Streptococcus agalactiae, more commonly known as Group B Streptococcus (GBS), is amongst the leading infection-related causes of adverse pregnancy and neonatal outcomes (Shabayek and Spellerberg, 2018). Adverse maternal complications include chorioamnionitis, preterm premature rupture of membranes (PPROM), preterm birth, stillbirth, and maternal sepsis (Goldenberg et al., 2008; Koumans et al., 2012). For the newborn, GBS infections can result in early- and late- onset neonatal sepsis, meningitis, and endocarditis. Early onset disease (EOD) occurs in neonates up to a week after birth (Verani et al., 2010). Neonates with EOD usually present with pneumonia and sepsis. In contrast, late onset disease (LOD) defines infection between 1-week and 3 months after birth and most commonly manifests as sepsis and meningitis. Newborns who survive LOD frequently suffer from neurodevelopmental impairments (Russell et al., 2017).

GBS is a gram-positive encapsulated bacterium, and a commensal member of the human microflora in the gastrointestinal tract. While GBS asymptomatically colonizes 20-30% of adults, the bacterium may traverse from the lower gastrointestinal tract to the vagina and infect the neonate through ascending infection or ingestion/inhalation of infectious fluids during childbirth (Verani et al., 2010). Indeed, the primary risk factor for EOD is rectovaginal colonization of pregnant women with GBS during delivery (Seale et al., 2017b). The ability of GBS to colonize and persist in the maternal urogenital tract to cause disease is related to its ability to form biofilms (Rosini and Margarit, 2015). Colonization rates differ worldwide, spanning between 6.5-36% (Shabayek and Spellerberg, 2018). Some recent reports include colonization rates of 13.2% in a cohort in Ethiopia (Ali et al., 2020), 16.6% in the Western Cape region of South Africa (Africa and Kaambo, 2018), and 21.6% over a twelve-year span in North Carolina, USA (Edwards et al., 2019).

GBS strains can be divided into 10 distinct serotypes (Ia, Ib, and II to IX) based on a serological reaction directed against the polysaccharide capsule (Teatero et al., 2017). The streptococcal polysaccharide capsule facilitates evasion of the innate immune response by protecting the bacterial cell from deposition of complement, opsonization, and phagocytosis (Winkelstein et al., 1980; Brown et al., 1983; Abeyta et al., 2003). A recent study from our laboratory revealed that the GBS capsule aids in biofilm formation and ascending infection of the reproductive tract during pregnancy (Noble et al., 2021). Moreover, the capsule across all serotypes shares terminal sialic acid (Sia) residues that allow molecular mimicry of human cell surface sialic acids. This allows interaction with Sia-receptors, Siglecs, on innate immune cells that serve to dampen inflammatory responses (Carlin et al., 2007). Different capsular serotypes result in different range and severity in human disease. For instance, capsular serotype III strains are associated with higher rates of invasive neonatal disease (Alhhazmi et al., 2017) and account for the majority of late-onset meningitis cases in neonates (Bellais et al., 2012). In contrast, serotype Ia and V predominate among invasive isolates in non-pregnant cases (Phares et al., 2008). However, dominate serotypes fluctuate between regions and across time (Shabayek and Spellerberg, 2018).

Another method by which GBS strains are grouped and characterized is multi-locus sequence typing (MLST), which classifies strains into sequence types (STs) based on allelic variation within seven conserved housekeeping genes (Jones et al., 2003). Based on phylogenetic analysis, GBS STs can be grouped into multiple clonal complexes (CCs) with most human isolates belonging to CC1, CC10, CC17, CC19, CC23, and CC26 (Sørensen et al., 2010). Similar to capsular type, ST diversity also manifests in different disease outcomes and severities. For instance, a study with GBS from multiple continents revealed that STs 1 and 19 have been linked to asymptomatic colonization, while ST-17 predominately related to invasive neonatal disease. ST-23 was associated with both carriage and invasive GBS disease (Jones et al., 2003). All four STs, however, were found to colonize pregnant women at higher rates in different patient populations (Manning et al., 2009). ST-17 serotype III, alongside ST-291, belong to CC17, a group of GBS noted for its hypervirulence (Bellais et al., 2012). ST-17 is strongly linked to both EOD and LOD, as well as meningitis (Musser et al., 1989; Lin et al., 2006; Manning et al., 2008). Meanwhile, ST-291 belonging to serotype IV is strongly associated with EOD and septicemia (Tien et al., 2011; Wang et al., 2015).

In order to combat GBS, the immune system deploys a repertoire of antimicrobial peptides. These peptides aid in combatting infection through the process of nutritional immunity, or the sequestration of essential metals to starve bacteria (Hood and Skaar, 2012). Bacteria require these trace elements as cofactors for essential biological processes. One important example of a protein expressed in defense against GBS is lactoferrin (Kothary et al., 2017; Lu et al., 2020). Lactoferrin is a glycoprotein that contains two iron binding sites (Weinberg, 1975) and was shown to have antimicrobial activity against a wide range of bacterial, viral, and fungal pathogens (Lu et al., 2020). Indeed, our previous study demonstrated that human breast milk lactoferrin has antimicrobial and anti-biofilm activity against GBS and inhibits some GBS strains from adhering to human gestational membranes (Lu et al., 2021). In this study, we advanced our findings by analyzing the antimicrobial and anti-biofilm effects of lactoferrin against a larger panel of clinical GBS strains that vary by capsular serotype, ST, isolation source, and clinical presentation. We observed broad antimicrobial and antibiofilm action by lactoferrin against most GBS strains, though the maternal colonizing strains were more susceptible to inhibitory effects than the neonatal invasive strains.

In this present study, we expanded upon our previous work by increasing the panel of GBS strains to include phenotypically and genetically diverse clinical strains from diverse anatomical sites of isolation and assessing susceptibility to the antimicrobial and anti-biofilm activity of human milk lactoferrin. We discovered that lactoferrin possesses antimicrobial and antibiofilm properties against many diverse GBS strains. In particular, colonizing maternal strains were more susceptible to lactoferrin, compared to invasive neonatal strains.

Other studies have revealed that lactoferrin may contribute to improvement of reproductive tract infections and subsequent disease. For instance, vaginal lactoferrin supplementation in pregnant people with bacterial vaginosis reduced the rate of preterm birth (Miranda et al., 2019). Furthermore, other groups have identified lactoferrin as a critical component of cervicomucosal defense against a variety of lower genital tract infections caused by Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis (Pino et al., 2017). One plausible explanation for this phenomenon may be that lactoferrin protects by repressing Gardnerella, Prevotella, and Lachnospira species within the host microbiome, thus decreasing competition for Lactobacillus species and allowing them to prevent dysbiosis by dominating the vaginal microbiota (Valenti et al., 2018). Adding credence to this possibility is that Lactobacillus species have been recognized as one of the few bacterial populations that lack a strict nutrient requirement for iron (Imbert and Blondeau, 1998). Thus, it is plausible that the antimicrobial activity of lactoferrin associated with its role in nutritional immunity would be largely ineffective against these important commensals. Indeed, our previous work has shown various Lactobacillus strains and their secreted products modulate GBS interactions with cells of the extraplacental membranes. Specifically, Lactobacillus supernatants inhibited GBS growth, biofilm formation and invasion of host cells, though strain-dependent effects were observed. Notably, supernatant from L. reuteri 6475 broadly inhibited growth in 36 distinct GBS strains and inhibited GBS growth to an average of 46.6% of each GBS strain alone (Shiroda et al., 2020). Thus, there is merit in studying the use of human lactoferrin alone or in combination with Lactobacillus spp. in the prevention of GBS-mediated preterm births and adverse pregnancy outcomes as maternal colonizing GBS strains can infect the fetus by ascending the gravid reproductive tract. Here, we have shown that maternal colonizing GBS strains are greatly susceptible to the antimicrobial and antibiofilm action of lactoferrin.

Our results indicate that certain STs are more susceptible than others at 750 µg/mL of lactoferrin treatment. The MLST scheme uses seven loci that encode enzymes involved in intermediary metabolism to distinguish GBS STs (Jones et al., 2003). Because iron is an important cofactor for many enzymes involved in bacterial metabolism and physiology (Andrews et al., 2003), lactoferrin can help defend against invading bacteria by starving the prokaryotic cells of nutritional iron needed for optimal enzyme activity. However, some enzymes are promiscuous with their utilization of transition metal co-factors. As a result, it is plausible that we witnessed variable antimicrobial effects of lactoferrin because some of these housekeeping enzymes, or another enzyme up- or downstream of its respective pathways, require iron for full function while others may use other transitional metals under conditions of iron starvation (Foster et al., 2014). An alternate explanation is that other enzymes in some pathways with similar functions may be able to compensate for the absence of or limited iron-cofactors. Perhaps the number of loci used in the MSTL were too limited. It is plausible that the effect of lactoferrin is indirect and does not involve the specific genes loci used in the MSTL. Further examination at the whole genome phylogeny may better differentiate lactoferrin effects across close or distantly related genomes regardless of cps type or isolation source.

The capsule of GBS plays an important role in pathogenesis in humans (Vornhagen et al., 2017). As different capsular types have been correlated to varying disease outcomes and susceptibility to antimicrobial agents (Teatero et al., 2017), we hypothesized that different capsular serotypes might have variable responses to the antimicrobial activity of lactoferrin. We observed differences in biofilm formation but not bacterial growth between different GBS capsular types under a lower treatment of lactoferrin. Given that the capsule is an important virulence factor for pathogenesis and evasion of immune assault (Gendrin et al., 2018), it was not surprising that no differences were observed in our controlled in vitro studies free of immune stressors. However, we did observe differences in GBS biofilm formation. Our group previously described the role of capsule in biofilm formation in GBS (Noble et al., 2021), further bolstering our findings. It is plausible that iron starvation alters capsule-mediated biofilm formation in certain capsular types of GBS. Further in vitro work with host cells or in vivo experiments are needed to bridge our gap in knowledge of these relationships between capsular serotype and lactoferrin.

In our study, we further analyzed differences in biofilm inhibition by lactoferrin between strains that formed robust biofilms compared to those that formed low or weak biofilms. Lactoferrin inhibited biofilm formation in high biofilm forming isolates, which is consistent to with our previous study in a smaller cohort of strains (Lu et al., 2021). The intersection between iron and biofilm formation has also been studied in other bacterial pathogens. One study of interest by Trappetti and colleagues described the role of the mononuclear iron protein S-ribosylhomocysteine lyase (LuxS) in quorum sensing and biofilm formation in Streptococcus pneumoniae (Trappetti et al., 2011). Consistent with their work, we also observed that iron starvation results in inhibition of biofilm formation. Thus, it is plausible that GBS possesses similar iron-sensing pathways that govern biofilm formation. More work, however, is needed to elucidate and confirm the function of these pathways.

Colonization of the maternal genitourinary tract is the most important risk factor for neonatal GBS disease (Benitz et al., 1999). In a longitudinal study performed by Kwatra and colleagues, up to 50% of the study cohort was transiently colonized by GBS at some point during pregnancy, highlighting the dynamic nature of GBS colonization (Kwatra et al., 2014). Currently in the United States, pregnant individuals are screened for the presence of GBS between 35 and 37 weeks of gestation (Carrillo-Ávila et al., 2018). If a patient tests positive for GBS, then intrapartum antibiotic prophylaxis (IAP) is administered during labor and delivery to prevent neonatal EOD (Seale et al., 2017a). Though antibiotic treatment is the only current preventative strategy available, the efficacy of IAP against EOD is around 80% (Fairlie et al., 2013). Despite the availability of IAP, however, the rates of LOD have remained unchanged (Nanduri et al., 2019). Additional drawbacks associated with the use of IAP (Rao and Khanna, 2020) include hypersensitivities to first-line antibiotics (Matteson et al., 2008), alteration of the neonatal microbiota (Nogacka et al., 2017), and emergence of antibiotic resistant strains (Hayes et al., 2020). Hence, the discovery of alternative therapies is critical and may help overcome these IAP drawbacks. Breastfeeding has been associated with protection against infection and could be exploited as a risk-mitigation strategy. However, cases of GBS transfer by breast milk have been recorded (Ager et al., 2020). The protective effects of breast milk are likely derived from the antimicrobial and immunomodulatory molecules that comprise milk, including lactoferrin and human milk oligosaccharides (Andreas et al., 2015). Thus, utilization of these specific molecules could be leveraged to mitigate the risk of GBS transmission. In the study herein, we found that maternal colonizing GBS strains are most susceptible to lactoferrin, suggesting that the antimicrobial peptide may be a viable candidate to aid in the prevention of GBS disease.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

Ethics approval to carry out this study was provided by the Vanderbilt University Institutional Review Board (IRB #100897 for human milk donation).

KC, JL, and ST purified lactoferrin for the studies. JL performed bacterial culture experiments. SDM curated and validated the clinical strains for use in this study. JL, JF, MG, SC, RM, SS, KC, ST, and JG conceptualized and analyzed results and interpreted data, and wrote and edited the manuscript for critical content. All authors contributed to the article and approved the submitted version.

This work was funded by the National Institutes of Health grants HD090061 (to JG) and the Department of Veterans Affairs Office of Research BX005352 (to JG). which supported wet bench experiments, 2T32HL007411-39 (to JL), 2T32AI112541-06 (to JF), which supported their time and efforts, National Science Foundation 1847804 which supported reagents and wet bench work. Additional support was provided by the Vanderbilt Institute for Clinical and Translational Research program supported by the National Center for Research Resources, Grant UL1 RR024975-01, and the National Center for Advancing Translational Sciences, Grant 2 UL1 TR000445-06 which supports access to Core Facilities and Biostatistics support.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.