Analysis on the technical key points of Salmonella isolation,screening and identification based on routine detection process

doi:10.3969/j.issn.1673-8640.2020.06.003

Abstract

Abstract:

Objective To analyze and evaluate the technical key points for routine detection of Salmonella,using multi-dimensional composite process based on standard method. Method Process material included Salmonella selective agars and selective enrichment broth. Recovery ratios of 4 selective enrichment broths were detected by reference isolates,and the typical colony characteristics of 5 selective agars belonging to 3 kinds were verified. Suspected colony distinguishment,screening efficiency and sensitivity were compared using different selective agars and selective enrichment broth by actual samples. Results The recovery efficiency of Rappaport Vassiliadis broth（RV）,tetrathionate broth（TTB） and selenite brilliant green broth（SBG） was one dilution different from that of selenite cystine broth（SC）. The first 3 selective enrichment broths were effective for non-typhoidal Salmonella,and SC was effective for Salmonella typhi and Salmonellaparatyphoid. Screening method of double-sugar tubes accorded with the biochemical characteristics of confirmed reference isolates. Except CHORMagar Salmonella（CAS）,the others showed atypical colony characteristics,including H₂S negative isolates and small colonies. The 25 of 84 meat samples were positive,and 42 Salmonella isolates were isolated. The sensitivities of CAS and xylose lysine deoxycholate agar（XLD） combined with RV,TTB,SBG,SC were 88%,84%,72% and 0%,respectively. The sensitivities of RV with TTB,RV with SBG,TTB with SBG were 100%,92% and 88%,respectively. The sensitivities of RV,TTB,and SBG combined with CAS or XLD were 48.00% and 68.00%,respecitvely. The method of screening Salmonella like colonies had high specificity and positive predictive value by double-sugar tubes. Totally,42 Salmonella isolates were confirmed by biochemistry identifier,mass spectrometry and serotyping. Conclusions The effective combined processes could improve the sensitivities of Salmonella detection based on verification by double selective agar and double selective enrichment broths. The technical key points include the gradient selective enrichment,distinguish of the colonies and screening of biochemistry identifier by routine detection of Salmonella.

Key words: Salmonella, Enrichment broth, Selective agar, Screening, Technical key point

CLC Number:

R446.1

LIU Yue, ZHANG Hongzhi, GU Qifang, LIU Cheng, ZHU Yingying, XU Xuebin. Analysis on the technical key points of Salmonella isolation,screening and identification based on routine detection process[J]. Laboratory Medicine, 2020, 35(6): 524-530.

Figures/Tables 6

References 25

[1]	SAVELLI C J,BRADSHAW A,BEN EMBAREK P,et a1. The FAO/WHO international food safety authorities network in review,2004-2018:learning from the past and looking to the future[J]. Foodborne Pathog Dis,2019,16(7):480-488.
[2]	MAJOWICZ S E,MUSTO J,SCALLAN E,et al.The global burden of nontyphoidal Salmonella gastroenteritis[J]. Clin Infect Dis,2010,50(6):882-889.
[3]	中华人民共和国国家卫生和计划生育委员会,国家食品药品监督管理总局. GB 4789.4—2016 食品安全国家标准食品微生物学检验沙门氏菌检验[S]. 北京:中国标准出版社,2017.
[4]	中华人民共和国卫生部. WS 271—2007 感染性腹泻诊断标准附录B实验室诊断方法:沙门菌检验[S]. 北京:人民卫生出版社,2008.
[5]	ANDREWS W H,WANG H,JACOBSON A,et al. Bacteriological analytical manual（BAM） chapter 5：Salmonella[EB/OL]. (2019-12-01)[2019-12-15]. .
[6]	RUIZ J,NÚÑEZ M L,DÍAZ J,et al. Comparison of five plating media for isolation of Salmonella species from human stools[J]. J Clin Microbiol,1996,34(3):686-688.
[7]	周永明,陈秀华,徐闻,等. 沙门菌常规检测方法分段控制技术在网络实验室构建中基础作用的评估[J]. 中华流行病学杂志,2013,34(11):1105-1110.
[8]	ZHANG J,JIN H,HU J,et a1. Serovars and antimicrobial resistance of non-typhoidal Salmonella from human patients in Shanghai,China,2006-2010[J]. Epidemiol Infect,2014,142(4):826-832.
[9]	WEI Z,XU X,YAN M,et a1. Salmonella typhimurium and Salmonella enteritidis infections in sporadic diarrhea in children:source tracing and resistance to third-generation cephalosporins and ciprofloxacin[J]. Foodborne Pathog Dis,2018,16(4):1-12.
[10]	罗铭,顾桂敏,景春梅,等. 猪霍乱沙门菌在全球的流行和耐药特征[J]. 疾病监测,2018,33(8):690-699.
[11]	ZHAN Z,XU X,GU Z,et al.Molecular epidemiology and antimicrobial resistance of invasive non-typhoidal Salmonella in China,2007-2016[J]. Infect Drug Res,2019,12:2885-2897.
[12]	PAN H,ZHOU X,CHAI W,et al.Diversified sources for human infections by Salmonella enterica serovar newport[J]. Transbound Emerg Dis,2019,66(2):1044-1048.
[13]	LU X,ZENG M,XU J L,et al.Epidemiologic and genomic insights on mcr-1-harbouring Salmonella from diarrhoeal outpatients in Shanghai,China,2006-2016[J]. EBioMedicine,2019,42:133-144.
[14]	FEDER I,NIETFELD J C,KELLY B.Evaluation of enrichment techniques for the isolation of Salmonella choleraesuis from swine feces[J]. J Microb Methods,1998,33:143-151.
[15]	WANG Y,CAO C,ALALI W Q,et al.Distribution and antimicrobial susceptibility of foodborne Salmonella serovars in eight provinces in China from 2007 to 2012(except 2009)[J]. Foodborne Pathog Dis,2017,14(7):393-399.
[16]	许学斌,顾宝柯,杨兰萍,等. 5种沙门菌分离培养基的应用和比较[J]. 中国卫生检验杂志,2005,15(7):773-775.
[17]	杨兰萍,曹晓伟,黄峥,等. 1例乳糖发酵鼠伤寒沙门菌腹泻病例株的表型和分子特征研究[J]. 疾病监测,2016,31(1):34-39.
[18]	酒跃光. 猪源沙门菌耐药性及屠宰环节污染规律研究[D]. 武汉:华中农业大学,2017.
[19]	李兵兵,刘纯成,刘靓,等. 2015-2016年淮安市售禽畜肉中沙门菌污染及其病原学特征[J]. 卫生研究,2018,47(2):260-265.
[20]	YANG X,WU Q,ZHANG J,et al.Prevalence,bacterial load,and antimicrobial resistance of Salmonella serovars isolated from retail meat and meat products in China[J]. Front Microbiol,2019,10:2121.
[21]	刘玥,顾其芳,刘诚,等. 2016 年上海市市售肉制品中沙门菌耐药谱与分型研究[J]. 中国食品卫生杂志,2018,30(2):132-138.
[22]	HUMPHRIES R M,LINSCOTT A J.Laboratory diagnosis of bacterial gastroenteritis[J]. Clin Microbiol Rev,2015,28(1):3-31.
[23]	XIE J,WU F,XU X,et al. Antibiotic resistance and molecular characterization of the hydrogen sulfide-negative phenotype among diverse Salmonella serovars in China[J]. BMC Infect Dis,2018,18(1):292.
[24]	COLOMBE S,JERNBERG C,LÖF E,et al. Outbreak of unusual H2S-negative monophasic Salmonella typhimurium strain likely associated with small tomatoes,Sweden,August to October 2019[J]. Euro Surveill,2019,24(47):19000643.
[25]	蔡文诚. 实用临床微生物诊断学[M]. 南京:东南大学出版社,1998.

参比菌株名称	肠道菌筛选双糖管								CAS
参比菌株名称	氧化酶	葡萄糖	甘露醇	尿酶	蔗糖	动力	硫化氢	吲哚	CAS
鼠伤寒沙门菌	-	⊕	⊕	-	-	+	+	-	酒红色
伤寒沙门菌	-	+	+	-	-	+	+	-	酒红色
甲型副伤寒沙门菌	-	+	+	-	-	+	+	+	酒红色
猪霍乱沙门菌	-	+	+	-	-	+	-	-	酒红色
猪霍乱沙门菌孔成道夫变种	-	+	+	-	-	+	+	-	酒红色
肺炎克雷伯菌	-	⊕	⊕	-	+	-	-	-	蓝色
奇异变形杆菌	-	⊕	⊕	-	+	+	-	-	淡粉色
弗劳地枸橼酸杆菌	-	⊕	⊕	-	+	+	+	-	蓝色
大肠埃希菌	-	⊕	⊕	-	+	+	-	+	蓝色

参比菌株名称	肠道菌筛选双糖管								CAS
参比菌株名称	氧化酶	葡萄糖	甘露醇	尿酶	蔗糖	动力	硫化氢	吲哚	CAS
鼠伤寒沙门菌	-	⊕	⊕	-	-	+	+	-	酒红色
伤寒沙门菌	-	+	+	-	-	+	+	-	酒红色
甲型副伤寒沙门菌	-	+	+	-	-	+	+	+	酒红色
猪霍乱沙门菌	-	+	+	-	-	+	-	-	酒红色
猪霍乱沙门菌孔成道夫变种	-	+	+	-	-	+	+	-	酒红色
肺炎克雷伯菌	-	⊕	⊕	-	+	-	-	-	蓝色
奇异变形杆菌	-	⊕	⊕	-	+	+	-	-	淡粉色
弗劳地枸橼酸杆菌	-	⊕	⊕	-	+	+	+	-	蓝色
大肠埃希菌	-	⊕	⊕	-	+	+	-	+	蓝色

沙门菌型	SC		TTB		RV		SBG
沙门菌型	靶菌	加标	靶菌	加标	靶菌	加标	靶菌	加标
伤寒沙门菌	10^-10	10^-9	10^-9	10^-8	10^-9	10^-8	10^-9	10^-8
甲型副伤寒沙门菌	10^-10	10^-9	10^-9	10^-8	10^-9	10^-8	10^-9	10^-8
鼠伤寒沙门菌	10^-10	10^-9	10^-9	10^-8	10^-10	10^-9	10^-10	10^-9
猪霍乱沙门菌	10^-7	10^-6	10^-10	10^-8	10^-10	10^-8	10^-9	10^-8
猪霍乱沙门菌孔成道夫变种	10^-7	10^-6	10^-9	10^-8	10^-9	10^-8	10^-9	10^-8

沙门菌型	SC		TTB		RV		SBG
沙门菌型	靶菌	加标	靶菌	加标	靶菌	加标	靶菌	加标
伤寒沙门菌	10^-10	10^-9	10^-9	10^-8	10^-9	10^-8	10^-9	10^-8
甲型副伤寒沙门菌	10^-10	10^-9	10^-9	10^-8	10^-9	10^-8	10^-9	10^-8
鼠伤寒沙门菌	10^-10	10^-9	10^-9	10^-8	10^-10	10^-9	10^-10	10^-9
猪霍乱沙门菌	10^-7	10^-6	10^-10	10^-8	10^-10	10^-8	10^-9	10^-8
猪霍乱沙门菌孔成道夫变种	10^-7	10^-6	10^-9	10^-8	10^-9	10^-8	10^-9	10^-8