畜牧兽医学报  2019, Vol. 50 Issue (4): 851-860. DOI: 10.11843/j.issn.0366-6964.2019.04.018    PDF    
鉴别显色培养基在奶牛乳房炎病原菌快速鉴定中的应用
吕天星, 李松建, 郝永清     
内蒙古农业大学兽医学院微生物学与免疫学实验室, 呼和浩特 010018
摘要:为研发用于奶牛乳房炎常见病原菌分离鉴定的鉴别显色培养基,并检验其与普通分离鉴定培养基辅以生化鉴定和16S rRNA核酸序列测定两种传统方法鉴定结果的一致性,笔者以生物信息学方法筛出各种属细菌的特异性酶、可作为唯一碳源发酵产酸的碳源,然后合成酶显色底物,连同碳源、酸碱指示剂制备显色培养基,将各种属标准菌株、野生型菌株涂布培养,评估菌落形态、菌落颜色及培养基基质颜色变化。采集隐性乳房炎(CMT法检测)及临床型乳房炎病例的牛奶样品(絮状物、凝块、清亮状或血乳)共计482份,用鉴别显色培养基和两种传统方法进行病原菌的分离、鉴定。鉴别显色培养基上菌落纯化后提取DNA用于16S rRNA序列扩增,产物送去测序(n=194)。以两种传统方法为参考,判定鉴别显色培养基鉴定病原菌的可靠性,用SAS 9.4的FREQ程序计算鉴别显色培养基的简单科恩κ系数。鉴别显色培养基上常见乳房炎病原菌不同种属的菌落形态和培养基基质颜色明显不同,肉眼即可辨别。鉴别显色培养基与两种传统方法鉴定结果的一致性参数分别为κ=0.70、κ=0.96。鉴别显色培养基能够作为常见奶牛乳房炎病原菌的标准鉴定方法。
关键词奶牛    乳房炎    病原菌快速鉴定    鉴别显色培养基    
Application of a New On-farm Differential Chromogenic Medium for Fast Identification of Pathogens Associated with Mastitis in Milk of Dairy Cows
LÜ Tianxing, LI Songjian, HAO Yongqing     
Laboratory of Microbiology and Immunology, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China
Abstract: The present study aimed to develop a new differential chromogenic medium for fast identification of pathogens associated with mastitis in dairy cows, and check its identification consistency with two traditional approaches including normal isolation and identification media combined with biochemical identifications and 16S rRNA sequencing. Specific enzymes and the only carbon source that could be used to produce acid in bacteria of each genus were screened via bioinformatics tools. Synthesized chromogenic substrate of specific enzyme, carbon and acid-base indicator were mixed into basal enrichment medium to prepare differential chromogenic media. Standard strains and wild-type strains of related pathogens were inoculated on differential chromogenic media, and morphology of colonies and color changes of colony and medium that could be considered as identification standards were observed. Milk samples from mastitic quarters (samples diagnosed as subclinical mastitis by California Mastitis Test or samples of clinical mastitis with presence of flakes, clots, or serous milk; n=482) were cultured aerobically at 37℃ and identified using differential chromogenic media and traditional culture methods. Colonies from differential chromogenic media were purified and DNA was extracted for 16S rRNA sequencing (n=194). The reliability of identification by differential chromogenic media was diagnosed referring to results of two traditional approaches, based on which consistency inspection parameter, namely, simple Cohen's kappa coefficient was calculated via FREQ program in SAS Version 9.4. The colony and color of medium matrix from common bovine mastitis-associated pathogens of different genus grown on differential chromogenic media, regardless of standard strains or wild-type strains, were morphologically distinct and visible to the naked eyes. Consistency inspection parameter of differential chromogenic media referred to normal isolation and identification media combined with biochemical identifications and 16S rRNA sequencing was seperately κ=0.70 and κ=0.96. In conclusion, differential chromogenic media showed great potential in identifying common pathogens of bovine mastitis.
Key words: dairy cow     mastitis     fast identification of pathogens     differential chromogenic medium    

据报道,20%~30%的奶牛在泌乳期至少患一次临床型乳房炎[1]。瑞典农业科学大学的研究人员根据治疗奶牛乳房炎使用的抗生素、因抗生素疗法导致的弃奶量及药物残留检测、当地奶价和奶牛淘汰政策计算出每起乳房炎的治疗成本为179~488 $[2]。乳房炎的防治已成为奶牛养殖业中使用抗生素的主要原因[3-4],其病原菌涉及十多个属的细菌,其中葡萄球菌属、链球菌属和大肠埃希菌属在乳房炎牛奶的常见分离菌中约占90%[5-6]。又由于不同种属病原菌药物敏感性不同、治疗手段不同,如乳腺内抗生素疗法能够提高环境型链球菌性乳房炎的治愈率,但不建议用于大肠杆菌性乳房炎的治疗[7-8]。所以如需对奶牛乳房炎病原菌的流行、分布作出统计,分析病原菌来源并作出预防措施,同时参照已有的病原菌耐药图谱及治疗方案实现科学、合理的抗生素治疗或者选择不予治疗,避免抗生素滥用,降低生产成本,就需要以病原菌的种属鉴定为前提。基于病原菌鉴定结果进行奶牛乳房炎的抗生素治疗能减少50%的抗生素使用量,同时能降低复发率[9]。然而,传统的细菌生化实验及普通鉴别培养基虽然能将病原菌鉴定至种属级别,但操作复杂、耗时耗力[10-11]

因此,研发出一种操作简单、快速省力、检测参数满意的、能够同时分离鉴定牛奶中多种常见奶牛乳房炎病原菌的培养基,成为奶牛养殖业的需求,也符合当前各养殖业建立场内检测中心及国际OIE、WHO主张减少抗生素使用、降低抗生素耐药性发展的形势[12-13]。显色培养基已开始用于动物样品中微生物的鉴定[14],但在国内尚未用于乳房炎牛奶中多种病原菌的鉴定。本研究的目的在于评估鉴别显色培养基在乳房炎牛奶中病原菌分离鉴定的应用,并检验其与传统的实验室分离培养及生化鉴定、16S rRNA通用引物核酸序列测定鉴定病原菌的一致性。

1 材料与方法 1.1 菌种

ATCC标准菌株:大肠杆菌8739、肺炎克雷伯菌13883、奇异变形杆菌35659、铜绿假单胞菌15442、黏质沙雷菌8100、无乳链球菌12386、停乳链球菌35666、粪肠球菌29212、乳酸乳球菌11007、金黄色葡萄球菌25923、产色葡萄球菌43764,购自广东省微生物菌种保藏中心;解糖肠球菌、拟生葡萄球菌和马胃葡萄球菌的野生型菌株及上述ATCC标准菌株对应种属的野生型菌株,由内蒙古农业大学兽医学院微生物与免疫实验室分离、鉴定。

1.2 主要试剂和材料

胰蛋白胨大豆琼脂(TSA)、心脑浸出液(BHI)、麦康凯琼脂培养基(MCA)、高盐甘露醇琼脂培养基(MSA)、血琼脂平板(BAP)、胆汁七叶苷琼脂培养基(BAAA)、增菌基础培养基、卵磷脂等购于青岛海博生物技术有限公司,核糖、甘露醇等碳源及苯酚红、栀子苷等购于北京索莱宝科技有限公司,显色底物由深圳艾瑞生物科技有限公司合成,API生化鉴定条(API Staph,API 20E,APl 20STREP)购于生物梅里埃中国有限公司,TaKaRa提取细菌基因组DNA试剂盒购于宝生物工程(大连)有限公司,细菌16S rRNA通用引物(16S-F:5′-ACGCGTCGACAGAGTTTGATCCTGGCT-3′,16S-R:5′-CGCGGATCCGCTACCTTGTTACGACTT -3′,目的片段1 500 bp)合成及扩增产物的测序由生工生物工程(上海)股份有限公司完成,2×EasyTaq PCR SuperMix、DNA Gel Extraction Kit购于天根生化科技(北京)有限公司。

1.3 鉴别显色培养基的研制

首先将常见奶牛乳房炎病原菌分为三组进行鉴别,即革兰阴性菌、葡萄球菌、其他球菌,利用生物信息学数据库Integrated Microbial Genomes (http://img.jgi.doe.gov/)筛选出各组内病原菌各种属(或几个种属)表达的、编码基因保守性较高的特异性酶,并合成其显色底物;然后,对组内无法通过特异性酶鉴别或者无法合成酶显色底物的病原菌,依据其酶及其编码基因进行GO/KEGG功能分析注释,通过注释到的KEGG代谢通路确定各种属病原菌能够利用并产酸的碳源;其次,利用显色底物琼脂培养基(蛋白胨10 g·L-1,氯化钠5 g·L-1,琼脂15 g·L-1,显色底物50 mg,灭菌水1 L)、碳源发酵肉汤(蛋白胨10 g·L-1,氯化钠5 g·L-1,碳源10 g,苯酚红18 mg,灭菌水1 L)进行标准菌株种属特异性显色及可利用特异性碳源产酸显色的表型验证;最后,将对应的显色底物、碳源、酸碱指示剂加入增菌基础培养基,121 ℃高压灭菌15 min后分别倾入内含3个独立隔断的90 mm平皿,此部分在内蒙古农业大学兽医学院微生物与免疫实验室完成。

1.4 ATCC标准菌株、野生型菌株在鉴别显色培养基上菌落形态的评估

将常见的乳房炎相关病原菌标准菌株、野生型菌株用TSA活化,37 ℃有氧培养24 h后,挑取单一菌落至5 mL BHI混匀,37 ℃有氧培养12 h。用无菌棉签浸沾少许菌液在鉴别显色培养基上划线培养,观察其生长特性、菌落形态、菌落颜色及培养基基质颜色变化。

1.5 牛奶样品的采集

无菌采集CMT法检测为隐性乳房炎及临床型乳房炎(物理状态异常,如絮状、凝块、清亮、血乳)的牛奶样品:75%酒精湿巾擦拭乳头孔,弃去前三把奶后,挤取5 mL牛奶至无菌样品管中[15],样品置于冰盒送至实验室。2017年5月至2017年10月于呼和浩特市周边规模化牧场共计采集482份牛奶样品。

1.6 牛奶样品中病原菌的分离鉴定 1.6.1 鉴别显色培养基方法

无菌棉签浸沾牛奶样品在鉴别显色培养基三个区域划线接种后倒置于37 ℃恒温培养箱,培养16 h后按照“1.4”的评估结果进行判断。当两种以上不同的菌落类型同时出现在鉴别显色培养皿第二区域和第三区域,且该样品中没有诊断出金黄色葡萄球菌、链球菌等传染性病原时,则判定该样品污染;有氧培养48 h后仍无菌落生长迹象,则判定该样品中无病原菌。

1.6.2 普通分离鉴定培养基辅以生化鉴定方法

无菌棉签浸沾牛奶样品涂布于TSA、MCA、MSA、BAP、BAAA等培养基,倒置于37 ℃恒温培养箱培养24 h后,进行细菌的革兰染色、镜检,细菌生化鉴定参照伯杰氏细菌鉴定手册(第八版)及API鉴定条说明书。

1.6.3 16S rRNA核酸序列测定方法

挑取“1.6.1”中鉴别显色培养基上阳性单菌落划线接种至TSA,37 ℃培养24 h,单菌落连续纯化两次后接种至5 mL BHI中37 ℃摇床培养12 h。随后参照试剂盒说明进行DNA提取。使用细菌16S rRNA通用引物对提取的DNA进行序列扩增,反应体系(25 μL):1 μL DNA模板,12.5 μL 2×EasyTaq PCR SuperMix,上、下游引物(1 μmol·L-1)各1 μL,ddH2O 9.5 μL。反应条件:94 ℃预变性3 min;94 ℃变性45 s,35个循环,50 ℃退火1 min,72 ℃延伸90 s;72 ℃终延伸10 min。每批次序列扩增都增加空白对照组确保无细菌DNA污染,扩增产物用DNA Gel Extraction Kit回收、测序。

1.7 数据统计分析

简单科恩κ系数能够作为检验鉴别显色培养基与两种传统鉴定方法一致性的参数,可通过SAS 9.4中FREQ程序计算[16-17]。本检验中,零假设为两种鉴定方法的结果一致属于机遇造成,即κ=0时,表明两种方法的鉴定结果一致纯属偶然,κ=1时表明两种诊断方法的结果完全一致。在此零假设条件下,κ系数对应的P≤0.05时,视为一致性显著。“1.6.1”、“1.6.2”中被判定污染的样品不计入统计。最终结果以参数值和95%置信区间(confidence interval,CI)表示,CI基于二项分布的标准差(standard error,SE)计算:$SE = \frac{{\sqrt {p\left( {1 - p} \right)} }}{n}$CI=参数值±1.96×SE[18]。其中n为样品总量,p为实际阳性率,即真阳性和假阴性总量占样品总量的比例,鉴别显色培养基鉴定病原菌的可靠性数据(真阳性、假阳性、真阴性、假阴性的数量)均分别以2种传统方法的结果为参考判定获得。

2 结果 2.1 各种属病原菌表达的特异性酶、可发酵产酸的碳源

奶牛乳房炎常见革兰阴性病原菌(大肠埃希菌、克雷伯菌、变形杆菌、假单胞菌、沙雷菌)中,仅大肠埃希菌、克雷伯菌表达β-半乳糖苷酶,而后者能利用核糖作为唯一碳源产酸;奶牛乳房炎常见革兰阳性菌:仅链球菌、解糖肠球菌表达β-葡萄糖醛酸糖苷酶,仅肠球菌、乳酸球菌表达β-葡萄糖苷酶;金黄色葡萄球菌表达α-葡萄糖苷酶、磷脂酶,马胃葡萄球菌、拟生葡萄球菌表达α-葡萄糖苷酶,金黄色葡萄球菌、马胃葡萄球菌能够将甘露醇作为唯一碳源产酸。

酶显色底物(表 1)的病原菌种属特异性显色验证:β-半乳糖苷酶的①号底物被分解形成的鲑红色菌落不易与黏质沙雷菌自身产生红色色素形成的红色菌落辨别,②号底物抑制细菌生长,③号底物被分解形成翠绿色菌落。β-葡萄糖苷酶的①、②号底物被分解形成蓝灰色产物比③号慢6 h以上。β-葡萄糖醛酸糖苷酶的3种底物均可被分解形成绿色菌落,③号价格最低。α-葡萄糖苷酶的①号底物被分解后不显色,②号底物水溶性差,③号底物被分解形成绿色-蓝灰色菌落。

表 1 酶及其显色底物 Table 1 Screened enzymes and corresponding chromogenic substrates
2.2 标准菌株、野生型菌株在鉴别显色培养基上的菌落形态

粉色的第一区(图 1):革兰阳性菌不能生长;大肠埃希菌8739的菌落为翠蓝色,野生型菌株的菌落呈翠蓝色细沙状;肺炎克雷伯菌13883及其野生型菌株均形成深紫色菌落;奇异变形杆菌35659呈现棕褐色椭圆形菌落,菌落上有不规则迁徙圈,野生型菌株菌落上未见迁徙圈;铜绿假单胞菌15442及其野生型菌株形成绿褐色梭形或不规则形菌落;黏质沙雷菌8100及其野生型菌株形成红色菌落。

A.鉴别显色培养基;B.大肠埃希菌;C.肺炎克雷伯菌;D.奇异变形杆菌;E.铜绿假单胞菌;F.沙雷菌 A. Differential chromogenic media; B. Escherichia coli; C. Klebsiella pneumoniae; D. Proteus mirabilis; E. Pseudomonas aeruginosa; F. Serratia 图 1 常见乳房炎相关革兰阴性菌在鉴别显色培养基上的菌落形态 Fig. 1 Visual assessment of Gram-negative bacterial growth on differential chromogenic media

浅黄色的第二区(图 2A~D):革兰阴性菌和葡萄球菌属不能生长;无乳链球菌12386、停乳链球菌35666形成绿色菌落,野生型菌株形成绿色细沙样菌落;粪肠球菌29212及其野生型菌株形成白色菌落,培养基基质呈蓝灰色;乳酸乳球菌11007及其野生型菌株形成无色半透明、扁平菌落,培养基基质呈蓝灰色;野生型解糖肠球菌形成圆形、白色、中心有绿点的菌落。红色的第三区(图 2E~I):革兰阴性菌和葡萄球菌属以外的其他球菌不能生长;金黄色葡萄球菌25923及野生型菌株形成绿色菌落,菌落周围有不溶性白色沉淀圈,培养基基质由红变黄;产色葡萄球菌43764及野生型菌株形成米黄色至橙黄色菌落;野生型拟生葡萄球菌形成蓝灰色菌落;1株马胃葡萄球菌野生型菌株形成绿色菌落,培养基基质变黄,2株形成黄色菌落。

A.无乳链球菌;B.乳酸乳球菌;C.解糖肠球菌;D.粪肠球菌;E.金黄色葡萄球菌;F.产色葡萄球菌;G.表皮葡萄球菌;H.拟生葡萄球菌;I.马胃葡萄球菌 A. Streptococcus agalactiae; B. Lactococcus Lactis; C. Enterococcus saccharolyticus; D. Enterococcus faecalis; E. Staphylococcus aureus; F. Staphylococcus chromogenes; G. Staphylococcus epidermidis; H. Staphylococcus simulans; I. Staphylococcus equorum 图 2 常见乳房炎相关革兰阳性菌在鉴别显色培养基上的菌落形态 Fig. 2 Visual assessment of Gram-positive bacterial growth on differential chromogenic media
2.3 鉴别显色培养基与普通分离鉴定培养基辅以生化鉴定方法的一致性检验

鉴别显色培养基与普通分离鉴定培养基辅以生化鉴定方法(传统方法1)的鉴定结果的一致性为κ=0.70,二者高度一致,且P<0.001说明检验具有统计学意义、且一致性极显著(表 2)。鉴别显色培养基在分离、鉴定革兰阴性菌、大肠埃希菌、克雷伯菌、假单胞菌、沙雷菌方面,与传统方法1的一致性参数分别为0.90、0.95、0.98、0.92、0.92(表 2),结果均高度一致;在分离鉴定革兰阳性菌、链球菌、肠球菌属、乳球菌、凝固酶阴性葡萄球菌方面,与传统方法1的一致性参数分别为0.88、0.96、0.88、0.96、0.96,结果均高度一致,在鉴定金黄色葡萄球菌方面与传统方法1的一致性参数为1.00,结果完全一致(表 3)。

表 2 普通分离鉴定培养基辅以生化鉴定检验鉴别显色培养基鉴定乳房炎相关革兰阴性病原菌的检测特性 Table 2 Test characteristics of differential chromogenic media to identify Gram-negative bacteria associated with bovine mastitis referred to method using normal isolation and identification medium combined with biochemical identifications
表 3 普通分离鉴定培养基辅以生化鉴定检验鉴别显色培养基鉴定乳房炎相关革兰阳性病原菌的检测特性 Table 3 Test characteristics of differential chromogenic media to identify Gram-positive bacteria associated with bovine mastitis referred to method using normal isolation and identification medium combined with biochemical identifications
2.4 鉴别显色培养基与16S rRNA序列测定鉴定方法的一致性检验

鉴别显色培养基与16S rRNA序列测定(传统方法2)鉴定结果的一致性为κ=0.96,二者高度一致,P<0.001说明检验具有统计学意义、且一致性极显著(表 4)。鉴别显色培养基在分离、鉴定大肠埃希菌、沙雷菌方面,与传统方法2的一致性参数分别为0.98、0.96,结果高度一致,在鉴定克雷伯菌与传统方法2的一致性参数为1.00,结果完全一致(表 4);在分离鉴定链球菌、肠球菌属、乳球菌、葡萄球菌方面,与传统方法2的一致性参数均为1.0,结果均完全一致(表 5)。

表 4 16S rRNA核酸序列测定检验鉴别显色培养基鉴定乳房炎相关革兰阴性病原菌的检测特性 Table 4 Test characteristics of differential chromogenic media to identify Gram-negative bacteria associated with bovine mastitis referred to 16S rRNA sequencing
表 5 16S rRNA核酸序列测定检验鉴别显色培养基鉴定乳房炎相关革兰阳性病原菌的检测特性 Table 5 Test characteristics of differential chromogenic media to identify Gram-positive bacteria associated with bovine mastitis referred to 16S rRNA sequencing
3 讨论

准确快速地鉴定乳房炎病原的种属,是指导有效治疗的前提[19],且选择性治疗能够减少抗生素使用、降低弃奶量[1, 9, 20]。本研究研制的鉴别显色皿能够鉴别革兰阴性菌(大肠埃希菌属、克雷伯菌属、变形杆菌属、假单胞菌属、沙雷菌属)、革兰阳性菌(链球菌属、肠球菌属、乳酸乳球菌属、金黄色葡萄球菌属、凝固酶阴性葡萄球菌),涵盖了乳房炎牛奶常见分离菌属的80%以上[6, 21-22]。该鉴别显色培养基与普通分离鉴定培养基辅以生化鉴定的方法、16S rRNA核酸测序方法的鉴定结果均高度一致,证明该鉴别显色培养基可靠性很好。

奶牛乳腺金黄色葡萄球菌的感染需高强度的抗生素治疗,原因如韩博课题组[23]和其他学者报道,金黄色葡萄球菌分离株有38种毒力基因模式,44.7%分离株多重耐药[24-25]。金黄色葡萄球菌产生的α-葡萄糖苷酶能分解5-溴-4-氯-3-吲哚葡萄糖苷使得菌落呈现绿色,卵磷脂酶与卵磷脂反应形成不溶性甘油酯沉淀圈,该菌还能发酵甘露醇产酸使得培养基基质由红变黄(图 2E)。凝固酶阴性葡萄球菌致病性相对金黄色葡萄球菌较弱[26-27],但因其与亚临床型/中度临床型乳房炎相关,有着较高的自愈率[28],所以凝固酶阴性葡萄球菌的鉴定能够减少抗生素使用。每种凝固酶阴性葡萄球菌至多只能出现菌落颜色、培养基基质颜色、白色沉淀圈中的两种颜色变化,这使得鉴别显色培养基对金黄色葡萄球菌与凝固酶阴性葡萄球菌的鉴别与两种传统方法鉴定结果完全一致。而基于溶血型的鉴定方法需仔细辨认β溶血等细微差别[11],且20%~25%的奶牛乳房炎源金黄色葡萄球菌在初培养时β溶血都检测不到[29]

链球菌感染奶牛乳腺经及时诊断及治疗对牛群健康无影响,因漏诊、误诊导致的不予治疗或者错误治疗则对牛群健康危害较大[7]。鉴别显色培养皿第二区能够鉴别链球菌、肠球菌、乳酸球菌呈现三种菌落:链球菌产生的β-葡萄糖醛酸糖苷酶能分解培养皿中的5-溴-6-氯-3-吲哚-β-D-葡萄糖醛酸苷胺盐,使得链球菌菌落呈现绿色,肠球菌和乳酸乳球菌都能分解培养皿中的栀子苷产生可溶性蓝灰色产物,而肠球菌呈现白色圆形凸起菌落,乳酸乳球菌呈现无色透明扁平菌落,凭此即可实现鉴别。大肠埃希菌性乳房炎有着较高的自愈率,Suojala等[8]和Erskine等[30]指出轻度、中度案例不建议乳腺内抗生素治疗。因此,鉴别大肠埃希菌属也益于减少抗生素的使用。本研究研制的鉴别显色培养基在接种培养16 h后通过菌落颜色、培养基质颜色变化鉴定病原菌至种属级别,为乳房炎病原菌牧场内快速检测提供了一种可靠的方法,无需专业培训、操作简单快捷、结果判定直观、能够长期存储(2~8 ℃存储3个月不会影响菌落颜色的形成),能为抗生素治疗提供参考,值得作为鉴定方法推广。

4 结论

以生物信息学方法筛出各种属细菌的特异性酶、可作为唯一碳源发酵产酸的碳源,然后合成酶显色底物,连同碳源、酸碱指示剂制备显色培养基,将各种属标准菌株、野生型菌株涂布培养,评估菌落形态、菌落颜色及培养基基质颜色变化。鉴别显色培养基上常见乳房炎病原菌不同种属的菌落形态和培养基基质颜色明显不同,肉眼即可辨别。鉴别显色培养基与传统的实验室分离培养及生化鉴定、16S rRNA通用引物核酸序列测定鉴定结果的一致性参数分别为κ=0.70、κ=0.96。鉴别显色培养基能够作为常见奶牛乳房炎病原菌的标准鉴定方法。

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