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。     

肉鸡屠宰场多重耐药沙门氏菌Ⅰ类整合子与磺胺类耐药基因（sul1、sul2和sul3）的检测

目的：了解肉鸡屠宰场多重耐药沙门氏菌Ⅰ类整合子及磺胺类耐药基因携带情况。方法：采用纸片扩散法对肉鸡屠宰场84 株沙门氏菌分离株进行10 种抗生素敏感性实验;应用聚合酶链式反应（polymerase chain reaction,PCR）方法检测多重耐药沙门氏菌sul1、sul2、sul3和int1基因。结果：84 株沙门氏菌对氨苄西林和萘啶酸耐药率高达100%,对环丙沙星、四环素、甲氧苄啶/磺胺甲噁唑、大观霉素、氟苯尼考、庆大霉素耐药率分别为39.29%、35.71%、35.71%、35.71%、22.62%、14.29%。38 株沙门氏菌对3 种及以上抗生素耐药,属于多重耐药菌株。38 株多重耐药菌株中,有20 株携带Ⅰ类整合子。30 株对甲氧苄啶/磺胺甲噁唑耐药的菌株中,均能检测到sul1或sul2或sul3基因,与表型耐药100%符合,其检出率分别为40%、100%、63.3%。结论：肉鸡屠宰场中沙门氏菌耐药现象已不容乐观,沙门氏菌多重耐药性与Ⅰ类整合子的携带之间关系密切,且Ⅰ类整合子在磺胺类耐药菌株的产生中起到重要作用。     

发酵奶中乳酸菌菌种检出及活菌计数调查

目的分析北京市场发酵奶(酸奶)在保质期内的乳酸菌数及乳酸菌菌种的检出率。方法对11个酸奶厂家的20种不同酸奶产品进行乳酸菌活菌计数及所用菌种的检验。结果在保质期间,双歧杆菌和嗜酸乳杆菌检出率分别为23.08%和27.27%;保加利亚乳杆菌与嗜热链球菌分别为72.22%和94.44%。在保质期末,嗜热链球菌的平均活菌数为3.23×106CFU/ml,保加利亚乳杆菌为4.17×105CFU/ml,双歧杆菌为1.12×104CFU/ml,嗜酸乳杆菌为1.32×104CFU/ml。结论酸奶中嗜热链球菌和保加利亚乳杆菌的检出率及活菌数均高于双歧杆菌和嗜酸乳杆菌。     

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%)。     

大黄鱼中哈维氏弧菌毒力及耐药特性分析

目的分析鉴定海产养殖中主要病原哈维氏弧菌的毒力及耐药特性。方法采集来自于网箱养殖区病死大黄鱼,分离幵鉴定哈维氏弧菌,利用微量肉汤稀释法分析分离株对13种抗生素的敏感特性,PCR鉴定耐药基因(tetA、tetB、ermA、ermB、ermC、mecA、aac(6’)-Ib、oqxA、sul1、sul2、mcr-1)及其毒力基因。结果从病料中分离幵鉴定出54株哈维氏弧菌,耐药分析表明对硫酸粘杆菌素、庆大霉素、四环素、氨苄西林、复方新诺明和阿莫西林均耐药, 96.2%的分离株对头孢噻呋, 92.6%的分离株对链霉素、阿米卡星表现为耐药, 88.9%的分离株对恩诺沙星, 87.1%的分离株对卡那霉素呈现高度耐受特性。所有分离株同时对9种抗生素显示100%耐药,同时耐受10种抗生素的菌株有94.4%,同时对11和12种抗生素耐受的菌株分别达到85.2%和64.8%,对13种抗生素均产生耐药的菌株达到37.0%(20/54)。耐药基因分析表明四环素类tetA为主要耐药基因,检出率高达81.5%;氨基糖苷类aac(6’)-Ib,β-内酰胺类mecA,磺胺类sul1、sul2,喹诺酮类oqxA和大环内酯类ermA、ermC分别检出4～7株。毒力基因分析表明toxR携带率高达75.9%。结论本研究分析了海产养殖中主要病原哈维氏弧菌感染状况,其耐药严重且普遍携带毒力基因,表明哈维氏弧菌耐药和毒力对海产养殖造成重要威胁。同时本研究为弧菌病的有效防治提供参考依据。     

蒙古国传统发酵乳制品中保加利亚乳杆菌耐药性的研究

保加利亚乳杆菌是发酵乳制品中的常用菌种之一,因此对保加利亚乳杆菌安全性菌株的选育非常关键。以蒙古国传统发酵乳制品中的5株保加利亚乳杆菌为研究对象,对其耐药性进行了初步研究。首先采用肉汤稀释法分析其耐药性,其次利用聚合酶链式反应(PCR)技术对其耐药基因进行了检测。结果表明,保加利亚乳杆菌对利奈唑胺、氨苄西林、克林霉素、利福平、万古霉素、甲氧苄啶及奎奴普丁/达福普汀全部敏感,对四环素、卡那霉素、氯霉素、庆大霉素、链霉素、环丙沙星、红霉素及新霉素均表现出不同程度的耐药性。基于PCR技术在这些菌株中检测到5种不同的耐药基因(erm(B)、vanX、aac(6')-aph(2')、parC、rpoB),每株都携带2种或2种以上耐药基因。本研究结果可为后续的菌种选育提供参考。     

内蒙古传统发酵乳制品中乳酸菌的分离及其耐药性研究

以内蒙古通辽地区自制的奶豆腐和酸性奶油（乌日莫）为材料,采用稀释涂布法从中分离乳酸菌,通过分子生物学技术对其进行菌种鉴定,并对其耐药性进行研究。结果表明,共分离纯化得到80株乳酸菌,经鉴定,归属于13个种,分别为短乳杆菌（Lactobacillus brevis）、粪肠球菌（Enterococcus faecalis）、乳酸片球菌（Lactobacillus acidilacticii）、乳酸乳球菌（Lactococcus lactis）、瑞士乳杆菌（Lactobacillus helveticus）、屎肠球菌（Enterococcus faecium）、戊糖片球菌（Pediococcus pentosaceus）、副干酪乳杆菌（Lactobacillus paracasei）、食二酸乳杆菌（Lactobacillus digitalis）、奥塔基乳杆菌（Lactobacillus ottaki）、耐久肠球菌（Enterococcus durans）、植物乳杆菌（Lactobacillus plantarum）、开菲尔乳杆菌（Lactobacillus kefir）,表现出高的生物多样性。80株乳酸菌对萘啶酸的耐药率为100%;对链霉素、环丙沙星、万古霉素的耐药率较高,分别为95.00%、80.00%、72.50%;对利福平和四环素的耐药率中等,分别为51.25%,45.00%;对红霉素、氯霉素的耐药率较低,分别为26.25%、23.75%。此外,76株（95%）的乳酸菌还具有多重耐药性。     

泡菜发酵乳酸菌的分离鉴定及耐药性分析

对吉林地区朝鲜族自制泡菜中的发酵乳酸菌进行分离鉴定,并对菌株耐药情况进行分析,以期为泡菜发酵菌的风险评估奠定基础。采集吉林省多个不同地区的泡菜制品,通过MRS培养基分离,16S rDNA测序,分析其乳酸菌种类及数量,同时采用平板琼脂稀释法进一步检测了分离菌对10 种临床常用抗生素的药物敏感性。结果表明,从15 份不同厂家泡菜中共分离得到34 株乳酸菌,分别为植物乳杆菌12 株,短乳杆菌5 株,鼠李糖乳杆菌3 株,干酪乳杆菌2 株,清酒杆菌2 株,肠膜明串珠菌2 株,嗜热链球菌2 株,食窦魏斯氏乳杆菌1 株,弯曲乳杆菌1 株,棒状乳杆菌1 株,嗜酸乳杆菌1 株,坚强肠球菌1 株,Lactobacillus namurensis 1 株。药敏实验结果表明,所有菌株对氨苄西林和氯霉素敏感,对其他抗生素均有不同程度的耐药性,其中对环丙沙星耐药率最高。     

传统发酵食品中乳酸菌的抗生素耐药性评估及耐药基因分析

对传统发酵食品中分离的97株乳酸菌的抗生素耐药性进行评估及耐药基因分析,结果表明:所有分离菌株对万古霉素、庆大霉素、卡那霉素、链霉素、四环素和替考拉宁的耐药性较强,耐药率达50%以上;对氨苄青霉素、红霉素、甲氧苄啶、头孢呋肟较敏感,耐药率为10%左右。携带最多的耐药基因是tetM(91.75%)和ermB(86.60%)基因,其次为aac(6,)-aph(2')(53.61%),lnuA(45.36%),tetK(39.18%),ermC(36.08%),strA(34.02%)和blaZ(32.99%)基因,携带率相对较低的基因为strA(27.84%),msrA/B(22.22%)和aph(3')-III(7.22%)基因。另外,所有乳酸菌中均未检测出氯霉素(cat)和万古霉素(vanX)耐药基因。多重耐药菌株在发酵乳制品中的分离率最高,且多出现在植物乳杆菌(45.83%)、凝结芽孢杆菌(20.83%)和耐久肠球菌(12.28%)中。MTP试验结果显示70%的菌株都具有产生物被膜的能力,其多重耐药性显著高于不产生物被膜的菌株。综上所述,乳酸菌的耐药性可能成为重要的食品安全隐患,急需出台一系列用药规定,减少抗生素的选择压力,加强食用菌株的安全性检测。     

基于特征风味定向筛选酸奶用乳酸菌

针对国内酸奶发酵剂成本高,菌剂被国外垄断的问题。本实验基于特征风味物质乙醛和双乙酰,从市售酸奶产品和菌剂中定向筛选出高风味物质含量菌株,经复配和感官评定,得到适合生产菌株。实验共分离鉴定出58株乳酸菌,包括嗜酸乳杆菌(9株)、鼠李糖乳杆菌(2株)、德氏乳杆菌(4株)、嗜热链球菌(39株)、乳酸乳球菌乳酸亚种/乳酸乳球菌(4株)。产乙醛能力呈现出德氏乳杆菌嗜热链球菌乳酸乳球菌嗜酸乳杆菌鼠李糖乳杆菌,最高达到25.57μg/g。产双乙酰能力呈现出鼠李糖乳杆菌嗜酸乳杆菌嗜热链球菌乳酸乳球菌德氏乳杆菌,最高达到3.57μg/g。德氏乳杆菌和鼠李糖乳杆菌产酸能力较强。菌株复配后大多风味物质含量提高,在一定条件下,呈现出乙醛/双乙酰比例越高,乙醛含量越高,感官风味越好。     

Antibiotic resistance of lactic acid bacteria isolated from Chinese yogurts

The aim of this study was to evaluate the susceptibility of 43 strains of lactic acid bacteria, isolated from Chinese yogurts made in different geographical areas, to 11 antibiotics (ampicillin, penicillin G, roxithromycin, chloramphenicol, tetracycline, chlortetracycline, lincomycin, kanamycin, streptomycin, neomycin, and gentamycin). The 43 isolates (18 Lactobacillus bulgaricus and 25 Streptococcus thermophilus) were identified at species level and were typed by random amplified polymorphic DNA analysis. Thirty-five genotypically different strains were detected and their antimicrobial resistance to 11 antibiotics was determined using the agar dilution method. Widespread resistance to ampicillin, chloramphenicol, chlortetracycline, tetracyclines, lincomycin, streptomycin, neomycin, and gentamycin was found among the 35 strains tested. All of the Strep. thermophilus strains tested were susceptible to penicillin G and roxithromycin, whereas 23.5 and 64.7% of Lb. bulgaricus strains, respectively, were resistant. All of the Strep. thermophilus and Lb. bulgaricus strains were found to be resistant to kanamycin. The presence of the corresponding resistance genes in the resistant isolates was investigated through PCR, with the following genes detected: tet(M) in 1 Lb. bulgaricus and 2 Strep. thermophilus isolates, ant(6) in 2 Lb. bulgaricus and 2 Strep. thermophilus isolates, and aph(3')-IIIa in 5 Lb. bulgaricus and 2 Strep. thermophilus isolates. The main threat associated with these bacteria is that they may transfer resistance genes to pathogenic bacteria, which has been a major cause of concern to human and animal health. To our knowledge, the aph(3')-IIIa and ant(6) genes were found in Lb. bulgaricus and Strep. thermophilus for the first time. Further investigations are required to analyze whether the genes identified in Lb. bulgaricus and Strep. thermophilus isolates might be horizontally transferred to other species.     

Assessment of antibiotic susceptibility within lactic acid bacteria strains isolated from wine

Susceptibility to 12 antibiotics was tested in 75 unrelated lactic acid bacteria strains of wine origin of the following species: 38 Lactobacillus plantarum, 3 Lactobacillus hilgardii, 2 Lactobacillus paracasei, 1 Lactobacillus sp, 21 Oenococcus oeni, 4 Pediococcus pentosaceus, 2 Pediococcus parvulus, 1 Pediococcus acidilactici, and 3 Leuconostoc mesenteroides. The Minimal Inhibitory Concentrations of the different antibiotics that inhibited 50% of the strains of the Lactobacillus, Leuconostoc and Pediococcus genera were, respectively, the following ones: penicillin (2, < or =0.5, and < or =0.5 microg/ml), erythromycin (< or =0.5 microg/ml), chloramphenicol (4 microg/ml), ciprofloxacin (64, 8, and 128 microg/ml), vancomycin (> or =128 microg/ml), tetracycline (8, 2, and 8 microg/ml), streptomycin (256, 32, and 512 microg/ml), gentamicin (64, 4, and 128 microg/ml), kanamycin (256, 64, and 512 microg/ml), sulfamethoxazole (> or =1024 microg/ml), and trimethoprim (16 microg/ml). All 21 O. oeni showed susceptibility to erythromycin, tetracycline, rifampicin and chloramphenicol, and exhibited resistance to aminoglycosides, vancomycin, sulfamethoxazole and trimethoprim, that could represent intrinsic resistance. Differences were observed among the O. oeni strains with respect to penicillin or ciprofloxacin susceptibility. Antibiotic resistance genes were studied by PCR and sequencing, and the following genes were detected: erm(B) (one P. acidilactici), tet(M) (one L. plantarum), tet(L) (one P. parvulus), aac(6')-aph(2") (four L. plantarum, one P. parvulus, one P. pentosaceus and two O. oeni), ant(6) (one L. plantarum, and two P. parvulus), and aph(3')-IIIa (one L. plantarum and one O. oeni). This is the first time, to our knowledge, that ant(6), aph(3')-IIIa and tet(L) genes are found in Lactobacillus and Pediococcus strains and antimicrobial resistance genes are reported in O. oeni strains.     

Characterization of Antibiotic Resistance Genes from Lactobacillus Isolated from Traditional Dairy Products

Lactobacilli are widely used as starter cultures or probiotics in yoghurt, cheese, beer, wine, pickles, preserved food, and silage. They are generally recognized as safe (GRAS). However, recent studies have shown that some lactic acid bacteria (LAB) strains carry antibiotic resistance genes and are resistant to antibiotics. Some of them may even transfer their intrinsic antibiotic resistance genes to other LAB or pathogens via horizontal gene transfer, thus threatening human health. A total of 33 Lactobacillus strains was isolated from fermented milk collected from different areas of China. We analyzed (1) their levels of antibiotic resistance using a standardized dilution method, (2) their antibiotic resistance gene profiles by polymerase chain reaction (PCR) using gene‐specific primers, and (3) the transferability of some of the detected resistance markers by a filter mating assay. All Lactobacillus strains were found to be resistant to vancomycin, but susceptible to gentamicin, linezolid, neomycin, erythromycin, and clindamycin. Their susceptibilities to tetracycline, kanamycin, ciprofloxacin, streptomycin, quinupristin/dalfopristin, trimethoprim, ampicillin, rifampicin, and chloramphenicol was different. Results from our PCR analysis revealed 19 vancomycin, 10 ciprofloxacin, and 1 tetracycline‐resistant bacteria that carried the van(X), van(E), gyr(A), and tet(M) genes, respectively. Finally, no transferal of the monitored antibiotic resistance genes was observed in the filter mating assay. Taken together, our study generated the antibiotic resistance profiles of some milk‐originated lactobacilli isolates and preliminarily assessed their risk of transferring antibiotic gene to other bacteria. The study may provide important data concerning the safe use of LAB.     

不同来源的植物乳杆菌基因组和抗生素抗性

为了研究不同分离源对植物乳杆菌基因组和功能的影响,选取了来自发酵酱、泡菜、粪便3种环境的33株植物乳杆菌,通过比较基因组学手段研究菌株的基因组基本特征、直系同源基因、系统进化关系,并结合功能基因注释结果与表型结果分析菌株对抗生素的耐药性。系统发育树揭示了分离源对植物乳杆菌的遗传进化具有较为显著的影响;同源基因结果表明粪便源的菌株其特殊基因数量要高于泡菜源和发酵酱源菌株的数量。33株植物乳杆菌均含有环丙沙星、四环素、氯霉素、甲氧苄氨嘧啶和万古霉素的抗性基因,菌株对这些抗生素也表现出抗性;绝大多数菌株对庆大霉素、链霉素、卡那霉素、新霉素、氨苄西林敏感,在基因组中也没有相关抗性基因;而红霉素和克林霉素的基因和表型并不对应。抗生素实验结果说明,分离源对菌株影响较小,大部分基因型与表型可以对应,基因组学对研究植物乳杆菌的生理特性起一定的指导作用。     

牛乳源金黄色葡萄球菌耐药性变迁及β内酰胺类药物耐药基因分析

采用肉汤微量稀释法,对203 株源于2006—2011年四川牛乳源金黄色葡萄球菌(Staphylococcus aureus,Sa)进行10 种常用抗生素药敏性检测,多重聚合酶链式反应(multiplex polymerase chain reaction,M-PCR)法对其耐甲氧西林基因(mecA)和β-内酰胺类药物耐药基因(blaZ)进行分析,了解Sa耐药性变迁。结果表明:牛乳源Sa菌株对青霉素、氨苄西林耐药率一直居高不下;对阿莫西林/克拉维酸、红霉素、甲氧苄啶/磺胺甲噁唑耐药率有增强趋势;对林可霉素、四环素耐药率呈降低趋势;对环丙沙星耐药率不定;对苯唑西林和头孢噻呋敏感;Sa分离株多重耐药率介于72.1%～100.0%,显示出不尽相同的耐药谱,却呈现出不断增宽的趋势。M-PCR检测显示,2006—2011年分离Sa菌株未从基因水平扩增出mecA基因;不同年度分离株均不同程度携带blaZ基因,不同年度分离株blaZ+检出率介于49.1%～87.5%。药敏实验结果与相关耐药基因检测结果存在对应关系,部分菌株的耐药性与耐药基因的检出率不一致,推测还存在其他耐药机制。结论:四川地区牛乳源金黄色葡萄球菌耐药性不容乐观。     

食源性沙门氏菌耐药表型与耐药基因的研究

为了解食源性沙门氏菌（Salmonella）的耐药状况以及耐药基因与耐药表型的关系,从基因水平上探究Salmonella的耐药机制。本研究对215 株分离自1 093 份市售新鲜肉类中的Salmonella进行了耐药状况检测,设计了16 种引物对耐药基因进行聚合酶链式反应（polymerase chain reaction,PCR）扩增,并对扩增产物进行克隆测序。结果显示,分离株对磺胺异恶唑耐药率最高（71.7%）,其次为四环素（69.8%）、甲氧嘧啶（67.9%）和复方新诺明（52.8%）,分离株对环丙沙星、呋喃妥因和头孢类比较敏感。三重及以上耐药的菌株占60.5%,最多可以耐受13 种抗生素;PCR共扩增到10 种耐药基因,测序结果表明扩增到的序列与GenBank上相应参考序列的相似性均在99%以上;β-酰胺类以及四环素类的耐药基因检出情况与耐药表型具有很高的一致性,符合率均在90%以上,其次为磺胺类（81.6%）。说明本研究中的食源性Salmonella分离株的耐药率高,多重耐药情况较为严重,耐药表型与耐药基因检测结果基本一致。     

Identification and antimicrobial susceptibility of lactic acid bacteria from retail fermented foods

One important safety criterion of using lactic acid bacteria (LAB) in food applications is to ensure that they do not carry transferable antimicrobial resistance (AR) determinants. In this study, 63 LAB belonging to six genera, Streptococcus, Lactobacillus, Lactococcus, Enterococcus, Leuconostoc, and Pediococcus, were recovered from 28 retail fermented food products in Maryland, identified to species with 16S-23S rRNA spacer PCRs, and characterized for antimicrobial susceptibility against eight antimicrobials. Besides intrinsic resistance to ciprofloxacin or vancomycin in some lactobacilli, tetracycline resistance was observed in two Streptococcus thermophilus isolates from one cheese and one sour cream sample and was associated with the presence of a nonconjugative tet(S) gene. The results indicated a low level of AR among naturally occurring and starter LAB cultures in fermented dairy and meat products in the United States; therefore, the probability for foodborne LAB to serve as reservoirs of AR is low. Further studies involving a larger sample size are needed to assess the potential risk of AR gene transfer from LAB in fermented food products.     

食源性乳酸菌安全性的评价

从不同来源的样品中分离得到36 株乳酸菌,对其进行常见抗生素如氯霉素、四环素、红霉素抗药性分析,结果表明,26 株菌具有抗药性,其中4 株携带抗生素抗性基因,并且菌株Enterococcus faecium KN9所携带的四环素抗性基因tet（M）和tet（L）能通过接合作用,转移到受体菌Lactococcus lactis MG1614中。对这些乳酸菌产生有害代谢产物分析结果表明,部分菌株可以产生生物胺,所有的菌株都不产生硝基还原酶和偶氮还原酶。所以,本实验分离到的大部分乳酸菌是安全的,只有携带有抗四环素基因的E. faecium KN9具有潜在的安全隐患。因此,需要在食用前对乳酸菌进行安全性评价,以减少可能的安全隐患。