First report of Streptococcus agalactiae isolated from a healthy captive sichuan golden snub-nosed monkey (Rhinopithecus roxellana) in China.

Streptococcus agalactiae (S. agalactiae) is an opportunistic pathogen, and to date, studies have mainly focused on S. agalactiae strains isolated from humans, dairy cows, and fish. We reported one S. agalactiae strain, named CFFB, which was isolated from a healthy Sichuan golden snub-nosed monkey. Classical bacteriological approaches, as well as, next-generation sequencing, comparative genomics, and mice challenge test were used to characterize this strain. CFFB was identified as serotype III, ST19 combination which is a common type found in human strains. Phylogenetic analysis showed that the genome of CFFB was closely related to human clinical isolates, rather far away from animal strains. In total, CFFB contained fewer virulence-associated genes and antibiotic resistance genes than human isolates that were close to CFFB in evolutionary relationships. In the mice challenge test, CFFB had a relative weak virulence that just caused death in 33 % of ICR mice at a dose of 108 CFU by intraperitoneal injection, and CFFB was reisolated from the cardiac blood of the dead mice. Meanwhile, two intact prophages (prophage 1 and 2) were identified in the CFFB genome and shared high similarities with phage Javan52 and Javan29 which from human S. agalactiae isolate Gottschalk 1002A and RBH03, respectively. Moreover, the type II-A CRISPR-Cas system was detected in the CFFB genome, and the spacers from CFFB were the same to the streptococci isolates from human. These results suggest that CFFB isolated from healthy Sichuan golden snub-nosed monkeys may have its origin in human S. agalactiae. Our results suggested some genomic similarities between the S. agalactiae colonized in Sichuan golden snub-nosed monkey and those in infected humans.

The cadDX operon contributes to cadmium resistance, oxidative stress resistance, and virulence in zoonotic streptococci.

Mobile genetic elements (MGEs) enable bacteria to acquire novel genes and traits. However, the functions of cargo genes within MGEs remain poorly understood. The cadmium resistance operon cadDX is present in many gram-positive bacteria. Although cadDX has been reported to be involved in metal detoxification, its regulatory mechanisms and functions in bacterial pathogenesis are poorly understood. This study revealed that cadDX contributes to cadmium resistance, oxidative stress resistance, and virulence in Streptococcus suis, an important zoonotic pathogen in pigs and humans. CadX represses cadD expression by binding to the cadDX promoter. Notably, cadX responds to H2O2 stress through an additional promoter within the cadDX operon, mitigating the harmful effect of excessive cadD expression during oxidative stress. cadDX resides within an 11 K integrative and mobilizable element that can autonomously form circular structures. Moreover, cadDX is found in diverse MGEs, accounting for its widespread distribution across various bacteria, especially among pathogenic streptococci. Transferring cadDX into another zoonotic pathogen, Streptococcus agalactiae, results in similar phenotypes, including resistance to cadmium and oxidative stresses and increased virulence of S. agalactiae in mice. The new functions and regulatory mechanisms of cadDX shed light on the importance of the cadDX system in driving evolutionary adaptations and survival strategies across diverse gram-positive bacteria.

Unveiling the genetic landscape of Streptococcus agalactiae bacteremia: emergence of hypervirulent CC1 strains and new CC283 strains in Tehran, Iran.

BACKGROUND: The emergence of Streptococcus agalactiae infections in patients with bacteremia is increasing. It is crucial to investigate the epidemiology, molecular characteristics, biofilm status, and virulence analysis of Streptococcus agalactiae in these patients. METHODS: In this cross-sectional study, 61 S. agalactiae isolated from blood infection were subjected to characterization through antimicrobial susceptibility tests, biofilm formation, multilocus sequence typing (MLST), and PCR analysis for detecting resistance (tet and erm family) and virulence genes (alp2/3, alp4, bca, bac, eps, rib, lmb, cylE, and pilus island genes). RESULTS: Overall, 32.7% of the isolates demonstrated an inducible clindamycin resistance phenotype. The results showed that 49.2, 24.6, and 8.2% of confirmed Streptococcus agalactiae strains were classified as strong, intermediate, and weak biofilm-forming strains, respectively. tet(M) (57.1%) was recovered most, followed by tet(M) + tet(L) (14.3%), tet(S) + tet(K) (10.7%), tet(M) + tet(K) (8.9%), tet(M) + tet(K) + tet(O) (5.4%), and tet(S) + tet(L) + tet(O) (3.6%). Three virulence gene profiles of cylE, lmb, bca, rib (24.6%; 15/61), cylE, lmb, rib, alp3 (19.7%; 12/61), and cylE, lmb, bac, rib (16.4%; 10/61) were detected in approximately two-thirds of the isolates. MLST revealed that the 61 isolates belonged to six clonal complexes, including CC1 (49.2%), followed by CC12 (18%), CC19 (13.1%), CC22 (9.8%), CC17 (6.6%), and CC283 (3.3%), and 11 different sequence types (STs), including ST1 (24.6%), ST7 (14.8%), ST918 (13.1%), ST2118 (9.8%), ST19 (9.8%), ST48 (6.6%), ST1372 (4.9%), ST22 (4.9%), ST40 (4.9%), ST734 (3.3%), and ST283 (3.3%). Remarkably, all CC1 and CC12 isolates, three-fourths of CC19, and half of CC22 were confirmed as biofilm producers. Conversely, CC17 and CC28 isolates were found to be nonproducers. The occurrence of strong biofilm formation was limited to specific CCs, namely CC1 (34.4%), CC12 (8.2%), CC19 (3.3%), and CC22 (3.3%). CONCLUSION: The high prevalence of CC1 and CC12 clones among S. agalactiae strains reflects the emergence of these lineages as successful clones in Iran, which is a serious concern and poses a potential threat to patients.

Streptococcus agalactiae isolated from clinical mastitis cases on large dairy farms in north China: phenotype, genotype of antimicrobial resistance and virulence genes.

Streptococcus agalactiae (Strep. agalactiae) is bovine mastitis pathogen and has thus became a matter of concern to dairy farms worldwide in terms of economic loss. The aims of this study were to (a) determine virulence genes, and (b) characterize the antimicrobial resistance (AMR) profiles and AMR genes and (c) figure out the relationship between AMR phenotypes and genotypes of Strep. agalactiae isolated from dairy cows in north China. A total of 20 virulence genes and 23 AMR genes of 140 isolates collected from 12 farms in six provinces were studied. The antimicrobial susceptibility of 10 veterinary commonly used antimicrobials were tested using the broth microdilution method. Results showed that all the isolates harbored the virulence genes lacIV, gapC, and dltA. The isolates that harbored the genes lacIII, fbsA, hylB, and cfb exhibited the high prevalence (99.29%), followed by isolates that harbored lacI (98.57%), bibA (97.86%), cylE (97.14%), lacII (92.14%), cspA (52.14%), pavA (25%), bca (2.14%), and scpB (0.71%). The fbsB, lmb, spbI, bac, and rib genes were not detected. The virulence patterns of B (fbsA_cfb_cylE_ hylB_bibA_cspA_ gapC_dltA_lacIII/IV) and C (fbsA_cfb_ bibA _ gapC_ dltA_lacIV) were dominant, accounting for 97.86% of the isolates. The following AMR genes were prevalent: pbp1A (97.14%), tet(M) (95.00%), lnu (A) (80.71%), erm (B) (75.00%), tet(O) (72.14%), blaZ (49.29%), tet(S) (29.29%), blaTEM (25.71%), erm (A) (17.14%), erm (C) (13.57%), tet (L) (10.71%), linB (2.86%), and erm (TR) (2.86%). The pbp2b, mecA1, mecC, lnu (D), erm (F/G/Q), and mef (A) genes were not detected. Eighty percent of the isolates harbored AMR genes and were highly resistant to tetracycline, followed by macrolides (10.71%), lincosamides (9.29%) and ß-lactams (4.29%). In conclusion, isolates only exhibited well correlation between tetracyclines resistance phenotype and genotype, and almost all isolates harbored intact combination of virulence genes.

The SaeRS two-component system regulates virulence gene expression in group B Streptococcus during invasive infection.

Group B Streptococcus (GBS) is a pathobiont responsible for invasive infections in neonates and the elderly. The transition from a commensal to an invasive pathogen relies on the timely regulation of virulence factors. In this study, we characterized the role of the SaeRS two-component system in GBS pathogenesis. Loss-of-function mutations in the SaeR response regulator decrease virulence in mouse models of invasive infection by hindering the ability of bacteria to persist at the inoculation site and to spread to distant organs. Transcriptome and in vivo analysis reveal a specialized regulatory system specifically activated during infection to control the expression of only two virulence factors: the PbsP adhesin and the BvaP secreted protein. The in vivo surge in SaeRS-regulated genes is complemented by fine-tuning mediated by the repressor of virulence CovRS system to establish a coordinated response. Constitutive activation of the SaeRS regulatory pathway increases PbsP-dependent adhesion and invasion of epithelial and endothelial barriers, though at the cost of reduced virulence. In conclusion, SaeRS is a dynamic, highly specialized regulatory system enabling GBS to express a restricted set of virulence factors that promote invasion of host barriers and allow these bacteria to persist inside the host during lethal infection. IMPORTANCE: Group B Streptococcus (or GBS) is a normal inhabitant of the human gastrointestinal and genital tracts that can also cause deadly infections in newborns and elderly people. The transition from a harmless commensal to a dangerous pathogen relies on the timely expression of bacterial molecules necessary for causing disease. In this study, we characterize the two-component system SaeRS as a key regulator of such virulence factors. Our analysis reveals a specialized regulatory system that is activated only during infection to dynamically adjust the production of two virulence factors involved in interactions with host cells. Overall, our findings highlight the critical role of SaeRS in GBS infections and suggest that targeting this system may be useful for developing new antibacterial drugs.

Genomic and functional determinants of host spectrum in Group B Streptococcus.

Group B Streptococcus (GBS) is a major human and animal pathogen that threatens public health and food security. Spill-over and spill-back between host species is possible due to adaptation and amplification of GBS in new niches but the evolutionary and functional mechanisms underpinning those phenomena are poorly known. Based on analysis of 1,254 curated genomes from all major GBS host species and six continents, we found that the global GBS population comprises host-generalist, host-adapted and host-restricted sublineages, which are found across host groups, preferentially within one host group, or exclusively within one host group, respectively, and show distinct levels of recombination. Strikingly, the association of GBS genomes with the three major host groups (humans, cattle, fish) is driven by a single accessory gene cluster per host, regardless of sublineage or the breadth of host spectrum. Moreover, those gene clusters are shared with other streptococcal species occupying the same niche and are functionally relevant for host tropism. Our findings demonstrate (1) the heterogeneity of genome plasticity within a bacterial species of public health importance, enabling the identification of high-risk clones; (2) the contribution of inter-species gene transmission to the evolution of GBS; and (3) the importance of considering the role of animal hosts, and the accessory gene pool associated with their microbiota, in the evolution of multi-host bacterial pathogens. Collectively, these phenomena may explain the adaptation and clonal expansion of GBS in animal reservoirs and the risk of spill-over and spill-back between animals and humans.

The hypervirulent Group B Streptococcus HvgA adhesin promotes central nervous system invasion through transcellular crossing of the choroid plexus.

BACKGROUND: Group B Streptococcus (GBS) is the leading cause of neonatal meningitis responsible for a substantial cause of death and disability worldwide. The vast majority of GBS neonatal meningitis cases are due to the CC17 hypervirulent clone. However, the cellular and molecular pathways involved in brain invasion by GBS CC17 isolates remain largely elusive. Here, we studied the specific interaction of the CC17 clone with the choroid plexus, the main component of the blood-cerebrospinal fluid (CSF) barrier. METHODS: The interaction of GBS CC17 or non-CC17 strains with choroid plexus cells was studied using an in vivo mouse model of meningitis and in vitro models of primary and transformed rodent choroid plexus epithelial cells (CPEC and Z310). In vivo interaction of GBS with the choroid plexus was assessed by microscopy. Bacterial invasion and cell barrier penetration were examined in vitro, as well as chemokines and cytokines in response to infection. RESULTS: GBS CC17 was found associated with the choroid plexus of the lateral, 3rd and 4th ventricles. Infection of choroid plexus epithelial cells revealed an efficient internalization of the bacteria into the cells with GBS CC17 displaying a greater ability to invade these cells than a non-CC17 strain. Internalization of the GBS CC17 strain involved the CC17-specific HvgA adhesin and occurred via a clathrin-dependent mechanism leading to transcellular transcytosis across the choroid plexus epithelial monolayer. CPEC infection resulted in the secretion of several chemokines, including CCL2, CCL3, CCL20, CX3CL1, and the matrix metalloproteinase MMP3, as well as immune cell infiltration. CONCLUSION: Our findings reveal a GBS strain-specific ability to infect the blood-CSF barrier, which appears to be an important site of bacterial entry and an active site of immune cell trafficking in response to infection.

Molecular epidemiology and virulence factors of group B Streptococcus in South Korea according to the invasiveness.

BACKGROUND: Group B Streptococcus (GBS) causes invasive infections in newborns and elderly individuals, but is a noninvasive commensal bacterium in most immunocompetent people. Recently, the incidence of invasive GBS infections has increased worldwide, and there is growing interest in the molecular genetic characteristics of invasive GBS strains. Vaccines against GBS are expected in the near future. Here, we aimed to analyze the molecular epidemiology of GBS according to the invasiveness in South Korea. METHODS: We analyzed GBS isolates collected and stored in two hospitals in South Korea between January 2015 and December 2020. The invasiveness of these isolates was determined via a retrospective review of clinical episodes. Totally, 120 GBS isolates from 55 children and 65 adults were analyzed. Serotype and sequence type (ST) were determined using multiplex polymerase chain reaction (PCR) and multilocus sequence typing, respectively. Fourteen virulence factor-encoding genes of GBS were analyzed using multiplex PCR. RESULTS: Forty one (34.2%) were invasive infection-related GBS isolates (iGBS). The most frequently detected serotype was III (39/120, 32.5%), and it accounted for a high proportion of iGBS (21/41, 51.2%). The most frequent ST was ST19 (18/120, 15.0%), followed by ST2 (17/120, 14.2%). Serotype III/ST17 was predominant in iGBS (12/41, 29.3%), and all 17 ST2 strains were noninvasive. The distribution of most of the investigated virulence factors was not significantly related to invasiveness; noteworthily, most of the serotype III/ST17 iGBS carried pilus island (PI) 2b (10/12, 83.3%), and the prevalence of fbsB was significantly low compared with noninvasive GBS isolates (P = 0.004). Characteristically, the combination of bca(+)-cspA(+)-pavA(+)-fbsB(-)-rib(+)-bac(-) was predominant in iGBS (24.4%, 10/41). CONCLUSIONS: Serotype III/ST17 GBS carrying PI-2b was frequently detected in iGBS. There was no significant association between invasiveness and the pattern of virulence factors; however, a specific combination of virulence factors was predominant in iGBS.

The serine-rich repeat glycoprotein Srr2 mediates Streptococcus agalactiae interaction with host fibronectin.

BACKGROUND: Group B Streptococcus (GBS) is a commensal of healthy adults and an important pathogen in newborns, the elderly and immunocompromised individuals. GBS displays several virulence factors that promote colonisation and host infection, including the ST-17 strain-specific adhesin Srr2, previously characterised for its binding to fibrinogen. Another common target for bacterial adhesins and for host colonization is fibronectin, a multi-domain glycoprotein found ubiquitously in body fluids, in the extracellular matrix and on the surface of cells. RESULTS: In this study, fibronectin was identified as a novel ligand for the Srr2 adhesin of GBS. A derivative of the ST-17 strain BM110 overexpressing the srr2 gene showed an increased ability to bind fibrinogen and fibronectin, compared to the isogenic wild-type strain. Conversely, the deletion of srr2 impaired bacterial adhesion to both ligands. ELISA assays and surface plasmon resonance studies using the recombinant binding region (BR) form of Srr2 confirmed a direct interaction with fibronectin with an estimated Kd of 92 nM. Srr2-BR variants defective in fibrinogen binding also exhibited no interaction with fibronectin, suggesting that Srr2 binds this ligand through the dock-lock-latch mechanism, previously described for fibrinogen binding. The fibronectin site responsible for recombinant Srr2-BR binding was identified and localised in the central cell-binding domain of the protein. Finally, in the presence of fibronectin, the ability of a Δsrr2 mutant to adhere to human cervico-vaginal epithelial cells was significantly lower than that of the wild-type strain. CONCLUSION: By combining genetic and biochemical approaches, we demonstrate a new role for Srr2, namely interacting with fibronectin. We characterised the molecular mechanism of this interaction and demonstrated that it plays a role in promoting the adhesion of GBS to human cervico-vaginal epithelial cells, further substantiating the role of Srr2 as a factor responsible for the hypervirulence of GBS ST-17 strains. The discovery of the previously undescribed interaction between Srr2 and fibronectin establishes this adhesin as a key factor for GBS colonisation of host tissues.

Group B Streptococcus transcriptome when interacting with brain endothelial cells.

Bacterial meningitis is a life-threatening infection of the central nervous system (CNS) that occurs when bacteria are able to cross the blood-brain barrier (BBB) or the meningeal-cerebrospinal fluid barrier (mBCSFB). The BBB and mBCSFB comprise highly specialized brain endothelial cells (BECs) that typically restrict pathogen entry. Group B Streptococcus (GBS or Streptococcus agalactiae) is the leading cause of neonatal meningitis. Until recently, identification of GBS virulence factors has relied on genetic screening approaches. Instead, we here conducted RNA-seq analysis on GBS when interacting with induced pluripotent stem cell-derived BECs (iBECs) to pinpoint virulence-associated genes. Of the 2,068 annotated protein-coding genes of GBS, 430 transcripts displayed significant changes in expression after interacting with BECs. Notably, we found that the majority of differentially expressed GBS transcripts were downregulated (360 genes) during infection of iBECs. Interestingly, codY, encoding a pleiotropic transcriptional repressor in low-G + C Gram-positive bacteria, was identified as being highly downregulated. We conducted qPCR to confirm the codY downregulation observed via RNA-seq during the GBS-iBEC interaction and obtained codY mutants in three different GBS background parental strains. As anticipated from the RNA-seq results, the [Formula: see text]codY strains were more adherent and invasive in two in vitro BEC models. Together, this demonstrates the utility of RNA-seq during the BEC interaction to identify GBS virulence modulators. IMPORTANCE: Group B Streptococcus (GBS) meningitis remains the leading cause of neonatal meningitis. Research work has identified surface factors and two-component systems that contribute to GBS disruption of the blood-brain barrier (BBB). These discoveries often relied on genetic screening approaches. Here, we provide transcriptomic data describing how GBS changes its transcriptome when interacting with brain endothelial cells. Additionally, we have phenotypically validated these data by obtaining mutants of a select regulator that is highly down-regulated during infection and testing on our BBB model. This work provides the research field with a validated data set that can provide an insight into potential pathways that GBS requires to interact with the BBB and open the door to new discoveries.

Distributions of candidate vaccine Targets, virulence Factors, and resistance features of invasive group B Streptococcus using Whole-Genome Sequencing: A Multicenter, population-based surveillance study.

BACKGROUND: Group B Streptococcus (GBS) is a leading cause of morbidity and mortality in young infants worldwide. This study aimed to investigate candidate GBS vaccine targets, virulence factors, and antimicrobial resistance determinants. METHODS: We used whole-genome sequencing to characterize invasive GBS isolates from infants < 3 months of age obtained from a multicenter population-based study conducted from 2015 to 2021 in China. RESULTS: Overall, seven serotypes were detected from 278 GBS isolates, four (Ia, Ib, III, V) of which accounted for 97.8 %. We detected 30 sequence types (including 10 novel types) that were grouped into six clonal complexes (CCs: CC1, CC10, CC17, CC19, CC23 and CC651); three novel ST groups in CC17 were detected, and the rate of CC17, considered a hyperinvasive neonatal clone complex, was attached to 40.6 % (113/278). A total of 98.9 % (275/278) of isolates harbored at least one alpha-like protein gene. All GBS isolates contained at least one of three pilus backbone determinants and the pilus types PI-2b and PI-1 + PI-2a accounted for 79.8 % of the isolates. The 112 serotype III/CC17 GBS isolates were all positive for hvgA. Most of the isolates (75.2 %) were positive for serine-rich repeat glycoprotein determinants (srr1or srr2). Almost all isolates possessed cfb (99.6 %), c1IE (100 %), lmb (95.3 %) or pavA (100 %) gene. Seventy-seven percent of isolates harboured more than three antimicrobial resistance genes with 28.4 % (79/278) gyrA quinoloneresistancedeterminants mutation, 83.8 % (233/278) carrying tet cluster genes and 77.3 % (215/278) carrying erm genes which mediated fluoroquinolone, tetracycline and clindamycin resistance, respectively." CONCLUSIONS: The findings from this large whole-genome sequence of GBS isolates establish important baseline data required for further surveillance and evaluating the impact of future vaccine candidates.

Comparative analysis of Streptococcus agalactiae serotypes Ia and II isolates from China and Pakistan in a murine model: A focus on pathogenesis and immune response.

Bovine mastitis, caused by Streptococcus agalactiae (Group B Streptococcus; GBS), poses significant economic challenges to the global dairy industry. Mouse models serves as valuable tools for assessing GBS-induced infections as an alternative to large animals. This study aimed to investigate the LD50 dose, organ bacterial load, and quantification of peritoneal leukocyte populations for GBS serotypes Ia and II isolates from China and Pakistan. Additionally, we measured indicators such as lactoferrin, albumin, and myeloperoxidase (MPO) activity. Pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-2) and anti-inflammatory cytokines (IL-10 and TGF-ß) in serum and tissue samples were evaluated using ELISA and qPCR, respectively. BALB/c mice (4 mice per group) received individual intraperitoneal injections of 100 µl containing specific bacterial inoculum concentrations (ranging from 105 to 109 CFU per mouse) of Chinese and Pakistani GBS isolates (serotypes Ia and II). Control groups received 100 µL of sterile PBS. Results revealed that the LD50 bacterial dose causing 50 % mortality in mice was 107 CFU. The highest bacterial load in all experimental groups was quantified in the peritoneum, followed by blood, mammary gland, liver, spleen, lungs, and brain. The most significant bacterial dissemination was observed in mice inoculated with Pakistani serotype Ia at 24 h, with a subsequent notable decline in bacterial counts at day 3. Notably, infection with Pakistani serotype Ia showed a trend of increased total leukocyte counts, significantly higher than Pakistani serotype II, Chinese Serotype Ia, and Chinese serotype II. A substantial influx of neutrophils and lymphocytes was observed in response to all tested serotypes, with Pakistani serotype Ia inducing a significantly higher influx compared to other groups (Pakistani serotype II, Chinese serotype Ia, and Chinese serotype II). Furthermore, TNF-α, IL-1ß, IL-2, and IL-6 expressions were significantly increased in mice one day after infection with the Pakistani serotype Ia. Compared to mice infected with the Pakistani serotype II, Chinese Serotype Ia, and Chinese serotype II, those infected with the Pakistani serotype Ia isolate exhibited the highest production of IL-10 and TGF-ß, along with significantly increased concentrations of lactoferrin, albumin, and MPO. These findings suggest that the persistence and severity of infection caused by the Pakistani serotype Ia may be linked to its ability to spread to deeper tissues. This study enhances our understanding of the clinical characteristics of bovine mastitis caused by S. agalactiae in China and Pakistan.

Genomic insights into the diversity, virulence, and antimicrobial resistance of group B Streptococcus clinical isolates from Saudi Arabia.

Introduction: Detailed assessment of the population structure of group B Streptococcus (GBS) among adults is still lacking in Saudi Arabia. Here we characterized a representative collection of isolates from colonized and infected adults. Methods: GBS isolates (n=89) were sequenced by Illumina and screened for virulence and antimicrobial resistance determinants. Genetic diversity was assessed by single nucleotide polymorphisms and core-genome MLST analyses. Results: Genome sequences revealed 28 sequence types (STs) and nine distinct serotypes, including uncommon serotypes VII and VIII. Majority of these STs (n=76) belonged to the human-associated clonal complexes (CCs) CC1 (33.71%), CC19 (25.84%), CC17 (11.24%), CC10/CC12 (7.87%), and CC452 (6.74%). Major CCs exhibited intra-lineage serotype diversity, except for the hypervirulent CC17, which exclusively expressed serotype III. Virulence profiling revealed that nearly all isolates (94.38%) carried at least one of the four alpha family protein genes (i.e., alphaC, alp1, alp2/3, and rib), and 92.13% expressed one of the two serine-rich repeat surface proteins Srr1 or Srr2. In addition, most isolates harbored the pilus island (PI)-2a alone (15.73%) or in combination with PI-1 (62.92%), and those carrying PI-2b alone (10.11%) belonged to CC17. Phylogenetic analysis grouped the sequenced isolates according to CCs and further subdivided them along with their serotypes. Overall, isolates across all CC1 phylogenetic clusters expressed Srr1 and carried the PI-1 and PI-2a loci, but differed in genes encoding the alpha-like proteins. CC19 clusters were dominated by the III/rib/srr1/PI-1+PI-2a (43.48%, 10/23) and V/alp1/srr1/PI-1+PI-2a (34.78%, 8/23) lineages, whereas most CC17 isolates (90%, 9/10) had the same III/rib/srr2/P1-2b genetic background. Interestingly, genes encoding the CC17-specific adhesins HvgA and Srr2 were detected in phylogenetically distant isolates belonging to ST1212, suggesting that other highly virulent strains might be circulating within the species. Resistance to macrolides and/or lincosamides across all major CCs (n=48) was associated with the acquisition of erm(B) (62.5%, 30/48), erm(A) (27.1%, 13/48), lsa(C) (8.3%, 4/48), and mef(A) (2.1%, 1/48) genes, whereas resistance to tetracycline was mainly mediated by presence of tet(M) (64.18%, 43/67) and tet(O) (20.9%, 14/67) alone or in combination (13.43%, 9/67). Discussion: These findings underscore the necessity for more rigorous characterization of GBS isolates causing infections.

Streptococcus agalactiae glycolipids promote virulence by thwarting immune cell clearance.

Streptococcus agalactiae [group B Streptococcus (GBS)] is a leading cause of neonatal meningitis, with late-onset disease (LOD) occurring after gastrointestinal tract colonization in infants. Bacterial membrane lipids are essential for host-pathogen interactions, and the functions of glycolipids are yet to be fully elucidated. GBS synthesizes three major glycolipids: glucosyl-diacylglycerol (Glc-DAG), diglucosyl-DAG (Glc2-DAG), and lysyl-Glc-DAG (Lys-Glc-DAG). Here, we identify the enzyme, IagB, as responsible for biosynthesis of Glc-DAG, the precursor for the two other glycolipids in GBS. To examine the collective role of glycolipids to GBS virulence, we adapted a murine model of neonatal meningitis to simulate LOD. The GBS∆iagB mutant traversed the gut-epithelial barrier comparable to wild type but was severely attenuated in bloodstream survival, resulting in decreased bacterial loads in the brain. The GBS∆iagB mutant was more susceptible to neutrophil killing and membrane targeting by host antimicrobial peptides. This work reveals an unexplored function of GBS glycolipids with their ability to protect the bacterial cell from host antimicrobial killing.

Identification of ClpB, a molecular chaperone involved in the stress tolerance and virulence of Streptococcus agalactiae.

Bacterial ClpB is an ATP-dependent disaggregate that belongs to the Hsp100/Clp family and facilitates bacterial survival under hostile environmental conditions. Streptococcus agalactiae, which is regarded as the major bacterial pathogen of farmed Nile tilapia (Oreochromis niloticus), is known to cause high mortality and large economic losses. Here, we report a ClpB homologue of S. agalactiae and explore its functionality. S. agalactiae with a clpB deletion mutant (∆clpB) exhibited defective tolerance against heat and acidic stress, without affecting growth or morphology under optimal conditions. Moreover, the ΔclpB mutant exhibited reduced intracellular survival in RAW264.7 cells, diminished adherence to the brain cells of tilapia, increased sensitivity to leukocytes from the head kidney of tilapia and whole blood killing, and reduced mortality and bacterial loads in a tilapia infection assay. Furthermore, the reduced virulence of the ∆clpB mutant was investigated by transcriptome analysis, which revealed that deletion of clpB altered the expression levels of multiple genes that contribute to the stress response as well as certain metabolic pathways. Collectively, our findings demonstrated that ClpB, a molecular chaperone, plays critical roles in heat and acid stress resistance and virulence in S. agalactiae. This finding provides an enhanced understanding of the functionality of this ClpB homologue in gram-positive bacteria and the survival strategy of S. agalactiae against immune clearance during infection.

Protease SfpB plays an important role in cell membrane stability and immune system evasion in Streptococcus agalactiae.

Bacteria possess the ability to develop diverse and ingenious strategies to outwit the host immune system, and proteases are one of the many weapons employed by bacteria. This study sought to identify S. agalactiae additional serine protease and determine its role in virulence. The S. agalactiae THN0901 genome features one S8 family serine peptidase B (SfpB), acting as a secreted and externally exposed entity. A S8 family serine peptidase mutant strain (ΔsfpB) and complement strain (CΔsfpB) were generated through homologous recombination. Compared to the wild-type strain THN0901, the absorption of EtBr dyes was significantly reduced (P < 0.01) in ΔsfpB, implying an altered cell membrane permeability. In addition, the ΔsfpB strain had a significantly lower survival rate in macrophages (P < 0.01) and a 61.85 % lower adhesion ability to the EPC cells (P < 0.01) compared to THN0901. In the in vivo colonization experiment using tilapia as a model, 210 fish were selected and injected with different bacterial strains at a concentration of 3 × 106 CFU/tail. At 6, 12, 24, 48, 72 and 96 h post-injection, three fish were randomly selected from each group and their brain, liver, spleen, and kidney tissues were isolated. Subsequently, it was demonstrated that the ΔsfpB strain exhibited a markedly diminished capacity for colonization in tilapia. Additionally, the cumulative mortality of ΔsfpB in fish after intraperitoneal injection was reduced by 19.92-23.85 %. In conclusion, the findings in this study have demonstrated that the SfpB plays a significant role in S. agalactiae cell membrane stability and immune evasion. The immune evasion is fundamental for the development and transmission of invasive diseases, the serine protease SfpB may be a promising candidate for the development of antimicrobial agents to reduce the transmission of S. agalactiae.

Unravelling Streptococcus agalactiae: Serotype distribution, virulence factors, obstetrics history and clinical presentation correlations in tertiary care hospitals of the western province of Sri Lanka.

PURPOSE: This study investigated to detect serotypes and virulence genes of Group B Streptococcus (GBS) isolated from pregnant women. METHODS: Forty-five samples of GBS isolates from January to August 2019 at antenatal clinics of 4 teaching hospitals in Western Province, Sri Lanka were included. Isolated GBS were carried to identify 9 serotypes by multiplex PCR. Different virulence determinants, including bac, rib and scp(B) have been detected by PCR. RESULTS: Among GBS-positive culture isolates most abundant serotype detected was type III 12/45 (26.7%) while serotype VII, VIII and IX were not seen. Furthermore, serotype Ia (15.6%); II (20%); V (17.8%); VI (15.6%); Ib (2.2%) and IV (2.2%) were identified. Among 5 rectal isolates, 1 isolate was serotype Ia, 2 isolates were serotype II and 2 isolates were serotype III. Forty (40/45) isolates expressed scpB gene (88.8%). Presence of rib gene was confirmed in 17.8%, bac in 13.3% isolates. ScpB, rib and bac were identified in 4.4% isolates, 8.9% isolates were scpB, rib positive and bac negative, 8.9% isolates were scpB, bac positive and rib negative. These three-virulence genes did not express in 8.9% isolates. ScpB gene was found once in serotype Ib and IV and all serotype VI expressed scpB gene. Rib gene was more common among serotype II and it was not found in serotype Ib, IV and VI. Bac gene was more common in serotype V and it was not found in serotype Ia, Ib and IV. There was not significant association between serotypes and virulence gene (p > 0.05). CONCLUSION: Serotype III is the most abundant serotype. In formulation of vaccine against GBS for Sri Lanka, serotype III should be targeted. Prevalence of vaccine candidate virulence protein such as ß antigens of the C protein (bac) and surface protein Rib (rib) genes were low in this study.

Alterations in cell arrangements of group B streptococcus due to virulence factor expression can bias estimates of bacterial populations based on colony count measures.

Group B streptococcus (GBS) is a chain-forming commensal bacterium and opportunistic pathogen that resides in the gastrointestinal and genitourinary tract of healthy adults. GBS can cause various infections and related complications in pregnant and nonpregnant women, adults, and newborns. Investigations of the mechanisms by which GBS causes disease pathogenesis often utilize colony count assays to estimate bacterial population size in experimental models. In other streptococci, such as group A streptococcus and pneumococcus, variation in the chain length of the bacteria that can occur naturally or due to mutation can affect facets of pathogenesis, such as adherence to or colonization of a host. No studies have reported a relationship between GBS chain length and pathogenicity. Here, we used GBS strain 874391 and several derivative strains displaying longer chain-forming phenotypes (874391pgapC, 874391ΔcovR, 874391Δstp1) to assess the impact of chain length on bacterial population estimates based on the colony-forming unit (c.f.u.) assay. Disruption of GBS chains via bead beating or sonication in conjunction with fluorescence microscopy was used to compare chaining phenotypes pre- and post-disruption to detect long- and short-chain forms, respectively. We used a murine model of GBS colonization of the female reproductive tract to assess whether chaining may affect bacterial colonization dynamics in the host during chronic infection in vivo. Overall, we found that GBS exhibiting long-chain form can significantly affect population size estimates based on the colony count assay. Additionally, we found that the length of chaining of GBS can affect virulence in the reproductive tract colonization model. Collectively, these findings have implications for studies of GBS that utilize colony count assays to measure GBS populations and establish that chain length can affect infection dynamics and disease pathogenesis for this important opportunistic pathogen.

An opportunistic pathogen under stress: how Group B Streptococcus responds to cytotoxic reactive species and conditions of metal ion imbalance to survive.

Group B Streptococcus (GBS; also known as Streptococcus agalactiae) is an opportunistic bacterial pathogen that causes sepsis, meningitis, pneumonia, and skin and soft tissue infections in neonates and healthy or immunocompromised adults. GBS is well-adapted to survive in humans due to a plethora of virulence mechanisms that afford responses to support bacterial survival in dynamic host environments. These mechanisms and responses include counteraction of cell death from exposure to excess metal ions that can cause mismetallation and cytotoxicity, and strategies to combat molecules such as reactive oxygen and nitrogen species that are generated as part of innate host defence. Cytotoxicity from reactive molecules can stem from damage to proteins, DNA, and membrane lipids, potentially leading to bacterial cell death inside phagocytic cells or within extracellular spaces within the host. Deciphering the ways in which GBS responds to the stress of cytotoxic reactive molecules within the host will benefit the development of novel therapeutic and preventative strategies to manage the burden of GBS disease. This review summarizes knowledge of GBS carriage in humans and the mechanisms used by the bacteria to circumvent killing by these important elements of host immune defence: oxidative stress, nitrosative stress, and stress from metal ion intoxication/mismetallation.

Serotype distribution, virulence and antibiotic resistance of Streptococcus agalactiae isolated from cultured tilapia Oreochromis niloticus in Lake Volta, Ghana.

Streptococcus agalactiae infection is one of the major factors limiting the expansion of tilapia farming globally. In this study, we investigated the serotype distribution, virulence and antimicrobial resistance of S. agalactiae isolates from tilapia farmed in Lake Volta, Ghana. Isolates from 300 moribund fish were characterised by Gram staining, MALDI-TOF/MS and 16S rRNA sequencing. Serotype identification was based on multiplex polymerase chain reaction (PCR) amplification of the capsular polysaccharide genes. Detection of virulence genes (cfb, fbsA and cspA) and histopathology were used to infer the pathogenicity of the isolates. The susceptibility of isolates to antibiotics was tested using the Kirby-Bauer disk diffusion assay. All 32 isolates identified as S. agalactiae were of serotype Ia. This was notably different from isolates previously collected from the farms in 2017, which belonged to serotype Ib, suggesting a possible serotype replacement. The prevalence of the pathogen was related to the scale of farm operation, with large-scale farms showing higher S. agalactiae positivity. Data from histopathological analysis and PCR amplification of targeted virulence genes confirmed the virulence potential and ability of the isolates to cause systemic infection in tilapia. Except for gentamicin, the majority of the isolates were less resistant to the tested antibiotics. All isolates were fully sensitive to oxytetracycline, erythromycin, florfenicol, enrofloxacin, ampicillin and amoxicillin. This study has improved our understanding of the specific S. agalactiae serotypes circulating in Lake Volta and demonstrates the need for continuous monitoring to guide the use of antimicrobials and vaccines against streptococcal infections in Ghanaian aquaculture systems.