Acidic pH Strongly Enhances In Vitro Biofilm Formation by a Subset of Hypervirulent ST-17 Streptococcus agalactiae Strains

Abstract

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30 to 76% of the cases of neonatal meningitis. In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low-, and non-biofilm-forming strains, and to facilitate interpretation of data. This protocol was used to screen the biofilm-forming abilities of 366 GBS clinical isolates from pregnant women and from neonatal infections of different serotypes in relation to medium composition and pH. The results identified a subset of isolates of serotypes III and V that formed strong biofilms under acidic conditions. Importantly, the best biofilm formers belonged to serotype III hypervirulent clone ST-17. Moreover, the abilities of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm initiation and contribute to biofilm structural stability. Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a leading cause of invasive neonatal infections worldwide. It is a common colonizer of the gastrointestinal and urogenital tracts of up to 40% of healthy individuals (1). However, under certain circumstances, GBS can become a life-threatening pathogen causing invasive infections in human neonates (2, 3). Earlyonset group B streptococcal disease occurs in infants less than 7 days old, and late-onset disease (LOD) occurs in infants between 7 and 89 days old. GBS is usually transmitted from mothers to newborns during childbirth (4), but it can also penetrate the human placenta (5), and in the case of LOD, it can be nosocomially acquired.Historically, GBS isolates have been classified into 10 different serotypes according to their capsular polysaccharide composition (6, 7). Multiple surveillance studies have indicated that all serotypes are able to colonize the vagina and perianal region of pregnant women, but five serotypes (Ia, Ib, II, III, and V) are predominant and are also the most frequent in human infections (8-12). In particular, serotype III accounts for 30 to 76% of neonatal disease cases (13,14). The use of multilocus sequence typing (MLST) allowed the classification of GBS isolates independently from their capsular serotypes and the identification of the bacterial genogroups more often associated with invasive infections in newborns (15). Serotype III isolates of a particular genotype cluster, sequence type 17 (ST-17), disproportionately cause late-onset GBS disease (15-19) and more frequently cause meningitis than other STs do (20). The precise mechanism by which ST-17 causes LOD more frequently than other STs do is not well unde...

Maternal colonization with Streptococcus agalactiae and associated stillbirth and neonatal disease in coastal Kenya

“…In contrast, neonatal GBS disease is increased with HIV-exposure,40 with reduced maternal GBS capsular antibody in HIV-1 infection,41,42 and/or because, as shown here, the most virulent clone, CC17, is more frequently found in HIV-infected GBS colonised women, compared to other non-CC17 types. There have been a number of virulence factors (adhesins, invasins and immune evasins) associated with increased ability of GBS to colonise and cause disease,43 with the more homogeneous CC17 having acquired its own set of virulence genes,38 and increased ability to form biofilms in acidic conditions 44…”

Biofilm formation on different pH conditions byStreptococcus agalactiaeisolated from bovine mastitic milk

“…In this study, CTII bovine S. agalactiae isolates recovered from subclinical mastitis increased biofilm formation when 1% glucose was added to the culture medium, as previously reported for S. agalactiae CTIII human isolates of the hypervirulent ST‐17 lineage (D'Urzo et al . ). Accordingly to these authors, the production of organic acids associated with the metabolism of glucose may induce a pH decrease and be the direct cause of the observed effect on S. agalactiae biofilm formation in glucose‐rich medium.…”

“…Bacterial pellets were suspended in THB medium at different pH levels (pH 4·2, 5·5, 6·5 and 7·2) or THB supplemented with 1% glucose at pH 7·2 (D'Urzo et al . ). Briefly, aliquots of 200 μ l per well of bacterial suspensions were incubated aerobically for 24, 48 and 72 h at 37°C and measurements of pH levels were performed in all opportunities.…”

Perinatal Group B Streptococcal Infections: Virulence Factors, Immunity, and Prevention Strategies

“…For instance, the GBS CovR/S system responds to pH wherein increased CovR/S regulation was observed under low (acidic) pH [65, 71–73]. Changes in pH also influences GBS adhesion [72, 74], survival [75], and biofilm formation [76, 77]. Of note, high vaginal pH and a non-lactobacilli dominated vaginal microbiome [78] are associated with women of African descent with higher incidence of GBS vaginal colonization [15, 21, 22] and neonatal disease [23–25].…”