Lancefield Group Research Articles

Clinical Laboratory Perspective on Streptococcus halichoeri, an Unusual Nonhemolytic, Lancefield Group B Streptococcus Causing Human Infections

Abstract Streptococcus halichoeri is a relatively newly identified species of pyogenic streptococci that causes zoonotic infection in humans. S. halichoeri was first described in 2004 as indigenous to seals, and only 8 reports of human S. halichoeri infection have been published. S. halichoeri grows as small, white, nonhemolytic colonies and may be strongly catalase-positive on routine blood agar media, which can lead to isolates being misidentified as coagulase-negative staphylococci. S. halichoeri tests positive for Lancefield group B antigen, like S. agalactiae, but can be identified with matrix-assisted laser desorption/ionization time of flight mass spectrometry or partial 16S rRNA sequencing. We describe 3 cases of S. halichoeri bone and joint infections in patients in the United States with underlying health conditions. In addition, we examine the microbiologic characteristics of S. halichoeri and discuss the importance of fully identifying this organism that might otherwise be disregarded as a skin commensal.

Clinical Laboratory Perspective on Streptococcus halichoeri, an Unusual Nonhemolytic, Lancefield Group B Streptococcus Causing Human Infections.

Streptococcus halichoeri is a relatively newly identified species of pyogenic streptococci that causes zoonotic infection in humans. S. halichoeri was first described in 2004 as indigenous to seals, and only 8 reports of human S. halichoeri infection have been published. S. halichoeri grows as small, white, nonhemolytic colonies and may be strongly catalase-positive on routine blood agar media, which can lead to isolates being misidentified as coagulase-negative staphylococci. S. halichoeri tests positive for Lancefield group B antigen, like S. agalactiae, but can be identified with matrix-assisted laser desorption/ionization time of flight mass spectrometry or partial 16S rRNA sequencing. We describe 3 cases of S. halichoeri bone and joint infections in patients in the United States with underlying health conditions. In addition, we examine the microbiologic characteristics of S. halichoeri and discuss the importance of fully identifying this organism that might otherwise be disregarded as a skin commensal.

English

This study assessed the microbiological quality of meat from free rage-produced chicken processed in an informal slaughter facility. The total viable counts (TVC), total coliform counts, coagulase positive Staphylococcus aureus, Streptococcus species, Salmonella species and Campylobacter species were used as indicators. A cross-sectional sampling of chicken carcasses at informal slaughter facility was carried out. Whole carcass rinse fluid was prepared from 40 randomly obtained freshly dressed carcasses. Fluid samples were cultured in selective media to isolate and enumerate the specific bacteria. S. aureus was further identified by coagulase test, Streptococci by serotyping into Lancefield groups, Campylobacter by DNA analysis and Salmonella by biochemical tests and serology. Bacterial concentrations in the carcasses were calculated as colony forming units (CFU) per ml and CFU/cm2. The mean carcass CFU/ml concentration was 1.59 × 107, 1.44 ×105, 3.2 × 104 and 1.06 × 104 for TVC, Coliforms, S. aureus and Streptococci, respectively. All the mean concentration values were higher than the limits recommended by the Codex Alimentarius Commission (CAC). Coagulase-positive Staphylococcus was isolated from 12 (30%) carcasses and Streptococci from 35 (87%). Majority Streptococci were Lancefield Group D (48.57%) followed by Group G (17.14%), and Group F (14.28%). Campylobacter genus was identified in 11 carcasses (27.5%) and Campylobacter jejuni in three (7.5%). On the other hand, Salmonella was not isolated from any carcass. The results of the study indicated that the low hygienic standard in non-regulated slaughter houses exposed the chicken meat to microbial contaminants which may pose a risk to the consumers. Improvement of slaughter infrastructure and capacity-building of slaughter personnel is therefore critically required to ensure food safety and enable access to high value markets. Key words: Slaughter, free-range chicken, bacterial quality.

Effectiveness of adjunctive clindamycin in β-lactam antibiotic-treated patients with invasive β-haemolytic streptococcal infections in US hospitals: a retrospective multicentre cohort study

Clindamycin is strongly recommended as an adjunctive treatment to β-lactam antibiotics in patients with severe invasive group A β-haemolytic streptococcal (iGAS) infections. However, there is little evidence of a benefit in the use of clindamycin in humans, and its role, if any, in treating patients with invasive non-group A/B β-haemolytic streptococcal (iNABS) infections is unclear. For this retrospective multicentre cohort study, we used a dataset from patients in the Cerner Health Facts database, which contains electronic health-based data from 233 US hospitals. We queried the Cerner Health Facts database for inpatients (no age restriction) admitted to hospital in 2000-15, with any clinical cultures positive for β-haemolytic streptococcal taxa of interest, and who had received β-lactam antibiotics within 3 days either side of culture sampling. This group of patients was then queried for those who had also received intravenous or oral clindamycin within 3 days either side of culture sampling. Patients were excluded if they had polymicrobial growth or clindamycin non-susceptible isolates, received linezolid, or had missing variable data needed for analysis. Patients were categorised by Lancefield group (iGAS or iNABS); β-lactam antibiotic-treated patients who had received clindamycin were propensity-matched (1:2) to those who did not receive clindamycin separately for iGAS and iNABS cohorts, and logistic regression was then used to account for residual confounding factors. The primary outcome was the adjusted odds ratio (aOR) of in-hospital mortality in propensity-matched patients treated with adjunctive clindamycin versus those not treated with clindamycin in the iGAS and iNABS infection cohorts. We identified 1956 inpatients with invasive β-haemolytic streptococcal infection who had been treated with β-lactam antibiotics across 118 hospitals (1079 with iGAS infections and 877 with iNABS infections). 459 (23·4%) of these patients had received adjunctive clindamycin treatment (343 [31·7%] patients with iGAS infections and 116 [13·2%] patients with iNABS infections). The effect of adjunctive clindamycin therapy on in-hospital mortality differed significantly and showed the opposite trend in iGAS and iNABS infection cohorts (p=0·013 for an interaction). In the iGAS cohort, in-hospital mortality in propensity-matched patients who received adjunctive clindamycin (18 [6·5%] of 277 patients) was significantly lower than in those who did not (55 [11·0%] of 500 patients; aOR 0·44 [95% CI 0·23-0·81]). This survival benefit was maintained even in patients without shock or necrotising fasciitis (six [2·6%] of 239 patients treated with adjunctive clindamycin vs 27 [6·1%] of 422 patients not treated with adjunctive clindamycin; aOR 0·40 [0·15-0·91]). By contrast, in the iNABS infection cohort, in-hospital mortality in propensity-matched patients who received adjunctive clindamycin (ten [9·8%] of 102) was higher than in those who did not (nine [4·6%] of 193), but this difference was not significant (aOR 2·60 [0·94-7·52]). Several subset analyses found qualitatively similar results. Real-world data suggest that increased use of adjunctive clindamycin for invasive iGAS infections, but not iNABS infections, could improve outcomes, even in patients without shock or necrotising fasciitis. Intramural Research Program of the National Institutes of Health Clinical Center and the National Institute of Allergy and Infectious Disease.

Meningitis Caused by Streptococcus agalactiae in Nile Tilapia (Oreochromis niloticus): Infection and Inflammatory Response.

Simple SummaryStreptococcus agalactiae (group B Streptococcus, GBS) is the main pathological agent in meningitis in Nile tilapia (Oreochromis niloticus). In this study, we describe the mechanism of infection and the immune response in the brain tissue of experimentally infected tilapia. This study understanding of the pathophysiology of meningitis in this species and bring the possibility of using tilapia as a study model for meningitis.Streptococcus agalactiae (Sta) of Lancefield group B is the primary etiological agent of bacterial meningitis in Nile tilapia and newborn humans. Thus, the study of this disease is of fundamental importance for aquaculture and human medicine. Additionally, elucidation of the mechanisms involved in the host–pathogenic response is important for the success of new therapies. In the present study, we elucidated important aspects of the innate immune response in the brain tissue of Nile tilapia (Oreochromis niloticus) infected by Sta. The neuroinflammatory process in the meninges started with the migration of MHC class II and CD68 + cells, production of TNF-alpha, and the effective immune response to Sta was mediated by the increased iNOs+. In conclusion, the present study brings a partial understanding of the pathophysiological and neuroinflammatory mechanisms in meningitis in Sta infected tilapia, enabling important advances in the therapy of this disease as well as the possibility of using this biological model to understand human meningitis.

Group C Streptococcus dysgalactiae infection in fish.

Streptococcus dysgalactiae subsp. dysgalactiae (GCSD) is a Gram-positive, facultative anaerobic bacterium and mostly non-β-haemolytic with Lancefield group C antigen. GCSD infection has been identified in various vertebrates. From 2002 to the present, GCSD infection of fish has been reported to cause severe economic losses in aquaculture farms around the world. Moreover, GCSD isolates from teleosts have been identified in patients with ascending upper limb cellulitis. Therefore, the economic and clinical significance of GCSD has increased in aquaculture, livestock and human health. Many studies have been presented, from the first report of isolated GCSD in fish, to the pathogenesis, characterization, immune responses and vaccine development. In this review, we present the current knowledge of GCSD in teleosts.

Neonatal Exposure to Commensal-Bacteria-Derived Antigens Directs Polysaccharide-Specific B-1 B Cell Repertoire Development

B-1 B cells derive from a developmental program distinct from that of conventional B cells, through B cell receptor (BCR)-dependent positive selection of fetally derived precursors. Here, we used direct labeling of B cells reactive with the N-acetyl-D-glucosamine (GlcNAc)-containing Lancefield group A carbohydrate of Streptococcus pyogenes to study the effects of bacterial antigens on the emergent B-1 B cell clonal repertoire. The number, phenotype, and BCR clonotypes of GlcNAc-reactive B-1 B cells were modulated by neonatal exposure to heat-killed S.pyogenes bacteria. GlcNAc-reactive B-1 clonotypes and serum antibodies were reduced in germ-free mice compared with conventionally raised mice. Colonization of germ-free mice with a conventional microbiota promoted GlcNAc-reactive B-1 B cell development and concomitantly elicited clonally related IgA+ plasma cells in the small intestine. Thus, exposure to microbial antigens in early life determines the clonality of the mature B-1 B cell repertoire and ensuing antibody responses, with implications for vaccination approaches and schedules.

Characterization of Endolysin LyJH307 with Antimicrobial Activity Against Streptococcus Bovis.

Simple SummaryDevelopment of endolysin is a promising strategy because of having the ability to control problematic bacteria specifically. In this study, we developed and characterized the endolysin having lytic activity against Streptococcus bovis (S. bovis), which is one of the initiators of ruminal acidosis. Based on our findings, endolysin LyJH307 showed potent lytic activity in ruminal pH range and ruminal temperature. In addition, LyJH307 was effective against not only S. bovis isolated from rumen, but also several S. bovis groups. We suggest that LyJH307 may have a lytic effect in the ruminal condition and prevent acute ruminal acidosis by controlling S. bovis specifically.Streptococcus bovis (S. bovis) is one of the critical initiators of acute acidosis in ruminants. Therefore, we aimed to develop and characterize the endolysin LyJH307, which can lyse ruminal S. bovis. We tested the bactericidal activity of recombinant LyJH307 against S. bovis JB1 under a range of pH, temperature, NaCl, and metal ion concentrations. In silico analyses showed that LyJH307 has a modular design with a distinct, enzymatically active domain of the NLPC/P60 superfamily at the N-terminal and a cell wall binding domain of the Zoocin A target recognition domain (Zoocin A_TRD) superfamily at the C-terminal. The lytic activity of LyJH307 against S. bovis JB1 was the highest at pH 5.5, and relatively higher under acidic, than under alkaline conditions. LyJH307 activity was also the highest at 39 °C, but was maintained between 25°C and 55°C. LyJH307 bactericidal action was retained under 0-500 mM NaCl. While the activity of LyJH307 significantly decreased on treatment with ethylenediaminetetraacetic acid (EDTA), it was only restored with supplementation of 10 mM Ca2+. Analyses of antimicrobial spectra showed that LyJH307 lysed Lancefield groups D (S. bovis group and Enterococcus faecalis) and H (S. sanguinis) bacteria. Thus, LyJH307 might help to prevent acute ruminal acidosis.

Antimicrobial activity of Cannabis sativa extracts on Lancefield Group A Streptococcus species associated with streptococcal pharyngitis (strep throat)

Antimicrobial activity of Cannabis sativa extracts on Lancefield Group A Streptococcus species associated with streptococcal pharyngitis (strep throat)

Prevalence and pattern of beta-haemolytic streptococcal throat infections among primary school children in a rural community in rivers state

Background: Group A beta-haemolytic Streptococcus (GABHS) remains a major cause of morbidity and mortality in humans where it causes symptomatic pharyngitis and the non-suppurative sequelae of acute rheumatic fever and post-streptococcal acute glomerulonephritis. Regional evaluation of streptococcal throat infections may be helpful in disease surveillance. The aim of this study was to evaluate the prevalence and pattern of GABHS throat infections among primary school children in a rural community in Rivers State, Nigeria. Methodology: This cross-sectional study was carried out between May and July 2015 in Emohua local government area of Rivers State, Nigeria. Pupils were recruited using a multistaged random sampling technique. Throat swabs obtained were cultured using sheep blood and sent for microscopy and Lancefield grouping. Results: A total of 456 pupils aged 6–12 years were recruited, of which 54 (11.8%) had a positive throat culture of beta-haemolytic Streptococcus (BHS). The isolates were Lancefield Groups B (23; 42.6%), A (18; 33.3%), D (9; 19.6%), C (3; 5.6%) and F (1; 1.8%). No Group G BHS was isolated. Age, sex, socioeconomic class, classroom and household overcrowding did not significantly influence the prevalence of BHS throat infections. Conclusion: The prevalence of BHS throat infections in this rural community was similar to that in the urban areas in Nigeria. There was no significant difference between the prevalence of BHS in symptomatic and asymptomatic pupils.

Molecular detection of <i>C5a</i> Peptidase (<i>scp</i>B) Gene in Group B Streptococcus Isolated from Pregnant Women and the Correspondence with Adverse Pregnancy Outcome

Group B Streptococcus is Gram-positive cocci non motile encapsulated bacteria which produce an arrow zone of beta-hemolysis on blood agar. It belongs to Lancefield group B antigen. Group B Streptococcus is the mainly cause of neonate’s invasive bacterial disease such as neonatal sepsis meningitis, septicemia and pneumonia. The study planned to evaluate the vaginal colonization rate of Streptococcus agalactiae and virulence genes in pregnant women with adverse pregnancy outcome. A cross-sectional study was designed included 200 pregnant women at 34–37 weeks of gestation. A total of two hundred vaginal swabs was taken from all pregnant women enrolled in this project by the gynecologist. GBS isolated bacteria was evaluated by means of using classical microbiological approach, after DNA extraction the isolated GBS strains were screened for the presence of scpB gene by polymerase chain reaction. Thirty-six (18%) out of 200 pregnant women enrolled in this project were positive for group B Streptococcus by culture method, the majority of them were from age ranged (18-36 years), 20 (20.6%) of them with a history of abortion, females with positive vaginal colonization indicated that they had a history of rupture membrane only in 10 (9.9%) with prolonged rupture membrane >18hr in 3 (30%).The specific PCR primer was used for the detection of scpB gene. It was found that scpB gene was observed in 17 (47%) isolate.It can be conclude that there is no statistical significance between repeated abortions with the presence of scpBgene as a virulence factor in GBS while there are statistically significant between the presences of these virulence gene with rupture membrane.

Group A, B, C, and G Streptococcus Lancefield antigen biosynthesis is initiated by a conserved α-d-GlcNAc-β-1,4-l-rhamnosyltransferase

Group A carbohydrate (GAC) is a bacterial peptidoglycan-anchored surface rhamnose polysaccharide (RhaPS) that is essential for growth of Streptococcus pyogenes and contributes to its ability to infect the human host. In this study, using molecular and synthetic biology approaches, biochemistry, radiolabeling techniques, and NMR and MS analyses, we examined the role of GacB, encoded in the S. pyogenes GAC gene cluster, in the GAC biosynthesis pathway. We demonstrate that GacB is the first characterized α-d-GlcNAc-β-1,4-l-rhamnosyltransferase that synthesizes the committed step in the biosynthesis of the GAC virulence determinant. Importantly, the substitution of S. pyogenes gacB with the homologous gene from Streptococcus agalactiae (Group B Streptococcus), Streptococcus equi subsp. zooepidemicus (Group C Streptococcus), Streptococcus dysgalactiae subsp. equisimilis (Group G Streptococcus), or Streptococcus mutans complemented the GAC biosynthesis pathway. These results, combined with those from extensive in silico studies, reveal a common phylogenetic origin of the genes required for this priming step in >40 pathogenic species of the Streptococcus genus, including members from the Lancefield Groups B, C, D, E, G, and H. Importantly, this priming step appears to be unique to streptococcal ABC transporter–dependent RhaPS biosynthesis, whereas the Wzx/Wzy-dependent streptococcal capsular polysaccharide pathways instead require an α-d-Glc-β-1,4-l-rhamnosyltransferase. The insights into the RhaPS priming step obtained here open the door to targeting the early steps of the group carbohydrate biosynthesis pathways in species of the Streptococcus genus of high clinical and veterinary importance.

Incidence, clinical characteristics, and outcomes of Streptococcus dysgalactiae subspecies equisimilis bacteremia in a tertiary hospital: comparison with S. agalactiae bacteremia

The clinical characteristics and outcomes of Streptococcus dysgalactiae subspecies equisimilis (SDSE) bacteremia cases have not been adequately evaluated. We retrospectively enrolled consecutive adult patients with SDSE or S. agalactiae (group B streptococci, GBS) bacteremia at a tertiary care hospital (Republic of Korea) from August 2012 to December 2016. We compared the incidence, seasonality, clinical characteristics, and outcomes of 52 SDSE bacteremia cases with 151 GBS bacteremia cases. The incidence of SDSE and GBS bacteremia in these patients was 1.28/100,000 and 4.22/100,000 person-days, respectively. Most SDSE bacteremia cases were of community-onset infection (SDSE 94.2% vs GBS 83.4%; p = 0.052). Lancefield group G was the most common bacteria type among SDSE isolates (43/47; 91.5%). Patients with SDSE bacteremia were older (median, 68.0years vs 61.0years; p = 0.03). In both groups, solid tumor was the most common underlying disease, and more than half of the patients were immunocompromised (51.9% vs 54.3%; p = 0.77). Chronic kidney disease was more common in the SDSE group (19.2% vs 5.3%; p < 0.01). Cellulitis was the most common clinical syndrome of SDSE bacteremia and was more common in the SDSE group (59.6% vs 29.1%; p < 0.01). SDSE bacteremia cases occurred more frequently in the warm season compared with GBS bacteremia cases (65.4% vs 37.1%; p < 0.01); in-hospital mortalities were not significantly different between the groups (3.8% vs 10.6%; p = 0.17). In conclusion, SDSE bacteremia is commonly associated with cellulitis, especially in older and immunocompromised patients during the warm season.

Complete genome sequence and characterization of virulence genes in Lancefield group C Streptococcus dysgalactiae isolated from farmed amberjack (Seriola dumerili).

Lancefield group C Streptococcus dysgalactiae causes infections in farmed fish. Here, the genome of S. dysgalactiae strain kdys0611, isolated from farmed amberjack (Seriola dumerili) was sequenced. The complete genome sequence of kdys0611 consists of a single chromosome and five plasmids. The chromosome is 2,142,780 bp long and has a GC content of 40%. It possesses 2061 coding sequences and 67 tRNA and 6 rRNA operons. One clustered regularly interspaced short palindromic repeat, 125 insertion sequences, and four predicted prophage elements were identified. Phylogenetic analysis based on 126 core genes suggested that the kdys0611 strain is more closely related to S. dysgalactiae subsp. dysgalactiae than to S. dysgalactiae subsp. equisimilis. The genome of kdys0611 harbors 87 genes with sequence similarity to putative virulence-associated genes identified in other bacteria, of which 57 exhibit amino acid identity (>52%) to genes of the S. dysgalactiae subsp. equisimilis GGS124 human clinical isolate. Four putative virulence genes, emm5 (FGCSD_0256), spg_2 (FGCSD_1961), skc (FGCSD_1012), and cna (FGCSD_0159), in kdys0611 did not show significant homology with any deposited S. dysgalactiae genes. The chromosomal sequence of kdys0611 has been deposited in GenBank under Accession No. AP018726. This is the first report of the complete genome sequence of S. dysgalactiae isolated from fish.

Pathogenicity Factors in Group C and G Streptococci.

Initially recognized zoonoses, streptococci belonging to Lancefield group C (GCS) and G (GGS) were subsequently recognised as human pathogens causing a diverse range of symptoms, from asymptomatic carriage to life threatening diseases. Their taxonomy has changed during the last decade. Asymptomatic carriage is <4% amongst the human population and invasive infections are often in association with chronic diseases such as diabetes, cardiovascular diseases or chronic skin infections. Other clinical manifestations include acute pharyngitis, pneumonia, endocarditis, bacteraemia and toxic-shock syndrome. Post streptococcal sequalae such as rheumatic fever and acute glomerulonephritis have also been described but mainly in developed countries and amongst specific populations. Putative virulence determinants for these organisms include adhesins, toxins, and other factors that are essential for dissemination in human tissues and for interference with the host immune responses. High nucleotide similarities among virulence genes and their association with mobile genetic elements supports the hypothesis of extensive horizontal gene transfer events between the various pyogenic streptococcal species belonging to Lancefield groups A, C and G. A better understanding of the mechanisms of pathogenesis should be apparent by whole-genome sequencing, and this would result in more effective clinical strategies for the pyogenic group in general.

Genetics and Pathogenicity Factors of Group C and G Streptococci.

Of the eight phylogenetic groups comprising the genus Streptococcus, Lancefield group C and G streptococci (GCS and GGS, resp.) occupy four of them, including the Pyogenic, Anginosus, and Mitis groups, and one Unnamed group so far. These organisms thrive as opportunistic commensals in both humans and animals but may also be associated with clinically serious infections, often resembling those due to their closest genetic relatives, the group A streptoccci (GAS). Advances in molecular genetics, taxonomic approaches and phylogenomic studies have led to the establishment of at least 12 species, several of which being subdivided into subspecies. This review summarizes these advances, citing 264 early and recent references. It focuses on the molecular structure and genetic regulation of clinically important proteins associated with the cell wall, cytoplasmic membrane and extracellular environment. The article also addresses the question of how, based on the current knowledge, basic research and translational medicine might proceed to further advance our understanding of these multifaceted organisms. Particular emphasis in this respect is placed on streptokinase as the protein determining the host specificity of infection and the Rsh-mediated stringent response with its potential for supporting bacterial survival under nutritional stress conditions.

“Epidemiology and aetiology of influenza-like illness among households in metropolitan Vientiane, Lao PDR”: A prospective, community-based cohort study

Respiratory diseases are a major contributor to morbidity and mortality in many tropical countries, including Lao PDR. However, little has been published regarding viral or bacterial pathogens that can contribute to influenza-like illness (ILI) in a community setting. We report on the results of a community-based surveillance that prospectively monitored the incidence of ILI and its causative pathogens in Vientiane capital in Lao PDR. A cohort of 995 households, including 4885 study participants, were followed-up between May 2015 and May 2016. Nasopharyngeal swabs, throat swabs, and sputum specimens were collected from ILI cases identified through active case-finding. Real-Time PCR was used to test nasopharyngeal swabs for 21 respiratory pathogens, while throat and sputum samples were subjected to bacterial culture. Generalized linear mixed models were used to assess potential risk factors for associations with ILI. In total, 548 episodes of ILI were reported among 476 (9.7%) of the study participants and 330 (33.2%) of the study households. The adjusted estimated incidence of ILI within the study area was 10.7 (95%CI: 9.4–11.9) episodes per 100 person-years. ILI was significantly associated with age group (p<0.001), sex (p<0.001), and number of bedrooms (p = 0.04) in multivariate analysis. In 548 nasopharyngeal swabs, the most commonly detected potential pathogens were Streptococcus pneumoniae (17.0%), Staphylococcus aureus (11.3%), influenza A (11.1%; mostly subtype H3N2), rhinovirus (7.5%), and influenza B (8.0%). Streptococci were isolated from 42 (8.6%) of 536 throat swabs, most (27) of which were Lancefield Group G. Co-infections were observed in 132 (24.1%) of the 548 ILI episodes. Our study generated valuable data on respiratory disease burden and patterns of etiologies associated with community-acquired acute respiratory illness Laos. Establishment of a surveillance strategy in Laos to monitor trends in the epidemiology and burden of acute respiratory infections is required to minimize their impact on human health.

Biofilm development and computational screening for new putative inhibitors of a homolog of the regulatory protein BrpA in Streptococcus dysgalactiae subsp. dysgalactiae

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry. Adherence and biofilm production are important factors in streptoccocal pathogenesis. We have previously described the adhesion and internalization of SDSD isolates in human cells and now we describe the biofilm production capability of this bacterium. In this work we integrated microbiology, imaging and computational methods to evaluate the biofilm production capability of SDSD isolates; to assess the presence of biofilm regulatory protein BrpA homolog in the biofilm producers; and to predict a structural model of BrpA-like protein and its binding to putative inhibitors. Our results show that SDSD isolates form biofilms on abiotic surface such as glass (hydrophilic) and polystyrene (hydrophobic), with the strongest biofilm formation observed in glass. This ability was mainly associated with a proteinaceous extracellular matrix, confirmed by the dispersion of the biofilms after proteinase K and trypsin treatment. The biofilm formation in SDSD isolates was also confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Under SEM observation, VSD16 isolate formed cell aggregates during biofilm growth while VSD9 and VSD10 formed smooth and filmy layers. We show that brpA-like gene is present and expressed in SDSD biofilm-producing isolates and its expression levels correlated with the biofilm production capability, being more expressed in the late exponential phase of planktonic growth compared to biofilm growth. Fisetin, a known biofilm inhibitor and a putative BrpA binding molecule, dramatically inhibited biofilm formation by the SDSD isolates but did not affect planktonic growth, at the tested concentrations. Homology modeling was used to predict the 3D structure of BrpA-like protein. Using high throughput virtual screening and molecular docking, we selected five ligand molecules with strong binding affinity to the hydrophobic cleft of the protein, making them potential inhibitor candidates of the SDSD BrpA-like protein. These results warrant further investigations for developing novel strategies for SDSD anti-biofilm therapy.

Streptococcal Lancefield polysaccharides are critical cell wall determinants for human Group IIA secreted phospholipase A2 to exert its bactericidal effects.

Human Group IIA secreted phospholipase A2 (hGIIA) is an acute phase protein with bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic mice have suggested the importance of hGIIA as an innate defense mechanism against the human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon library to recombinant hGIIA and compared relative mutant abundance with library input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in the ΔgacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is conserved across different GAS serotypes. Increased resistance is associated with delayed penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. This suggests that the streptococcal Lancefield antigens, which are critical determinants for streptococcal physiology and virulence, are required for the bactericidal enzyme hGIIA to exert its bactericidal function.

Streptococcus pyogenes Causing Skin and Soft Tissue Infections Are Enriched in the Recently Emerged emm89 Clade 3 and Are Not Associated With Abrogation of CovRS.

Although skin and soft tissue infections (SSTI) are the most common focal infections associated with invasive disease caused by Streptococcus pyogenes (Lancefield Group A streptococci - GAS), there is scarce information on the characteristics of isolates recovered from SSTI in temperate-climate regions. In this study, 320 GAS isolated from SSTI in Portugal were characterized by multiple typing methods and tested for antimicrobial susceptibility and SpeB activity. The covRS and ropB genes of isolates with no detectable SpeB activity were sequenced. The antimicrobial susceptibility profile was similar to that of previously characterized isolates from invasive infections (iGAS), presenting a decreasing trend in macrolide resistance. However, the clonal composition of SSTI between 2005 and 2009 was significantly different from that of contemporary iGAS. Overall, iGAS were associated with emm1 and emm3, while SSTI were associated with emm89, the dominant emm type among SSTI (19%). Within emm89, SSTI were only significantly associated with isolates lacking the hasABC locus, suggesting that the recently emerged emm89 clade 3 may have an increased potential to cause SSTI. Reflecting these associations between emm type and disease presentation, there were also differences in the distribution of emm clusters, sequence types, and superantigen gene profiles between SSTI and iGAS. According to the predicted ability of each emm cluster to interact with host proteins, iGAS were associated with the ability to bind fibrinogen and albumin, whereas SSTI isolates were associated with the ability to bind C4BP, IgA, and IgG. SpeB activity was absent in 79 isolates (25%), in line with the proportion previously observed among iGAS. Null covS and ropB alleles (predicted to eliminate protein function) were detected in 10 (3%) and 12 (4%) isolates, corresponding to an underrepresentation of mutations impairing CovRS function in SSTI relative to iGAS. Overall, these results indicate that the isolates responsible for SSTI are genetically distinct from those recovered from normally sterile sites, supporting a role for mutations impairing CovRS activity specifically in invasive infection and suggesting that this role relies on a differential regulation of other virulence factors besides SpeB.