华北地区乳房炎奶样中大肠杆菌的耐药性研究

为了解华北地区乳房炎奶样中大肠杆菌的耐药情况,本试验采集华北地区乳房炎奶样240批次,采用EMB培养基和菌悬液法对大肠杆菌进行分离鉴定,通过纸片扩散法对常用的八类29种抗生素进行药敏试验。试验结果表明共分离大肠杆菌17株(7.08%)。药敏试验结果显示大肠杆菌分离株表现出高度耐药和多重耐药。分离株对至少4种抗生素产生耐药性,最多耐药19种,耐药5种以上的菌株较多(58.82%);针对单一抗生素,分离菌株对青霉素、苯唑西林、林可霉素3种药物耐药率高达100%。针对不同种类抗生素,大肠杆菌分离株对β-内酰胺类药物和林可酰胺类药物的耐药率分别为55.15%,61.76%,均超过半数。本研究旨在发现乳房炎奶样中大肠杆菌的耐药性规律,并根据数据对奶牛乳房炎精准防控,减少牛乳中兽药残留,提升牛乳质量。     

发酵酸鱼中乳酸菌的鉴定及特性分析

该研究以分离自酸鱼发酵后期的乳酸菌菌株LP224为研究对象，采用形态学观察、生理生化试验和分子生物学技术对其进行菌种鉴定，采用K-B药敏纸片扩散法和聚合酶链式反应（PCR）法对其耐药性和抗性基因进行检测，并研究其生长特性及耐受性。结果表明，菌株LP224被鉴定为植物乳杆菌（Lactobacillus plantarum），其对头孢曲松、链霉素、环丙沙星、磺胺异噁唑、万古霉素、林可霉素等6类15种抗生素耐药，基因组DNA检测出ermC和tetG2种抗性基因，而细菌质粒未检测到抗性基因，说明该菌株相对安全。菌株LP224经55℃水浴处理30 min后存活率为35.06%;p H2.5环境下存活率为52.60%;0.3%胆盐浓度下存活率为31.67%，说明该菌株具有较强的耐热、耐酸、耐胆盐能力。     

高产林可霉素链霉菌的筛选及发酵条件的优化

采用平板划线,摇瓶复筛等方法从福清近海海域泥土中分离筛选到一株林可霉素产率较高的链霉菌株6204;采用单因素试验和正交设计试验,从葡萄糖添加量、磷酸二氢钾条件两及金属离子对菌株6204产林可霉素影响,并对其发酵条件进行优化。结果表明,最适的发酵条件为葡萄糖添加量10.0%,镁离子质量浓度0.10 g/L,磷酸二氢钾添加量0.020%,在此优化发酵条件下,菌株6204产林可霉素的含量可达3 907.738 μg/mL,与优化前相比较,林可霉素的含量提高了1 823.082 μg/mL。     

黄精多糖脱色和脱蛋白工艺研究

目的:研究脱色和脱蛋白工艺中不同条件对黄精中多糖纯化效果的影响,得出黄精多糖的最佳脱色和脱蛋白工艺。方法:通过用双氧水和活性炭对黄精多糖进行脱色,在此基础上研究浓度差异对脱色效果的影响,确定最佳脱色工艺。采用Sevage法和TCA法对黄精多糖进行脱蛋白,研究不同条件对脱蛋白效果的影响。结果:双氧水法为黄精多糖的最佳脱色方法,其最佳脱色浓度为1%;Sevage法为黄精多糖的最佳脱蛋白方法,其最佳比例为氯仿:正丁醇为6:1。     

2015年分离自中国大陆食品的1 070株沙门菌耐药性分析

目的 了解我国2015年食源性沙门菌的耐药状况及mcr-1基因在硫酸粘菌素(conlistin,CT)耐药菌株中的分布情况。方法 采用微量肉汤稀释法测定1 070株食源性沙门菌对10类16种抗生素的药物敏感性,采用荧光定量聚合酶链式反应(PCR)方法检测196株CT耐药菌株中是否存在mcr-1基因。结果 71.9%(769/1 070)的沙门菌对受试的16种抗生素呈现不同程度的耐药性,其中萘啶酸(NAL)、四环素(TET)、氨苄西林(AMP)、氨苄西林/舒巴坦(SAM)4种抗生素的耐药率较高,均在40%以上,未见碳青霉烯类耐药菌株,47.5%(508/1 070)的沙门菌同时耐受3类或3类以上抗生素,表现为多重耐药,同时耐受抗生素种类最高为9类。共存在141种耐药谱,优势耐药谱型为NAL、AMP-SAM-NAL-CT和TET。196株CT耐药沙门菌检出1株携带mcr-1基因且超广谱β-内酰胺酶(ESBLs)阳性的菌株,该菌株为伦敦血清型,是所有测试菌株中唯一1株九重耐药株。部分省份沙门菌耐药率较高。生禽肉和生畜肉来源沙门菌耐药率较高,分别为80.6%(349/433)和73.5%(283/385)。结论 我国2015年食源性沙门菌整体耐药水平较高,且多重耐药情况严重,生禽畜肉是耐药沙门菌的主要来源,我国食源性沙门菌中存在携带mcr-1基因的严重耐药菌株,应引起关注。     

2016年中国26个省市食源性沙门菌耐药性特征分析

目的 了解2016年中国26个省、直辖市和自治区食源性沙门菌的耐药状况。方法 采用微量肉汤稀释法测定755株食源性沙门菌对10类16种抗生素的药物敏感性,采用实时荧光定量聚合酶链式反应方法检测mcr-1基因的存在情况。结果 72.7%(549/755)的沙门菌对受试的16种抗生素呈现不同程度的耐药性,其中萘啶酸(NAL)、四环素(TET)、氨苄西林(AMP)、氨苄西林/舒巴坦(SAM)4种抗生素的耐药率较高,均在34%以上,未见碳青霉烯类[亚胺培南(IPM)、美罗培南(MEM)]耐药菌株,44.4%(335/755)的沙门菌同时耐受3类或3类以上抗生素,表现为多重耐药,同时耐受抗生素种类最高为8类。共存在134种耐药谱,优势耐药谱型为NAL、TET和AMP-SAM-NAL。全部菌株中检出2株携带mcr-1基因的菌株,分别为八重耐药的德尔卑沙门菌(Salmonella Derby)和七重耐药的鼠伤寒沙门菌(Salmonella Typhimurium)。部分省份沙门菌耐药率较高。结论 2016年中国26个省、直辖市和自治区食源性沙门菌整体耐药水平较高,多重耐药情况严重,我国食源性沙门菌中存在携带mcr-1基因的多重耐药菌株,应引起关注。     

蚯蚓抗菌肽的提取与抗菌活性研究

用30%～80%不同浓度硫酸铵沉淀蚯蚓蛋白,用考马斯亮蓝测定蛋白含量,采用管磲法和微量稀释方法分别测定蚯蚓抗菌肽和苯甲酸钠在pH值分别是4.0、7.0、9.0下的抑菌活性及最小抑菌浓度(MIC).结果显示40%、50%、60%、70%硫酸铵沉淀出的蛋白都表现出抗菌活性,并对G (金黄色葡萄球菌)和G-(大肠杆菌)均有抑菌性.同时证实蚯蚓抗菌肽抗菌性受pH影响不大.     

大米类蛋白反应条件优化及其产物的氨基酸组成分析

以大米蛋白的碱性蛋白酶水解产物为原料,研究了反应条件对类蛋白反应产率的影响,采用正交试验优化了类蛋白反应的条件,并分析了最佳条件下类蛋白的氨基酸组成.结果表明,合成用蛋白酶的种类及其添加量、底物浓度、反应体系的pH、反应温度和时间均对类蛋白反应产率有明显影响,在底物浓度为150%(w/v)、胃蛋白酶添加量5%、pH 6.0下,于65 ℃保温反应6 h,类蛋白产率最高(54.85%).类蛋白的氨基酸组成与大米蛋白、大米蛋白的碱性蛋白酶水解产物的氨基酸组成存在差异,类蛋白中苏氨酸、异亮氨酸、苯丙氨酸和赖氨酸等必需氨基酸含量高于大米蛋白和大米蛋白的碱性蛋白酶水解产物的,类蛋白反应可改善大米蛋白的营养价值.     

乳品生产链金黄色葡萄球菌的污染、耐药性分析及β-内酰胺类药物耐药基因的检测

为分析乳品生产链中金黄色葡萄球菌(Sa)的污染及耐药情况,从部分乳品生产企业采集乳样(生鲜牛乳、生产车间半成品牛乳、成品牛乳)523份,按照GB 4789.10-2010及PCR方法分离鉴定Sa菌株1;采用微量肉汤稀释法,测定Sa分离株对12种抗生素的药敏性,并利用多重PCR方法分析Sa对β-内酰胺类药物耐药性的产生与其耐药相关基因(mecA、blaZ)的关联性。结果显示,乳样Sa总分离率为24.9%(129株),其中生鲜牛乳、中间半成品牛乳及成品牛乳Sa污染率分别为37.5%、7.1%和0.0%;受试Sa分离株对青霉素的耐药率(97.7%)最高,其它依次是氨苄西林(95.4%)、甲氧苄啶/磺胺甲噁唑(61.9%)、阿莫西林/克拉维酸(61.7%)、红霉素(39.7%)、四环素(36.6%)、盐酸林可霉素(35.2%);所有菌株均对苯唑西林、头孢噻呋、氟苯尼考敏感;多重耐药Sa分离率为69.8%,主要对青霉素类、磺胺类、大环类脂类、林可胺类及四环素类表现出多重耐药;多重PCR检测结果显示,129株测试Sa的nuc、blaZ、mecA阳性率分别为100.0%、60.5%及0.0%;不同生产环节乳源Sa携带blaZ基因与其对β-内酰胺类药物的耐药表型有一定的对应关系。结果表明,乳品生产链Sa污染及耐药现状已不容乐观,应引起重视,避免食源性疾病的发生。     

酸菜汁来源的几株乳杆菌功能特性研究

以自然发酵酸菜汁中分离获得的几株乳杆菌为研究对象,首先通过模拟乳杆菌在不同酸度、不同氯化钠浓度以及胆盐浓度、人工胃液和人工肠液等环境中的生长状态,评价其耐受性。结果表明,菌株可耐受pH4.0和pH5.0的环境,2%~6%的氯化钠浓度以及0.1%和0.2%的胆盐条件。通过稀释涂布法发现LS-9菌株在人工肠液中的存活率最高,8 h达到98.34%。采用液相色谱法测定菌株对MRS培养基中的胆固醇降解率在16.65%~29.33%之间。自聚集和共聚集实验中,LS-5菌株20 h的聚集能力显著高于其余菌株(p0.01)。LS-6菌株表现出最明显的疏水性,疏水率为45.16%。采用纸片扩散法对菌株进行药敏性分析,均对青霉素、红霉素、氨苄西林、氯霉素和四环素这5种抗生素具有极敏的性质。以上实验结果为进一步认识并应用这些乳杆菌提供参考。     

Antimicrobial activity and antibiotic resistance of lactic acid bacteria isolated from Armada cheese (a Spanish goats’ milk cheese)

Thirty-one lactic acid bacteria isolated from Armada cheese and previously selected in accordance with their technological properties, were screened for antimicrobial activity one against another. Four strains showed inhibitory activity against 14 strains when tested by well diffusion assay after the effects of organic acid and hydrogen peroxide were eliminated. Extracts of the strains did not show inhibitory activity after treatment with proteinase K, trypsin, α-chymotrypsin or pepsin. None of the strains showed antimicrobial activity against several pathogenic and spoilage reference strains. Eighteen representative strains were tested for their antibiotic resistance. None of the strains were totally susceptible to all antibiotics tested and multiple resistance was observed. Most of the tested strains were resistant to cefotoxin, oxacillin, vancomycin, teicoplanin, nitrofurantoin and trimethoprim.     

2016年广西壮族自治区食源性沙门菌的耐药性与耐药谱研究

目的 了解广西壮族自治区食源性沙门菌耐药状况,建立广西壮族自治区食源性沙门菌耐药性数据库。方法 对2016年从广西壮族自治区各市县收集的分离自食品的70株沙门菌和分离自腹泻患者的234株沙门菌采用微量肉汤法对8类14种抗生素进行耐药性试验,开展耐药谱研究。结果 所有沙门菌菌株均对亚胺培南敏感,对其余13种抗生素产生不同程度的耐药。70株食品来源的沙门菌对四环素耐药率最高(45.71%,32/70);234株腹泻患者来源的沙门菌对氨苄西林耐药率最高(70.94%,166/234),其次为四环素(70.09%,164/234)。腹泻患者来源沙门菌对氨苄西林、氨苄西林/舒巴坦、四环素、萘啶酸、头孢噻肟和头孢唑啉的耐药率均高于食品分离株,差异有统计学意义(P<0.05)。2种来源的沙门菌耐药谱广泛,共同耐药谱为ACTT/S(耐氨苄西林-氯霉素-四环素-甲氧苄啶/磺胺甲噁唑)。结论 广西壮族自治区食源性沙门菌耐药状况不容乐观,特别是对青霉素类、氯霉素类、磺胺类、四环素类抗生素严重耐药,提示应对其加强监测,建立科学的防控策略。     

Antibiotic resistance in gut bacteria from dairy calves: a dose response to the level of antibiotics fed in milk

Dairy calves are commonly fed milk from cows treated with antibiotics. The concentration of beta-lactam antibiotic residues found in milk from treated cows was used to determine the range of concentrations of penicillin used in a dose-regulated experiment. Thirty-one Holstein calves were randomly assigned to milk with penicillin G added at concentrations of 0, 6.25, 12.5, 25, and 50 microl/kg. Fecal swabs were taken from each calf twice weekly. Resistance to penicillin was tested by measuring the zone of inhibition in bacterial growth around a disk impregnated with the antibiotic. Inhibition was greatest for bacteria from calves fed milk with no penicillin (2.89 +/- 0.14 mm), and declined as the penicillin dose provided in the milk increased (to a low of 0.70 +/- 0.10 for the 50 microl/kg treatment group). In conclusion, resistance of gut bacteria to antibiotics increases with increasing concentrations of penicillin in the milk fed to dairy calves.     

2019年我国不同地区牧场生牛乳中致病菌污染及抗生素耐药情况研究

目的 了解我国不同地区牧场生牛乳中致病菌污染情况和细菌耐药特征。方法 对2019年9—11月采集自43家不同地区牧场的53份生牛乳样品进行6种致病菌的分离鉴定,对分离株开展抗生素耐药性检测。结果 53份样品中大肠埃希氏菌、金黄色葡萄球菌、蜡样芽孢杆菌和单核细胞增生李斯特氏菌(单增李斯特菌)的污染率分别为62.26%(33/53)、20.75%(11/53)、5.66%(3/53)和3.77%(2/53),克罗诺杆菌和沙门氏菌均未检出。选取117株分离株进行药物敏感性实验,其中58株菌耐药,总体耐药率为49.57%(58/117);90株革兰氏阴性菌中有33株耐药,耐药率为36.67%(33/90);27株革兰氏阳性菌中有25株耐药株,耐药率为92.59%(25/17)。结论 不同地区牧场采集的生牛乳中细菌污染情况差异较大,污染随气温降低呈下降趋势。同时生乳中细菌分离株多重耐药率高,需进一步严格控制兽用抗生素的使用。     

双歧杆菌的药敏性及其质粒DNA的检测

利用药敏纸片琼脂扩散法检测了10株具有粘附性的双歧杆菌对临床常用的12大类38种抗菌药物的药敏性。结果显示，对于同一菌株，其对同类抗菌药物表现出基本一致的药物敏感性；不同菌株间在对于同一类药物的敏感性上也有着较好的吻合，如对青霉素类、喹诺酮类、氨基糖甙类及多肽类的表现几乎全为耐药，对头孢类、大环内酯类、磺胺、氯霉素、克林霉素则基本表现为敏感。但在此基础上，不同来源的菌株问也存在着一定的差异。采用碱裂解法提取并检测了受试双歧杆菌的耐药性质粒，仅在一株受试双歧杆菌H-3中发现有1条质粒带，其对应的核酸分子量参照在5148～21226bps之间，其他受试菌株中未发现有质粒存在。     

Antibiotic Resistance Patterns of Gram‐Negative Psychrotrophic Bacteria from Bulk Tank Milk

Bacterial resistance to antibiotics is a major global health problem and resistance of Pseudomonadaceae and Enterobacteriaceae is a serious concern. We investigated the prevalence of drug‐resistance in a total of 80 psychrotrophic strains from bulk milk belonging to Pseudomonas genus (n. 63) and Enterobacteriaceae group (n. 17). All the strains were tested against 16 antibiotics. Pseudomonas were further investigated for their sensitivity against 12 additional antibiotics. Pseudomonas showed a high susceptibility toward fluoroquinolones, aminoglycosides, and piperacillin and, to a lesser extent, to imipenem, ceftazidime, cefepime. Thirty‐five out of 63 Pseudomonas strains were susceptible to meropenem, while among antibiotics for which recommended breakpoints are not yet available, 55% of Pseudomonas strains had no inhibition halo in presence of nitrofurantoin, highlighting a resistance toward this drug. The results obtained in this study indicate a high efficiency of fluoroquinolones, chloramphenicol (94%), and kanamycin (76%) for Enterobacteriaceae while a high prevalence of resistant strains was found to ampicillin (13/17). Serratia marcescens is highly susceptible to fluoroquinolones, chloramphenicol, and kanamycin. Moreover, mupirocin seems to be the new antibiotic with the less efficacy for Enterobacteriaceae, with 41% of strains without halo, pointing out an important resistance. Further knowledge on resistance to known and new antibiotics among Pseudomonas species and Enterobacteriaceae of milk origin was acquired.     

Insights into antibiotic use in Brazilian dairy production

Milk and dairy products are important sources of proteins, fats and vitamins. Although Brazil is the fourth largest milk producer in the world, mastitis, metritis, enteritis and respiratory diseases are still important in this industry. A number of antibiotics are employed for treatment and prophylaxis for these diseases, including cephalosporins, lincosamides, aminoglycosides, penicillins, tetracyclines and macrolides. Vaccination offers an important opportunity to reduce the demand for antibiotics. In this review, we present insights into milk production, antibiotic use in the Brazilian dairy industry, the consequences of these activities and perspectives for the control and surveillance of antibiotic resistance.     

鲜牛奶中耐药性细菌的分离与鉴定

目的:分离鉴定鲜牛奶中耐药性细菌的分布。方法:利用含有四环素(16μg/mL)、环丙沙星(4μg/mL)或庆大霉素(16μg/mL)的Luria-Bertani平板,分离样品中的耐药性细菌;采用K-B纸片法对分离菌株的耐药性进行确认;利用血平板测定分离菌株的溶血性;利用16S rRNA方法对分离菌株进行鉴定。结果:对30份采自河北张家口地区新鲜无菌牛奶样品的平板筛选结果显示,具有耐受1种以上抗菌素细菌的样品为23份(76.67%),具有耐受2种以上抗菌素细菌的样品为7份(23.33%),具有耐受3种以上抗菌素细菌的样品为1份(3.33%);共分离得到耐受四环素菌株37株、耐受环丙沙星8株、耐受庆大霉素菌株7株;随机挑选6株(每种抗菌素耐受细菌选取2株)进行16S rRNA鉴定,结果为黏质沙雷氏菌(Serratia marcescens)1株、铜绿假单胞菌(Pseudomonas aeruginosa)2株、琼氏不动杆菌(Acinetobacter junii)1株、阪崎肠杆菌(Cronobacter sakazakii)1株和金黄色葡萄球菌(Staphylococcus aureus)1株;6株细菌中α溶血3株,β溶血1株,γ溶血2株;纸片法验证结果显示,只有1株分离细菌对庆大霉素敏感。结论:鲜牛奶样品中普遍存在多种耐药性细菌,抗菌素平板可以用于初步分析牛奶样品的耐药性细菌。     

Comparison of the incidence of virulence determinants and antibiotic resistance between Enterococcus faecium strains of dairy, animal and clinical origin

Enterococci are part of the dominant microbiota of several dairy products. They are also present in the gut of humans and animals. Their presence in traditional raw milk cheeses is probably due to faecal contamination of milk during milking. Due to their importance as a cause of nosocomial infections, enterococci are acquiring increased significance. Such infections are becoming more and more difficult to treat as resistance to antibiotics increases. The aim of this investigation was to compare the potential virulence of Enterococcus faecium isolated from different ecological habitats and to establish if strains isolated from dairy products should really be considered as potential pathogens. In the present work, the antibiotic resistance pattern of 40 E. faecium strains isolated from dairy products, 26 E. faecium isolated from ewes' faeces and 28 clinical isolates of the same species was studied, and checks were made to see if known virulence determinants were present. Resistance to 12 different antibiotics commonly used in the treatment of human infections was tested using the broth microdilution method as described by the NCCLS. In addition, polymerase chain reaction (PCR) tests were carried out to see if genes for vancomycin resistance were present. The presence of the aggregation substance (AS) gene, the surface protein gene esp, the accessory colonisation factor ace, the Enterococcus faecalis endocarditis antigen efaA and the gelatinase gelE gene, which are involved in the virulence of enterococci, were also tested by PCR. The results of this study clearly indicate that E. faecium strains isolated from both cheese and sheep faeces are less pathogenic than those isolated from clinical samples. A similar pattern of resistance to antibiotics was observed in both dairy and animal strains. It was also found that there was difference in the kind of virulence determinants present in dairy and clinical isolates, while no virulence traits were found in sheep faeces strains. The results of this study suggest that E. faecium from traditional Sardinian raw milk cheeses should not be considered to be the main source of untreatable nosocomial enterococcal infections in humans in the island of Sardinia.     

Antibiotic residues in milk from small dairy farms in rural Peru

The use of antibiotics in livestock can pose a public health threat, especially if antibiotic residues remain in the food product. Understanding how often and why farmers sell products with antibiotic residues is critical to improving the quality of these products. To understand how often milk with antibiotic residues is sold on small farms in a major dairy-producing region of Peru and identify factors associated with selling milk with antibiotic residues, we tested milk samples for antibiotic residues from every provider on three routes of commercial milk companies and from bulk tanks of farmers currently treating cows with antibiotics. We also asked farmers if they sold milk from treated cows and examined factors associated with the tendency to do so. The prevalence of milk contamination with antibiotic residues on commercial routes was low (0–4.2%); however, 33/36 farmers treating their animals with antibiotics sold milk that tested positive for antibiotic residues. The self-reported sale of milk from treated cows had a sensitivity, specificity, and positive and negative predictive values of 75.8%, 100%, 100% and 27.2%, respectively (with testing of milk for residues as the gold standard). Finally, 69/156 randomly selected farmers reported selling milk from treated cows, and farmers’ knowledge of antibiotics and the milk purchaser were significantly associated with a farmer’s tendency to report doing so. Educating farmers on the risks associated with antibiotics and enforcement of penalties for selling contaminated milk by milk companies are needed to improve milk quality.