不同中药提取物对耐甲氧西林金黄色葡萄球菌的抑菌作用和逆转机制

采用(K-B)药敏纸片扩散法检测耐甲氧西林金黄色葡萄球菌(MRSA)的药物敏感性,并用二倍稀释法分别测定10种中药提取物对MRSA的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),选取抑菌效果最好的中药提取物(黄芩)通过棋盘法测定其与5种常用抗生素的联合抑菌效果。观察黄芩不同亚抑菌浓度作用下对MRSA耐药性的逆转效果,并通过荧光实时定量PCR法检测耐药基因mec A在mRNA表达水平上的变化。结果表明,MRSA具有多重耐药性,10种中药提取物里黄芩的抑菌和杀菌效果最为显著,MIC和MBC均为156 mg/L,但其与5种抗生素没有显著的联合抗菌作用,在黄芩亚抑菌浓度130 mg/L作用20 h后,MRSA主要耐药基因mec A的表达量下调并恢复了对药物的相对敏感性。     

地锦草不同组方对大肠杆菌的体外抑菌试验

为研制以地锦草为主药,组方治疗临床大肠杆菌病,提供理论依据,自拟中药处方6个,经水煮处理制成浓度为1g/mL的受试溶液,采用试管倍比稀释法和平板打孔法,对猪源致病性大肠杆菌O20、O48和O161进行体外抑菌试验。试验结果:组方Ⅲ抑菌作用最强,对O48和O161的最低抑菌浓度(MIC)和最小杀菌浓度(MBC)均为62.5mg/mL,对O20的最低抑菌浓度(MIC)为62.5mg/mL,最小杀菌浓度(MBC)为125mg/mL。     

功劳叶提取物的体外抗菌活性研究

采用水提法和乙醇回流加热法分别提取功劳叶,应用牛津杯法和试管二倍稀释法分别检测功劳叶提取物对试验菌的抑菌圈直径、最低抑菌浓度(MinimumInhibitory Consistency,MIC)和最低杀菌浓度(Minimum Bactericidal Consistency,MBC)。结果显示,功劳叶醇提物金黄色葡萄球菌和大肠杆菌的抑菌圈直径分别为20.69 mm、15.40 mE;MIC值分别是31.3 mg/mL、125 mg/mL;MBC值分别是62.5 mg/mL、500 mg/mL。功劳叶水提物对金黄色葡萄球菌的抑菌圈13.54mm,其MIC值、MBC值分别是62.5 mg/mL、500 mg/mL,对大肠杆菌抗菌活性较弱,其MIC值为500 mg/mL,MBC500 mg/mL。功劳叶不同提取物对细菌的抗菌活性有所差异,功劳叶醇提物对金黄色葡萄球菌和大肠杆菌均由不同程度的抑菌或杀菌能力。     

4种清热解毒复方中草药体外抑菌及抗PRRSV活性研究

【目的】 探索泻心汤、三黄虎杖汤、黄连解毒汤和清瘟败毒散4种清热解毒复方中草药体外抑制大肠杆菌、金黄色葡萄球菌及抗猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)的效果, 为临床应用提供参考。【方法】 采用传统水煎法对4种复方中草药进行提取, 通过二倍稀释法测定中药对大肠杆菌标准菌ATCC 259222和金黄色葡萄球菌标准菌ATCC 25923的体外抑菌活性; 在测定复方中草药对Marc-145细胞最大安全浓度的基础上, 通过实时荧光定量PCR鉴定复方中草药对PRRSV的抑制作用。【结果】 泻心汤对大肠杆菌的体外抑菌活性最好, 其最小抑菌浓度(minimum inhibitory concentration, MIC)和最小杀菌浓度(minimum bactericidal concentration, MBC)均为7.81 mg/mL; 三黄虎杖汤和黄连解毒汤次之, 其MIC和MBC为31.25~125 mg/mL; 清瘟败毒散抑制大肠杆菌效果较差: MIC和MBC均>250 mg/mL, 而金黄色葡萄球菌对泻心汤、黄连解毒汤和三黄虎杖汤非常敏感, 其MIC和MBC为0.24~0.98 mg/mL。在体外抗PRRSV作用的试验中, 泻心汤、三黄虎杖汤、黄连解毒汤和清瘟败毒散对Marc-145细胞最大安全浓度分别为0.49、0.49、3.90和1.95 mg/mL。4种复方中草药对PRRSV的抑制与直接灭活作用较好, 黄连解毒汤和清瘟败毒散均在0.98 mg/mL时对PRRSV有阻断作用, 泻心汤和三黄虎杖汤在0.24和0.12 mg/mL时对PRRSV有阻断效果。【结论】 4种复方中草药中泻心汤的体外抑菌及抗PRRSV的效果最好, 其次是三黄虎杖汤、黄连解毒汤和清瘟败毒散, 4种清热解毒复方均有一定的抑菌和抗病毒活性, 可以为临床应用提供参考依据。     

百里香酚和植酸对猪源致病菌的抑菌效果研究

研究旨在探究百里香酚和植酸对猪源大肠杆菌和沙门氏菌的抑制效果，为进一步改善猪舍环境和猪肠道功能研究提供参考。试验采用牛津杯法、二倍稀释法和平板涂布法，分别测定百里香酚和植酸对两种猪源致病菌的抑菌圈直径、最小抑菌浓度（MIC）和最小杀菌浓度（MBC）的影响。结果表明：猪源大肠杆菌和沙门氏菌对50 mg/mL百里香酚表现为高度敏感，对17.5%植酸表现为极度敏感。百里香酚对猪源大肠杆菌MIC和MBC分别为1.56 mg/mL和3.12 mg/mL，对猪源沙门氏菌MIC和MBC均为1.56 mg/mL；植酸对猪源沙门氏菌的抑制效果强于猪源大肠杆菌，对猪源大肠杆菌MIC和MBC均为4.37%，对猪源沙门氏菌MIC和MBC均为2.18%。说明百里香酚和植酸对猪源大肠杆菌和沙门氏菌具有较好的抑菌效果，在抑菌活性物质筛选中具备较好的应用前景。     

动物源大肠杆菌及沙门氏菌抗药性的快速检测

利用本室自主研制的新型96点阵药敏检测盒检测山东地区动物源大肠杆菌、沙门氏菌的药物敏感性,试验结果表明,大肠杆菌对氨苄西林和喹诺酮类药物的抗药率达98%以上,禽源大肠杆菌的抗药率高于猪源大肠杆菌,猪源大肠杆菌的抗药谱广于禽源大肠杆菌;沙门氏菌抗药率略低于大肠杆菌,但同样呈现多重抗药,主要集中在5抗-7抗和13抗-14抗两个范围。96点阵抗生素药敏检测盒是适合于试验室进行大规模抗药性监测的新工具,具有简易、准确、快速、大通量、节约的优点,为临床治疗中抗生素的使用、细菌病的治疗提供了技术保障,从而达到避免盲目使用抗生素,降低细菌抗药率的目的。     

石榴皮水提物对猪源多重耐药大肠杆菌的抑菌作用研究

【目的】筛选腹泻仔猪中携毒力基因且多重耐药的大肠杆菌菌株,评估中药水提物对该菌株的抑菌活性,并探索中药抑菌机制。【方法】通过PCR方法与Kirby-Bauer （K-B）药敏纸片法评估临床大肠杆菌菌株的肠毒素基因携带情况和耐药性;通过微量肉汤稀释法评估中药水提物对多重耐药大肠杆菌菌株的抑菌活性,确定最小抑菌浓度（minimal inhibitory concentration,MIC）和最小杀菌浓度（minimal bactericidal concentration,MBC）;通过电导率仪以及相关试剂盒检测评估石榴皮水提物对多重耐药菌株作用不同时间后菌液电导率、碱性磷酸酶（alkaline phosphatase,AKP）含量及菌体内ATP含量的变化;通过SDS-PAGE和蛋白含量检测评估菌体内可溶性蛋白的含量变化;通过扫描电镜观察大肠杆菌形态变化。【结果】PCR检测14株临床大肠杆菌中肠毒素基因携带率达到78.57%,抗生素药敏试验结果显示,所有菌株至少对2种抗生素耐药,均属于多重耐药菌株。中药药敏试验结果显示,黄连、黄芩、石榴皮水提物对多重耐药菌株抑菌活性较好,其中石榴皮水提物的MIC和MBC分别为31.25和62.5 mg/mL。石榴皮水提物在1/2MIC和MIC浓度下能显著抑制质控菌株和多重耐药菌株的生长繁殖。在石榴皮水提物作用大肠杆菌3 h内,菌液电导率相比于对照组极显著上升（P<0.01）。AKP与ATP检测结果显示,与对照组相比,石榴皮水提物在作用大肠杆菌6 h内,菌液中AKP含量极显著增加（P<0.01）,菌体内ATP含量极显著降低（P<0.01）。SDS-PAGE和蛋白含量检测结果显示,随着石榴皮水提物刺激时间增加,菌体内可溶性蛋白含量减少。扫描电镜结果显示,石榴皮刺激后菌体形态发生碎裂和溶解。【结论】本研究结果表明,石榴皮水提物能显著抑制多重耐药且携带肠毒素基因的大肠杆菌生长,为临床治疗大肠杆菌性仔猪腹泻提供理论参考和药物开发依据。     

四种兽用抗生素的体外抑菌效果评价

本实验用试管药物稀释了四种兽用抗生素对猪常见病原菌和正常菌的最小抑菌浓度 （minimal inhibitory concentration, MIC） 和最小杀菌浓度 （minimal bactericidal concentration, MBC）。通过实验发现：各种菌对四种药物的敏感性存在很大的差异,大多数细菌对药物C和D较敏感而对药物A和B存在较严重的耐药。     

中药对猪源大肠杆菌O_8体外抑菌试验研究

为了探讨猪源大肠杆菌O8对黄连等10种中药的体外敏感性,采用琼脂扩散法和微量二倍稀释法对10种中药水提物进行体外抑菌试验,测定抑菌圈直径和最小抑菌浓度(MIC)。结果表明:黄连对大肠杆菌O8呈高度敏感,抑菌圈直径为21.3 mm,其MIC值为7.813mg/mL,抑菌效果最好;黄芩、五倍子呈中度敏感,其MIC值为31.25mg/mL;而苦参不敏感,其MIC值为500mg/mL,抑菌效果差。     

5种中药对大肠杆菌的抑菌作用

为研究黄连、黄芩、五倍子、黄柏和鱼腥草提取物对大肠杆菌的抑菌效果,采用超声波处理,乙醇回流,水浴加热的方法提取5种中药的有效成分,采用二倍稀释法分别测定提取物对大肠杆菌的最小抑菌浓度(Minimum inhibitory concentration,MIC)和最小杀菌浓度(Minimum bactericidal concentration,MBC),采用肉汤稀释棋盘法,测定提取物对大肠杆菌的联合抑菌效果。结果显示,鱼腥草和五倍子单独抑菌时的MIC值较其他中药的MIC值低,且鱼腥草与黄芩和五倍子联合用药时MIC值由15.63 mg/m L降到3.91 mg/m L,同时五倍子与黄芩和鱼腥草联合用药时MIC值也由15.63 mg/m L降到3.91 mg/m L。黄芩与黄连、黄柏、五倍子、鱼腥草联合用药时MIC值均降低。结果表明:鱼腥草、黄芩、五倍子对大肠杆菌有较好的抑菌作用,鱼腥草、黄芩、五倍子联用时,抑菌效果为协同或相加作用;黄连与其他4种中药联用时抑菌效果为无关作用;黄柏与黄芩、五倍子及鱼腥草联用时抑菌效果为无关作用。     

Antimicrobial susceptibility testing of Australian isolates of Brachyspira hyodysenteriae using a new broth dilution method.

The antimicrobial susceptibilities of 76 field isolates of Brachyspira hyodysenteriae from different states of Australia were tested in a newly developed broth dilution procedure. The antimicrobial agents used were tiamulin, valnemulin, tylosin, erythromycin, lincomycin and clindamycin. The results from the broth dilution susceptibility testing of 39 of the isolates were compared with results obtained for the same isolates using the agar dilution method. Amongst the isolates tested by broth dilution, 17 were from three farms and had been collected over a number of years. Their pulsed field gel electrophoresis pattern previously had been determined. The broth dilution technique was simple to use, less labor intensive than agar dilution, and gave clear end points. The results obtained using the two methods generally corresponded well, although in a few cases the MIC obtained by broth dilution were lower than those with agar dilution. For the 76 isolates tested by broth dilution, the MIC(90) (mg/l) was: tiamulin, 1; valnemulin, 0.5; tylosin>256; erythromycin>256; lincomycin, 64 and clindamycin, 16. Only minor differences in susceptibility patterns were found amongst isolates from different Australian states. Over all the isolates, and also amongst the isolates obtained from different years on the three farms, there was no trend for the susceptibility of the isolates to alter with time.     

Comparison of Agar Dilution and E-test for antimicrobial susceptibility testing of Campylobacter coli isolates recovered from 80 Ontario swine farms

The primary aim of this study was to evaluate the level of agreement of the E-test for in vitro antimicrobial susceptibility testing of Campylobacter coli using the agar dilution technique, which is the approved method. A convenience sample of 80 Ontario swine farms was chosen for this study; each farm was visited from January to June 2004. A total of 233 isolates of C. coli were tested for susceptibility to 10 antimicrobials by agar dilution and the E-test. Performance of the tests was evaluated using 7 quality control strains: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Campylobacter jejuni ATCC 33560, and Campylobacter coli ATCC 33559 for the E-test and E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and C. jejuni ATCC 33560 for the agar dilution test. Weighted Cohen's kappa and prevalence-adjusted bias-adjusted kappa (PABAK) tests were used for statistical analysis. The E-test and agar dilution test results had a strong agreement when resistance to streptomycin and tetracycline were evaluated (weighted kappa: 0.68 and 0.66, respectively). However, marked disagreement was detected when testing susceptibility to nalidixic acid and ampicillin (0.15 and 0.22, respectively). Almost perfect agreement was detected by PABAK when testing susceptibility to gentamicin (0.99). Agreement was found to be moderate for ciprofloxacin, azithromycin, clindamycin, erythromycin, and chloramphenicol. Although the level of agreement between the E-test and agar dilution depended on the antimicrobial being tested, the E-test always detected a lower proportion of resistant isolates compared to agar dilution.     

Antimicrobial susceptibility of Brachyspira hyodysenteriae isolated from 21 Polish farms

Swine dysentery (SD) is a common disease among pigs worldwide, which contributes to major production losses. Antimicrobial susceptibility testing of B. hyodysenteriae, the etiological agent of SD, is mainly performed by the agar dilution method. This method has certain limitations due to difficulties in interpretation of results. The aim of this study was the analysis of antimicrobial susceptibility of Brachyspira hyodysenteriae (B. hyodysenteriae) Polish field isolates by broth microdilution procedure. The study was performed on 21 isolates of B. hyodysenteriae, collected between January 2006 to December 2010 from cases of swine dysentery. VetMIC Brachyspira panels with antimicrobial agents (tiamulin, valnemulin, doxycycline, lincomycin, tylosin and ampicillin) were used for susceptibility testing of B. hyodysenteriae. The minimal inhibitory concentration (MIC) was determined by the broth dilution procedure. The lowest antimicrobial activity was demonstrated for tylosin and lincomycin, with inhibition of bacterial growth using concentrations > 128 microg/ml and 32 microg/ml, respectively. In the case of doxycycline, the MIC values were < or = 2.0 microg/ml. No decreased susceptibility to tiamulin was found among the Polish isolates and MIC values for this antibiotic did not exceed 1.0 microg/ml. The results of the present study confirmed that Polish B. hyodysenteriae isolates were susceptible to the main antibiotics (tiamulin and valnemulin) used in treatment of swine dysentery. Further studies are necessary to evaluate a possible slow decrease in susceptibility to tiamulin and valnemulin of B. hyodysenteriae strains in Poland.     

Antimicrobial susceptibility of 51 strains of Haemophilus pleuropneumoniae.

Fifty-one strains of Haemophilus pleuropneumoniae were tested for susceptibility to 27 antimicrobial agents using agar disc diffusion, broth-tube dilution and microdilution methods. There was generally good agreement between the interpretation of the disc diffusion inhibition zones and the actual minimal inhibitory concentrations obtained with the dilution methods. The agreement between the results obtained with the broth-tube dilution method and the microdilution method was very good. Three strains were resistant to penicillin, ampicillin, carbenicillin, methicillin and tetracycline. One of those was also resistant to chloramphenicol. Forty strains were resistant to streptomycin, 23 strains were resistant to novobiocin and seven were resistant to triple sulfa. It is thus necessary to consider resistance development against antimicrobial agents chosen for the treatment of pleuro-pneumonia in pigs caused by Haemophilus pleuropneumoniae.     

Bactericidal activity of standard bovine serum against coliform bacteria isolated from udders and the environment of dairy cows.

Coliform organisms were isolated from bedding, udders of cows exhibiting acute or chronic mastitis, and milk of cows tested for udder pathogens. The bactericidal activity of standard bovine serum was tested on 362 isolates, of which 2.4% were killed at a serum dilution of less than 1:5; 2.7% were killed at a dilution of 1.5; 3.0% were killed at a dilution of 1:12.5; and 4.4% were sensitive to a dilution of 1:25. Organisms isolated from mastitic udders were not killed by serum. Sensitivity to agglutination in 1:500 solution of acriflavine was tested on 157 isolates, and only 54 had any degree of agglutination. Of these 54, only 4 organisms had any susceptibility to killing by serum. Results suggest that most organisms in the environment of the cow are resistant to serum, few serum-sensitive coliforms cause mastitis, and agglutination in acriflavine (indicating organisms in the rough phase) does not correlate with sensitibity to serum.     

Tiamulin resistance in porcine Brachyspira pilosicoli isolates

There are few studies on antimicrobial susceptibility of Brachyspira pilosicoli, therefore this study was performed to investigate the situation among isolates from pigs. The tiamulin and tylosin susceptibility was determined by broth dilution for 93 and 86 porcine B. pilosicoli isolates, respectively. The isolates came from clinical samples taken in Swedish pig herds during the years 2002 and 2003. The tylosin minimal inhibitory concentration (MIC) was >16 microg/ml for 50% (n=43) of the isolates tested. A tiamulin MIC >2 microg/ml was obtained for 14% (n=13) of the isolates and these were also tested against doxycycline, salinomycin, valnemulin, lincomycin and aivlosin. For these isolates the susceptibility to salinomycin and doxycycline was high but the MICs for aivlosin varied. The relationship between the 13 tiamulin resistant isolates was analyzed by pulsed-field gel electrophoresis (PFGE). Among the 13 isolates 10 different PFGE patterns were identified.     

Comparison of methods for antimicrobial susceptibility testing and MIC values for pleuromutilin drugs for Brachyspira hyodysenteriae isolated in Germany

In Germany treatment of swine dysentery is hampered by Brachyspira hyodysenteriae strains showing elevated MIC values to the few antibiotics licensed. Therefore, susceptibility testing of clinical isolates is an important service to the swine practitioner. This study compares the established agar dilution procedure for antimicrobial susceptibility testing of this fastidious anaerobe to the broth microdilution test newly developed [Anim. Health Res. 2 (2001) 59; Vet. Microbiol. 84 (2002) 123; J. Clin. Microbiol. 41 (2003) 2596]. A total of 221 isolates were examined twice with either test procedure using tiamulin and valnemulin as antibiotics. Both methods gave reproducible results, and the MIC values for the reference strains B. hyodysenteriae B204 and Staphylococcus aureus ATCC 29213 corresponded to previously published data. However, the results for individual strains differed significantly for both tests (P < 0.001) with MIC values being on average one dilution step lower in the broth dilution method. The 221 strains used for comparing test procedures were isolated between 1989 and 2001. An additional 102 strains isolated in 2002 were tested only with the broth dilution procedure. A significant rise in the average MIC value for both pleuromutilins could be demonstrated when comparing earlier isolates to those from 2000 to 2001 (P < 0.05), while in 2002 the average MIC significantly decreased when compared to the value in 2000 (P < 0.05). However, strains with MIC values for tiamulin as high as 8 microg/ml (broth dilution) could still be isolated.     

Comparison between diffusion and dilution methods for testing antimicrobial susceptibility of Staphylococcus aureus cultures derived from bovine mastitis

One hundred Staphylococcus aureus strains from bovine mastitis were tested for susceptibility to eight antimicrobials by the paper disc diffusion method (Kirby-Bauer) and a commercially prepared dilution system. Except in the case of penicillin and possibly ampicillin, comparison of the two methods showed excellent correlation. Agreement was perfect with methicillin, chloramphenicol, gentamicin, and kanamycin.     

Antibiotic susceptibility testing of Mycoplasma bovis using Tween 80 hydrolysis as an indicator of growth.

Lysis and precipitation of polyoxyethylene sorbitan monoleate (Tween 80) was used as an indicator of growth in quantitative antibiotic dilution susceptibility tests in microtiter plates. This allowed test results to be read after 2 or 3 days. Antibiotic sensitivity levels obtained with this method were homogenous and tightly clustered. No acquired resistance was found among 16 field strains isolated in 1990.     

广东地区2007～2009年禽源大肠杆菌的耐药性演变

通过对2007～2009年内从广东省10多个地市分离得到的378株禽源大肠杆菌进行15种兽医临床常用抗生素的药物敏感性试验(MIC),调查该区域禽类大肠杆菌的耐药性演变情况.采用琼脂稀释法对临床分离株开展MIC测试.结果显示临床分离株对多数兽医临床常用抗生素耐药率在30 ％以上,且有逐年上升的趋势,多数药物的半数抑菌浓度( MIC50)和有效抑菌浓度( MIC90)水平高且保持逐年升高的趋势;细菌的多重耐药情况严重,多集中在5～12耐,提示细菌的耐药能力逐渐提高.表明,广东地区禽源大肠杆菌的耐药水平已经达到了较为严重的程度,应加强食用动物源细菌耐药性的监测.