms by metal ions released from orthodontic appliances

To evaluate the impact of metal ions commonly shed from orthodontic appliances on the virulence of Streptococcus mutans ATCC®25175™ biofilms. Biofilms were grown in the presence of Ni(2+), Fe(3+), Cr(3+), Co(2+), and a metal ion pool at concentrations similar to those released in saliva of orthodontic patients for 72 hours. Once mature, biofilms were treated for up to 12 hours with 5% glucose. Ions interfered with the growth of S mutans by reducing its biomass (Ni(2+), Fe(3+), Cr(3+)), raising its rates of sugar metabolism (Ni(2+), Fe(3+), Cr(3+)), and raising its secretion of lactate (Ni(2+), Fe(3+), Cr(3+), pool). The laboratory data presented here point to the possibility of virulence increase of S mutans by metal ions commonly released during orthodontic therapy.