我国东北与西部地区乳房炎牛奶中链球菌的鉴定及耐药性比对

为了了解我国奶牛由链球菌引起的乳房炎感染现状,本论文系统地分离鉴定了我国两个地区(东北和西部)170个乳房炎乳的链球菌,并对它们进行了抗生素的耐药性比对。结果表明:我国东北和西部地区的乳房炎乳中分别分离出18株和11株链球菌;药敏试验显示,该两个地区的所有链球菌对29种抗生素药物均表现出不同程度的耐药性,其中,在东北地区链球菌中甲氧苄啶耐药菌(90.0%)、磺胺异噁唑耐药菌(80.0%)、苯唑西林耐药菌(78.5%)、林可霉素耐药菌(74.6%)、克林霉素耐药菌(66.9%)和头孢西丁耐药菌(62.3%)检出率较高;西部地区链球菌检出菌对甲氧苄啶(90.9%)、苯唑西林(81.8%)、青霉素G和磺胺异噁唑(72.7%)耐药性较高;该两个地区的乳房炎乳对抗生素都表现出多重的耐药现象。     

直接提取快速测定牛奶及奶粉中四环素残留量

以草酸甲醇液直接提取残留药物,应用反相高效液相色谱-二极管检测器(DAD),外标法定量,测定了乳及奶粉中土霉素、四环素、金霉素、强力霉素的含量.土霉素、四环素浓度在1～100ng、金霉素、强力霉素浓度在2～200 ng范围内呈良好线性关系,r=O.9996以上,平均加标回收率为79.6%～102.0%,CV为2.28%～4.57%,该方法简便,准确,重现性好,可用于乳及奶粉中土霉素、四环素、金霉素、强力霉素质量分数的测定.     

酸乳用乳酸菌抗生素耐药性研究

旨在通过纸片扩散法对乳酸菌菌株耐药情况进行研究。对48份酸乳发酵剂中的乳酸菌进行分离鉴定,共分离出63株乳酸菌,其中嗜热链球菌43株、德氏乳杆菌保加利亚亚种20株。结果表明:63株乳酸菌对临床使用率较高的12种抗生素呈现不同程度的耐药性,其中,青霉素(36.51%)、复方新诺明(33.33%)、链霉素(28.57%)、氨苄西林(26.98%)、环丙沙星(20.63%),同时52.38%(33/63)的菌株具有多重耐药性,其中嗜热链球菌对12种抗生素的多重耐药性(30.23%)显著低于德氏乳杆菌保加利亚亚种(100%)。     

广州市售酸奶中乳酸菌的耐药性评估

研究广州市售酸奶中乳酸菌对不同种类抗生素的药物敏感性。从市售酸奶中分离并鉴定乳酸菌,采用纸片扩散法检测分离菌株对18种抗生素的药物敏感性;E-test方法对耐药菌株进行最低抑菌浓度测定。从31份市售保质期内商标注册酸奶中分离鉴定出48株乳酸菌,其中25株嗜热链球菌(Streptococcus thermophilus)、20株保加利亚乳杆菌(Lactobacillus bulgaricus)、3株副干酪乳杆菌(Lactobacillus paracasei)。48株乳酸菌对卡那霉素、链霉素均耐药;对头孢他啶、头孢噻肟、红霉素均敏感;对环丙沙星、庆大霉素、万古霉素等呈现出不同程度的耐受性;79.2%(38/48)表现出多重耐药性;E-test分析表明,除了保加利亚乳杆菌MR10的MIC为256μg/mL以外,所有万古霉素耐药菌株对万古霉素的MIC均大于1024μg/mL。不同品牌酸奶分离菌株对抗生素具有不同的耐受性,均有多重耐受性出现,应加强其耐药性连续监测及安全评价。     

超高效液相色谱-串联质谱法同时测定水产品中4种酰胺醇类抗生素残留

目的 建立了一种超高效液相色谱-串联质谱同时测定水产品中酰胺醇类抗生素氯霉素、甲砜霉素、氟苯尼考及代谢物氟苯尼考胺的方法。方法 水产品样品经乙腈提取,正己烷除脂后,在Capcell Pak ADME (100×2.1 mm, 2.7 μm, 1.7 ?m)色谱柱上分离,于多反应监测模式,以氯霉素-D5和氟苯尼考胺-D3为内标进行定量测定。结果 氯霉素在0.5～25 ng/mL范围内线性关系良好（r>0.999）,甲砜霉素、氟苯尼考和氟苯尼考胺在5～250 ng/mL范围内线性关系良好（r>0.998）。方法检测限为0.1~1.0 μg/kg,定量限为0.2~2.0 μg/kg。当氯霉素添加水平为0.2、1和10 μg/kg时,回收率为82.5%～104%,相对标准偏差在2.2%~11.8%之间;当甲砜霉素、氟苯尼考和氟苯尼考胺添加水平为2、10和100 μg/kg时,回收率为80.9%～102%,相对标准偏差在0.8%~8.4%之间。结论 该方法快速简单,准确,成本低,适用于水产品中酰胺醇类抗生素及代谢物残留的检测。     

超高效液相色谱-串联质谱结合谱库检索快速测定鸡蛋中氯霉素、氟苯尼考和甲砜霉素残留

采用超高效液相色谱-三重四级杆/复合线性离子阱质谱(QTrap UPLC-MS/MS)技术结合谱库检索分析,建立了鸡蛋中氯霉素、氟苯尼考和甲砜霉素的快速检测确证方法。样品加入氯霉素-D5后,经乙腈提取,浓缩至干,3.00 mL水复溶,正己烷去脂净化,以乙腈-水为流动相,经Phenomenex Kinetex F5色谱柱(100 mm×3.0 mm,2.6 μm)分离,用QTrap UPLC-MS/MS进行多反应监测(MRM)、信息依赖性扫描(IDA)、增强子离子扫描(EPI)和谱库检索分析。氯霉素内标法定量,氟苯尼考、甲砜霉素外标法定量。结果表明,氯霉素和氟苯尼考在0.03~50.0 μg/L,甲砜霉素在0.06~50.0 μg/L的线性范围内线性关系良好,相关系数r>0.999;氯霉素、氟苯尼考和甲砜霉素在3个加标水平下,平均回收率为90.1%~120.0%,精密度为4.2%~11.9%,检出限为0.05~0.10 μg/kg。该方法高效、简便、准确,可用于鸡蛋中酰胺醇类抗生素定性和定量分析。     

内蒙古牧区传统乳制品中潜在益生植物乳杆菌的药物敏感性研究

采用纸片琼脂扩散法(K-B法),对从内蒙古牧区传统乳制品中分离到的16株对pH3及0.3%牛胆酸钠有一定耐受性的植物乳杆菌的药物敏感性进行了测定。结果显示,所测试的16株植物乳杆菌均对青霉素G、万古霉素、诺氟沙星、氧氟沙星、环丙沙星不敏感;对头孢哌酮、乙酰螺旋霉素中度敏感;对哌拉西林、阿莫西林、头孢唑啉、克林霉素、磺胺甲恶唑、利福平、呋喃妥因敏感;对其他抗生素的敏感性因菌株不同而有差异。所有菌株对阿奇霉素、新霉素表现为敏感或中度敏感;多数菌株对氨苄青霉素敏感(11/16),对头孢噻肟敏感(8/16)或中度敏感(6/16),对多黏菌素B不敏感(10/16)。     

高效液相色谱-串联质谱法测定动物肌肉组织中氯霉素、甲砜霉素和氟苯尼考残留量

采用液相色谱- 串联质谱(LC-MS/MS)法测定动物肌肉组织中氯霉素,甲砜霉素和氟苯尼考三种药物的残留量。添加氘代氯霉素(d5- 氯霉素)作为内标,碱化乙酸乙酯提取,正己烷去除脂肪,省去固相萃取步骤直接上机定量。选择电喷雾离子源(ESI),负离子扫描多反应监测(MRM)模式,并优化了质谱参数,使仪器的灵敏度达到最高。氯霉素、甲砜霉素和氟苯尼考的检出限均达到0.01 μ g/kg。空白样品中添加氯霉素、甲砜霉素和氟苯尼考水平为0.02～1 μ g/kg 时,三种药品的线性相关系数均大于0.9990。氯霉素平均回收率为92.8%～97.0%,甲砜霉素平均回收率为93.5%～96.6%,氟苯尼考平均回收率为91.2%～95.9%。     

食源性气单胞菌属种水平监测和表型特征研究

调查上海市浦东新区食源性气单胞菌的属种分布和表型特征。方法 2011年5~11月,对采集自上海市浦东新区2个农贸市场的3种生鲜食品(共291份)进行气单胞菌属的选择性分离和属种水平鉴定;分析菌株表型特征的差异性。结果 291份生鲜食品样品中,74份阳性,总检出率25.4%。 其中肉制品阳性检出率最高,为34.1%(43/126);水产品和蔬菜阳性检出率分别为18.9%(17/90)和18.7%(14/75)。经系统生化鉴定的74株气单胞菌属菌株包括:嗜水气单胞菌23株,维隆气单胞菌温和生物变种35株,豚鼠气单胞菌16株。溶血试验和蛋白酶表达测试显示分别有51株菌β溶血素阳性,59株菌蛋白酶试验阳性,两者皆阳性的菌株有48株。抗生素敏感性试验表明,多数菌株对头孢噻肟、头孢他啶、头孢吡肟、庆大霉素、环丙沙星、左氧氟沙星呈现高度敏感,但对氨苄西林呈现高度耐药;对含青霉素酶药物(奥格门丁)的耐药能力存在种水平的差异;3种气单胞菌均存在低水平的多重耐药(MDR)。结论 上海市浦东新区农贸市场流行季节供应的主要生鲜食品气单胞菌属污染程度较高,除嗜水气单胞菌外的其他气单胞菌株也具有毒力因子,虽然目前耐药水平较低,但仍需加强基于食物链的食源性疾病的综合监测。     

不同来源乳酸菌的药敏试验

从不同来源的材料中分离并鉴定12株乳酸菌菌株,并进行10种常用抗生素药敏检测。结果表明:耐药率较高药物为阿米卡星(100%)、氟哌酸(83.33%),敏感率最高是氯霉素(100%),中介率最高为氟哌酸(16.67%)。不同来源的乳杆菌药敏性各不相同,其中新疆来源的菌株对阿米卡星和氟哌酸的耐药率为100%。不同菌种的药敏性也有差异:干酪乳杆菌的耐药性较广,为33.33%～91.67%。所有菌种均对氟哌酸和阿米卡星有耐药性,对氯霉素敏感。在不同来源和菌种这两种因素中,来源是影响菌株药敏性的更主要因素。乳酸乳球菌乳亚种LQ-12、瑞士乳杆菌15019、戊糖乳杆菌SC-1、布氏乳杆菌SC-2、干酪乳杆菌S1、发酵乳杆菌A1、发酵乳杆菌B3、发酵乳杆菌B5含有质粒,这几株乳杆菌可能含有可转移抗药性基因。本试验结果为所分离到的乳酸菌应用于生产实践及合理地使用抗生素提供了指导意义。     

Evaluation and validation of a multi-residue method based on biochip technology for the simultaneous screening of six families of antibiotics in muscle and aquaculture products

The Evidence Investigator? system (Randox, UK) is a biochip and semi-automated system. The microarray kit II (AM II) is capable of detecting several compounds belonging to different families of antibiotics: quinolones, ceftiofur, thiamphenicol, streptomycin, tylosin and tetracyclines. The performance of this innovative system was evaluated for the detection of antibiotic residues in new matrices, in muscle of different animal species and in aquaculture products. The method was validated according to the European Decision No. EC/2002/657 and the European guideline for the validation of screening methods, which represents a complete initial validation. The false-positive rate was equal to 0% in muscle and in aquaculture products. The detection capabilities CCβ for 12 validated antibiotics (enrofloxacin, difloxacin, ceftiofur, desfuroyl ceftiofur cysteine disulfide, thiamphenicol, florfenicol, tylosin, tilmicosin, streptomycin, dihydrostreptomycin, tetracycline, doxycycline) were all lower than the respective maximum residue limits (MRLs) in muscle from different animal origins (bovine, ovine, porcine, poultry). No cross-reactions were observed with other antibiotics, neither with the six detected families nor with other families of antibiotics. The AM II kit could be applied to aquaculture products but with higher detection capabilities from those in muscle. The detection capabilities CCβ in aquaculture products were respectively at 0.25, 0.10 and 0.5 of the respective MRL in aquaculture products for enrofloxacin, tylosin and oxytetracycline. The performance of the AM II kit has been compared with other screening methods and with the performance characteristics previously determined in honey.     

Prevalence and resistance to antibiotics for Aeromonas species from retail fish in Malaysia

A total of 87 market fish samples representing five types of fish were evaluated for the presence of Aeromonas spp. Of the samples examined, 69%, 55%, 11.5% and 2.3% harbored Aeromonas spp., A. veronii biovar sobria, A. hydrophila and A. caviae, respectively. The 60 isolated Aeromonas spp. strains were further examined for hemolytic activity, resistance to antimicrobial agents and presence of plasmids. Hemolytic activity varied widely among the isolated strains. Though all the isolates demonstrated resistance to three or more of the antibiotics tested, all were susceptible to ceptazidime. Thirty-four (56.7%) of the sixty isolates harbored plasmids, with sizes ranging from 2.3 to 15.7 kb. These results indicate that hemolytic, multiple antibiotic resistant and genetically diverse aeromonads are easily recovered from fish in this region.     

固相萃取-高效液相色谱-串联质谱法同时测定蜂蜡中氯霉素、甲砜氯霉素、氟苯尼考和其代谢产物氟苯尼考胺残留

首次建立了一种同时测定蜂蜡中氯霉素、甲砜氯霉素、氟苯尼考和其代谢物氟苯尼考胺等药物残留的固相萃取-高效液相色谱-串联质谱分析方法。根据蜂蜡脂溶性的特点,试样采用正己烷溶剂预溶解,用水提取2次后经混合型阳离子交换固相萃取柱（MCX）净化,用InertSustain Swift C₁₈柱（100 mm×2.1 mm, 1.9 μm）分离,多反应监测（MRM）模式串联质谱进行测定,以内标法定量。结果表明,在0.1~10 ng/mL范围内4种测定物均具有较好的线性关系,相关系数大于0.99,方法定量限（S/N>10）为0.1 μg/kg。对阴性蜂蜡在0.1、0.3和1.0 μg/kg等3个添加水平下分别进行加标回收试验,实际样品中4种测定物质的平均回收率在65.0%~113.2%之间,相对标准偏差（RSD,n=6）小于20%。应用本方法对15个蜂蜡样品进行检测,结果表明有4批次样品检出氯霉素类药物残留。本方法快速、灵敏、准确,适用于日常大批量蜂蜡样品中氯霉素类药物及其代谢物残留的定性、定量分析。     

2008~2017年间北京进出口水产品中副溶血性弧菌耐药性分析

目的 了解北京进出口水产品中副溶血性弧菌(Vibrio parahaemolyticus, VP)的耐药情况。方法 对2008~2017年间北京进出口水产品中分离出的320株副溶血性弧菌应用药敏纸片扩散法(K-B法)进行21种常用抗生素药敏测试。结果 89.1%的实验菌株同时对氨苄西林耐药, 而对环丙沙星、诺氟沙星、庆大霉素、多粘菌素B、亚胺培南全部敏感; 分别有高达94.7%和82.1%的菌株对红霉素和链霉素表现为中介。结论 实验菌株存在大量中介耐药和异质性耐药的情况, 应进一步加强水产品中副溶血性弧菌耐药性的监测工作, 以期指导临床和水产养殖中抗生素的使用。     

Impact of environmental stress desiccation, acidity, alkalinity, heat or cold on antibiotic susceptibility of Cronobacter sakazakii

Cronobacter sakazakii is an emerging foodborne pathogen that has been implicated in severe forms of meningitis, septicemia or necrotizing colitis in pre-term neonates. Although illness outbreaks (primarily associated with powdered infant formula, PIF) caused by this pathogen are rare, the case-fatality rate may reach 50%. Successful treatment of C. sakazakii infection is reliant upon clinical use of antibiotics (AB) such as ampicillin. Recent reports showed increased resistance of C. sakazakii to broad-spectrum antibiotics. The objective of this study was to evaluate the effect of extreme pH (3.5 for 30 min or 11.25 for 5 min), cold (4 °C for 24 h), heat (55 °C for 5 min), and desiccation (cells were dried at 40 °C for 2 h and held at 21 °C for 4 d) stresses on susceptibility of five isolated strains of C. sakazakii to streptomycin, gentamicin, kanamycin, neomycin, tetracycline, doxycycline, tilmicosin, florfenicol, ampicillin, amoxicillin, vancomycin, ciprofloxacin and enrofloxacin. All unstressed strains of C. sakazakii were sensitive to streptomycin, gentamycin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and amoxicillin, but were moderately resistant or resistant to the rest. Exposing cells to alkaline or acidic stress did not change their sensitivity toward streptomycin, gentamycin, kanamycin or ciprofloxacin, but their resistance toward the other AB was increased. Cells stressed by desiccation showed increased sensitivity toward streptomycin, gentamicin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and doxycycline, but showed resistance toward the others. Cold-stressed cells were more sensitive to streptomycin, gentamicin, kanamycin, and ciprofloxacin compared with heat-stressed cells, but both heat and cold-stressed cells showed increased resistance toward all the other AB. Results obtained will help in understanding the effect of environmental stresses during processing on C. sakazakii susceptibility to AB.     

Antibiotic resistance pattern of motile aeromonads from farm raised fresh water fish

Motile aeromonads isolated from the intestines of farm-raised freshwater fish such as Catla catla, Labeo rohita and Ctenopharyngodon idella have been characterized to species level. Morphological and physiological grouping revealed 61% Aeromonas hydrophila, 30% Aeromonas caviae, 7% Aeromonas sobria and 2% which remained unidentified. Hemolytic activity was detected mostly in A. hydrophila, while only half of the A. sobria and A. caviae showed this activity. Antibiotic resistance patterns of the strains revealed that they had acquired a relatively higher resistance to oxytetracycline, amoxycillin, ampicillin, novobiocin and polymixin-B, implicating possible use of these antibiotics in the aquaculture systems.     

Evaluation of the Rapid Strep system for identification of gram-positive, catalase-negative cocci isolated from bovine intramammary infections

The Rapid Strep system (Analytab Products, Plainview, NY) was used to identify 199 gram-positive, catalase-negative cocci isolated from bovine intramammary infections. The system accurately identified 88.4% of isolates. The system identified 100% of 46 Streptococcus agalactiae, 100% of 48 Streptococcus dysgalactiae, 54.5% of 11 Streptococcus equinus, and 96.2% of 53 Streptococcus uberis isolates. Enterococcus spp. were identified correctly 83.3% of the time. One of 4 Streptococcus saccharolyticus strains was identified as Streptococcus bovis, the previous classification for this organism, and 8 Streptococcus equi ssp. equi strains were misidentified as Streptococcus dysgalactiae. The Rapid Strep system was determined to be an acceptable alternative to conventional methods for identification of gram-positive, catalase-negative cocci isolated from bovine intramammary infections.     

石家庄市肉鸡屠宰和市售环节弯曲菌污染状况及耐药分析

目的了解石家庄市肉鸡屠宰和市售环节中弯曲菌的污染状况及对12种抗生素的耐药谱和耐药特征。方法采集屠宰前肉鸡肛拭子、鸡酮体涂抹拭子标本和市售鸡肉、鸡肝脏样品,参照GB 4789.9—2014《食品安全国家标准食品微生物学检验空肠弯曲菌检验》和多重聚合酶链式反应(PCR)方法进行弯曲菌的分离和鉴定,用K-B纸片法对分离弯曲菌进行6类12种抗生素药敏测定。结果在439份标本/样品中共有17份检出弯曲菌,总检出率为3.9%(17/439)。空肠弯曲菌对萘啶酸、左氧氟沙星、环丙沙星和四环素的耐药率均为85.7%(6/7),对庆大霉素、链霉素、丁胺卡那、阿奇霉素、氟苯尼考全部敏感。结肠弯曲菌除对阿奇霉素、多西环素、氟苯尼考的耐药率小于50.0%外,对环丙沙星、左氧氟沙星、萘啶酸、庆大霉素、丁胺卡那、四环素、链霉素、克林霉素的耐药率均高于80.0%。17株弯曲菌多重耐药率为70.6%(12/17),呈11种耐药谱型,市售环节分离株耐药谱型多于屠宰环节分离株,并具有各自不同的优势耐药谱型。结论石家庄市肉鸡屠宰和市售环节均有一定程度的弯曲菌污染,分离株多重耐药情况严重,呈多种耐药谱型。     

Characterisation of Aeromonas spp. isolated from frozen fish intended for human consumption in Mexico

A total of 82 strains of presumptive Aeromonas spp. were identified biochemically and genetically (16S rDNA-RFLP). The strains were isolated from 250 samples of frozen fish (Tilapia, Oreochromis niloticus niloticus) purchased in local markets in Mexico City. In the present study, we detected the presence of several genes encoding for putative virulence factors and phenotypic activities that may play an important role in bacterial infection. In addition, we studied the antimicrobial patterns of those strains. Molecular identification demonstrated that the prevalent species in frozen fish were Aeromonas salmonicida (67.5%) and Aeromonas bestiarum (20.9%), accounting for 88.3% of the isolates, while the other strains belonged to the species Aeromonas veronii (5.2%), Aeromonas encheleia (3.9%) and Aeromonas hydrophila (2.6%). Detection by polymerase chain reaction (PCR) of genes encoding putative virulence factors common in Aeromonas, such as aerolysin/hemolysin, lipases including the glycerophospholipid-cholesterol acyltransferase (GCAT), serine protease and DNases, revealed that they were all common in these strains. Our results showed that first generation quinolones and second and third generation cephalosporins were the drugs with the best antimicrobial effect against Aeromonas spp. In Mexico, there have been few studies on Aeromonas and its putative virulence factors. The present work therefore highlights an important incidence of Aeromonas spp., with virulence potential and antimicrobial resistance, isolated from frozen fish intended for human consumption in Mexico City.