Molecular epidemiology, characterization of virulence factors and antibiotic resistance profile of Streptococcus agalactiae isolated from dairy farms in China and Pakistan

Streptococcus agalactiae is one of the most common pathogens that cause bovine mastitis worldwide. Identifying pathogen prevalence and virulence factors is critical for developing prevention and control approaches. Herein, 1 161 milk samples from various dairy farms in China(n=558) and Pakistan(n=603) were collected between 2019–2021 and were subjected to S. agalactiae isolation. Prevalence, serotyping, virulence genes, and antibiotic-resistant genes of S. agalactiae were evaluated by PCR assay. All isolates were characterized for haemolysis, biofilm production, cytotoxicity, adhesion, and invasion on bovine mammary epithelial cells. The prevalence of S. agalactiae-induced mastitis in cattle was found to be considerably higher in Pakistan than in China. Jiangsu and Sindh provinces had the highest area-wise prevalence in China and Pakistan, respectively. Serotypes Ia and II were prevalent in both countries, whereas serotype III was found only in Pakistan. Moreover, all isolates tested positive for PI-2b gene but negative for PI-1 and PI-2a genes. All isolates harboured cfb, cylE, hylB, and fbsB virulent genes, whereas many of them lacked bibA, rib and bca. However, the absence of bac and scp genes in Chinese isolates and cspA in Pakistani isolates was noted, while spb1 and lmb were not detected in isolates of both countries. Pakistani isolates, particularly serotype Iapositive, had a considerably higher ability to produce biofilm, haemolysis, cytotoxicity, adhesion, and invasion than Chinese isolates. Most of the isolates were phenotypically resistant to tetracycline, erythromycin, and clindamycin and genotypic resistance was confirmed by the presence of ermA, ermB, tetM and tetO genes. Our study highlights the antimicrobial resistance profile and virulence-related factors contributing to the epidemiological spread of mastitis-causing S. agalactiae in China and Pakistan. The findings may facilitate future studies designed to develop improved treatment and control strategies against this pathogen.

Antibacterial Activity of Herbal Preparations against Staphylococcus aureus and Streptococcus agalactiae in Cow Mastitis

[Objective] This study aimed to analyze the antibacterial activity of herbal preparations against Staphylococcus aureus and Streptococcus agalactiae in cow mastitis. [Method] The crude drug solutions of four different prescriptions for Zengrujianniusan were prepared through reflux extraction. Their antibacterial activity in vitro against Staphylococcus aureus and Streptococcus agalactiae in cow mastitis were investigated. [Result] All the four different prescriptions exhibited antibacterial activity against S. aureus and S. agalactiae. Among them, prescription Ⅲ was extremely sensitive, and had the best bactericidal effect. The other three prescriptions were highly sensitive. [Conclusion] This study provides a theoretical basis for the development of herbal preparations for the treatment of cow mastitis.

Trend of Invasive Streptococcus agalactiae at Tertiary Care Hospital in Japan

Streptococcus agalactiae causes various severe infectious diseases such as sepsis, meningitis, and streptococcal toxic shock-like syndrome. Though Streptococcus agalactiae infection has been increasing recently, the comprehensive characteristic investigation of invasive Streptococcus agalactiae isolated in tertiary care hospitals has not been nearly performed in Japan. In this study, we investigated the clinical characteristics and antimicrobial susceptible patterns of 88 Streptococcus agalactiae isolated at two tertiary care hospitals during 2009-2015 in Japan. There was no significant differences between genders in our study. Two-third Streptococcus agalactiae were isolated from over age 60. Total mortality rate was 19% and invasive Streptococcus agalactiae-associated death cases have occurred every year after 2011. All Streptococcus agalactiae were completely susceptible toampicillin. Total non-susceptible rates of erythromycin, minocycline, levofloxacin and trimethoprim-sulfamethoxazole in this study were approximately 30%, 44%, 37%, and 7%, respectively. Our results suggest the need for continuous antimicrobial susceptibility survey of Streptococcus agalactiae.

Streptococcus agalactiae:Identification methods,antimicrobial susceptibility, and resistance genes in pregnant women

BACKGROUND Group B Streptococcus(GBS)is a normal component of the gastrointestinal and genital microbiota in humans and can lead to important infections in newborns.AIM To compare GBS isolation and identification methods as well as to assess the antibiotic susceptibility and to identify resistance genes in GBS strains from pregnant women attended in healthcare services from the city of Vitória da Conquista,in Bahia State,Brazil.METHODS From January 2017 to February 2018,vaginorectal swabs were obtained from 186 participants and the samples were seeded onto chromogenic agar for GBS before and after inoculation in selective broth.Confirmatory identification using 3 CAMP and latex tests was performed in samples with GBS-suggestive colonies.Then,disk diffusion antibiograms were performed in GBS-positive samples,and the detection of the resistance genes ermB,ermTR,mefA,and linB in the clindamycin and/or erythromycin-resistant samples was carried out.RESULTS Thirty-two samples(17.2%)were GBS-positive.The culture in chromogenic agar after sample incubation in selective broth was the most sensitive method(96.9%)for GBS detection.All isolates were susceptible to penicillin,ampicillin,cefotaxime,and vancomycin.Clindamycin resistance was observed in 6 samples(18.8%),while 8 samples(25%)were erythromycin-resistant.All erythromycin and/or clindamycin-resistant GBS strains had negative D-tests.Two strains(25%)presented an M phenotype and 6 isolates(75%)presented a cMLSB phenotype.The ermB gene was identified in 4 samples(44.4%),the mefA gene was also found in 4 samples(44.4%),the ermTR gene was identified in 1 isolate(11.1%),and the linB gene was not found in any isolate.CONCLUSION This study evidenced that the screening for SGB can be performed by means of various methods,including chromogenic media,and that the chemoprophylaxis for pregnant women who cannot use penicillin must be susceptibility-guided.

In vitro Antibacterial Activity of Palmitoleic Acid Isolated from Filamentous Microalga Tribonema minus Against Fish Pathogen Streptococcus agalactiae

Filamentous microalgae from genus Tribonema are promising sustainable sources of omega-7 palmitoleic acid,but their ability to accumulate this compound varies among species and depends on the initial nitrogen concentration(INC)supply.In this study,the palmitoleic acid accumulation capacities of five Tribonema species were examined under three different INCs to select the alga species with the highest production.Results showed that a high INC was associated with increased palmitoleic acid accumulation but led to decreased biomass concentration in all tested species.In particular,T.minus grown at 18 mmol L^(−1)INC had the highest palmitoleic acid content(20.72%of dry weight)and productivity(90.88 mg L^(−1)d−1).The combination of alkali metal freezing precipitation(AMFP)and urea complexation successfully isolated and enriched palmitoleic acid from T.minus and obtained a purity of 80.11%and a yield of 7.39 g(100 g)^(−1) of algal powder.The compound was identified as(9Z)-hexadecenoic acid(C16:1ω-7).Antibacterial activity evaluation for the highly concentrated palmitoleic acid(10 mg mL^(−1))against Streptococcus agalactiae revealed the formation of a 12.10 mm-diameter inhibition zone and the minimum inhibitory concentration of 31.25μg mL^(−1),indicating that palmitoleic acid is an effective antibacterial agent.This study is the first to report that palmitoleic acid derived from T.minus can antagonize S.agalactiae,which further broadens the potential application of Tribonema biomass in green aquaculture.

The Streptococcus agalactiae Ribose Binding Protein B (RbsB) Mediates Quorum Sensing Signal Uptake via Interaction with Autoinducer-2 Signals

Understanding aquatic pathogen in sediments or aquacultural water is crucial to protect public health from soilborne and waterborne diseases.Quorum sensing(QS)was increasingly reported in biological wastewater treatment processes because of their inherent roles in biofilm development,bacterial aggregation and so on.The widely QS signals was Antoinducer-2(AI-2),primarily involved to allow the possibility of interspecies communication.However,the cellular components that mediate the response of Streptococcus agalactiae to AI-2 have not been fully characterized.Analysis of the complete genome sequence of S.agalactiae indi-cated that its RbsB protein has similarity to Escherichia coli LsrB and Aggregatibacter actinomycetemcomitans RbsB proteins that bind AI-2.We hypothesized that RbsB protein mediates quorum sensing signal uptake via interaction with AI-2.To evaluate the regulatory effect of RbsB on QS system,the recombinant plasmid pGEX-6p-1-RbsB was constructed and RbsB protein was purified with GST-tag.To further elucidate the role of RbsB protein binding to DPD(AI-2 precursor dihydroxypentanedione),the systemati-cally throughput circular dichroism(CD)spectroscopy,isothermal titration calorimetry200(ITC200)and molecular docking methods were employed.The high expression of soluble RbsB protein with molecular weight of 33 kDa was obtained.The thermodynamics results(ΔH<0,ΔS<0,ΔG<0)with ITC determination indicated that the binding process between DPD and RbsB was exothermic and spontaneous,with hydrogen bonds and van der Waals forces as the main binding forces.Obviously,DPD can be more easily combined with RbsB in a dose-dependent manner,suggesting that RbsB was changed in the microenvironment of DPD when the DPD concentration was between 0.8-1.0mmolL−1 and reaching the maximum binding amount.According to molecular docking,3 hydrophobic residues involved in DPD and RbsB protein stable binding were be found,and also hydrogen bonding plays a key role in the formation of the new complex.RbsB efficiently inhibited V.harveyi bioluminescence induced by both S.agalactiae AI-2 and V.harveyi AI-2 in a dose-dependent manner.However,our results suggest that RbsB may play a role in the response of S.agalactiace to AI-2.

PCR Detection of Streptococcus agalactiae in Dairy Cows Suffering from Mastitis

[ Objective ] This study aimed to identify Streptococcus agalactiae and lay the foundation for the prevention and control of mastiffs in dairy cows. [ Method] Ten strains were isolated from milk samples produced by diseased dairy cows suffering from mastitis for morphologic observation, culture characteristic investigation, biochemical identification and Lancefield grouping. The isolated strains were identified at the molecular level by nested-PCR. [ Result] Among the ten isolates, six strains were 13-hemolytic and Gram-positive on blood agar, belonging to Lancefield group B, which were identified as Streptococcus agalactiae by biochemical identification and nested-PCR. After overnight incubation, the coincidence rate between results of nested-PCR detection and biochemical identification reached 100%. [ Conclusion ] Bacterial incubation, rapid DNA extraction and specific PCR can provide basis for early epidemiological survey of Streptococcus agalactiae infection in cattle.

A Pathological Study of GIFT Strain of Nile Tilapia(Oreochromis niloticus)Infected by Streptococcus agalactiae

[Objectives] This study aimed to determine the infection pathway and target organs of Streptococcus agalactiae in GIFT strain of Nile tilapia, thus providing theoretical basis for the breeding of disease-resistant tilapia and development of S. agalactiae vaccines.[Methods] GIFT strain of Nile tilapia was inoculated by S. agalactiae through intraperitoneal injection, oral gavage and in vitro immersion. The gill, spleen, liver and small intestine tissues of infected tilapia were collected for pathomorphological observation. Immunohistochemical localization was conducted using rabbit anti- S. agalactiae serum to identify the distribution pattern of S. agalactiae in various tissues of tilapia and its target organs via different infection pathways.[Results] GIFT strain of Nile tilapia could be infected by S. agalactiae via three artificial inoculation modes. Specifically, pathological changes occurred at 2 h post-inoculation in intraperitoneal injection and oral gavage groups, whereas tilapia in in vitro immersion group showed pathological changes at 5 h post-inoculation, and the lesion intensity in in vitro immersion group was slighter than that in intraperitoneal injection and oral gavage groups. Immunohistochemical localization indicated that the appearance time of positive signals in intraperitoneal injection group demonstrated an order of spleen→liver and gill→small intestine; positive signals in oral gavage group appeared in the order of small intestine→gill and spleen→liver; the appearance time of positive signals in in vitro immersion group showed an order of gill→spleen→liver and small intestine.[Conclusions] GIFT strain of Nile tilapia could be infected by S. agalactiae via intraperitoneal injection, oral gavage and in vitro immersion. The corresponding positive signals for pathogen infection were preferentially present in the spleen, intestine and gill tissues. Thus, preventing S. agalactiae contamination in aquaculture water and food sources is an effective measure to control the outbreak of S. agalactiae infections in tilapia under natural aquaculture conditions.

Streptococcus agalactiae:Sensitivity profile in pregnant women attending health units in northeastern Brazil

BACKGROUNDG roup B Streptococcus agalactiae (GBS) is the main etiologic agent associated withearly-onset neonatal sepsis, and of all newborns of parturients colonized by GBS,approximately 1%-2% develop invasive, early-onset disease. The risk of infectionincreases to 15.2% in premature neonates, to 10.7% when the parturient haschorioamnionitis or premature rupture of membranes for more than 24 h and to9.7% if the mother has postpartum bacteremia. In addition to causing perinatal,neonatal and postnatal deaths, neonatal hospital infection is associated with highcosts, as hospitalization is three times longer than in uninfected children. Theidentification of pregnant women colonized by GBS, through universal screening,associated with the adoption of appropriate antibiotics at the time of delivery arethe most successful preventive measures.AIMTo evaluate the sensitivity profile of GBS isolated from pregnant womenattending Vitória da Conquista-BA.METHODS This is a cross-sectional study with a quantitative approach carried out in themunicipality of Vitória da Conquista-Bahia between February 2017 and March2018. The study population was composed of 210 pregnant women, with agestational age of 32 to 40 wk, who were aged 18 years or older living in the urbanarea of the municipality of Vitória da Conquista. After a brief explanation aboutthe research and obtaining a signed an informed consent form, data andvaginorectal swabs were collected from the women for GBS research. Examinationof the samples in order to identify the presence of GBS was by culture on sheep blood agar and chromogenic agar for GBS and then, seeded on plates containingstreptococcal culture medium and incubated for 18 h to 24 h at 35°C. Theantimicrobial sensitivity profile of positive GBS samples was determined by thedisk diffusion technique, according to the Clinical and Laboratory StandardsInstitute manual (2017). The data obtained were stored in a database usingMicrosoft Office Excel spreadsheets and a descriptive analysis was performedwith the aid of the EPI-INFO statistical package (version 3.5.2).RESULTSAmong the 210 pregnant women participating in the study, 38 (18.1%) had apositive GBS culture. All strains isolated from GBS were sensitive to 10 Upenicillin, 10 μg ampicillin, 30 μg cefotaxime and 30 μg vancomycin. Seven strains(18.4%) resistant to clindamycin 2 μg and eight (21.1%) resistant to erythromycin15 μg were found. Of these, six were concomitantly resistant to erythromycin andclindamycin, two resistant only to erythromycin and one resistant only toclindamycin. All nine GBS isolates that showed resistance to erythromycin and/orclindamycin showed negative results on the D-test. Two thirds of the isolatesshowed cMLSB phenotype and resistance only to erythromycin in specimens inthis study (02), refers to strains with phenotype M and resistance to clindamycin(01) only with phenotype L.CONCLUSIONChemoprophylaxis for GBS in pregnant women, especially for those allergic topenicillin, should be guided by an antimicrobial susceptibility test as resistantGBS strains were reported in this study.

无乳链球菌(Streptococcus agalactiae)三重PCR快速检测方法的建立与应用

参照GenBank发表的无乳链球菌sip、cpsE这两个高度保守基因设计两组特异性引物,应用已报道的无乳链球菌cpsL基因高度保守序列的引物作为阳性对照引物,经反应条件和反应体系参数优化,建立一种检测无乳链球菌的三重PCR方法,并应用本三重PCR方法检测40尾人工感染罗非鱼样本和22尾临床上收集的疑似无乳链球菌感染鱼样本。结果表明,所建立的三重PCR方法具有良好特异性,检测无乳链球菌DNA样本时出现三条扩增条带,检测其他7种供试菌株DNA则不出现出任何扩增条带,对无乳链球菌DNA样本的最低检测浓度为0.32ng/μl,最适Mg2+浓度为37.5mmol/L,整个检测过程耗时100min左右。40尾人工感染罗非鱼的肝脏、肾脏组织DNA样本阳性检出率分别为100%,22尾疑似无乳链球菌感染鱼的肝脏、肾脏组织DNA样本阳性检出率分别为72.7%,以上两批样本检测结果与常规细菌鉴定方法结果一致。

罗非鱼源无乳链球菌(Streptococcus agalactiae)Sip基因的克隆、鉴定及分子特性分析

利用设计的特异性引物,扩增出了分离自患病罗非鱼无乳链球菌强毒株Sip基因,并将其克隆到pMD19-T载体上,之后对重组质粒进行了PCR和双酶切(BamHⅠ+HindⅢ)鉴定,并利用多种生物信息学软件对Sip基因进行分子特性分析。结果显示,罗非鱼源无乳链球菌Sip编码氨基酸序列具有极高保守性,与人源、哺乳动物源无乳链球菌亲缘性达100%,存在1个由25个氨基酸组成的信号肽,具有1个与免疫调节功能相关的LysM结构域,具有与蛋白翻译后修饰功能相关的磷酸化位点33个和N-糖基化位点2个,编码多肽链中疏水区大于亲水区,是一种膜外蛋白。密码子偏爱性分析表明,罗非鱼源无乳链球菌Sip基因密码子使用频率差异较大,密码子偏爱性与真核生物较为接近。

无乳链球菌(Streptococcus agalactiae)荚膜多糖合成基因研究进展

无乳链球菌是一种人畜鱼共患的重要病原菌,菌体表面的荚膜多糖是公认的毒力因子,其合成过程受荚膜多糖合成基因的调控。荚膜多糖合成基因存在于荚膜多糖操纵子中,参与无乳链球菌荚膜多糖的合成启动、寡糖和多糖的聚合以及外输并锚定于菌体表面,在新型诊断技术和减毒突变株的构建方面取得良好的应用。本文首次就cps基因的基本属性、转录调节、编码蛋白及其生物功能、对荚膜多糖合成的调控机理、在血清分型和突变株构建的应用这六个方面进行深入分析和讨论,以期为GBScps基因的新功能研究和创新应用提供理论参考。

Epidemiological observations of invasive group B Streptococcus infections in six major hospitals in Peninsular Malaysia

Objective:To address the lack of research on invasive group B Streptococcus(GBS)infections in Malaysia and Southeast Asia through a comprehensive analysis of GBS isolates obtained from hospitals.Methods:Medical records from patients with GBS infection isolated from the sterile site,such as blood and cerebrospinal fluid from 14 July 2019 to 15 December 2020,were reviewed from six major hospitals in Peninsular Malaysia.Inclusion criteria were invasive GBS,sterile sites and non-repeated GBS isolated from the same patients in the same admission.Viable isolates were re-identified for GBS and serotyped.Results:A total of 118 patients were eligible,with a majority of non-pregnant adults(76.3%).Over half of the patients(62.7%)had underlying medical conditions,with diabetes as the most common disease,followed by respiratory disease,renal disease,cardiovascular disease and skin and soft tissue disease.The most common manifestations were sepsis,followed by soft tissue abscess,diabetic foot ulcer,wet gangrene and cellulitis.The overall mortality was 7.6%.The most common serotype was serotype桋.Conclusions:Invasive GBS infection among non-pregnant adults showed a rising trend,particularly among diabetic individuals.The study underscores the importance of reducing risk factors and highlights the necessity of developing GBS vaccination as a preventive strategy for both infants and adults.

Study of Serotypes, Susceptibility to Macrolide and Virulence and Resistance Molecular Profiles in Invasive Strains of <i>Streptococcus agalactiae</i>in Two Argentine Provinces

A study of invasive strains of Streptococcus agalactiae (GBS) from Cordoba and Misiones, Argentina;was conducted to determine serotypes, the susceptibility to macrolides and molecular profiles of virulence and resistance. We studied 17 strains, recovered from cerebrospinal fluid, blood and cellulite and, a strain of trophoblastic remnants from Misiones. The serotypes were determined by agglutination with sera and phenotypes of resistance to macrolide-lincosamide-streptogramin B (MLSB), were determined with the double-disk test (D-test). The confirmation was performed by E-test by ERI and CLI respectively that determined the minimum inhibitory concentration (MIC). Results were interpreted as recommended by the Clinical and Laboratory Standards Institute (CLSI) 2013. Resistance genes: ermB, ermTR and mefA and the virulence genes: bac, bca, rib, lmb, hylB, scpB, fbsA, fbsB and cylB were investigated by conventional PCR. Serotype III (50%) and Ia (50%) were detected in Cordoba. One strain showed cMLSB phenotype, confirmed by MIC. The same strains showed a resistance gene ermB. All studied virulence genes were detected in 100% of these strains. In Misiones, serotypes were III (72.7%), Ia (18.2%) and Ib (9.1%). All strains were susceptible to CLI and ERI by D-test, confirmed by MIC. None of the strains showed resistance genes. Virulence genes bca, rib, hylB, lmb, fbsA, fbsB and cylB were detected in 100% of the strains, bac in 81.8% and scpB in 90.9%. Our results are in accordance with international data, associating higher frequency of serotype III of invasive neonatal disease followed by Ia. The presence of serotype Ib could indicate a regional difference for Misiones. We highlight the macrolides susceptibility in strains of Misiones and consistency in the results for D-test, MIC and PCR for the single strain resistant phenotype cMLSB from Cordoba. The virulence genes studied were presented with high frequency as expected for invasive strains.

Molecular Profiles and Antimicrobial Susceptibility of First Isolates of <i>Streptococcus agalactiae</i>Serotype IX in Argentina

Streptococcus agalactiae (GBS) is considered as the most important cause of invasive bacterial disease in infants. There are few current data from serotype IX of GBS worldwide. The present work has been done in order to make a contribution to the knowledge of serotype IX through phenotypic and genotypic study of virulence and resistance. Of 200 strains tested, 5.5% were serotype IX and all were colonizing. In 63.6% of the strains the presence of the bac, bca and hylB genes was determined, and in 54.5% of lmb and rib. All strains were susceptible to Clindamycin and Erythromycin however five isolates showed the resistance genes: ermB, ermTR and/or mefA. The presence of serotype IX in Misiones, a province situated in the northeaster of Argentina, which limits with Paraguay and Brazil in South America, gives the region a particular situation. Currently, public health efforts are aimed at prevention and treatment, study of the virulence mechanisms, surveillance of resistance to antibiotics and the development of effective vaccines to prevent GBS infection.

罗非鱼源无乳链球菌(Streptococcus agalactiae)新型AI-2信号分子受体RbsB蛋白结晶生长研究

为了开展无乳链球菌(Streptococcus agalactiae)核糖结合蛋白(Ribose binding protein B,RbsB)结构功能的研究,本实验根据已知无乳链球菌ZQ0910全基因组序列设计相关引物。采用PCR方法扩增其RbsB基因,随后将该基因定向克隆到原核表达载体pGEX-6p-1中,在大肠杆菌BL21(DE3)感受态细胞中进行IPTG诱导表达;采用HRV 3C蛋白酶切除GST标签,分子筛分离获得RbsB蛋白;运用生物信息学软件对RbsB基因序列进行分析,并对RbsB蛋白二级和三级结构进行预测;采用NeXtal Tubes JCSG Core Suite结晶试剂盒筛选蛋白结晶条件。研究结果表明,该基因全长为969碱基,编码322个氨基酸,RbsB蛋白理论分子量33.9ku,等电点为9.41,二级结构中α螺旋结构所占比重最高,建立RbsB蛋白三维结构模式图;经IPTG诱导后表达的融合蛋白分子量为59ku,筛选RbsB蛋白的初始结晶条件为(0.2mol/L di-Potassium hydrogen phosphate,20%(W/V)PEG3350;1.5mol/L ammonium sulfate,25%(V/V)Glycerol),获得RbsB蛋白结晶体。本研究结果可为无乳链球菌核糖结合蛋白(RbsB)的功能解析提供实验及理论基础。

Strain-specific effect of Streptococcus thermophilus consumption on host physiology

Streptococcus thermophilus is one of the most prevalent species in stool samples of westernized populations due to continuous exposure to fermented dairy products.However,few studies have explored the effect on host physiology by multiple S.thermophilus strains and considered the inter-strain differences in regulating host.In the present study,we investigated how four S.thermophilus strains influenced the gut microbiota,mucin changes,and host metabolism after 28 days of intervention in conventional mice.The results indicated that the consumption of S.thermophilus affected the host with strain specificity.Among four S.thermophilus strains,DYNDL13-4 and DQHXNQ38M61,especially DQHXNQ38M61,had more effect on host physiology by modulating gut microbiota and host metabolism than LMD9 and 4M6.Ingestion of strains DYNDL13-4 and DQHXNQ38M61 resulted in more remarkable changes in amino acid metabolism and lipid metabolism than that of strains LMD9 and 4M6,which may be related to the elevation of intestinal Bifidobacterium by DYNDL13-4 and DQHXNQ38M61.The enriched Coriobacteriaceae UCG-002,Rikenellaceae RC9 gut group,and Lactobacillus only in the DQHXNQ38M61 group,had a close relationship with the prominent effect of DQHXNQ38M61 on regulating amino acid and lipid metabolism.In addition,DQHXNQ38M61 had a strong influence on degrading colonic mucin fucose by decreasedα-fucosidase activity in feces,and improving mucin sulfation by upregulated Gal3ST2 expression.Comparative genomic analysis revealed that the four S.thermophilus strains belonged to different branches in the phylogenetic tree,and DYNDL13-4 and DQHXNQ38M61 had more genes involved in carbohydrate metabolism,amino acid metabolism,membrane transport,and signal transduction,which may confer the capacity of nutrient utilization and gastrointestinal adaptation of the strains and be associated with their strong regulation in host.Our study provides valuable information for understanding the regulation of host metabolism after consuming different S.thermophilus strains and could facilitate potential personalized applications of S.thermophilus based on strain varieties.

Antibiotic Resistance Profile of Serotypes of Streptococcus pneumoniae Strains in Bangui, from 2017 to 2022: Case of Serotype 1

Goals: The aim of this study was to determine the antibiotic resistance profile of serotypes of Streptococcus pneumoniae strains circulating in Bangui. Methodology: A prospective and analytical analysis was carried out at the National Laboratory of Clinical Biology and Public Health from 2017 to 2022. The strains came from our study on the contribution to the study of antibiotic sensitivity of Streptococcus pneumoniae strains. The multiplex PCR test was used for its cost-effectiveness in terms of amplifiers which can be purified in order to be sequenced. It also makes it possible to detect several germs as well as their serotypes. For a PCR reaction, several elements are involved in the reaction medium or Master Mix. These are the desoxyribonucleotides (dNTPs), the magnesium ions (MgCl2) and the primers. A set of 14 primers divided into 3 classes were used. Class 1 primers served as an internal control by targeting the cpsA gene. It is a highly conserved gene found in capsular loci characterized to date. The primers of the second class were used to target specific serotypes by specific reactions (out of six possibilities). The group reaction was carried out using the primers of the third class in order to carry out an initial screening of the samples and to classify the pneumococcal isolates. Related serotypes were grouped based on the amplification of common genes. Using the technique of electrophoresis on agarose gel and an ultraviolet radiation device, the migration bands are then visualized and analyzed. The data collected had been entered into Excel 2010 and analyzed with Epi info 7. The exact Fischer chi2 test at the 5% threshold, the relative risk and its 95% confidence interval were used to compare the proportions and determine the associations. Results: 187 antibiotic-resistant strains of Streptococcus pneumoniae were collected. The average frequency of serotypes 1, 9A, 4 and untypeable identified were 43.59%, 18.18%, 18.27% and 39.57% respectively. The frequency of serotype 1 was predominant for the age group over five years old with 56.88%. The male sex was predominant with 55.08% for serotype 1. Resistance to penicillin and gentamicin for serotype 1 during this study, for the age group under 5 years old, was 77%. For serotypes 19A and 4, tetracycline resistance was predominant with 20% for the age group under 5 years. The resistance to penicillin and gentamicin of non-typeable serotypes was 33% for the age group under 5 years old. For the age group over 5 years old, resistance to erythromycin predominated at 37%. The distribution of serotypes by sex depending on antibiotic resistance was variable. There was a statistically significant association between identified serotypes and antibiotic resistance (p Conclusion: The study determined serotypes 1, serotypes 19A, serotypes 4 and non-typeable serotypes. These results would be due to the quality of vaccination or poor protection of vaccines.

Protective eff ect and mechanism of nanoantimicrobial peptide ND-C14 against Streptococcus pneumoniae infection

BACKGROUND:Streptococcus pneumoniae(S.pneumoniae)is a common pathogen that causes bacterial pneumonia.However,with increasing bacterial resistance,there is an urgent need to develop new drugs to treat S.pneumoniae infections.Nanodefensin with a 14-carbon saturated fatty acid(ND-C14)is a novel nanoantimicrobial peptide designed by modifying myristic acid at the C-terminus of humanα-defensin 5(HD5)via an amide bond.However,it is unclear whether ND-C14 is effective against lung infections caused by S.pneumoniae.METHODS:In vitro,three groups were established,including the control group,and the HD5 and ND-C14 treatment groups.A virtual colony-count assay was used to evaluate the antibacterial activity of HD5 and ND-C14 against S.pneumoniae.The morphological changes of S.pneumoniae treated with HD5 or ND-C14 were observed by scanning electron microscopy.In vivo,mice were divided into sham,vehicle,and ND-C14 treatment groups.Mice in the sham group were treated with 25μL of phosphate-buffered saline(PBS).Mice in the vehicle and ND-C14 treatment groups were treated with intratracheal instillation of 25μL of bacterial suspension with 2×108 CFU/mL(total bacterial count:5×10^(6) CFU),and then the mice were given 25μL PBS or intratracheally injected with 25μL of ND-C14(including 20μg or 50μg),respectively.Survival rates were evaluated in the vehicle and ND-C14 treatment groups.Bacterial burden in the blood and bronchoalveolar lavage fluid were counted.The lung histology of the mice was assessed.A propidium iodide uptake assay was used to clarify the destructive eff ect of ND-C14 against S.pneumoniae.RESULTS:Compared with HD5,ND-C14 had a better bactericidal eff ect against S.pneumoniae because of its stronger ability to destroy the membrane structure of S.pneumoniae in vitro.In vivo,ND-C14 significantly delayed the death time and improved the survival rate of mice infected with S.pneumoniae.ND-C14 reduced bacterial burden and lung tissue injury.Moreover,ND-C14 had a membrane permeation eff ect on S.pneumoniae,and its destructive ability increased with increasing ND-C14 concentration.CONCLUSION:The ND-C14 may improve bactericidal eff ects on S.pneumoniae both in vitro and in vivo.

Membrane vesicles derived from Streptococcus suis serotype 2 induce cell pyroptosis in endothelial cells via the NLRP3/Caspase-1/GSDMD pathway

Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different organs,S.suis 2 must colonize the host,break the blood barrier,and cause exaggerated inflammation.In the last few years,most studies have focused on a single virulence factor and its influences on the host.Membrane vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host interactions.Gram-negative bacteria-derived outer membrane vesicles(OMVs)were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide(LPS),causing host cell pyroptosis.However,little is known about the effect of the MVs from S.suis 2(Gram-positive bacteria without LPS)on cell pyroptosis.Thus,we investigated the molecular mechanism by which S.suis 2 MVs participate in endothelial cell pyroptosis.In this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by S.suis 2.First,we demonstrated that S.suis 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were identified.Then,we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis.The S.suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 pathway.More importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by S.suis 2 MVs.The results in this study suggest for the first time that MVs from S.suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage,which produced mtDNA and ROS under induction,leading to the pyroptosis of endothelial cells.