耐药猪链球菌膜蛋白图谱分析及能量抑制剂对其敏感性的影响

为证实国内临床分离的动物源链球菌菌株中是否存在与红霉素耐药性相关的主动外排机制,选取含有mefA基因和ermB基因和不含mefA/ermB基因的链球菌耐药菌株和青霉素、红霉素均敏感菌株,在能量抑制剂氰氯苯腙(CCCP)存在时,采用棋盘法分析红霉素、阿齐霉素、替米考星、青霉素对分离菌MIC的影响,并采用SDS-PAGE电泳分析分离株的膜蛋白图谱.结果显示:CCCP可对含有mefA基因的耐药菌株产生影响,可增强其对红霉素、阿齐霉素的敏感性,但不影响其对替米考星和青霉素的敏感性.含有ermB基因、不含mefA/ermB基因的耐药菌,对红霉素、阿齐霉素、替米考星和青霉素的敏感性不受CCCP的影响.菌株膜蛋白的SDS-PAGE电泳图谱显示,含有mefA基因的耐药菌株与其他菌株的膜蛋白图谱差异较大,其膜蛋白条带多而深浓,在45 000处有明显的条带,其他菌株无该条带,推测该蛋白可能是膜主动外排的相关蛋白.本试验结果佐证了在链球菌内存在与红霉素耐药相关的主动外排机制.     

猪链球菌2型福建株的分离鉴定

从临床表现为败血症的濒死病猪肝脏、肺脏、淋巴结中分离到一株链球菌,进行形态学、生化特性、小白鼠感染试验及PCR鉴定和CPS2 J序列测定.结果表明:该分离菌的形态、培养和生化特性基本符合链球菌的特点;人工感染试验显示,小白鼠腹腔接种0.2 mL分离菌培养物不发病,但接种1 mL能在48 h内致小白鼠死亡,并能从脏器中分离到接种菌;分离菌的PCR扩增产物与猪链球菌2型阳性参考菌株JA070109大小一致、长度为675 bp的特异性条带,其PCR产物的核苷酸序列与GeneBank上公布的猪链球菌2型不同菌株的同源性高达98%-100%.可见,该分离菌为猪链球菌2型,并首次证实福建存在猪链球菌2型.     

上海部分地区猪链球菌2型的流行病学调查及其耐药性

猪链球菌2型是一种重要的人兽共患病病原,由其引起的猪链球菌病给养猪业带来巨大的危害。本研究通过PCR方法扩增谷氨酸脱氢酶(GDH)基因和荚膜多糖(CPS)基因检测猪链球菌2型,对上海部分地区猪链球菌2型的流行病学进行调查,并对分离到的菌株进行生化鉴定及耐药性研究。结果显示,上海部分地区猪链球菌的检出率为41.2%(103/250),猪链球菌2型的检出率为12.4%(31/250),猪链球菌2型占猪链球菌的30.1%。猪链球菌2型菌株对四环素、链霉素和氯霉素的耐药性最为严重,多数菌株呈多重耐药性,其中以4重耐药性为主。这为上海地区猪链球菌病的监管提供了依据,并对临床治疗使用抗生素具有参考价值。     

猪链球菌2型及7型分离株对16种抗生素的耐药性分析

[目的]有效控制猪链球菌的传播。[方法]测定121株2型和7型湖南地区分离株对16种抗生素的耐药性。[结果]结果发现,猪链球菌湖南分离株（2型和7型）对红霉素、万古霉素、克林霉素和四环素具有高频的抗性,对氟哌酸、左氟沙星、环丙沙星、克拉霉素和青霉素G等抗生素较为敏感。细菌对敏感抗生素的抗性有上升趋势。[结论]该研究为养猪业临床治疗猪链球菌感染的用药提供依据。     

人工诱导猪链球菌氟喹诺酮耐药株的靶位突变分析

以4株临床分离的对环丙沙星和恩诺沙星敏感的猪链球菌2型菌株为研究对象,采用体外递增药物浓度的方法分别诱导了其对环丙沙星和恩诺沙星耐药的菌株,按CLSI推荐方法测定了环丙沙星和恩诺沙星对亲本敏感株和诱导耐药株的MIC,测定了亲本株和诱导耐药株的生长曲线,并采用PCR和基因测序的方法分析了诱导耐药株的DNA回旋酶(GyrA和GyrB)和拓扑异构酶Ⅳ(ParC和ParE)耐药决定区(QRDR)的基因突变和氨基酸序列变化.结果表明:浓度递增法成功诱导了猪链球菌对环丙沙星和恩诺沙星耐药性,其MIC分别由05 mg·L-1上升至128 mg·L-1;与敏感株比较,恩诺沙星与环丙沙星诱导的耐药菌在gyrA和gyrB,或parC和parE耐药决定区的氨基酸序列有突变,除了已报道的与氟喹诺酮耐药相关的ParC的Ser79Phe,GyrA的Ser81Arg,GyrB的Asp315Asn、Ser285Leu和Glu354Lys及ParE的Pro278Ser点突变外,在诱导菌中还出现了一些不曾报道的突变位点和氨基酸缺失,如GyrA的Gln118His和ParE的Asn297Tyr突变,GyrB的288～291位和ParC的62位氨基酸缺失.结果提示:逐步增加药物浓度可以诱导猪链球菌对氟喹诺酮类抗菌药耐药性,并导致主要靶位发生突变.     

猪链球菌的分离鉴定和药敏试验

从豫北的安阳县猪场采集的病料中分离出了5株猪链球菌。经细菌分离培养、生化试验及兰氏分群A—G诊断试剂鉴定,分离出E群链球菌2株,C群链球菌2株,D群链球菌1株。药敏试验结果表明：分离的链球菌对氨苄西林、头孢他啶和头孢曲松高度敏感,对青霉素、复方新诺明和土霉素等药物产生耐药性。     

链霉素抗性筛选吸水链霉菌高产农用抗生素菌株研究

[目的]筛选吸水链霉菌的高产农用抗生素菌株。[方法]从海南土壤中筛选出1株吸水链霉菌,以经过自然分离和2次紫外诱变的S6-7为出发菌株,经过紫外光照射辅以链霉素抗性筛选后,再进行摇瓶发酵复筛。[结果]从62株随机挑选的单菌落中初筛出7株较好菌株,经斜面连续传代3次后复筛,发现5株菌株具有较好的正突变,正突变率可达8.06%,效价最高提高25.11%。[结论]将链霉素筛选法与传统紫外诱变法相结合,使菌株的产素能力有较大提高,而且极大地提高了菌种正向突变的筛选效率。     

链霉素抗性筛选吸水链霉菌高产农用抗生素菌株研究（英文）

[Objective] The research aimed to screen Streptomyces hygroscopicus strains with high production of agricultural antibiotics.[Method] A strain of S.hygroscopicus was screened from the soil of Hainan Island.After natural screening and consecutive ultraviolet induced mutation twice,S6-7 strain was obtained as the original strain then treated by UV irradiation and streptomycin resistance screening,and finally rescreened through shake-flask fermentation.[Result] 7 better strains were selected by primary screening from 62 single colonies which were picked out randomly.After 3 generations of consecutive cultivation on slant media and rescreening,5 strains presented obvious forward mutation.The forward mutation rate reached 8.06%,and the largest production increasing rate came up to 25.11%.[Conclusion] By combining streptomycin resistance screening and conventional ultraviolet induced mutation,both the antibiotic-producing capacity and forward mutation screening efficiency of the original strain were greatly enhanced.     

链霉素抗性筛选吸水链霉菌高产农用抗生素菌株研究

农用抗生素作为一类生物农药,具有使用剂量低,环境相容性好,选择性强的特点,因此受到普遍重视。目前已发现的天然抗生素有约2／3来源于链霉菌。利用链霉菌产抗生素能力与链霉素抗性基因之间的对应关系来定向筛选正向突变株,是目前农用抗生素科研领域的研究热点。     

Genetic characterization of antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis cases in Northwest China

Staphylococcus aureus is the most common etiological pathogen of bovine mastitis. The resistant strains make the disease difficult to cure. The aim of this study was to characterize the genetic nature of the antimicrobial resistance in S. aureus cultured from bovine mastitis in Northwest China in 2014. A total of 44 S. aureus were isolated for antimicrobial resistance and resistance-related genes. Antimicrobial resistance was determined by disc diffusion and the corresponding resistance genes were detected by PCR. Phenotype indicated that S. aureus isolates were resistant to penicillin(84.09%), erythromycin(20.45%), tetracycline(15.91%), gentamicin(9.09%), tobramycin(6.82%), kanamycin(6.82%) and methicillin(2.27%). 9.09% of the S. aureus isolates were classified as multidrug resistant. In addition, genotypes showed that the isolates were resistant to rifampicin(100%, rpo B), penicillin(95.45%, bla Z), tetracycline(22.73%, tet K, tet M, alone or in combination), erythromycin(22.73%, erm B or erm C), gentamicin/tobramycin/kanamycin(2.27%, aac A-aph D), methicillin(2.27%, mec A) and vancomycin(2.27%, van A). Resistance to tetracycline was attributed to the genes tet K and tet M(r=0.558, P 0.001). This study noted high-level geno- and phenotypic antimicrobial resistance in S. aureus isolates from bovine mastitis cases in Northwest China.     

Detection of antimicrobial resistance and virulence-related genes in Streptococcus uberis and Streptococcus parauberis isolated from clinical bovine mastitis cases in northwestern China

The objectives of this study were to investigate antimicrobial resistance of Streptococcus uberis and Streptococcus parauberis isolated from cows with bovine clinical mastitis in China and to examine the distribution of resistance- and virulence-related gene patterns. Antimicrobial susceptibility was determined by the E-test. Genes encoding antimicrobial resistance and invasiveness factors were examined by PCR. A total of 27 strains were obtained from 326 mastitis milk samples. Streptococcus parauberis isolates (n=11) showed high resistance to erythromycin (90.9%), followed by tetracycline (45.5%), chloramphenicol (36.4%) and clindamycin (27.3%). Streptococcus uberis isolates (n=16) were highly resistant to tetracycline (81.3%) and clindamycin (62.5%). Both species were susceptible to ampicillin. The most prevalent resistance gene in S. uberis was tetM (80.0%), followed by blaZ (62.5%) and ermB (62.5%). However, tetM, blaZ, and ermB genes were only found in 27.3, 45.5, and 27.3%, respectively, of S. parauberis. In addition, all of the isolates carried at least one selected virulence-related gene. The most prevalent virulence-associated gene pattern in the current study was sua+pauA/skc+gapC+hasC detected in 22.2% of the strains. One S. uberis strain carried 7 virulence-associated genes and belonged to the sua+pauA/skc+gapC+cfu+hasA+hasB+hasC pattern. More than 59.3% of analysed strains carried 4 to 7 virulence-related genes. Our findings demonstrated that S. parauberis and S. uberis isolated from clinical bovine mastitis cases in China exhibited diverse molecular ecology, and that the strains were highly resistant to antibiotics commonly used in the dairy cow industry. The data obtained in the current study contribute to a better understanding of the pathogenesis of bacteria in mastitis caused by these pathogens, and the findings are relevant to the development of multivalent vaccines and targeted prevention procedures.     

临床分离猪链球菌和副猪嗜血杆菌多重耐药性监测

为了解我国规模化猪场链球菌和副猪嗜血杆菌的耐药情况,用纸片扩散法对临床分离的112株猪链球菌和92株副猪嗜血杆菌进行23种药物的敏感性试验。结果表明,猪链球菌对林可胺类、大环内酯类、硝基呋喃类药物的耐药率均在90%以上,对链霉素、庆大霉素、卡那霉素、阿米卡星、多西环素的耐药率在60%～90%之间,而对β-内酰胺类和氟喹诺酮类的耐药率相对较低。副猪嗜血杆菌对四环素耐药率最高(78.3%),对螺旋霉素、复方磺胺甲噁唑、克林霉素、阿米卡星、林可霉素、新霉素的耐药率在50%～70%之间,对头孢类和氟喹诺酮类耐药率相对较低。2种细菌对不同种类药物的敏感性各异,并且多重耐药性严重,主要耐药谱具有多样性。     

人工感染猪链球菌对小鼠肝脏药物代谢酶的抑制作用

 【目的】评价猪链球菌感染对小鼠肝脏药物代谢酶活性的影响。【方法】120只雄性ICR小鼠,随机分为对照组和2个感染组,后者分别人工感染猪链球菌2型强毒力株HA9801和弱毒力株SS2-H,于感染不同时间测定小鼠体内肝脏药物代谢酶活性的变化。【结果】感染HA9801菌株的小鼠,细胞色素P450（CytP450）、细胞色素b5（Cytb5）、NADP-细胞色素C还原酶（NCCD）活性从第3日开始极显著（P＜0.01）或显著(P＜0.05)降低,第10日时降至最低（NCCD于第5天时最低）,第15日时活性有所增强,但仍显著低于对照组;红霉素-N-脱甲基酶（ERND）活性从第5日开始极显著地降低（P＜0.01）,变化趋势与CytP450、Cytb5相似;氨基比林-N-脱甲基酶（AND）活性虽有变化,但与对照组比较,差异不显著（P＞0.05）;苯胺-4-羟化酶（AH）活性在第10日时开始极显著地下降（P＜0.01）,第15日时稍有上升。感染SS2-H的小鼠,CytP450、Cytb5的活性从第5日开始显著降低(P＜0.05),第10日降至最低;NCCD、ERND和AH活性第10日时显著降低(P＜0.05),而AND活性在感染期间未发生显著变化（P＞0.05）。HA9801感染组与SS2-H感染组小鼠的谷胱甘肽巯基转移酶（－GSH-S-T）活性从感染后第3日开始即显著(P＜0.05)降低。HA9801感染组与SS2-H感染组比较,CytP450、Cytb5活性在感染前期均有极显著（第3日、第5日）差异（P＜0.01）,而在感染后期,差异不显著（P＞0.05）。微粒体蛋白浓度各组差异不显著（P＞0.05）。【结论】猪链球菌感染可调节小鼠肝脏药物代谢酶CytP450、Cytb5、NCCD、ERND、AH和GSH-S-T活性,故在感染期间选择药物进行治疗时,应制定合理的给药方案,避免药物毒性反应产生,加重病情。     

绍兴市售酸奶中乳酸菌的分离鉴定及耐药性分析简

[目的]从绍兴市售酸奶中分离并鉴定乳酸菌,研究乳酸菌分离株对6种抗生素的耐药性。[方法]利用MRS培养基迎过稀释法分离酸奶样品中的乳酸菌并进行菌落计数,对分离菌株进行革兰染色和生化反应鉴定,同时采用纸片扩散法进行耐药性分析。[结果]从15种绍兴市售酸奶样品中共分离出31株菌,生化反应鉴定为嗜酸乳杆菌（6株）、保加利亚乳杆菌（7株）、嗜热链球菌（11株）和双歧杆菌（7株）;活菌计数结果显示,4份样品（26．7％）中乳酸菌计数低于国家标准,11份样品（73．3％）中活菌计数符合国家标准规定;耐药性试验结果显示,31株乳酸菌对4种抗生素均有一定程度的耐受性,且90．3％的菌株具有多重耐药性,能耐受2～3种抗生素。[结论]绍兴市售酸奶中乳酸菌已具有一定程度的耐药性,对酸奶产品进行耐药性监测和安全评价已势在必行。     

1株猪链球菌的分离·鉴定及其毒力因子研究

[目的]对1株疑似猪链球菌菌株进行鉴定,为进一步研究猪链球菌的毒力提供理论基础。[方法]采用革兰氏染色镜检、形态观察和16S rDNA测序后NCBI数据库Blast比对鉴定菌株;另外选取猪链球菌7种主要毒力因子设计引物,利用PCR方法检测其毒力基因分布情况。[结果]显微镜下该疑似菌株为革兰氏阳性菌,多数呈链状,少见单个排列。16S rDNA测序结果与猪链球菌2型同源性达到99%。毒力因子检测均含有7种毒力因子。[结论]该疑似菌株为猪链球菌2型,其毒力基因型为：gdh＋/ef＋/mrp＋/sly＋/fbps＋/orf2＋/cps2J＋/。     

一株北方地区鲟鱼源病原细菌金黄杆菌B8致病性研究

从西伯利亚鲟(Acipenser baerii)病灶处筛选病原菌,结合药敏性试验、侵染路径及宿主相关指标分析,以期了解该病原菌的致病性。通过人工回归感染试验,从病鱼肾脏部位分离得到1株病原菌B8,对鲟鱼、罗非鱼和斑马鱼均有较强的致病性。经16S rDNA序列分析鉴定为金黄杆菌,B8菌对庆大霉素和氯霉素等10种常用抗生素具有耐药性,对新霉素和红霉素敏感。侵染路径结果显示B8菌首先定殖在粘膜组织(鳃和肠),然后是淋巴组织(肝、脾和肾),各组织定殖菌量在48 hpi达到峰值,然后呈缓慢下降。肠道炎症因子IL-1β表达水平呈先上升(24 hpi为峰值)后下降趋势,在12～48 hpi间较未感染组差异显著(P0.05),头肾溶菌酶和ROS活性表现出类似趋势(48 hpi为峰值),在感染的24～48 hpi间较未感染组差异显著(P0.05)。首次报道了鲟鱼源致病株金黄杆菌,通过其致病性研究为西伯利亚鲟金黄杆菌引发的病害防控提供参考。     

Prevalence of Streptococcus suis Isolated from Clinically Healthy Sows in China

Streptococcus suis is an important pathogen in pigs. Transmission of this pathogen is generally believed to occur between healthy carrier sows and their offspring, so the carrier status of S. suis in healthy sows is important for the control of S. suis infections in pigs, especially in suckling and growing pigs. In this study, the prevalence of S. suis isolated from clinically healthy sows in China was studied for the first time. A total of 1 043 tonsil samples were collected from clinically healthy sows from 10 regions in China from 2005 to 2007. Among the 421 S. suis isolates, 31 strains were identified as capsular type 2. The results showed that S. suis was widespread in swine herds in China with the carrier rates in different herds ranging from 19.5 to 93.9%. Overall, 40.4 and 3.0% of clinically healthy sows harbored S. suis and capsular type 2 in their palatine tonsils, respectively. Statistically significant differences of carrier rates of S. suis and capsular type 2 between the different farms were observed, which was independent of herd sizes and geographic distributions of different herds.     

猪链球菌的分离鉴定及自家灭活苗研制

为确诊猪链球菌病,筛选敏感药物和研制自家灭活苗防治该病,从送检病猪体内分离病原菌,通过对分离菌的形态染色、分离培养、生化试验、血清型鉴定等方法鉴定菌株;并通过药敏试验筛选敏感药物,配制铝胶佐剂,制备自家铝胶灭活苗对其进行防治。实验结果表明,分离鉴定的菌株为2型猪链球菌,分离菌对恩诺沙星、氨苄青霉素、庆大霉素、阿莫西林、头孢拉定高敏,用研制的猪链球菌自家铝胶灭活苗防治该病安全有效。结果提示,猪场链球菌病须经实验室分离鉴定病原才可确诊,临床治疗用药应筛选敏感药物,用自家灭活苗防治有较好效果。     

茯砖茶对肠道4种常住微生物的影响

用致泻剂番泻叶建立小鼠腹泻模型,研究茯砖茶水提物高、中、低剂量(分别为210、105、52.5 mg/mL对肠道大肠杆菌、肠球菌、双歧杆菌、乳杆菌数量及肠道状况的影响。结果显示：高、中剂量茯砖茶能促进肠道有益微生物的生长,抑制有害微生物增殖,对抗番泻叶所致的肠道微生物紊乱,其中对乳杆菌增殖的影响最为显著,预防组高、中、低剂量可促进乳杆菌繁殖,试验第7天比试验开始前的数量对数值分别增加3.309 0、3.237 3、2.690 5,且呈剂量依懒关系;治疗组高剂量可促进乳杆菌的增殖,试验第11天的数量对数值比试验第7天的增加3.541 9,比阳性药小檗碱促进乳杆菌增殖的效果(对数值增加1.466 9)更好;中、低剂量可减少肠球菌数量,试验第11天的数量对数值比第7天的分别减少0.423 6、0.231 5,而阳性药小檗碱反而使肠球菌数量增加,高、中、低剂量组试验第11天的大肠杆菌数量的对数值与试验第7天的相比分别增加0.471 0、0.335 3、0.510 9,与阳性药组(0.556 2)差异较小;灌服茶汤的小鼠的肠腔状况与正常空白对照小鼠的无异样。     

Improvement of spergualin production by mutation and medium optimization

In order to screen a high-yield spergualin-production strain and an optimal fermentation medium,large numbers of isolates were selected after ultraviolet(UV)mutation and self-tolerant mutation of Bacillus laterosporus A7.A high-yield strain A-94-7 was obtained and the spergualin productivity was 4.1-fold of the parent strain.The genetic stability of the high-yield strain Bacillus laterosporus A-94-7 was very good.After fermentation medium optimization,Bacillus laterosporus A-94-7 was able to produce 380 mg?L-1.The spergualin productivity in Bacillus laterosporus A7 was increased 4.75-fold by mutation and medium optimization.