PulseNet standardized protocol for subtyping Listeria monocytogenes by macrorestriction and pulsed-field gel electrophoresis

PulseNet is a national network of pubic health and food regulatory laboratories established in the US to detect clusters of foodborne disease and respond quickly to foodborne outbreak investigations. PulseNet laboratories currently subtype Escherichia coli O157:H7, non-typhoidal Salmonella, and Shigella isolates by a highly standardized 1-day pulsed-field gel electrophoresis (PFGE), and exchange normalized DNA "fingerprint" patterns via the Internet. We describe a standardized molecular subtyping protocol for subtyping Listeria monocytogenes that was recently added to PulseNet. The subtyping can be completed within 30 h from the time a pure culture of the bacteria is obtained.     

A pulsed-field gel electrophoresis typing scheme for Vibrio parahaemolyticus isolates from fifteen countries

Vibrio parahaemolyticus is an important foodborne pathogen in Taiwan and many other maritime Asian countries where seafood is frequently consumed. A total of 535 strains of V. parahaemolyticus were recovered mostly (97%) from clinical samples obtained in Taiwan or in 14 other countries. These strains were typed by pulsed-field gel electrophoresis following SfiI digestion and a typing scheme was generated. The 115 different patterns identified were grouped into 13 types with dissimilarity values less than 15, plus 16 miscellaneous patterns not grouped into any of the types. Types I, A, D and J contained the most patterns, with the numbers of patterns being 17, 13, 12, and 11, respectively. However, types I, B, D, A, H and C contained the most strains, with the numbers of strains being 204, 73, 71, 54, 29 and 25, respectively. Type I consisted exclusively of the pandemic O3:K6 strains and genetically closely related strains. This PFGE typing scheme for V. parahaemolyticus could be used for the characterization of pathogenic isolates.     

云南省肠炎沙门菌及德尔卑沙门菌脉冲场凝胶电泳分子分型及耐药性研究

目的 了解云南省沙门菌患者中分离最多的肠炎沙门菌及食品中分离最多的德尔卑沙门菌脉冲场凝胶电泳(pulse field gel electrophoresis, PFGE)分子分型及耐药状况。方法 参照中国细菌性传染病分子分型实验室监测网络Pulse Net China的沙门菌PFGE分子分型方法进行分子分型。分析耐药板最低抑菌浓度(minimal inhibitory concentration, MIC)值, 根据美国临床实验室标准化协会(Clinical and Laboratory Standard Institute, CLSI)的相应标准获得S、I、R结果。结果 56株肠炎沙门菌呈11种PFGE带型, 有4种优势带型, 对氨苄舒的耐药率最高, 为57.14%, 对亚胺培南最敏感。27株德尔卑沙门菌呈25种PFGE带型, 对复合磺胺的耐药率最高, 为64.29%, 对亚胺培南最敏感。结论 肠炎沙门菌分子分型有明显的优势带型, 本地区肠炎沙门菌对氨苄舒的耐药率最高。德尔卑沙门菌分子分型呈多样分布, 复方磺胺是本地区德尔脾沙门菌最耐药抗生素。2种沙门菌都对亚胺培南最敏感。     

进出口食品中不同血清型沙门氏菌PFGE和 MLST分型比较研究

目的：通过PFGE和MLST两种方法对进出口食品中不同血清型沙门氏菌的分辨力和应用价值进行了比较研究。方法：采用PFGE和MLST两种方法对进出口食品中分离得到的鼠伤寒及肠炎两种血清型共30株沙门氏菌进行分型研究。结果：两种血清型共30株沙门氏菌经PFGE分型可得到23种型别,DI值为0.9563;鼠伤寒和肠炎两种血清型菌分别经PFGE分型,DI值分别为1.000和0.9048。MLST对30株沙门氏菌分型得到4种ST型,DI值为0.5793;鼠伤寒和肠炎两种血清型菌分别经MLST分型,DI值分别为0.2571和0.1333。结论：在分辨力方面,PFGE更适合于同一沙门氏菌血清型的分型,MLST适合于不同沙门氏菌血清型间的分型。MLST在替代、补充血清型分型方面有潜在的优势,PFGE在溯源研究方面似乎更胜一筹。     

我国四省肉鸡屠宰厂沙门氏菌脉冲场凝胶电泳分子分型结果分析

目的 了解我国四省肉鸡屠宰厂沙门氏菌脉冲场凝胶电泳分子分型情况。方法 参照美国疾病预防控制机构PulsNet实验方法,对2010年监测四省肉鸡屠宰厂分离到的167株沙门氏菌,运用XbaⅠ酶进行酶切并完成PFGE分析,利用BioNumerics 软件对分离株的指纹图谱进行聚类分析并建立数据库。结果 167株沙门氏菌共分为18个群,包括了75个不同的带型。相同血清型具有相似带型,基本归于同一群。65株印地安纳沙门氏菌分成了38个带型,54株肠炎沙门氏菌分成了16个带型,16株奥尔巴尼沙门氏菌分成1个带型。结论 各省沙门氏菌的带型具有地区性差异, 又具有优势型别的交叉。屠宰厂存在严重的交叉污染,加强加工环节关键控制点沙门氏菌的监测和干预,从而降低肉鸡及其产品中沙门氏菌的污染,这是从源头控制污染的根本措施。     

我国4省肉鸡屠宰场沙门氏菌脉冲场凝胶电泳分子分型

目的 了解我国四省肉鸡屠宰场沙门氏菌脉冲场凝胶电泳分子分型情况.方法 参照美国疾病预防控制中心PulseNet实验方法,对2010年监测4省肉鸡屠宰场分离到的167株沙门氏菌,运用Xba Ⅰ酶进行酶切并完成PFGE分析,利用BioNumerics软件对分离株的指纹图谱进行聚类分析并建立数据库.结果 167株沙门氏菌共分为18个群,包括了75个不同的带型.相同血清型具有相似带型,基本归于同一群.65株印地安纳沙门氏菌有38个带型,54株肠炎沙门氏菌有16个带型,16株奥尔巴尼沙门氏菌仅有1个带型.结论 各省沙门氏菌的带型具有地区性差异,又具有优势型别的交叉.屠宰场存在严重的交叉污染,加强加工环节中的关键控制点—沙门氏菌的监测和干预,从而降低肉鸡及其产品中沙门氏菌的污染,这是从源头控制污染的根本措施.     

限制性内切酶组合对阪崎克罗诺杆菌的脉冲场凝胶电泳基因分型效果的影响

目的利用多种限制性内切酶提高脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)技术对阪崎克罗诺杆菌(Cronobacter Sakazakii)的分型效果,找到更多的以PFGE为基础的阪崎克罗诺杆菌分子分型信息。方法本研究选用XbaⅠ、SpeⅠ、NheⅠ和BlnⅠ分别酶切34株克罗诺杆菌分离株的DNA,并经PFGE得到相应的基因图谱,通过束平均数、束菌株百分比、结与菌株比值与Simpson多样性指数对各限制内切酶以及结合酶的相对分辨率进行评价。结果 NheⅠ是PFGE单酶切分型中效果最好的一种限制性内切酶;4种限制性内切酶相结合的分型方法可提高PFGE阪崎克罗诺杆菌基因分型效果。结论运用PFGE技术对菌株进行亚分型时,选用的酶种类越多,则结果越可靠,使用XbaⅠ、SpeⅠ、NheⅠ、BlnⅠ4种内切酶结合分析,可以得到理想的菌株亚分型结果。     

Antimicrobial susceptibilities and epidemiological analysis of Salmonella enteritidis isolates in Korea by phage typing and pulsed-field gel electrophoresis

A total of 81 isolates of Salmonella Enteritidis were analyzed by antibiotic susceptibility, phage typing, and pulsed-field gel electrophoresis (PFGE). Thirty-two isolates came from broiler carcasses and pig feces, and 49 isolates were from humans in Seoul and suburbs of Seoul, Korea. Antibiotic resistance was most prevalent among human isolates. Of human isolates, 89.8% were resistant to more than two antibiotics, while 64.7% of poultry isolates and 13.3% of pig isolates showed multiple resistance to more than two antibiotics. The most common phage type (PT) was PT1, followed by PT30 or 33, PT21 and PT20a. The isolates showed six PFGE patterns with XbaI or SpeI digestion, and five PFGE patterns with NotI digestion. But a single pattern, PFGE X1, S1, or N1, was predominant and the rest of the PFGE patterns differed by only one or two bands. Results indicated the spread of a genetically related clone of Salmonella Enteritidis in foods and humans in Korea and that phage typing as well as PFGE may offer an improved level of discrimination for the epidemiological investigation of Salmonella Enteritidis.     

脉冲场电泳在两起同期异地肠炎沙门菌食物中毒分子流行病学调查中的应用研究

目的 探讨两起相距210多公里乡镇同期发生的肠炎沙门菌食物中毒分离株之间的分子流行病学关系.方法 将两起食物中毒事件患者和剩余食物分离到的8株肠炎沙门菌进一步鉴定培养,挑取单个菌落增菌,用限制性内切酶Xba I消化酶切后进行脉冲场凝胶电泳,获得的分子分型指纹图谱,运用Bionumerics 5.1进行聚类分析.结果 指纹图谱聚类显示8株肠炎沙门菌分为两个分子型,D乡食物中毒分离到的5株肠炎沙门菌有相同的指纹图谱;P镇的3株分离株图谱也一致;两起食物中毒分离株图谱之间有4个条带的差异.结论两起食物中毒的暴发虽然都是肠炎沙门菌引起的,但具有不同的分子型别,食物中毒不是由同一种污染的食物源引起;脉冲场凝胶电泳可有效应用于食物中毒溯源分析及肠炎沙门菌分子流行病学研究.     

Technical note: a rapid pulsed-field gel electrophoresis method for analysis of bifidobacteria

Pulsed-field gel electrophoresis (PFGE) is a widely used and highly discriminatory molecular typing method that has been applied to bifidobacteria. However, published PFGE protocols used with bifidobacteria require between 5 and 7 d to complete. A rapid PFGE method was developed that can be completed within 24 h.     

Use of pulsed-field gel electrophoresis to characterize the genetic diversity and clonal persistence of Salmonella senftenberg in mussel processing facilities

Salmonella senftenberg was detected in association with persistent contamination events in mussel processing facilities between 1998 and 2002 in Spain. A total of 110 isolates from 8 facilities were subjected to molecular typing by Pulsed-Field Gel Electrophoresis (PFGE). Additionally, a selection of epidemiologically unrelated isolates of this serovar originating from human, animal, feed and environmental sources was included in the study. PFGE analysis proved to be a useful tool for studying the persistence and dissemination of S. senftenberg in factory environments. Facilities that used brine in their processing lines had greater genetic diversity among their S. senftenberg populations, which supports the hypothesis that imported salt used for brine preparation could have been the origin of the contamination. The XbaI type X19 was the most prevalent among the panel, and it was found persisting exclusively in one facility during the 5-year study. In general, isolates from mussel processing plants were clearly different from those of clinical and environmental sources. However, one of the human isolates showed an indistinguishable restriction pattern to an isolate from a frozen mussel sample, this could indicate the potential for food-borne transmission of this serovar via consumption of contaminated seafood products. Isolates in the study were largely sensitive to antimicrobials. Only 9 isolates (6 from mussel processing facilities, 1 from soy flour and 2 from meat meal) showed antimicrobial resistance.     

四川省鸭和猪源鼠伤寒沙门菌脉冲场凝胶电泳分型与耐药比较分析

目的比较研究四川省鸭和猪源鼠伤寒沙门菌脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)分型与耐药特征,为分析鼠伤寒沙门菌食品来源提供支持。方法将鸭和猪源监测中分离的47株鼠伤寒沙门菌进行PFGE分型,并采用最小抑菌浓度法测定9种药物敏感性。结果鸭和猪源中分离的鼠伤寒沙门菌在PFGE分型和耐药上存在差异。鸭源菌株聚集于簇I,而猪源菌株聚集于簇II。簇II菌株对氨苄西林、环丙沙星、氯霉素、庆大霉素、四环素、复方新诺明6种药物的耐药率高于簇I菌株。不同的PFGE分型鼠伤寒沙门菌在监测地区和耐药谱存在差异。SCSTm-10型菌株均分离于绵阳地区,SCSTm-11型主要分离于资阳地区。SCSTm-10型菌株对复方新诺明和环丙沙星的耐药率高于SCSTm-11型菌株。结论 PFGE分型可以获取四川省鼠伤寒沙门菌分离来源、分离地点和耐药的聚类信息,为鼠伤寒沙门菌食物中毒提供流行病学调查支持。     

Raw and processed fish show identical Listeria monocytogenes genotypes with pulsed-field gel electrophoresis

A total of 257 raw fish samples at two different sites were examined for the presence of Listeria monocytogenes. The prevalence of L. monocytogenes was 4%. From 11 positive samples, nine different L. monocytogenes pulsed-field gel electrophoresis genotypes were recovered. From nine pulsotypes recovered from raw fish and 32 pulsotypes shown by 101 fish product isolates, two raw fish and fish product pulsotypes were indistinguishable from each other. Although the prevalence of L. monocytogenes in raw fish is low, the range of L. monocytogenes strains entering the processing plant in large amounts of raw material is wide. This indicates that the raw material is an important initial contamination source of L. monocytogenes in fish processing plants. This postulation is supported by the identical pulsotypes recovered from both raw and processed fish. Some L. monocytogenes strains entering a plant may thus contaminate and persist in the processing environment, causing recurrent contamination of the final products via processing machines.     

Unveiling contamination sources and dissemination routes of Salmonella sp. in pigs at a Portuguese slaughterhouse through macrorestriction profiling by pulsed-field gel electrophoresis

Sixty-nine isolates of Salmonella sp. isolated from the ileum, tonsils, carcass and mandibular and ileocolic lymph nodes of individual pigs slaughtered for consumption in one abattoir were analyzed using serotyping and macrorestriction profiling by Pulsed-Field Gel Electrophoresis (RFLP-PFGE), in order to identify clonal relationships. XbaI macrorestriction was able to distinguish 18 genotypes among the eight identified serotypes: Salmonella Typhimurium (4 genotypes), Salmonella Rissen (3), Salmonella Tennessee (2), Salmonella Enteritidis (2), Salmonella 4,[5],12:i:- (4), Salmonella Give (1), Salmonella Anatum (1), and Salmonella Derby (1). Except for one sample, the serotype and the genotype identified in the samples from the same pork were always the same, allowing to unravel possible dissemination routes of Salmonella sp. through these pork tissues and equate presumptive sources of contamination or infection. Highly significant associations (p < 0.001) were observed for the presence of Salmonella sp. in the ileum and in the ileocolic lymph nodes, as well as between the carcass contamination and the presence of Salmonella sp. in others samples of the correspondent slaughtered pig, such as the ileum, the ileocolic and mandibular lymph nodes and the tonsils. Moreover, 80% of the pigs with ileum and ileocolic lymph nodes positive samples also presented the same salmonella genotype in the correspondent tonsils and, among pigs with positive tonsils, 70% also carried the same genotype in the corresponding mandibular lymph nodes. The occurrence of cross-contamination was also detected, since a genotype identified in other pigs slaughtered in the same day was found in 31% of positive carcasses. The global analysis of the genotypes suggested three different sources of pig infection: the farm of origin, the transportation and the lairage. A particular attention should be paid to the last one, since the majority of the isolates from pig samples were related to infection in the lairage. Since the presence of Salmonella sp. in the ileum of pigs and faeces ingestion promotes tonsils infection and internal dissemination of the agent through the mandibular lymph nodes, as well as drainage to the ileocolic lymph nodes, a potential risk exists at slaughter for Salmonella sp. contamination in the carcasses during pork processing. This risk may be increased by incorrect evisceration techniques and by hygienically inappropriate meat inspection procedures, especially those concerned to the mandibular lymph nodes incisions.     

进出口食品中不同血清型沙门氏菌PFGE和MLST分型比较研究

目的通过脉冲场凝胶电泳(PFGE)和多位点序列分型(MLST)两种方法对进出口食品中不同血清型沙门氏菌的分辨力和应用价值进行了比较研究。方法采用PFGE和MLST两种方法对进出口食品中分离得到的鼠伤寒及肠炎两种血清型共30株沙门氏菌进行分型研究。结果两种血清型共30株沙门氏菌经PFGE分型可得到23种型别,DI值为0.9563;鼠伤寒和肠炎两种血清型菌分别经PFGE分型,DI值分别为1.000和0.9048。MLST对30株沙门氏菌分型得到4种ST型,DI值为0.5793;鼠伤寒和肠炎两种血清型菌分别经MLST分型,DI值分别为0.2571和0.1333。结论 在分辨力方面,PFGE更适合于同一沙门氏菌血清型的分型,MLST则适合于不同沙门氏菌血清型间的分型。MLST在替代、补充血清型分型方面有潜在的优势,PFGE在溯源研究方面更胜一筹。     

进出境食品中单核增生李斯特菌的血清分型与脉冲场凝胶电泳分型分析

目的研究进出境食品中的单核增生李斯特菌(LMO)的血清分型与脉冲场凝胶电泳(PFGE)分型的特性及其关系。方法采用标准方法对39株分离的LMO进行PFGE分型,分别用ApaI和Asc I酶切,电泳图谱进行聚类分析,以上菌株同时进行血清分型检测。结果 39株LMO分成6个血清型;经ApaI酶切后,分成29种带型,相似度在57.4%～100%;Asc I酶切后,分成34种带型,相似度在48.6%～100%。讨论 AscI酶切的PFGE分离效果优于ApaI酶切,与血清分型的一致性低于ApaI酶切;PFGE分型效果优于血清分型。     

Epidemiological analysis of Salmonella enterica from beef sampled in the slaughterhouse and retailers in Dakar (Senegal) using pulsed-field gel electrophoresis and antibiotic susceptibility testing

Seventy-eight isolates of Salmonella spp. isolated from beef sampled from the official city slaughterhouse and from retailers in Dakar, Senegal were analyzed using serotyping, antimicrobial testing and macrorestriction profiling by Pulsed-Field Gel Electrophoresis (PFGE). These analyses were done to identify clonal relationships and potential transmission routes in beef channel. XbaI macrorestriction allowed defining 17 genotypes among the six main analyzed serotypes: Salmonella bredeney (3 genotypes), S. muenster (6), S. waycross (1), S. corvallis (3), S. kentucky (1) and S. brandenburg (3). The cross analysis of PFGE profiles and origin of the beef samples reveals a wide range of contamination sources in the beef channel in Dakar. Comparison of PFGE and antimicrobial resistance types shows that the Salmonella contamination sources are equally shared by the slaughterhouse (56% of the isolates) and by the distribution channel (44% of the isolates) by handlings and houseflies.     

Characterization of Listeria monocytogenes strains isolated from food and humans in Italy by pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was used to type 90 strains of Listeria monocytogenes isolated from clinical cases and from meat products in Italy in the period 1987–95. The objective of this retrospective study was to compare the genetic profiles to determine the existence of predominant clones and to evaluate their association with the sporadic cases of listeriosis reported in recent years in Italy. A total of 32 distinct PFGE types were identified: PFGE types 1 and 9 were identified both for strains isolated from clinical samples and those isolated from food. The use of the PFGE molecular method in surveillance projects and epidemiological investigations could contribute to better understanding of microbial populations and could also be useful, as part of the HACCP programme, in conducting controls along different points of the food production line.     

Restriction fragment length polymorphisms analysis by pulsed-field gel electrophoresis for discrimination of Staphylococcus aureus isolates from foodborne outbreaks

A number of outbreaks of disease due to Staphylococcus aureus occurring in Aichi-ken, Japan, have provided the opportunity to investigate aspects of the molecular epidemiology of this and related organisms. Coagulase types, enterotoxin types, phage types, and restriction fragment length polymorphisms (RFLPs) as assessed by pulsed-field gel electrophoresis (PFGE) was performed for S. aureus infections diagnosed in the area of Aichi-ken. Among the 56 isolates of S. aureus from 30 outbreaks, 15 distinctive RFLP types were found by digestion with the restriction enzyme, SmaI. A total of 32 isolates from patients, foodstuffs and cooks on six occasions had the same RFLP types, coagulase types, enterotoxin types and phage types in the same outbreaks. Moreover, the coagulase and phage types could be separated in terms of RFLP. In one outbreak, ten isolates, which were derived from six patients, two foodstuffs and two cooks, had the same coagulase type, enterotoxin type, phage type, and RFLP type. This PFGE method may therefore prove useful for subclassifying S. aureus and differentiating isolates of the same coagulase types and phage types derived from sporadic cases and those derived from foodborne outbreaks.     

Detection and differentiation of Salmonella serotypes using surface enhanced Raman scattering (SERS) technique

This research was conducted to prove that developed silver biopolymer nanoparticle substrate for surface enhanced Raman scattering (SERS) technique could detect and differentiate three different serotypes of Salmonella. Nanoparticle was prepared by adding 100 mg of silver nitrate to a 2 % polyvinyl alcohol solution, then adding 1 % trisodium citrate to reduce silver nitrate and produce silver encapsulated biopolymer nanoparticles. Then, nanoparticle was deposited on a stainless steel plate and used as SERS substrate. Fresh cultures of Salmonella typhimurium, Salmonella enteritidis and Salmonella infantis were washed and suspended in 10 mL of sterile deionized water. Approximately 5 μl of the bacterial suspensions were placed on the substrate individually and exposed to 785 nm laser excitation. SERS spectral data were recorded between 400 and 1,800 cm^?1. SERS signals were collected from 15 different spots on the substrate for each sample. PCA model was developed to classify Salmonella serotypes. PC1 identified 92 % of the variation between the Salmonella serotypes, and PC2 identified 6 % and in total 98 % between the serotypes. Soft independent modeling of class analogies of validation set gave an average correct classification of 92 %. Comparison of the SERS spectra of Salmonella serotypes indicated that both isolates have similar cell walls and cell membrane structures which were identified by spectral regions between 520 and 1,050 cm^?1. However, major differences were detected in cellular genetic material and proteins between 1,200 and 1,700 cm^?1. SERS with silver biopolymer nanoparticle substrate could be a promising tool in pathogen detection and it would potentially be used to classify them.