Selenium Exerts Antimicrobial Activity against the Perinatal Pathogen Streptococcus agalactiae and Perturbs Bacterial Interactions with Human Gestational Membranes

Abstract

Streptococcus agalactiae also known as Group B Streptococcus (GBS) is a Gram-positive, encapsulated, pathogenic bacterium. GBS causes severe perinatal infections that lead to chorioamnionitis, funisitis, premature rupture of membranes, preterm birth, maternal sepsis, neonatal sepsis, stillbirth, and maternal demise. Epidemiological data indicate that the nutrient selenium provides protection against infection and adverse disease outcomes and is a critical nutrient for development of a healthy pregnancy. We hypothesized that selenium could have antimicrobial activity against GBS. To test this, we employed a panel of colonizing and invasive GBS strains and evaluated the bacterial growth in response to selenium exposure. Our results indicate that selenium can inhibit GBS growth and adherence to gestational tissues and that colonizing strains are more sensitive to selenium than invasive strains. Together, these results indicate that selenium could be deployed as a cost-effective intervention to ameliorate the risk of GBS perinatal infections.

Streptococcus agalactiae (GBS) is a Gram-positive pathogen that is one of the primary causes of adverse pregnancy outcomes. GBS infections are associated with premature rupture of membranes, preterm birth, neonatal sepsis, maternal sepsis and death. − In 2021 the U.S. experienced a 10% incidence of preterm birth (birth before 37 weeks of gestation); the highest level of preterm birth since 2007. Additionally, maternal mortality is on the rise in the U.S.

Maternal nutrition is a significant environmental factor that can affect adverse pregnancy outcomes. Epidemiological studies have revealed that inadequate intake of micronutrients such as zinc and selenium during pregnancy is associated with enhanced risk of adverse pregnancy outcomes such as miscarriage, small-for-gestational age births, and preterm birth. Micronutrients serve as crucial cofactors and coenzymes for a variety of cellular processes within metabolism, including modulating the electron transport chain, regulation of DNA transcription, and combating oxidative stress. This underscores that micronutrients play a crucial role in mitigating oxidative stress in a cellular environment and the importance of maintaining micronutrient homeostasis.

Selenium is an essential micronutrient that serves a variety of functions. Inorganic selenium is initially reduced to hydrogen selenide in the cell and is involved in various redox reactions and antioxidant activity. , In particular, selenocysteine serves a critical role in glutathione peroxidase, an enzyme involved in defense against oxidative stress. Glutathione peroxidase reduces hydrogen peroxide, a reactive oxygen species, and limits the harmful effects of cellular oxidative damage. During fetal development, oxidative damage can lead to various complications such as miscarriage, preeclampsia, and fetal growth restriction or low birth weight.

Additionally, deficiencies in micronutrients have been associated with enhanced susceptibility to infections. Infection during pregnancy is a strong driver of adverse pregnancy outcomes. Micronutrient metals such as zinc, copper, nickel, and cobalt have antimicrobial activities against GBS. , With this knowledge that certain micronutrients reduce GBS growth, it is critical to explore how other micronutrients may affect bacterial growth during infection. However, the antimicrobial role of micronutrient selenium against GBS remains understudied.

We hypothesized that selenium could have antimicrobial activity against GBS and that this perinatal pathogen might deploy micronutrient resistance determinants to evade the impact of selenium toxicity. To test this, we utilized a variety of in vitro assays and ex vivo host–pathogen interaction experiments to interrogate how selenium impacts GBS.

Previous work has indicated that GBS strains exhibit varying susceptibility to micronutrient metal intoxication; hence, we sought to investigate the effects of selenium intoxication on a diverse set of GBS strains. A strain survey to identify the minimum inhibitory concentration (MIC) of selenium against a diverse set of GBS strains with varying capsular and multilocus sequence types (STs) revealed a broad range of MICs between 78 μM to over 5000 μM (Table S1). Strains GB00285, GB00377, GB00571, GB0651, and GB0663 were found to have the lowest MIC of 78 μM, encompassing both colonizing and invasive strains. However, strains GB00012, GB00037, GB00066, GB00079, and NEM316 were found to have the highest MICs of selenium, all greater than 5000 μM. These strains, which exhibited the least susceptibility to selenium intoxication, also represented both colonizing and invasive strains, capsular serotypes cpsV and cpsIII, and multiple STs, similar to the GBS strains, which were found to be the most susceptible to selenium intoxication.

We then sought to ascertain whether colonizing versus invasive strains exhibited differences in MIC of selenium. Analysis revealed that invasive strains of GBS have a significantly higher MIC compared to colonizing strains of GBS (Figure , unpaired Student’s t test with Welch’s correction, P = 0.0188). While individual variability was observed within the categories of colonizing and invasive strains, it is evident that selenium intoxication exerts a larger inhibitory effect on colonizing GBS strains than invasive GBS strains. Interestingly, previous work demonstrated that colonizing strains of GBS exhibited significantly enhanced susceptibility to zinc intoxication compared to invasive strains, indicating invasive GBS strains may have undergone selection for enhanced fitness under high concentrations of micronutrient ions.

1.

Analysis of the minimum inhibitory concentration (MIC) of selenium, which impacts growth of colonizing or invasive GBS strains. Invasive strains have a significantly higher MIC compared to colonizing isolates (P < 0.05, Student’s t test).

GBS requires cation resistance encoded in the cadD locus, to invade and ascend the gravid reproductive tract. An isogenic ΔcadD mutant exhibited significantly diminished growth in the presence of a metal cation challenge including copper, nickel, cobalt, cadmium, and zinc. We hypothesized that the ΔcadD mutant might exhibit altered susceptibility to the selenium challenge. To test this, the parental wild-type GB00112 strain (WT), ΔcadD mutant, and ΔcadD:C complemented isogenic derivative were grown in medium alone or medium supplemented with increasing concentrations of sodium selenite. All three strains exhibited similar growth patterns in medium alone over a 24 h growth period (Figure ). However, supplementation with 100, 250, or 500 μM selenium results in a significant lag of ΔcadD mutant growth compared to WT and complemented derivative (P < 0.0001, Two-way ANOVA with Šídák’s post hoc test). Additionally, at 750 μM of selenium the ΔcadD mutant growth is severely attenuated compared to WT and complemented derivative (P < 0.0001, Two-way ANOVA with Šídák’s post hoc test). At a concentration of 5000 μM selenium, all strains exhibit significant inhibition of bacterial growth, demonstrating the deleterious effects of extremely high selenium concentrations on GBS growth. These findings suggest that the antimicrobial activity of selenium is distinct and specific to certain concentrations of selenium and that the cation resistance determinant-encoding gene, cadD is crucial for GBS growth in the presence of selenium stress. Although the mechanism of selenium toxicity in GBS is unknown, there are strong data suggesting metal toxicity in streptococcal pathogens is associated with mismetalation of proteins that function in key metabolic pathways such as nucleotide synthesis. For example, excess copper ions can replace manganese ions in the active site of NrdF, a manganese-dependent ribonucleotide reductase that is crucial in the aerobic nucleotide synthesis pathway. Ultimately, this can impact cellular fitness and replication. It is possible that excess selenium could influence mismetalation in a similar fashion.

2.

Growth curve analysis of GBS strains challenged with selenium over time. Growth curve analysis of GBS was performed in increasing concentrations of selenium. Wild-type GB112 (WT), isogenic ΔcadD mutant, and isogenic ΔcadD:C complemented mutant exhibit differential growth in increasing concentrations of selenium. At concentrations of 100 uM selenium and higher, the isogenic ΔcadD mutant exhibits significant attenuation of growth compared to the wild-type (WT) parental strain (****P < 0.0001, Two-way ANOVA with Šídák’s post hoc test).

To better assess how selenium stress could influence GBS host–pathogen interactions at the maternal-fetal interface, we utilized our model of human gestational membrane ex vivo coculture with GBS (19–21) in tandem with selenium supplementation. Quantitative culture of GBS in human gestational membranes alone or supplemented with selenium demonstrated that selenium exposure represses association of GBS with gestational membranes consistently among the wild-type GB00112 (WT), the ΔcadD deletion mutant, and the ΔcadD:C complemented mutant strains (Figure ). In each condition, the addition of selenium significantly represses association of GBS with gestational membranes, but importantly, the ΔcadD mutant is repressed to undetectable levels upon treatment with selenium, underscoring that cadD expression is crucial for GBS to resist selenium stress at the host–pathogen interface.

3.

Quantitative culture of GBS in human gestational membranes. Treatment with 100 μM selenium represses GBS association with gestational membranes, but the isogenic ΔcadD mutant has no detectable bacterial cells associated with gestational membranes in conditions of high selenium compared to wild-type and complemented derivatives. **P < 0.01, Two-way ANOVA with Šídák’s post hoc test.

A potential mechanism for selenium supplementation to disrupt GBS association with gestational membranes may be a result of selenium’s antiadhesive and antibiofilm properties as GBS biofilm formation is crucial for bacterial association with gestational membranes and pathogenesis in a pregnant host. − Selenium-containing compounds have been demonstrated to repress adherence and biofilm formation by a variety of pathogens including methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Staphylococcus epidermidis, and Enterococcus faecalis. , To test this, we utilized a standard crystal violet colorimetric biofilm assay. Results demonstrated that selenium indeed inhibits GBS biofilm formation in vitro at concentrations as low as 5 μM (Figure S1).

Selenium is a required nutrient for optimum fetal and placental development during pregnancy. In a study of 1586 mother-offspring dyads selenium levels were positively associated with cognitive development in offspring Additionally, selenium supplementation has been associated with protective effects. For example, supplementation with 100 mg of selenium per day during pregnancy resulted in a significant increase in mean serum selenium concentration at term (p < 0.001), a result that was not observed in the control group. Concomitantly, the incidence of premature rupture of membranes (PROM) was significantly lower in the selenium-supplemented group. These findings indicate that selenium supplementation in pregnant patients can alter adverse pregnancy outcomes such as PROM. It is important to note that 60–400 mg selenium supplementation is considered the therapeutic window with anything above 400 mg/day being considered a toxic dose in pregnant humans. In this study, we utilized a “supranutritional” (meaning above adequate but below toxic) level of selenium supplementation to study its effect on bacteria in vitro and at the maternal-fetal interface ex vivo. Other factors, such as reactive oxygen species, antimicrobial peptides, peroxide stress, and nutritional challenges, likely enhance the activity of selenium against microorganisms in vivo.

Previous studies have shown that sodium selenite has antimicrobial activity against a variety of bacterial pathogens including Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Klebsiella planticola. Additionally, selenium nanoparticles have also demonstrated significant antibacterial efficacy, particularly against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus strains. Our work is an extension of this foundational work, adding important information about the utility of sodium selenite against the perinatal pathogen GBS.

Taken together, our results indicate that selenium exerts antimicrobial effects on GBS in vitro and in the context of gravid reproductive tract tissues. Future work will examine a larger number of diverse strains and elucidate whether selenium could be exploited as a supplement or chemotherapeutic intervention to ameliorate GBS-associated adverse perinatal outcomes in nutritionally compromised hosts. Additionally, genomic or transcriptomic data from high strains versus low strains could reveal key selenium resistance determinants to better understand the molecular mechanism of selenium susceptibility in GBS.

Materials and Methods

Bacterial Strains and Culture Conditions

Clinical strains of GBS utilized for this study are listed in Table S1. Strains were previously characterized by MLST, capsular serotyping, and were recovered from pregnant patients (colonizing) or newborns with disease (invasive) as previously described. , Isogenic ΔcadD mutant or complemented derivative (ΔcadD:C) were generated as previously described in the GB00112 parental strain. All strains were stored in 30% glycerol stocks at −70 °C until required for assays. Strains were streaked from glycerol stocks onto tryptic soy agar plates supplemented with 5% sheep blood (blood agar) and incubated in room air at 37 °C overnight. The following day, strains were subcultured into Todd-Hewitt broth (THB) and incubated in shaking conditions (180 rpm) at 37 °C overnight.

Analysis of Bacterial Growth in Response to Selenium Challenge

Overnight cultures of GBS strains grown in THB broth were utilized to inoculate brain-heart infusion broth alone or supplemented with increasing concentrations (0, 78, 156, 312, 625, 1250, 2500, or 5000 μM) of sodium selenite. Cultures were incubated at 37 °C (180 rpm shaking) for 24 h, and bacterial density was measured by optical density at 600 nm (OD₆₀₀). Minimum inhibitory concentration was calculated as the lowest concentration at which significant inhibition of bacterial growth compared to medium alone controls (P < 0.05, paired Student’s t test). To evaluate the contribution of cadD to GBS resistance to selenium, GB00112 wild-type (WT), the isogenic ΔcadD mutant, or the complemented derivative (ΔcadD:C) overnight cultures in THB broth were utilized to inoculate brain-heart infusion broth alone or supplemented with increasing concentrations (0, 100, 250, 500, 750, or 5000 μM) of sodium selenite. These concentrations were chosen because they encompass the MIC values observed across diverse strains of GBS in Table S1. Cultures were incubated at 37 °C (180 rpm shaking) for 24 h, and bacterial density was measured by OD₆₀₀ every 30 min.

Bacterial Coculture with Human Gestational Tissues

Gestational membrane tissues were isolated from placenta delivered from healthy, term, nonlaboring caesarean section live births. Human tissue samples (gestational membranes) were collected after informed consent was obtained in accordance with a protocol approved by the Vanderbilt University Medical Center Institutional Review Board (IRB) #181998. Tissues were cut into 12 mm diameter sections and placed into Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum, 25 mM HEPES and l-glutamine (RPMI 1640+) plus penicillin G sodium salt and streptomycin sulfate (100 u/mL). Tissues were incubated at 37 °C in room air supplemented with 5% carbon dioxide overnight. The following day, supernatants were removed, and tissues were washed three times with sterile phosphate buffered saline (PBS) and finally placed in 1 mL of RPMI 1640+ without antibiotics in 12 well plates. Tissues were cultured in RPMI 1640+ without antibiotics alone (medium alone) or supplemented with 100 μM of sodium selenite for 4 h prior to infection with 10⁵ colony forming units of either wild-type GB00112, ΔcadD, or ΔcadD:C. This concentration of selenium challenge was selected because growth curve analyses revealed that at 100 μM of sodium selenite, the isogenic ΔcadD mutant was impacted, but by 24 h all three isogenic strains exhibited similar cell density. Cocultures were incubated at 37 °C in room air supplemented with 5% carbon dioxide for 24 h. To determine bacterial burden, tissues were washed three times with sterile PBS, placed in 1 mL of sterile THB, and homogenized before being subjected to serial dilution and plating onto blood agar plates. Plates were incubated at 37 °C in room air conditions overnight and were visually surveyed for bacterial colony growth and determination of colony forming units (CFU) the following day.

Analysis of Bacterial Biofilm Formation in Response to Selenium Challenge

Biofilm analysis was performed as previously described. Briefly, an overnight culture of GBS (GB00112 wild-type) was grown in THB overnight, and the following day was used to inoculate fresh brain-heart infusion broth alone or supplemented with increasing concentrations of sodium selenite (0, 5, 10, 50, 100, and 250 μM). Additionally, the WT, isogenic ΔcadD mutant, or complemented derivative (ΔcadD:C)) were grown in THB overnight and the following day were utilized to inoculate fresh brain-heart infusion broth alone or supplemented with 100 μM of sodium selenite. Cultures were incubated in static conditions at 37 °C for 24 h to establish mature biofilms. Supernatants were decanted, and biofilms were washed three times with sterile phosphate buffered saline solution to remove nonadherent cells. Biofilms were stained with a 1% crystal violet solution for 30 min, washed three times with distilled water, and allowed to dry for 60 min. Crystal violet stain was solubilized in an 80%/20% solution of ethanol and acetone and biofilm quantity was measured spectrophotometrically at an absorbance of 580 nm (A₅₈₀).

Statistical Analyses

For all experiments, at least 3 biological replicates were performed to ensure rigor and reproducibility. Data with two experimental groups were analyzed by Student’s t test. Data with more than two experimental groups were analyzed by two-way ANOVA with Šdák’s post hoc test. All statistical analyses were performed using Graph Pad Prism software.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomedchemau.5c00112.

• Table S1 listing of clinical GBS strains used in this study, properties, and minimum inhibitory concentration of selenium (PDF)

R.C. conceptualization, investigation, formal analysis, validation, writing – original draft, writing - review and editing. K.H. investigation, formal analysis, writing review and editing W.A. investigation, formal analysis, writing review and editing S.D.M. conceptualization, methodology, writing – review and editing J.A.G. conceptualization, funding acquisition, investigation, methodology, supervision, validation, writing – original draft, writing – review and editing. S.M.D conceptualization, funding acquisition, methodology, supervision, writing – original draft, writing – review and editing. CRediT: Riya Chinni formal analysis, investigation, validation, writing - original draft, writing - review & editing; Kensley Horner formal analysis, investigation, writing - review & editing; Walter Moises Avila formal analysis, investigation, writing - review & editing; Shannon D. Manning conceptualization, methodology, writing - review & editing; Jennifer A. Gaddy conceptualization, funding acquisition, investigation, methodology, supervision, validation, writing - original draft, writing - review & editing; Steven Damo conceptualization, funding acquisition, methodology, supervision, writing - original draft, writing - review & editing.

The authors declare no competing financial interest.

Published as part of ACS Bio & Med Chem Au special issue “Juneteenth 2025”.