Jefferson Lab Research

Bacteria have the ability to switch from a single-celled, free-floating existence to a community-based, surface-associated mode of growth known as a biofilm.  Bacterial biofilm formation is a key determinant in the outcome of certain infections because bacteria that exist in this state are considerably more resistant to our immune defenses and to antimicrobial chemotherapy than their genotypically equivalent free-floating counterparts. In fact, biofilms can tolerate antibiotic concentrations 10-10,000-fold higher than those required to inhibit growth of free-floating bacteria. Bacterial biofilms that cause infections can be either composed of a single species or multiple species. My lab studies two clinically important types of biofilms: monospecies biofilms formed by Staphylococcus aureus, and multi-species biofilms that cause bacterial vaginosis.  

Project #1 Staphylococcus aureus biofilm formation. Patient hospitalization often involves the use of inserted medical devices, which can lead to the iatrogenic development of biofilm-related infections. Catheters and other medical devices breach the skin or mucosal barrier and serve as an ideal surface for bacterial adherence. The staphylococci are implicated in nosocomial infections, such as bloodstream and ventilator-associated infections, more frequently than any other bacterial species. When S. aureus assumes the biofilm phenotype, these infections are often extremely difficult to treat. The infection may fail to respond to antibiotic therapy or it may initially respond only to relapse weeks or months later. In such cases, invasive treatments, such as surgical removal and replacement of the infected tissue or device, may be required. 

TSBGThe urgent need to develop a more effective means to treat such infections calls for a thorough understanding of gene expression as it relates to the biofilm mode of growth, and is the inspiration behind our research. One of our projects investigates the effects of chelating agents, which were regarded as anti-biofilm agents, on biofilm formation in S. aureus. We found that while chelating agents are capable of preventing biofilm formation is many strains of S. aureus, they actually cause some strains to produce even thicker biofilms. This is because the agents induce a switch in the biofilm composition such that the bacteria adhere to one another through protein-protein interactions rather than depending upon exopolysaccharide.  More specifically, the chelating agents induce the expression of a surface protein called clumping factor B or ClfB.    

The second S. aureus project aims at unraveling the relationship between environmental cues and the molecular switches that turn on production of the biofilm polysaccharide matrix. Biofilm formation in vitro can be induced by a number of environmental factors that mimic conditions likely to be encountered in vivo, including glucose availability, iron deprivation, and high osmolarity. In vivo and under in vitro conditions that promote biofilm elaboration, S. aureus produces a cell surface-associated b-1-6-linked polymer of N-acetyl-glucosamine (PNAG) also referred to as polysaccharide intercellular adhesin (PIA). PNAG/PIA promotes adhesion to surfaces and to other bacterial cells, and is the major component of the biofilm matrix (Fig 1). It is synthesized by four proteins encoded within the intercellular adhesin (ica) locus, a glucosyltransferase (IcaA) and its chaperone (IcaD), a de-acetylase (IcaB) and the IcaC protein, which is thought to be involved in secretion of the polymer.  Not only is PNAG/PIA critical for biofilm formation, but it also plays an important role in virulence. Presence of the ica locus is important for certain types of human infections and animal infection models (2). PNAG/PIA inhibits phagocytosis in the absence of antibody whereas anti-PNAG antibodies mediate opsonophagocytosis and have demonstrated promise as a vaccine candidate (2). Very recent evidence indicates that a number of unrelated bacterial pathogens, including E. coli, Yersinia pestis, Y. pseutuberculosis, and Bordetella pertussis, harbor ica homologues, and we have found that these species produce an exo-polysaccharide that is antigenically indistinguishable from S. aureus PNAG. In summary, PNAG/PIA has an important and multi-faceted role in biofilm formation and virulence and we aim to elucidate its regulation in order to form a basis for anti-biofilm therapy.

Confocal Biofilm

Fig. 1. Use of confocal microscopy to analyze PNAG/PIA. The intercellular adhesin (ica) locus in S. aureus encodes the proteins necessary for PNAG/PIA synthesis so ica-positive and ica-negative mutants were used as positive and negative controls.  A) the strong PNAG-producing S. aureus strain MN8m produces a thick, mature biofilm with microcolonies separated by channels for nutrient and oxygen flow B) the PNAG-negative S. aureus strain MN8Dica::tet forms a homogenous mat of bacteria that is easily removed by washing. The bacteria were stained with WGA-Green, which binds to PNAG/PIA and imaged by confocal microscopy. The bacterial cells are presented in red. Each bar segment represents 20 mm.

Project #2 Preterm birth and bacterial vaginosis (BV).
AmnioticSacPreterm birth accounts for as much as 70% of neonatal mortality, 75% of neonatal morbidity, and nearly 50% of long-term neurologic sequelae. African American women are two times more likely to experience preterm birth (less than 37 weeks) and more than 3 times as likely to experience very preterm birth (less than 32 weeks) relative to Caucasian women. This racial disparity is mirrored in rates of bacterial vaginosis (BV), which affects 10-20% of Caucasian and 30-50% of African American women and doubles the relative risk for preterm birth in the overall population and more than triples the risk in African American women. The etiology of BV is complex and poorly understood because it is a polymicrobial disorder, involving tens or even hundreds of species of anaerobic bacteria, none of which is a clear pathogen. Sub-clinical intrauterine infections correlate strongly with preterm birth, especially very preterm birth, and chorioamniotic tissues from more than 75% of women presenting in spontaneous labor at <30 weeks are infected with bacteria. Vaginal bacteria are a source for these intrauterine infections, especially in women with BV, and the high prevalence of BV makes this an important consideration. BV is the most common vaginal disorder worldwide, and is more than twice as prevalent in African American versus Caucasian women. The Population Attributable Risk suggests that BV likely accounts for as much as 30% of the racial gap in preterm birth.

ChartThe BV-associated bacterial species most strongly associated with sub-clinical intrauterine invasion and preterm birth are Mycoplasma hominis, Ureaplasma urealyticum, Fusobacterium spp., Sneathia spp., and Gardnerella vaginalis.Lactobacillus crispatus correlates strongly with health and inversely with BV whereas women who are colonized with L. iners tend to get BV more frequently. We hypothesized that the increased relative risk for preterm birth in African American is due, in part, to decreased colonization by healthy vaginal species and increased vaginal colonization by utero-invasive species. We also hypothesized that Causasian women were more likely to get BV because they were more likely to be colonized by L. crispatus. To test this hypothesis, we analyzed the vaginal microbiomes of healthy women who visited the clinic for an annual exam and found that African American women were significantly more likely to be colonized with L. iners, Sneathia and G. vaginalis. These data support our hypothesis that the increased relative risk for preterm birth in African American women is partially attributable to a racial disparity in colonization by utero-invasive bacteria. Fusobacterium, Ureaplasma, and Mycoplasma colonized both populations equally; however, current techniques for the analysis of microbial populations, such as the Ribosomal Database Project (RDP), only resolve to the genus level and do not characterize the number of bacteria present. Therefore iti is possible that African American women are more likely to be colonized by more pathogenic species or strains of these organisms.
We hypothesize that colonization by utero-invasive strains or sub-species is greater in African American women and that colonization with these bacteria leads to preterm birth. The goals of this project are to identify bacterial virulence determinants that play a role in intrauterine invasion, or the “virulome of infectious preterm birth”, and to determine whether colonization by bacteria equipped with this virulome is associated with the elevated risk for preterm birth in African American women.

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VCU Department of Microbiology and Immunology Virginia Commonwealth University VCU Medical Center
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