RESUMO
Group B Streptococcus (GBS) asymptomatically colonizes the vagina but can opportunistically ascend to the uterus and be transmitted vertically during pregnancy, resulting in neonatal pneumonia, bacteremia, and meningitis. GBS is a leading etiologic agent of neonatal infection and understanding the mechanisms by which GBS persists within the polymicrobial female genital mucosa has the potential to mitigate subsequent transmission and disease. Type VIIb secretion systems (T7SSb) are encoded by Bacillota and often mediate interbacterial competition using LXG toxins that contain conserved N-termini important for secretion and variable C-terminal toxin domains that confer diverse biochemical activities. Our recent work characterized a role for the GBS T7SSb in vaginal colonization and ascending infection but the mechanisms by which the T7SSb promotes GBS persistence in this polymicrobial niche remain unknown. Herein, we investigate the GBS T7SS in interbacterial competition and GBS niche establishment in the female genital tract. We demonstrate GBS T7SS-dependent inhibition of mucosal pathobiont Enterococcus faecalis both in vitro using predator-prey assays and in vivo in the murine genital tract and found that a GBS LXG protein encoded within the T7SS locus (herein named group B streptococcal LXG Toxin A) contributes to these phenotypes. We identify BltA as a T7SS substrate that is toxic to E. coli and S. aureus upon induction of intracellular expression along with associated chaperones. Finally, we show that BltA and its chaperones contribute to GBS vaginal colonization. Altogether, these data reveal a role for a novel T7b-secreted toxin in GBS mucosal persistence and competition.IMPORTANCECompetition between neighboring, non-kin bacteria is essential for microbial niche establishment in mucosal environments. Gram-positive bacteria encoding T7SSb have been shown to engage in competition through the export of LXG-motif-containing toxins, but these have not been characterized in group B Streptococcus (GBS), an opportunistic colonizer of the polymicrobial female genital tract. Here, we show a role for GBS T7SS in competition with mucosal pathobiont Enterococcus faecalis, both in vitro and in vivo. We further find that a GBS LXG protein contributing to this antagonism is exported by the T7SS and is intracellularly toxic to other bacteria; therefore, we have named this protein group B streptococcal LXG Toxin A (BltA). Finally, we show that BltA and its associated chaperones promote persistence within female genital tract tissues, in vivo. These data reveal previously unrecognized mechanisms by which GBS may compete with other mucosal opportunistic pathogens to persist within the female genital tract.
Assuntos
Toxinas Bacterianas , Infecções Estreptocócicas , Streptococcus agalactiae , Sistemas de Secreção Tipo VII , Vagina , Feminino , Animais , Streptococcus agalactiae/metabolismo , Streptococcus agalactiae/genética , Streptococcus agalactiae/crescimento & desenvolvimento , Camundongos , Infecções Estreptocócicas/microbiologia , Vagina/microbiologia , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Sistemas de Secreção Tipo VII/metabolismo , Sistemas de Secreção Tipo VII/genética , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/efeitos dos fármacos , Genitália Feminina/microbiologia , Genitália Feminina/metabolismo , Interações Microbianas , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genéticaRESUMO
BACKGROUND: Rectovaginal group B Streptococcus (GBS) colonisation in pregnant individuals at the time of labour is a major risk factor for invasive GBS disease by age 7 days (early-onset disease). We aimed to investigate the prevalence of rectovaginal GBS colonisation at the time of labour among pregnant women and vertical transmission to their newborns across selected low-income and middle-income African and south Asian countries. METHODS: This prospective, observational study was undertaken at 11 maternity and obstetric care facilities based in Ethiopia, Kenya, Mozambique, Nigeria, Mali, South Africa, Bangladesh, India, and Bhutan. HIV-negative pregnant women aged 18-45 years who were in the early stages of labour and at least 37 weeks' gestation were eligible for inclusion. Lower vaginal and rectal swabs and urine were collected from the women, and swabs of the umbilicus, outer ear, axillary fold, rectum, and throat were obtained from their newborns, for GBS culture. Standardised sampling and culture using direct plating and selective media broth for detection of GBS colonisation was undertaken at the sites. Serotyping of GBS isolates was done in South Africa. The primary outcome was the prevalence of rectovaginal GBS among pregnant women, analysed in participants with available data. This study is registered with the South African National Clinical Trials Register, number DOH-27-0418-4989. FINDINGS: 6922 pregnant women were enrolled from Jan 10, 2016, to Dec 11, 2018, of whom 6514 (94·1%; 759-892 per country) were included in the analysis; data from Bhutan were not included in the study due to issues with specimen collection and processing. Overall, the prevalence of maternal GBS colonisation was 24·1% (95% CI 23·1-25·2; 1572 of 6514); it was highest in Mali (41·1% [37·7-44·6]; 314 of 764) and lowest in Ethiopia (11·6% [9·5-14·1]; 88 of 759). The overall rate of vertical transmission of GBS from women with rectovaginal GBS colonisation was 72·3% (70·0-74·4; 1132 of 1566); it was highest in Mozambique (79·2% [73·3-84·2]; 168 of 212) and lowest in Bangladesh (55·8%, 47·5-63·8; 77 of 138). The five most common GBS colonising serotypes were Ia (37·3% [34·9-39·7]; 586 of 1572), V (28·5% [26·3-30·8]; 448 of 1572), III (25·1% [23·0-27·3]; 394 of 1572), II (9·2% [7·8-10·7]; 144 of 1572), and Ib (6·5% [5·4-7·8]; 102 of 1572). There was geographical variability in serotype proportion distribution; serotype VII was the third most common serotype in India (8·6% [5·3-13·7]; 15 of 174) and serotype VI was mainly identified in Bangladesh (5·8% [3·0-11·0]; eight of 138) and India (5·7% [3·2-10·3]; ten of 174). INTERPRETATION: Our study reported a high prevalence of GBS colonisation in most settings, with some geographical variability even within African countries. Our findings suggest that serotypes not included in current multivalent capsular-polysaccharide GBS vaccines prevail in some regions, so vaccine efficacy and post-licensure effectiveness studies should assess the effect of vaccination on maternal GBS colonisation given the potential for replacement by non-vaccine serotypes. FUNDING: Bill & Melinda Gates Foundation.
Assuntos
Transmissão Vertical de Doenças Infecciosas , Complicações Infecciosas na Gravidez , Infecções Estreptocócicas , Streptococcus agalactiae , Humanos , Feminino , Gravidez , Estudos Prospectivos , Adulto , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/transmissão , Prevalência , Streptococcus agalactiae/isolamento & purificação , Streptococcus agalactiae/crescimento & desenvolvimento , Recém-Nascido , Adulto Jovem , Complicações Infecciosas na Gravidez/epidemiologia , Complicações Infecciosas na Gravidez/microbiologia , Adolescente , Reto/microbiologia , Vagina/microbiologia , África/epidemiologia , Moçambique/epidemiologia , África do Sul/epidemiologia , Índia/epidemiologia , Pessoa de Meia-Idade , Pobreza , Países em Desenvolvimento , Butão/epidemiologia , Ásia/epidemiologiaRESUMO
Group B Streptococcus (GBS) is the leading cause of bacterial neonatal sepsis. This study aimed to confirm the effect of Ligilactobacillus salivarius V4II-90 on GBS colonisation during pregnancy. A randomised, multicentre, double-blind, placebo-controlled, parallel-group study was conducted in seven hospitals in Madrid, Spain. The sample was broken down into two groups with 20 participants each (n = 40) in order to show reduced GBS colonisation frequency in the probiotic versus the placebo group. Pregnant participants positive for vaginal-rectal colonisation before or during the 13th week of gestation were randomly assigned to either the placebo or the probiotic group. The probiotic, L. salivarius V4II-90 at 1 × 109 cfu/day was administered for 12 weeks, starting at week 21-23 of gestation. The primary outcome was the percentage of participants with vaginal and/or rectal GBS colonisation at the end of the intervention period (35 weeks of gestation). Secondary outcomes were changes in the microbial composition of vaginal and rectal exudates; premature delivery; premature rupture of membranes; intrapartum antibiotics; new-borns with early or late-onset GBS sepsis; adverse events (AEs); and GBS test results performed at the hospital at week 35 of gestation. Of the 481 participants included, 44 were vaginal-rectal colonised with GBS and randomised. 43 completed the study (20 in the probiotic group and 23 in the placebo group). After intervention, GBS was eradicated in six participants (27%) from the placebo group and in twelve participants (63%) from the probiotic group ( P = 0.030). None of the 185 AEs reported were identified as possibly, probably, or definitely related to the investigational product. In conclusion, oral administration of L. salivarius V4II-90 is a safe and successful strategy to significantly decrease the rates of GBS colonisation at the end of pregnancy and, therefore, to reduce the exposure of subjects and their infants to intrapartum antibiotic prophylaxis. Trial registered at ClinicalTrials.gov: number NCT03669094.
Assuntos
Ligilactobacillus salivarius , Complicações Infecciosas na Gravidez , Probióticos , Reto , Infecções Estreptocócicas , Streptococcus agalactiae , Vagina , Humanos , Feminino , Gravidez , Probióticos/administração & dosagem , Método Duplo-Cego , Streptococcus agalactiae/crescimento & desenvolvimento , Streptococcus agalactiae/efeitos dos fármacos , Infecções Estreptocócicas/prevenção & controle , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/tratamento farmacológico , Adulto , Vagina/microbiologia , Reto/microbiologia , Ligilactobacillus salivarius/fisiologia , Complicações Infecciosas na Gravidez/microbiologia , Complicações Infecciosas na Gravidez/prevenção & controle , Complicações Infecciosas na Gravidez/tratamento farmacológico , Recém-Nascido , Espanha , Adulto JovemRESUMO
Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a predominant pathogen of neonatal sepsis, commonly associated with early-onset neonatal sepsis. GBS has also been associated with cases of late-onset sepsis potentially originating from the intestine. Previous findings have shown GBS can colonize the infant intestinal tract as part of the neonatal microbiota. To better understand GBS colonization dynamics in the neonatal intestine, we collected stool and milk samples from prematurely born neonates for identification of potential pathogens in the neonatal intestinal microbiota. GBS was present in approximately 10% of the cohort, and this colonization was not associated with maternal GBS status, delivery route, or gestational weight. Interestingly, we observed the relative abundance of GBS in the infant stool negatively correlated with maternal IgA concentration in matched maternal milk samples. Using a preclinical murine model of GBS infection, we report that both vertical transmission and direct oral introduction resulted in intestinal colonization of GBS; however, translocation beyond the intestine was limited. Finally, vaccination of dams prior to breeding induced strong immunoglobulin responses, including IgA responses, which were associated with reduced mortality and GBS intestinal colonization. Taken together, we show that maternal IgA may contribute to infant immunity by limiting the colonization of GBS in the intestine.
Assuntos
Translocação Bacteriana , Imunoglobulina A , Infecções Estreptocócicas , Streptococcus agalactiae , Streptococcus agalactiae/crescimento & desenvolvimento , Streptococcus agalactiae/imunologia , Animais , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/prevenção & controle , Infecções Estreptocócicas/imunologia , Feminino , Recém-Nascido , Humanos , Camundongos , Transmissão Vertical de Doenças Infecciosas , Fezes/microbiologia , Intestinos/microbiologia , Intestinos/imunologia , Leite Humano/microbiologia , Microbioma Gastrointestinal , Gravidez , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , MasculinoRESUMO
The vaginal microbiota plays a pivotal role in reproductive, sexual, and perinatal health and disease. Unlike the well-established connections between diet, metabolism, and the intestinal microbiota, parallel mechanisms influencing the vaginal microbiota and pathogen colonization remain overlooked. In this study, we combine a mouse model of Streptococcus agalactiae strain COH1 [group B Streptococcus (GBS)] vaginal colonization with a mouse model of pubertal-onset obesity to assess diet as a determinant of vaginal microbiota composition and its role in colonization resistance. We leveraged culture-dependent assessment of GBS clearance and culture-independent, sequencing-based reconstruction of the vaginal microbiota in relation to diet, obesity, glucose tolerance, and microbial dynamics across time scales. Our findings demonstrate that excessive body weight gain and glucose intolerance are not associated with vaginal GBS density or timing of clearance. Diets high in fat and low in soluble fiber are associated with vaginal GBS persistence, and changes in vaginal microbiota structure and composition due to diet contribute to GBS clearance patterns in nonpregnant mice. These findings underscore a critical need for studies on diet as a key determinant of vaginal microbiota composition and its relevance to reproductive and perinatal outcomes.IMPORTANCEThis work sheds light on diet as a key determinant influencing the composition of vaginal microbiota and its involvement in group B Streptococcus (GBS) colonization in a mouse model. This study shows that mice fed diets with different nutritional composition display differences in GBS density and timing of clearance in the female reproductive tract. These findings are particularly significant given clear links between GBS and adverse reproductive and neonatal outcomes, advancing our understanding by identifying critical connections between dietary components, factors originating from the intestinal tract, vaginal microbiota, and reproductive outcomes.
Assuntos
Dieta , Infecções Estreptocócicas , Streptococcus agalactiae , Vagina , Vagina/microbiologia , Feminino , Animais , Streptococcus agalactiae/crescimento & desenvolvimento , Camundongos , Infecções Estreptocócicas/microbiologia , Microbiota/fisiologia , Obesidade/microbiologia , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , HumanosRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Cloretos/farmacocinética , Sorogrupo , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/genética , Compostos de Zinco/farmacocinética , Antibacterianos/metabolismo , Cápsulas Bacterianas/classificação , Cápsulas Bacterianas/efeitos dos fármacos , Cloretos/metabolismo , Feminino , Humanos , Recém-Nascido , Testes de Sensibilidade Microbiana , Gravidez , Sorotipagem , Infecções Estreptocócicas/sangue , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/classificação , Streptococcus agalactiae/crescimento & desenvolvimento , Vagina/efeitos dos fármacos , Vagina/microbiologia , Compostos de Zinco/metabolismoRESUMO
The Gram-positive pathogen group B Streptococcus (GBS) is a leading cause of neonatal bacterial infections, preterm birth, and stillbirth. Although maternal GBS vaginal colonization is a risk factor for GBS-associated adverse birth outcomes, mechanisms promoting GBS vaginal persistence are not fully defined. GBS possesses a broadly conserved small molecule, CAMP factor, that is co-hemolytic in the presence of Staphylococcus aureus sphingomyelinase C. While this co-hemolytic reaction is commonly used by clinical laboratories to identify GBS, the contribution of CAMP factor to GBS vaginal persistence is unknown. Using in vitro biofilm, adherence and invasion assays with immortalized human vaginal epithelial VK2 cells, and a mouse model of GBS vaginal colonization, we tested the contribution of CAMP factor using GBS strain COH1 and its isogenic CAMP-deficient mutant (Δcfb). We found no evidence for CAMP factor involvement in GBS biofilm formation, or adherence, invasion, or cytotoxicity toward VK2 cells in the presence or absence of S. aureus. Additionally, there was no difference in vaginal burdens or persistence between COH1 and Δcfb strains in a murine colonization model. In summary, our results using in vitro human cell lines and murine models do not support a critical role for CAMP factor in promoting GBS vaginal colonization. IMPORTANCE Group B Streptococcus (GBS) remains a pervasive pathogen for pregnant women and their newborns. Maternal screening and intrapartum antibiotic prophylaxis to GBS-positive mothers have reduced, but not eliminated GBS neonatal disease, and have not impacted GBS-associated preterm birth or stillbirth. Additionally, this antibiotic exposure is associated with adverse effects on the maternal and neonatal microbiota. Identifying key GBS factors important for maternal vaginal colonization will foster development of more targeted, alternative therapies to antibiotic treatment. Here, we investigate the contribution of a broadly conserved GBS determinant, CAMP factor, to GBS vaginal colonization and find that CAMP factor is unlikely to be a biological target to control maternal GBS colonization.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Proteínas Hemolisinas/metabolismo , Mucosa/microbiologia , Streptococcus agalactiae/metabolismo , Vagina/microbiologia , Animais , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular , Células Epiteliais/microbiologia , Feminino , Deleção de Genes , Proteínas Hemolisinas/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Esfingomielina Fosfodiesterase/metabolismo , Streptococcus agalactiae/genética , Streptococcus agalactiae/crescimento & desenvolvimentoRESUMO
Camels are vital to food production in the drylands of the Horn of Africa, with milk as their main contribution to food security. A major constraint to camel milk production is mastitis, inflammation of the mammary gland. The condition negatively impacts milk yield and quality as well as household income. A leading cause of mastitis in dairy camels is Streptococcus agalactiae, or group B Streptococcus (GBS), which is also a commensal and pathogen of humans and cattle. It has been suggested that extramammary reservoirs for this pathogen may contribute to the occurrence of mastitis in camels. We explored the molecular epidemiology of GBS in camels using a cross-sectional study design for sample collection and phenotypic, genomic and phylogenetic analysis of isolates. Among 88 adult camels and 93 calves from six herds in Laikipia County, Kenya, GBS was detected in 20% of 50 milk samples, 25% of 152 nasal swabs, 8% of 90 oral swabs and 3% of 90 rectal swabs, but not in vaginal swabs. Per camel herd, two to four sequence types (ST) were identified using Multi Locus Sequence Typing (MLST). More than half of the isolates belonged to ST617 or its single-locus variant, ST1652, with these STs found across all sample types. Capsular serotype VI was detected in 30 of 58 isolates. In three herds, identical STs were detected in milk and swab samples, suggesting that extramammary sources of GBS may contribute to the maintenance and spread of GBS within camel herds. This needs to be considered when developing prevention and control strategies for GBS mastitis. The high nasal carriage rate, low recto-vaginal carriage rate, and high prevalence of serotype VI for GBS in camels are in stark contrast to the distribution of GBS in humans and in cattle and reveal hitherto unknown ecological and molecular features of this bacterial species.
Assuntos
Camelus/microbiologia , Infecções Estreptocócicas , Streptococcus agalactiae , Animais , Feminino , Humanos , Quênia/epidemiologia , Masculino , Glândulas Mamárias Animais/microbiologia , Leite/microbiologia , Tipagem de Sequências Multilocus , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus agalactiae/classificação , Streptococcus agalactiae/crescimento & desenvolvimentoRESUMO
Group B Streptococcus (GBS) causes serious neonatal infection via vertical transmission. The prenatal GBS screening test is performed at the late stage of pregnancy to avoid risks of infection. In this test, enrichment culture is performed, followed by GBS identification. Selective medium is used for the enrichment; however, Enterococcus faecalis, which is a potential contaminant in swab samples, can interfere with the growth of GBS. Such bacterial contamination can lead to false-negative results. Endolysin, a bacteriophage-derived enzyme, degrades peptidoglycan in the bacterial cell wall; it is a promising antimicrobial agent for selectively eliminating specific bacterial genera/species. In this study, we used the recombinant endolysin EG-LYS, which is specific to E. faecalis; the endolysin potentially enriched GBS in the selective culture. First, in the false-negative model (coculture of GBS and E. faecalis, which disabled GBS detection in the subsequent GBS identification test), EG-LYS treatment at 0.1 mg/ml improved GBS detection. Next, we used 548 vaginal swabs to test the efficacy of EG-LYS treatment in improving GBS detection. EG-LYS treatment (0.1 mg/ml) increased the GBS-positive ratio to 17.9%, compared to 15.7% in the control (phosphate-buffered saline [PBS] treatment). In addition, there were an increased number of GBS colonies under EG-LYS treatment in some samples. The results were supported by the microbiota analysis of the enriched cultures. In conclusion, EG-LYS treatment of the enrichment culture potentially improves the accuracy of the prenatal GBS screening test. IMPORTANCE Endolysin is a bacteriophage-derived enzyme that degrades the peptidoglycan in the cell wall of host bacteria; it could be used as an antimicrobial agent for selectively eliminating specific bacterial genera/species. Group B Streptococcus (GBS) causes neonatal infection via vertical transmission; prenatal GBS screening test, in which enrichment culture is followed by bacterial identification, is used to detect the presence of GBS in pregnant women. However, the presence of commensal bacteria such as Enterococcus faecalis in clinical specimens can inhibit GBS growth in the selective enrichment culture, resulting in false-negative result. Here, we demonstrated that the application of originally isolated endolysin in the enrichment culture improved the test accuracy by inhibiting unwanted E. faecalis growth and therefore avoiding false-negative results, not only in experimental settings, but also in tests using vaginal swabs.
Assuntos
Endopeptidases/farmacologia , Complicações Infecciosas na Gravidez/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/efeitos dos fármacos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Meios de Cultura/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Feminino , Humanos , Gravidez , Complicações Infecciosas na Gravidez/diagnóstico , Infecções Estreptocócicas/diagnóstico , Streptococcus agalactiae/crescimento & desenvolvimento , Streptococcus agalactiae/isolamento & purificação , Streptococcus agalactiae/metabolismo , Vagina/microbiologiaRESUMO
This study aimed to verify the effects of dietary supplementation with sodium butyrate and Lippia origanoides, combined and isolated, on the health and zootechnical performance of Nile tilapia juveniles Oreochromis niloticus. A total of 120 fish (5.38 ± 0.65 g) were randomly distributed in 12 experimental units and fed different experimental diets for 30 days, namely: commercial diet without supplementation (Unsupplemented); commercial diet supplemented with 0.5% sodium butyrate (Butyrate); commercial diet supplemented with 0.125% L. origanoides (Lippia) and commercial diet supplemented with a mixture of 0.5% sodium butyrate and 0.125% L. origanoides (Butyrate + Lippia). After preparing the experimental diets there was an increase in the pH of diet Butyrate when compared to the other diets. After 30 days the fish supplemented with Butyrate + Lippia showed reduction significate in the mean corpuscular haemoglobin, concentration of total heterotrophic bacteria in the intestine, and lymphocyte infiltrates in the liver. Besides that, the supplementation with Butyrate + Lippia promoted an increased number of intestinal villi compared to the fish Unsupplemented ones. Additionally, fish fed a diet containing only Lippia presented an increase in the villus perimeter in the posterior region of the gut and in the red blood cell number. Animals supplemented only with sodium butyrate demonstrated increased lactic acid bacterium in the gut and macrosteatosis in the liver, besides decreased melanomacrophages in the spleen. The use of sodium butyrate associated with essential oil had positive effects on the intestinal microbiota, intestinal structure, liver, and spleen integrity, suggesting a greater efficiency of the compounds when used together in the nutrition of Nile tilapia juveniles.
Assuntos
Ácido Butírico/farmacologia , Ciclídeos , Suplementos Nutricionais , Lippia , Óleos Voláteis/farmacologia , Animais , Ciclídeos/sangue , Ciclídeos/crescimento & desenvolvimento , Ciclídeos/imunologia , Ciclídeos/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Testes Hematológicos , Fígado/efeitos dos fármacos , Fígado/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Baço/efeitos dos fármacos , Baço/imunologia , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/crescimento & desenvolvimentoRESUMO
High-molecular-mass penicillin-binding proteins (PBPs) are enzymes that catalyze the biosynthesis of bacterial cell wall peptidoglycan. The Gram-positive bacterial pathogen Streptococcus agalactiae (group B streptococcus [GBS]) produces five high-molecular-mass PBPs, namely, PBP1A, PBP1B, PBP2A, PBP2B, and PBP2X. Among these, only PBP2X is essential for cell viability, whereas the other four PBPs are individually dispensable. The biological function of the four nonessential PBPs is poorly characterized in GBS. We deleted the pbp1a, pbp1b, pbp2a, and pbp2b genes individually from a genetically well-characterized serotype V GBS strain and studied the phenotypes of the four isogenic mutant strains. Compared to the wild-type parental strain, (i) none of the pbp isogenic mutant strains had a significant growth defect in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY) rich medium, (ii) isogenic mutant Δpbp1a and Δpbp1b strains had significantly increased susceptibility to penicillin and ampicillin, and (iii) isogenic mutant Δpbp1a and Δpbp2b strains had significantly longer chain lengths. Using saturated transposon mutagenesis and transposon insertion site sequencing, we determined the genes essential for the viability of the wild-type GBS strain and each of the four isogenic pbp deletion mutant strains in THY rich medium. The pbp1a gene is essential for cell viability in the pbp2b deletion background. Reciprocally, pbp2b is essential in the pbp1a deletion background. Moreover, the gene encoding RodA, a peptidoglycan polymerase that works in conjunction with PBP2B, is also essential in the pbp1a deletion background. Together, our results suggest functional overlap between PBP1A and the PBP2B-RodA complex in GBS cell wall peptidoglycan biosynthesis. IMPORTANCE High-molecular-mass penicillin-binding proteins (HMM PBPs) are enzymes required for bacterial cell wall biosynthesis. Bacterial pathogen group B streptococcus (GBS) produces five distinct HMM PBPs. The biological functions of these proteins are not well characterized in GBS. In this study, we performed a comprehensive deletion analysis of genes encoding HMM PBPs in GBS. We found that deleting certain PBP-encoding genes altered bacterial susceptibility to beta-lactam antibiotics, cell morphology, and the essentiality of other enzymes involved in cell wall peptidoglycan synthesis. The results of our study shed new light on the biological functions of PBPs in GBS.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Streptococcus agalactiae/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Deleção de Genes , Mutagênese , Mutagênese Insercional , Proteínas de Ligação às Penicilinas/química , Penicilinas/farmacologia , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/genética , Streptococcus agalactiae/crescimento & desenvolvimentoRESUMO
BACKGROUND: Group B streptococci (GBS) are important neonatal bacterial pathogens that can cause severe invasive disease in the newborn. It is thought that in many cases of invasive neonatal GBS disease, the bacteria ascend the vagina into the uterus and infect the amniotic fluid surrounding the fetus. Important constituents of this environment include the polyols or sugar alcohols of which erythritol, sorbitol and mannitol are examples. The aim of our study was to investigate the effect of polyols on GBS grown in media containing these sugar alcohols. RESULTS: GBS incubated in varying concentrations of polyols (erythritol, sorbitol or mannitol) did not display any significant enhancement or inhibition of bacterial growth. However, growth of GBS in the presence of erythritol significantly increased the surface expression of GBS-PGK (a plasminogen binding protein) 1.25 to 1.5-fold depending on the erythritol concentration and significantly enhanced the survival in human blood 3X to 18X depending on the concentration of polyol used. Interestingly, GBS grown in 1% erythritol significantly increased invasion by the bacteria of HeLa cells (epithelial cell line) (150% vs 100%) however, at higher concentrations (2% or 4% of polyol) the number of CFUs was significantly reduced (55-75% vs 100%) suggesting higher concentrations of polyols may inhibit invasion. Erythritol also increased GBS hemolytic activity as well as enhancing biofilm formation 1.4X to 3.3X depending on the concentration of polyol used. CONCLUSIONS: GBS grown in the presence of polyols alters the bacteria's phenotype resulting in changes associated with GBS virulence. This effect was greatest for the polyol erythritol.
Assuntos
Eritritol/metabolismo , Manitol/metabolismo , Polímeros/metabolismo , Sorbitol/metabolismo , Streptococcus agalactiae/crescimento & desenvolvimento , Células HeLa , Humanos , Fenótipo , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/metabolismo , Streptococcus agalactiae/patogenicidade , VirulênciaRESUMO
Streptococcus pneumoniae, S. pyogenes (Group A Streptococcus; GAS) and S. agalactiae (Group B Streptococcus; GBS) are major aetiological agents of diseases in humans. The cellular membrane, a crucial site in host-pathogen interactions, is poorly characterized in streptococci. Moreover, little is known about whether or how environmental conditions influence their lipid compositions. Using normal phase liquid chromatography coupled with electrospray ionization MS, we characterized the phospholipids and glycolipids of S. pneumoniae, GAS and GBS in routine undefined laboratory medium, streptococcal defined medium and, in order to mimic the host environment, defined medium supplemented with human serum. In human serum-supplemented medium, all three streptococcal species synthesize phosphatidylcholine (PC), a zwitterionic phospholipid commonly found in eukaryotes but relatively rare in bacteria. We previously reported that S. pneumoniae utilizes the glycerophosphocholine (GPC) biosynthetic pathway to synthesize PC. Through substrate tracing experiments, we confirm that GAS and GBS scavenge lysoPC, a major metabolite in human serum, thereby using an abbreviated GPC pathway for PC biosynthesis. Furthermore, we found that plasmanyl-PC is uniquely present in the GBS membrane during growth with human serum, suggesting GBS possesses unusual membrane biochemical or biophysical properties. In summary, we report cellular lipid remodelling by the major pathogenic streptococci in response to metabolites present in human serum.
Assuntos
Fosfolipídeos/metabolismo , Soro/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/metabolismo , Streptococcus pneumoniae/metabolismo , Streptococcus pyogenes/metabolismo , Membrana Celular/química , Membrana Celular/genética , Meios de Cultura/metabolismo , Humanos , Fosfolipídeos/química , Infecções Estreptocócicas/sangue , Streptococcus agalactiae/química , Streptococcus agalactiae/crescimento & desenvolvimento , Streptococcus pneumoniae/química , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pyogenes/química , Streptococcus pyogenes/crescimento & desenvolvimentoRESUMO
Zinc is an essential trace element for normal bacterial physiology but, divergently, can intoxicate bacteria at high concentrations. Here, we define the molecular systems for Zn detoxification in Streptococcus agalactiae, also known as group B streptococcus, and examine the effects of resistance to Zn stress on virulence. We compared the growth of wild-type bacteria and mutants deleted for the Zn exporter, czcD, and the response regulator, sczA, using Zn-stress conditions in vitro Macrophage antibiotic protection assays and a mouse model of disseminated infection were used to assess virulence. Global bacterial transcriptional responses to Zn stress were defined by RNA sequencing and quantitative reverse transcription-PCR. czcD and sczA enabled S. agalactiae to survive Zn stress, with the putative CzcD efflux system activated by SczA. Additional genes activated in response to Zn stress encompassed divalent cation transporters that contribute to regulation of Mn and Fe homeostasis. In vivo, the czcD-sczA Zn management axis supported virulence in the blood, heart, liver, and bladder. Additionally, several genes not previously linked to Zn stress in any bacterium, including, most notably, arcA for arginine deamination, also mediated resistance to Zn stress, representing a novel molecular mechanism of bacterial resistance to metal intoxication. Taken together, these findings show that S. agalactiae responds to Zn stress by sczA regulation of czcD, with additional novel mechanisms of resistance supported by arcA, encoding arginine deaminase. Cellular management of Zn stress in S. agalactiae supports virulence by facilitating bacterial survival in the host during systemic infection.IMPORTANCEStreptococcus agalactiae, also known as group B streptococcus, is an opportunistic pathogen that causes various diseases in humans and animals. This bacterium has genetic systems that enable zinc detoxification in environments of metal stress, but these systems remain largely undefined. Using a combination of genomic, genetic, and cellular assays, we show that this pathogen controls Zn export through CzcD to manage Zn stress and utilizes a system of arginine deamination never previously linked to metal stress responses in bacteria to survive metal intoxication. We show that these systems are crucial for survival of S. agalactiaein vitro during Zn stress and also enhance virulence during systemic infection in mice. These discoveries establish new molecular mechanisms of resistance to metal intoxication in bacteria; we suggest these mechanisms operate in other bacteria as a way to sustain microbial survival under conditions of metal stress, including in host environments.
Assuntos
Regulação Bacteriana da Expressão Gênica , Metais/farmacologia , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/patogenicidade , Estresse Fisiológico , Zinco/metabolismo , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Streptococcus agalactiae/genética , Streptococcus agalactiae/crescimento & desenvolvimento , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Transcrição Gênica , Células U937 , Virulência , Zinco/análiseRESUMO
OBJECTIVE: The objective of our study was to explore the possible graphene impact on microorganism growth as well as on laboratory animal overall condition. Materials and technique: The experiments applied samples of graphene three concentrations and two 15 × 15 mm quartz glasses one of which carrying deposited graphene lattice. We have also used 5% blood agar, thioglycollate broth, bacterial suspensions of standard turbidity containing pure clinical isolate of microorganisms. Three white male 6-month-old laboratory rats were used to estimate the graphene impact on the overall animal condition. RESULTS: Graphene did not contain any microorganisms, does not destroy erythrocytes placed within the artificial nutritional medium, graphene lattice did not add any properties to the quartz glasses which could allow the Proteus spread all over its surface. It was also established that graphene did not show any native antibacterial impact. No significant reaction was noticed in animals after graphene administration to laboratory rats neither at the injection spot nor at the overall level. CONCLUSION: Our data confirm the applicability of graphene both in scientific and practical biomedical purposes.
Assuntos
Grafite/toxicidade , Próteses e Implantes/efeitos adversos , Animais , Escherichia coli/crescimento & desenvolvimento , Grafite/química , Teste de Materiais , Medicina/métodos , Testes de Sensibilidade Microbiana , Ratos , Staphylococcus aureus/crescimento & desenvolvimento , Streptococcus agalactiae/crescimento & desenvolvimento , Testes de ToxicidadeRESUMO
Nutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal-binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Although GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here, we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon (Krmit) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative reverse transcriptase PCR (qRT-PCR) revealed a significant upregulation of genes encoding zinc-binding proteins, adcA, adcAII, and lmb, following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant (ΔadcAΔadcAIIΔlmb) compared to growth of the GBS wild-type (WT) strain. Furthermore, mice challenged with the ΔadcAΔadcAIIΔlmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery is important for combatting zinc chelation by calprotectin and establishing invasive disease.IMPORTANCE Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro and in a mouse model of infection. We observed that a global zinc uptake mutant was less virulent than the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by wild-type (WT) and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease.
Assuntos
Complexo Antígeno L1 Leucocitário/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus agalactiae/metabolismo , Zinco/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Feminino , Humanos , Complexo Antígeno L1 Leucocitário/genética , Meningites Bacterianas/genética , Meningites Bacterianas/metabolismo , Meningites Bacterianas/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/genética , Streptococcus agalactiae/crescimento & desenvolvimento , Streptococcus agalactiae/patogenicidade , VirulênciaRESUMO
Treatment of bacterial infections is a great challenge of our era due to the various resistance mechanisms against antibiotics. Antimicrobial peptides are considered to be potential novel compound as antibiotic treatment. However, some bacteria, especially many human pathogens, are inherently resistant to these compounds, due to the expression of BceAB-type ABC transporters. This rather new transporter family is not very well studied. Here, we report the first full characterization of the nucleotide binding domain of a BceAB type transporter from Streptococcus agalactiae, namely SaNsrF of the transporter SaNsrFP, which confers resistance against nisin and gallidermin. We determined the NTP hydrolysis kinetics and used molecular modeling and simulations in combination with small angle X-ray scattering to obtain structural models of the SaNsrF monomer and dimer. The fact that the SaNsrFH202A variant displayed no ATPase activity was rationalized in terms of changes of the structural dynamics of the dimeric interface. Kinetic data show a clear preference for ATP as a substrate, and the prediction of binding modes allowed us to explain this selectivity over other NTPs.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Farmacorresistência Bacteriana Múltipla , Streptococcus agalactiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacteriocinas/farmacologia , Sítios de Ligação , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Hidrólise , Modelos Moleculares , Simulação de Acoplamento Molecular , Nisina/farmacologia , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Espalhamento a Baixo Ângulo , Streptococcus agalactiae/química , Streptococcus agalactiae/genética , Streptococcus agalactiae/crescimento & desenvolvimento , Difração de Raios XRESUMO
Bacterial biofilms are an important virulence factor with a vital role in evasion from the host immune system, colonization and infection. The aim of the present study was to evaluate in vitro the effects of three environmental factors (H+, glucose and human plasma) in biofilm formation, by carrier and invasive Streptococcus agalactiae strains of ST17 and ST19 sequence types, including DNase producers and non-producers. Bacteria ability to assemble biofilms was classified based on crystal violet assay. Biofilm formation was also monitored by scanning electron microscopy. Depending on the growth medium used, each bacterial isolate could fit in different biofilm production categories. Our data showed that optimal conditions for S. agalactiae biofilm assembly were reached after 48 h incubation at pH 7.6 in the presence of glucose and inactivated human plasma. In the presence of inactivated human plasma, the biofilm biomass of ST19 strains experienced a higher increase than ST17 strains. The composition of the extracellular polymeric matrix of the three strongest biofilm producers (all from ST17) was accessed by enzymatic digestion of mature biofilms and proteins were shown to be the predominant component. The detailed identification of the extracellular protein components should contribute to the development of new therapeutic strategies to fight S. agalactiae infections.
Assuntos
Biofilmes/crescimento & desenvolvimento , Streptococcus agalactiae/crescimento & desenvolvimento , Meios de Cultura/metabolismo , Matriz Extracelular de Substâncias Poliméricas/química , Glucose , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Plasma , Streptococcus agalactiae/ultraestrutura , Fatores de Virulência/metabolismoRESUMO
Endometritis represents the main cause of reproductive failure in dromedary camels. In dromedary camels, associations between endometritis-causing pathogen-species, disease severity, and systemic changes in the immune system have not been evaluated. In the current study, there was use of flow cytometry and immunofluorescence of membrane proteins for the evaluation of leukocyte subsets and the cellular phenotype in blood of camels with clinical endometritis and evaluations of associations with disease severity and endometritis-causing pathogens. Animals with endometritis had markedly larger numbers of total leukocytes and neutrophils. Although total lymphocyte and monocyte counts did not differ between camels with and without clinical endometritis, there were lesser numbers of total and effector CD4-positive T cells in camels with endometritis. Among monocytes, number of camel inflammatory monocytes (Mo-II) was markedly greater, whereas Mo-III numbers were less in the blood of camels with clinical endometritis. Number of inflammatory monocytes was also indicative of endometritis severity grade. Among camels with clinical endometritis, E. coli- and S. aureus-infected animals had similar endometritis grades and comparable phenotype and composition patterns of leukocytes. Neutrophils and monocytes of camels with clinical endometritis had fewer cell adhesion molecules (i.e., CD11a and CD18). Collectively, the results from the current study allowed for identification of associations between endometritis severity grade and larger numbers of inflammatory monocytes. The results also indicate there is no association between endometritis pathogen-species and changes in phenotype or composition of blood leukocytes.
Assuntos
Camelus/sangue , Endometrite/veterinária , Leucócitos/classificação , Actinomycetaceae/isolamento & purificação , Animais , Endometrite/sangue , Endometrite/patologia , Endométrio/microbiologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Feminino , Citometria de Fluxo/veterinária , Leucócitos/citologia , Linfócitos/classificação , Linfócitos/citologia , Proteus/isolamento & purificação , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/isolamento & purificação , Streptococcus agalactiae/crescimento & desenvolvimento , Streptococcus agalactiae/isolamento & purificaçãoRESUMO
Streptococcus agalactiae (group B streptococcus, or GBS) is a common cause of bacteremia and sepsis in newborns, pregnant women, and immunocompromised patients. The molecular mechanisms used by GBS to survive and proliferate in blood are not well understood. Here, using a highly virulent GBS strain and transposon-directed insertion site sequencing (TraDIS), we performed genome-wide screens to discover novel GBS genes required for bacterial survival in human whole blood and plasma. The screen identified 85 and 41 genes that are required for GBS growth in whole blood and plasma, respectively. A common set of 29 genes was required in both whole blood and plasma. Targeted gene deletion confirmed that (i) genes encoding methionine transporter (metP) and manganese transporter (mtsA) are crucial for GBS survival in whole blood and plasma, (ii) gene W903_1820, encoding a small multidrug export family protein, contributes significantly to GBS survival in whole blood, (iii) the shikimate pathway gene aroA is essential for GBS growth in whole blood and plasma, and (iv) deletion of srr1, encoding a fibrinogen-binding adhesin, increases GBS survival in whole blood. Our findings provide new insight into the GBS-host interactions in human blood.