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.

A Microwave-Irradiated <i>Streptococcus</i><i>agalactiae</i>Vaccine Provides Partial Protection against Experimental Challenge in Nile Tilapia, <i>Oreochromis</i><i>niloticus</i>

Microwave irradiation, as opposed to formalin exposure, has not routinely been used in the preparation of killed vaccines despite the advantages of decreased chemical toxicity, ability to kill cells quickly, ease of completion requiring only a standard microwave, and potential increased protein conservation during irradiation. We evaluated the potential of microwave irradiation versus formalin fixation of bacteria to improve Streptococcus agalactiae vaccine efficacy in 5 gr fish by intraperitoneal (IP) injection and bath immersion (BI). There was no significant difference in the cumulative percent mortality (CPM) post-challenge between fish administered microwave-killed cells (MKC) or formalin killed cells (FKC) within the BI (p S. agalactiae antibody activity. Thirty days after vaccination and just prior to challenge, the optical density (OD) levels of the FKC and MKC groups in the IP trials were significantly higher (p < 0.0001) than that of the TSB group. None of the groups in the BI trial exhibited significantly different OD levels post vaccination. Fourteen days after the challenge, the OD levels of all groups in both trials increased significantly above their pre-challenge levels. Both the FKC and MKC IP groups (p < 0.0001) and only the FKC BI group (p < 0.0351) had significantly increased OD level above that of the corresponding post-challenge TSB group. These results indicate that the FKC vaccine provides marginally greater protection and increased antibody concentrations than the MKC vaccine by BI and the MKC vaccine may become a non-chemical alternative to FKC in vaccination.

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.

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.

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.

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.

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.

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.

Analysis of anti-infection therapeutic schedule analysis for neonatal Streptococcus agalactiae meningitis and discussion on the indications for drug withdrawal

The clinical manifestations of neonatal GBS meningitis are as follows:non-specificity,long hospital stay,easy to cause neurological complications,and sequelae.Clinical pharmacist participated in the anti-infective treatment of a neonatal Streptococcus agalactiae meningitis whose cerebrospinal fluid was still abnormal when the drug was stopped.According to the PK/PD characteristics of antibacterial drugs,the permeability of the blood-brain barrier,and the adjustment of the antibiotic dose,the reasons for the decrease in the number of cerebrospinal fluid cells after vancomycin treatment were analyzed.Clinical pharmacists also analyzed the efficacy of linezolid in the treatment of neonatal bacterial meningitis,and discussed the indications for stopping medication when the cerebrospinal fluid is abnormal,so as to improve the safety and effectiveness of antibiotics in the treatment of neonatal bacterial meningitis.

Streptococcus agalactiae Anterior Cervical Space and Superior Mediastinum Abscess in a Liver Transplant Recipient

Introduction Streptococcus agalactiae is most commonly associated with pregnancy-related infections,neonatal sepsis,and infections in patients with immunocompromised states,such as diabetes mellitus and malignancies.1 Although abscesses caused by S.agalactiae are sometimes encountered in immunocompromised patients,S.agalactiae abscess in transplant recipients is extremely rare.S.agalactiae abscesses have never been reported in liver transplant recipients.In this article,we report a case of S.agalactiae abscess in the anterior cervical space extending to the superior mediastinum in a liver transplant recipient.

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.

广东地区感染养殖罗非鱼的无乳链球菌分子分型研究

以广东省内5个罗非鱼Oreochromis niloticus主要养殖区内分离的无乳链球菌Streptococcus agalactiae为研究目标,通过毒力基因PCR检测、多位点序列分型、脉冲场凝胶电泳技术研究广东地区感染罗非鱼的无乳链球菌分子特征。结果发现170株分离无乳链球菌中毒力基因的携带率介于60.59%~100%之间,毒力基因携带形成10个谱系,dltR-bca-sod A-spb1-cfb-bac（38.24%,65/170）,dltr-bca-sod A-spb1-bac（23.53%,40/170）和dltr-bca-sod A-spb1-cfb-bac-scp B（22,35%,38/170）为主要的谱系,dltr-bca-sod A-spb1-bac-scpb（8.24%,14/170）和dltr-bca-sod A-spb1（4.71%,8/170）次之。多位点序列分型研究证实170株菌在7个位点均为单一的等位基因,为adh P（10）,phe S（1）,atr（2）,gln A（1）,sdh A（3）,glc K（2）,tkt（2）,序列型为ST7型。146株菌脉冲场凝胶电泳结果表明146株菌分为6个簇（A-D,F,G）,相似性介于86%~100%之间。A（18）,B（16）,C（103）,D（7）为主要的遗传簇,占菌株数的98.63%（144/146）。基因簇A,C在4个采样区均有发现;基因簇B分布在吴川、茂名和阳江;基因簇D分布在茂名、珠海和阳江;基因簇F和G分别来自茂名和珠海。在A-D遗传簇中均有发现来自不同地区的菌株呈现100%的相似性,为同一克隆株。结果表明广东省主要罗非鱼养殖区内感染链球菌携带毒力基因比率高,序列型均为高致病的ST7型别,不同地区间具有相同的PFGE型别,不同地区间交叉传播感染的可能高。

一株梭鲈源无乳链球菌的分离、鉴定及分子分型分析

为查明梭鲈(Lucioperca lucioperca)凸眼病病原,从患病梭鲈病灶眼、肝、脾和肾分离纯化病原菌,进行了生理生化特性测定及16S rRNA基因序列分析,并开展人工感染试验,测定其血清型及MLST分型,并利用纸片扩散法进行药敏特性分析。结果显示:分离菌株(命名为SL1701株)为本次引发梭鲈病害的致病菌,Ib-ST261型。回归感染实验证实,分离得到的细菌SL1701对健康梭鲈具有非常强的致病性,4.5×105CFU/m L时可使80%受感染梭鲈死亡,并可复制出自然发病鱼的症状。菌株SL1701革兰氏染色为阳性链球菌,γ溶血,生理生化特性与普通无乳链球菌基本一致,16S rRNA基因序列与无乳链球菌(Streptococcus agalactiae)同源性最高,综合判定分离菌株为无乳链球菌(Streptococcus agalactiae)。药敏试验结果显示:分离菌株SL1701对氟苯尼考、氟罗沙星等17种抗生素高度敏感,对青霉素、磷霉素等8种抗生素耐药。结果表明,分离菌株SL1701为Ib-ST261型无乳链球菌,养殖时可选用氟苯尼考及氟罗沙星等药物进行防控。

无乳链球菌LIP蛋白的截短表达及免疫保护性研究

为评价罗非鱼源无乳链球菌(Streptococcus agalactiae)BX2012株脂蛋白(lipoprotein)的免疫原性及其对奥利亚罗非鱼(Oreochromis aureus)和大菱鲆(Scophthalmus maximus)的保护效果,以无乳链球菌脂蛋白基因序列(GenBank序列号:CP000114.1,SAK_0321)的B细胞线性抗原表位区设计特异性引物进行扩增,构建重组表达载体,将截短表达的脂蛋白制备成亚单位疫苗,同时制备灭活疫苗进行免疫比对。结果显示:重组表达载体p ET-32a-LIP342在BL21(DE3)中获得了良好的可溶性表达,分子质量约为30 kDa,纯化使LIP342纯度由41.75%提至87.23%;Western blotting分析显示,LIP342可被兔抗无乳链球菌高免血清特异性识别;LIP342在目前已知不同种属来源或不同血清型的无乳链球菌分离株中同源性为90.35%~100%,在罗非鱼源分离株中同源性为100%;无乳链球菌LIP342蛋白和灭活疫苗均可显著提升奥利亚罗非鱼和大菱鲆血清抗体水平,而LIP342诱导的血清抗体水平均显著高于灭活疫苗;经0.1 mL 1×10~9cfu/mL的无乳链球菌攻毒后,LIP342蛋白和灭活疫苗对供试鱼的累积存活率均显著高于PBS对照组。结果表明,高度保守的LIP342具有较好的免疫原性,可作为无乳链球菌亚单位疫苗候选因子。

无乳链球菌不同途径感染吉富罗非鱼后病原菌在体内的分布规律研究

为探讨无乳链球菌(Streptococcus agalactiae)感染罗非鱼(Oreochromis spp)的致病途径。采用腹腔注射、灌胃和浸泡三种方式对吉富罗非鱼进行无乳链球菌(HN016菌株)胁迫感染,利用平板活菌计数法统计三种方式感染后病原菌在体内组织的分布。注射和灌胃两种方式感染后均出现典型的链球菌感染发病症状,其中注射组在感染24 h后出现死亡高峰,死亡率为92.5%;灌胃组感染48 h后出现死亡高峰,死亡率为90%;而浸泡组,感染后均没有出现明显的发病症状,也没有出现死鱼。注射组和灌胃组在感染后2 h,其脾脏、肝脏、前肾、胃、腮、皮肤和肌肉组织中均可分离出病原菌,5 h后在脑组织中均可分离出病原菌,8 h后各组织分离出的病原菌数达到峰值;而浸泡组在感染8 h后才从各组织中分离出病原菌,且它们的数量均低于同时期的注射组和灌胃组。注射和灌胃两种方式可使吉富罗非鱼快速感染无乳链球菌而发病,而浸泡方式感染后病原菌虽可以侵入机体,但不表现出症状。由此,我们推测在自然条件下养殖的罗非鱼是通过口腔采食携带无乳,链球菌的食物而被感染。

无乳链球菌GlnR因子DNA结合位点预测及其突变基因的构建

利用生物信息学的方法对无乳链球菌（Streptococcus agalactiae）GlnR因子与DNA结合的位点进行预测,筛选出GlnR因子中与DNA结合的关键位点的氨基酸残基R47和R48.然后利用重叠PCR的方法将glnR基因上的第139～144位上的碱基CGCCGC突变为GCGGCG,使其编码的2个精氨酸残基突变为丙氨酸残基,将突变基因片段TA克隆至PMD 18-T载体上进行测序验证.测序结果表明,利用重叠PCR成功突变了这6个碱基,为后续研究GlnR因子的突变位点对DNA结合的影响提供了参考.