Streptococcus agalactiae cadD Is Critical for Pathogenesis in the Invertebrate Galleria mellonella Model.

Group B Streptococcus (GBS) is a gram-positive bacterium that can cause invasive infections in immunocompromised, elderly, pregnant, or neonatal patients. The invertebrate model, Galleria mellonella, has emerged as an effective tool to study GBS-host interactions; specifically, those conserved within the innate arm of the immune system. We sought to determine the role of metal homeostasis functions in GBS infections of G. mellonella larvae and to validate this model as a tool to study GBS-host interactions. Our results indicate that wild-type GBS infects G. mellonella in a dose-dependent manner, replicates in the invertebrate host, induces larval melanization and larval killing. These results were significantly abrogated in cohorts of larvae infected with the isogenic cadD deletion mutant. Additionally, complementation restored GBS-dependent infection, bacterial burden, larval melanization, and killing to wild-type levels. Together, these results indicate that the G. mellonella model is a useful tool for studying GBS pathogenesis.

Streptococcus agalactiae npx Is Required for Survival in Human Placental Macrophages and Full Virulence in a Model of Ascending Vaginal Infection during Pregnancy.

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a Gram-positive encapsulated bacterium that colonizes the gastrointestinal tract of 30 to 50% of humans. GBS causes invasive infection during pregnancy that can lead to chorioamnionitis, funisitis, preterm prelabor rupture of membranes (PPROM), preterm birth, neonatal sepsis, and maternal and fetal demise. Upon infecting the host, GBS encounters sentinel innate immune cells, such as macrophages, within reproductive tissues. Once phagocytosed by macrophages, GBS upregulates the expression of the gene npx, which encodes an NADH peroxidase. GBS mutants with an npx deletion (Δnpx) are exquisitely sensitive to reactive oxygen stress. Furthermore, we have shown that npx is required for GBS survival in both THP-1 and placental macrophages. In an in vivo murine model of ascending GBS vaginal infection during pregnancy, npx is required for invading reproductive tissues and is critical for inducing disease progression, including PPROM and preterm birth. Reproductive tissue cytokine production was also significantly diminished in Δnpx mutant-infected animals compared to that in animals infected with wild-type (WT) GBS. Complementation in trans reversed this phenotype, indicating that npx is critical for GBS survival and the initiation of proinflammatory signaling in the gravid host. IMPORTANCE This study sheds new light on the way that group B Streptococcus (GBS) defends itself against oxidative stress in the infected host. The enzyme encoded by the GBS gene npx is an NADH peroxidase that, our study reveals, provides defense against macrophage-derived reactive oxygen stress and facilitates infections of the uterus during pregnancy. This enzyme could represent a tractable target for future treatment strategies against invasive GBS infections.

Streptococcus agalactiae cadD alleviates metal stress and promotes intracellular survival in macrophages and ascending infection during pregnancy.

Perinatal infection with Streptococcus agalactiae, or Group B Streptococcus (GBS), is associated with preterm birth, neonatal sepsis, and stillbirth. Here, we study the interactions of GBS with macrophages, essential sentinel immune cells that defend the gravid reproductive tract. Transcriptional analyses of GBS-macrophage co-cultures reveal enhanced expression of a gene encoding a putative metal resistance determinant, cadD. Deletion of cadD reduces GBS survival in macrophages, metal efflux, and resistance to metal toxicity. In a mouse model of ascending infection during pregnancy, the ΔcadD strain displays attenuated bacterial burden, inflammation, and cytokine production in gestational tissues. Furthermore, depletion of host macrophages alters cytokine expression and decreases GBS invasion in a cadD-dependent fashion. Our results indicate that GBS cadD plays an important role in metal detoxification, which promotes immune evasion and bacterial proliferation in the pregnant host.

The antimicrobial activity of zinc against group B Streptococcus is strain-dependent across diverse sequence types, capsular serotypes, and invasive versus colonizing isolates.

BACKGROUND: Streptococcus agalactiae or Group B Streptococcus (GBS) is an encapsulated gram-positive bacterial pathobiont that commonly colonizes the lower gastrointestinal tract and reproductive tract of human hosts. This bacterium can infect the gravid reproductive tract and cause invasive infections of pregnant patients and neonates. Upon colonizing the reproductive tract, the bacterial cell is presented with numerous nutritional challenges imposed by the host. One strategy employed by the host innate immune system is intoxication of bacterial invaders with certain transition metals such as zinc. METHODOLOGY: Previous work has demonstrated that GBS must employ elegant strategies to circumnavigate zinc stress in order to survive in the vertebrate host. We assessed 30 strains of GBS from diverse isolation sources, capsular serotypes, and sequence types for susceptibility or resistance to zinc intoxication. RESULTS: Invasive strains, such as those isolated from early onset disease manifestations of GBS infection were significantly less susceptible to zinc toxicity than colonizing strains isolated from rectovaginal swabs of pregnant patients. Additionally, capsular type III (cpsIII) strains and the ST-17 and ST-19 strains exhibited the greatest resilience to zinc stress, whereas ST-1 and ST-12 strains as well as those possessing capsular type Ib (cpsIb) were more sensitive to zinc intoxication. Thus, this study demonstrates that the transition metal zinc possesses antimicrobial properties against a wide range of GBS strains, with isolation source, capsular serotype, and sequence type contributing to susceptibility or resistance to zinc stress.

Antibiofilm Activity of Human Milk Oligosaccharides against Multidrug Resistant and Susceptible Isolates of Acinetobacter baumannii.

Acinetobacter baumannii is a serious threat to human health, per the Centers for Disease Control and Prevention's latest threat assessment. A. baumannii is a Gram-negative opportunistic bacterial pathogen that causes severe community and nosocomial infections in immunocompromised patients. Treatment of these infections is confounded by the emergence of multi- and pan-drug resistant strains of A. baumannii. A. baumannii colonizes abiotic and biotic surfaces and evades antimicrobial challenges by forming biofilms, which are three-dimensional architectural structures of cells adhered to a substrate and encased in an extracellular matrix comprised of polymeric substances such as polysaccharides, proteins, and DNA. Biofilm-inhibiting compounds have recently gained attention as a chemotherapeutic strategy to prevent or disperse A. baumannii biofilms and restore the utility of traditional antimicrobial strategies. Recent work indicates that human milk oligosaccharides (HMOs) have potent antibacterial and biofilm-inhibiting properties. We sought to test the utility of HMOs against a bank of clinical isolates of A. baumannii to ascertain changes in bacterial growth or biofilm formation. Our results indicate that out of 18 strains tested, 14 were susceptible to the antibiofilm activities of HMOs, and that the potent antibiofilm activity was observed in strains isolated from diverse anatomical sites, disease manifestations, and across antibiotic-resistant and susceptible strains.

Analysis of Susceptibility to the Antimicrobial and Anti-Biofilm Activity of Human Milk Lactoferrin in Clinical Strains of Streptococcus agalactiae With Diverse Capsular and Sequence Types.

Group B Streptococcus (GBS) is one of the leading infection-related causes of adverse maternal and neonatal outcomes. This includes chorioamnionitis, which leads to preterm ruptures of membranes and can ultimately result in preterm or stillbirth. Infection can also lead to maternal and neonatal sepsis that may contribute to mortality. Currently, treatment for GBS infection include a bolus of intrapartum antibiotic prophylaxis to mothers testing positive for GBS colonization during late pregnancy. Lactoferrin is an antimicrobial peptide expressed in human breast milk, mucosal epithelia, and secondary granules of neutrophils. We previously demonstrated that lactoferrin possesses antimicrobial and antibiofilm properties against several strains of GBS. This is largely due to the ability of lactoferrin to bind and sequester iron. We expanded upon that study by assessing the effects of purified human breast milk lactoferrin against a panel of phenotypically and genetically diverse isolates of GBS. Of the 25 GBS isolates screened, lactoferrin reduced bacterial growth in 14 and biofilm formation in 21 strains. Stratifying the data, we observed that colonizing strains were more susceptible to the growth inhibition activity of lactoferrin than invasive isolates at lactoferrin concentrations between 250-750 µg/mL. Treatment with 750 µg/mL of lactoferrin resulted in differences in bacterial growth and biofilm formation between discrete sequence types. Differences in bacterial growth were also observed between capsular serotypes 1a and III. Maternally isolated strains were more susceptible to lactoferrin with respect to bacterial growth, but not biofilm formation, compared to neonatal sepsis isolates. Finally, high biofilm forming GBS strains were more impacted by lactoferrin across all isolates tested. Taken together, this study demonstrates that lactoferrin possesses antimicrobial and antibiofilm properties against a wide range of GBS isolates, with maternally isolated colonizing strains being the most susceptible.

The Innate Immune Glycoprotein Lactoferrin Represses the Helicobacter pylori cag Type IV Secretion System.

Chronic infection with Helicobacter pylori increases risk of gastric diseases including gastric cancer. Despite development of a robust immune response, H. pylori persists in the gastric niche. Progression of gastric inflammation to serious disease outcomes is associated with infection with H. pylori strains which encode the cag Type IV Secretion System (cag T4SS). The cag T4SS is responsible for translocating the oncogenic protein CagA into host cells and inducing pro-inflammatory and carcinogenic signaling cascades. Our previous work demonstrated that nutrient iron modulates the activity of the T4SS and biogenesis of T4SS pili. In response to H. pylori infection, the host produces a variety of antimicrobial molecules, including the iron-binding glycoprotein, lactoferrin. Our work shows that apo-lactoferrin exerts antimicrobial activity against H. pylori under iron-limited conditions, while holo-lactoferrin enhances bacterial growth. Culturing H. pylori in the presence of holo-lactoferrin prior to co-culture with gastric epithelial cells, results in repression of the cag T4SS activity. Concomitantly, a decrease in biogenesis of cag T4SS pili at the host-pathogen interface was observed under these culture conditions by high-resolution electron microscopy analyses. Taken together, these results indicate that acquisition of alternate sources of nutrient iron plays a role in regulating the pro-inflammatory activity of a bacterial secretion system and present novel therapeutic targets for the treatment of H. pylori-related disease.

Group B Streptococcus cpsE Is Required for Serotype V Capsule Production and Aids in Biofilm Formation and Ascending Infection of the Reproductive Tract during Pregnancy.

Group B Streptococcus (GBS) is an encapsulated Gram-positive pathogen that causes ascending infections of the reproductive tract during pregnancy. The capsule of this organism is a critical virulence factor that has been implicated in a variety of cellular processes to promote pathogenesis. Primarily comprised of carbohydrates, the GBS capsule and its synthesis is driven by the capsule polysaccharide synthesis (cps) operon. The cpsE gene within this operon encodes a putative glycosyltransferase that is responsible for the transfer of a Glc-1-P from UDP-Glc to an undecaprenyl lipid molecule. We hypothesized that the cpsE gene product is important for GBS virulence and ascending infection during pregnancy. Our work demonstrates that a GBS cpsE mutant secretes fewer carbohydrates, has a reduced capsule, and forms less biofilm than the wild-type parental strain. We show that, compared to the parental strain, the ΔcpsE deletion mutant is more readily taken up by human placental macrophages and has a significantly attenuated ability to invade and proliferate in the mouse reproductive tract. Taken together, these results demonstrate that the cpsE gene product is an important virulence factor that aids in GBS colonization and invasion of the gravid reproductive tract.

Analysis of Antimicrobial and Antibiofilm Activity of Human Milk Lactoferrin Compared to Bovine Lactoferrin against Multidrug Resistant and Susceptible Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumannii is an opportunistic bacterial pathogen that causes severe infections in immunocompromised patients. The emergence of multi- and pan-drug resistant strains of A. baumannii from clinical sources has confounded treatment and enhanced morbidity and mortality associated with these infections. One way that A. baumannii circumnavigates environmental and antimicrobial challenge is by forming tertiary architectural structures of cells known as biofilms. Biofilm-inhibiting molecules could be deployed as a potential chemotherapeutic strategy to inhibit or disrupt A. baumannii biofilms and mitigate adverse outcomes due to infection. Lactoferrin is an innate immune glycoprotein produced in high concentrations in both human and bovine milk which has previously been shown to have antibacterial and antibiofilm activities. We sought to test lactoferrin against a bank of clinical isolates of A. baumannii to determine changes in bacterial growth or biofilm formation. Our results indicate that human lactoferrin has slightly more potent antibacterial activities than bovine lactoferrin against certain strains of A. baumannii and that these effects are associated with anatomical site of isolation. Additionally, we have shown that both bovine and human lactoferrin can inhibit A. baumannii biofilm formation and that these effects are associated with anatomical site of isolation and whether the strain forms robust or weak biofilms.

Antibacterial and Anti-biofilm Activity of the Human Breast Milk Glycoprotein Lactoferrin against Group B Streptococcus.

Group B Streptococcus (GBS) is an encapsulated Gram-positive human pathogen that causes invasive infections in pregnant hosts and neonates, as well as immunocompromised individuals. Colonization of the human host requires the ability to adhere to mucosal surfaces and circumnavigate the nutritional challenges and antimicrobial defenses associated with the innate immune response. Biofilm formation is a critical process to facilitate GBS survival and establishment of a replicative niche in the vertebrate host. Previous work has shown that the host responds to GBS infection by producing the innate antimicrobial glycoprotein lactoferrin, which has been implicated in repressing bacterial growth and biofilm formation. Additionally, lactoferrin is highly abundant in human breast milk and could serve a protective role against invasive microbial pathogens. This study demonstrates that human breast milk lactoferrin has antimicrobial and anti-biofilm activity against GBS and inhibits its adherence to human gestational membranes. Together, these results indicate that human milk lactoferrin could be used as a prebiotic chemotherapeutic strategy to limit the impact of bacterial adherence and biofilm formation on GBS-associated disease outcomes.

Analysis of virulence phenotypes and antibiotic resistance in clinical strains of Acinetobacter baumannii isolated in Nashville, Tennessee.

BACKGROUND: Acinetobacter baumannii is a gram-negative bacterium which causes opportunistic infections in immunocompromised hosts. Genome plasticity has given rise to a wide range of strain variation with respect to antimicrobial resistance profiles and expression of virulence factors which lead to altered phenotypes associated with pathogenesis. The purpose of this study was to analyze clinical strains of A. baumannii for phenotypic variation that might correlate with virulence phenotypes, antimicrobial resistance patterns, or strain isolation source. We hypothesized that individual strain virulence phenotypes might be associated with anatomical site of isolation or alterations in susceptibility to antimicrobial interventions. METHODOLOGY: A cohort of 17 clinical isolates of A. baumannii isolated from diverse anatomical sites were evaluated to ascertain phenotypic patterns including biofilm formation, hemolysis, motility, and antimicrobial resistance. Antibiotic susceptibility/resistance to ampicillin-sulbactam, amikacin, ceftriaxone, ceftazidime, cefotaxime, ciprofloxacin, cefepime, gentamicin, levofloxacin, meropenem, piperacillin, trimethoprim-sulfamethoxazole, ticarcillin- K clavulanate, tetracyclin, and tobramycin was determined. RESULTS: Antibiotic resistance was prevalent in many strains including resistance to ampicillin-sulbactam, amikacin, ceftriaxone, ceftazidime, cefotaxime, ciprofloxacin, cefepime, gentamicin, levofloxacin, meropenem, piperacillin, trimethoprim-sulfamethoxazole, ticarcillin- K clavulanate, tetracyclin, and tobramycin. All strains tested induced hemolysis on agar plate detection assays. Wound-isolated strains of A. baumannii exhibited higher motility than strains isolated from blood, urine or Foley catheter, or sputum/bronchial wash. A. baumannii strains isolated from patient blood samples formed significantly more biofilm than isolates from wounds, sputum or bronchial wash samples. An inverse relationship between motility and biofilm formation was observed in the cohort of 17 clinical isolates of A. baumannii tested in this study. Motility was also inversely correlated with induction of hemolysis. An inverse correlation was observed between hemolysis and resistance to ticarcillin-k clavulanate, meropenem, and piperacillin. An inverse correlation was also observed between motility and resistance to ampicillin-sulbactam, ceftriaxone, ceftoxamine, ceftazidime, ciprofloxacin, or levofloxacin. CONCLUSIONS: Strain dependent variations in biofilm and motility are associated with anatomical site of isolation. Biofilm and hemolysis production both have an inverse association with motility in the cohort of strains utilized in this study, and motility and hemolysis were inversely correlated with resistance to numerous antibiotics.

Vitamin D and Streptococci: The Interface of Nutrition, Host Immune Response, and Antimicrobial Activity in Response to Infection.

Streptococcus species are common causes of human infection. These Gram-positive, encapsulated bacterial pathogens infect diverse anatomic spaces, leading to infections including skin and soft tissue infection, endocarditis, pneumonia, meningitis, sinusitis, otitis media, chorioamnionitis, sepsis, and even death. Risk for streptococcal infection is highest in low- and middle-income countries where micronutrient deficiency is common. Epidemiological data reveal that vitamin D deficiency is associated with enhanced risk of streptococcal infection and cognate disease outcomes. Additionally, vitamin D improves antibacterial defenses by stimulating innate immune processes such as phagocytosis and enhancing production of reactive oxygen species (oxidative burst) and antimicrobial peptides (including cathelicidin and lactoferrin), which are important for efficient killing of bacteria. This review presents the most recent published work that studies interactions between the micronutrient vitamin D, the host immune system, and pathogenic streptococci as well as comparisons with other relevant infection models.