Real-time PCR von Enterobacter sakazakii in Säuglingsanfangsnahrung

Enterobacter sakazakii represents a rare opportunistic pathogen that causes a high mortality rate. Especially newborns are infected by contaminated infant formula. To reduce this risk, the EU directive 2073/2005 demands for the absence of the pathogen in 30 ×10 g infant formula. We describe a PCR-/real-time PCR that specifically detected 32 E. sakazakii strains among 21 species of Enterobacteriaceae as well as contaminated specimens among 30 different brands of infant formula. This real-time PCR, performed after a selective enrichment of E. sakazakii, reduces the working time by 1 to 3 days in comparison to the detection methods recommended by the US Food and Drug Administration (FDA) and ISO/DTS 22964 that is still under revision. In contrast to the latter procedure, we detected contaminated specimens of infant formula by the use of the procedure described in this report and the FDA method.

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Zusammenfassung: Enterobacter sakazakii repr?sentiert einen seltenen opportunistischen Erreger, der Infektionen mit hoher Mortalit?tsrate hervorruft. Insbesondere Neugeborene erkranken durch kontaminierte Anfangsnahrung. Zur Risikominimierung fordert die EU-Verordnung 2073/2005 die Abwesenheit des Erregers in 30 ×10 g S?uglingsanfangsnahrung. Ein PCR-/Real-Time PCR-Verfahren wird beschrieben, das zun?chst mit 32 E. sakazakii -St?mmen und weiteren 20 Arten der Enterobacteriaceen geprüft und anschlie?end zur Untersuchung 30 unterschiedlicher Marken von S?uglingsanfangsnahrung verwendet wurde. Nach der Selektivanreicherung führt das Real-Time PCR-Verfahren zu einer Ersparnis von 1 bis 3 Arbeitstagen beim Nachweis von E. sakazakii im Vergleich zum Verfahren der amerikanischen Food and Drug Administration (FDA) und dem noch in Bearbeitung befindlichen Verfahren ISO/DTS 22964. Im Gegensatz zur letztgenannten Untersuchungsmethode erfolgten mehrere Positivnachweise mit dem hier beschriebenen Verfahren und dem der FDA.

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Eingegangen: 18. Januar 2007     

婴儿配方粉中阪崎肠杆菌荧光定量PCR检测方法的建立

建立奶粉中阪崎肠杆菌荧光定量PCR检测方法,为准确、快速地定量检测阪崎肠杆菌奠定基础。根据阪崎肠杆菌16S rDNA基因序列,设计特异引物和荧光标记探针并合成,用PCR扩增产物克隆的重组质粒作为阳性对照,经紫外分光光度计测定重组质粒浓度．梯度稀释后作为标准品绘制标准曲线,其Ct值与模板浓度有良好的线形关系,相关系数R^2值为：0．997,可以用于阪崎肠杆菌浓度鉴定。用该方法检测阪崎肠杆菌样品21份,阳性4份,阳性检出率为19％,与常规检测方法结果完全符合。本文建立的检测阪崎肠杆菌的荧光定量PCR方法,较常规技术更为简便、快速,有很好的应用前景和研究价值。     

Characterization of the 16S-23S and 23S-5S rRNA intergenic spacer regions of dairy propionibacteria and their identification with species-specific primers by PCR.

In this study, the 16S-23S and 23S-5S rRNA intergenic spacer region sequences of Propionibacterium acidipropionici, P. freudenreichii ssp. freudenreichii and ssp. shermanii, P. jensenii and P. thoenii were determined. The sequences were shown to vary greatly between the species. Specific primer pairs were derived from the 16S-23S rRNA spacer sequences and used for the identification of the species by PCR.     

Detection of Enterobacter sakazakii strains by real-time PCR

A precise 5' nuclease (TaqMan) real-time PCR was developed and validated in house for the specific detection of Enterobacter sakazakii isolates. Specifically designed nonpatented primers and a hydrolysis (TaqMan) probe were used to target the 16S rRNA gene. All 27 E. sakazakii and 141 non-E. sakazakii strains tested with the real-time PCR were identified correctly. To monitor false-negative results, an internal amplification control was coamplified with the same primers used for the E. sakazakii DNA. The detection probability of the assay was 56% when an E. sakazakii cell suspension containing 10(2) CFU/ml was used as template in the PCR (0.5 CFU per reaction) and 100% with a 10(3) CFU/ml suspension. This PCR assay should be very useful for the diagnostic detection of E. sakazakii in foods, especially powdered infant formula, after cultural enrichment.     

A real-time PCR method targeting a gene sequence encoding 16S rRNA processing protein,rimM, for detection and enumeration of Streptococcus thermophilus in dairy products

A real-time PCR method targeting a gene sequence encoding 16S rRNA processing protein, rimM, for specific detection of Streptococcus thermophilus was developed. The designed real-time PCR primers and probe were specific for S. thermophilus JCM20026, LMG6896, LMG18311, OJT101, OJT102 but not Enteroccocus spp., Lactococcus lactis subsp. lactis, and Streptococcus salivarius which are phylogenetically closely related to S. thermophilus and are difficult to identify using culture-based methods. The linear range of the developed real-time PCR method was from 2.7 to 8.6 log CFU ml^?1 with an amplification efficiency of 96%. Minor differences (about 0.4 log CFU ml^?1) were observed between counts of S. thermophilus obtained by culture and real-time PCR method in plain yoghurt and yoghurt containing fruits. Therefore, the developed real-time PCR method could be of potential application in specific detection and accurate enumeration of S. thermophilus in a wide range of dairy products.     

食品中弓形菌16S rRNA特异性扩增检测方法的建立

针对弓形菌16SrRNA基因合成1对引物,通过对聚合酶链式反应(PCR)扩增条件的优化,建立了检测弓形菌的PCR方法。3株弓形菌标准菌株PCR产物测序结果与NCBI上公布的弓形菌16S rRNA基因序列进行比对,比对结果表明3株弓形菌测序结果与NCBI上公布的弓形菌16S rRNA基因序列同源性均在99%以上。3株弓形菌标准菌株均特异性地扩增出了长度为1202bp的片段,其他19株不同种类的菌株均无扩增产物出现。55份食品样品用Johnson-Murano肉汤增菌后用此法进行检测,其中6份样品为弓形菌阳性,阳性率为10.9%。上述实验结果表明,方法特异性强、操作简便,节省了检测时间,可用于食品中弓形菌的快速检测。     

An improved 16S rRNA based PCR method for the specific detection of Salmonella enterica

A molecular method for the detection of Salmonella enterica strains based on 16S rRNA sequence analysis was developed by a modification of the previously described PCR primer 16SFI [J. Appl. Bacteriol. 80 (1996) 659], which was combined with a newly developed primer annealing at the position 66-82. Only approximately two thirds of now determined Salmonella 16S rRNA sequences contained a region identical to the 16SFI primer sequence and the reverse primer 16SIII was also not specific. Combined, these two primers have been claimed to allow the specific detection of all Salmonella; however, in this study, they did not recognize S. bongori and 3 out of 78 tested S. enterica strains. They also identified some of the tested Enterobacter cloacae strains as Salmonella. On the contrary, the new primer pair, MINf and MINr, made it possible to recognize correctly all of the 78 tested S. enterica strains, representing 31 different Salmonella serovars. None of the 23 non-Salmonella strains from the related gamma-proteobacterial genera was incorrectly recognized as belonging to S. enterica.     

奶粉中阪崎肠杆菌PCR和荧光PCR检测方法的研究

建立了奶粉中可致婴幼儿高死亡率的阪崎肠杆菌的PCR和荧光PCR检测方法。利用细菌16S和23SrDNA的保守区设计通用引物,对6株阪崎肠杆菌16S-23SrDNA间区序列(ITS)进行扩增和测序,在比对阪崎肠杆菌ITS序列的基础上,设计了11条PCR和荧光PCR检测引物,组合成30对PCR引物,并筛选出一对种特异性引物,建立了奶粉中阪崎肠杆菌PCR和荧光PCR检测方法。用10株阪崎肠杆菌,18株近源菌株验证实验表明,本文所建立的PCR和荧光PCR方法特异性强;加菌实验表明,奶粉样品中阪崎肠杆菌检测低限为(2.2～5.4)CFU/100g,灵敏度高;新建的PCR和荧光PCR方法与FDABAM(美国食品及药品管理局微生物分析手册)方法比对实验表明,三种方法的检测结果完全一致。由于PCR和荧光PCR检测方法快速、可靠,因此可替代传统检验方法。     

Characterization of a collection of Enterobacter sakazakii isolates from environmental and food sources

Enterobacter sakazakii has emerged as a rare cause of neonatal meningitis, septicemia and enterocolitis. Contaminated infant milk formula (IMF) has been identified as one infection route. A small number of clinical outbreaks have been epidemiologically linked to IMF contaminated post-pasteurization during manufacture and/or mishandled when reconstituted. Currently no agreed standardized typing protocol has been developed to trace E. sakazakii. The objectives of this study were to apply biochemical and genetic methods to characterize 51 environmental and food E. sakazakii isolates and 6 E. sakazakii type strains. Isolates were presumptively identified using biochemical profiles based on API 20E and ID32E methods and by culture on differential selective Druggan Forsythe Iversen (DFI) agar. Identification was subsequently confirmed by real time polymerase chain reaction (PCR). All but one of the isolates was identified as E. sakazakii by biochemical profiling. One isolate was identified as Escherichia vulneris by ID 32E and as Pantoea agglomerans by API 20E. All isolates produced green/blue colonies on DFI medium characteristic of this organism. Real time PCR could differentiate between E. sakazakii, Enterobacter spp. and other Enterobacteriacae. Analysis of RAPD banding patterns revealed 3 major clusters of E. sakazakii. There was a large degree of diversity noted amongst the remaining isolates. Our findings indicate that RAPD may be applied as a useful and reliable tool for direct comparison of E. sakazakii isolates providing traceability through the infant formula food chain.     

乳粉中阪崎肠杆菌的检测方法

为乳品企业开展阪崎肠杆茵出厂检验寻找更加便捷、准确,且检测费用低廉的检测方法.参照SN/T1632.1-2005标准,在定性检测基础上有所改进.选择3种基础肠道分离培养基伊红美蓝、麦康凯、山梨醇麦康凯分离培养及生化鉴定阪崎肠杆菌,对两种修复培养基灵敏度进行比较及阪崎肠杆菌在5种肠道分离培养基上生长情况比较.结果表明.A和B两组修复培养对婴幼儿乳粉中阪崎肠杆菌均能起到修复作用,B组使用BP修复培养比A组蒸馏水修复培养灵敏度高出1个稀释度(10倍).阪崎肠杆菌在5种肠道分离培养基上均能生长.选择上述3种基础肠道分离培养基分离鉴定阪崎肠杆茵,费用低廉,用于乳品企业对每批次产品进行自检,有更大的经济效益和社会效益.     

Rapid, specific detection of Enterobacter sakazakii in infant formula using a real-time PCR assay

Enterobacter sakazakii is a rare cause of invasive infection with high mortality rates in neonates. Powdered milk-based infant formulas have been associated with the E. sakazakii-related outbreaks in premature or other immunocompromised infants. In this study, an assay was developed for the specific detection of E. sakazakii in infant formula using an application of the fluorogenic 5' nuclease assay (TaqMan). A set of primers and probe was designed using the E. sakazakii partial macromolecular synthesis operon: the rpsU gene 3' end and the primase (dnaG) gene 5' end. The specificity of the assay was evaluated using 68 Enterobacter and 55 non-Enterobacter strains. The newly developed assay enables us to detect 100 CFU/ ml in pure culture and in reconstituted infant formula in 50 cycles of PCR without enrichment. The assay was specific enough to discriminate E. sakazakii from all other Enterobacter and non-Enterobacter strains tested. The developed real-time PCR assay could save up to 5 days and eliminate the need for plating samples on selective or diagnostic agars and for biochemical confirmation steps. The real-time PCR assay could be used to rapidly screen infant formula samples for E. sakazakii and would be a boon to food industries and regulatory agencies.     

Evaluation of an automated repetitive sequence-based PCR system for subtyping Enterobacter sakazakii

Enterobacter sakazakii is regarded as a ubiquitous organism that can be isolated from a wide range of foods and environments. Infection in at-risk infants has been epidemiologically linked to the consumption of contaminated powdered infant formula. Preventing the dissemination of this pathogen in a powdered infant formula manufacturing facility is an important step in ensuring consumer confidence in a given brand together with the protection of the health status of a vulnerable population. In this study we report the application of a repetitive sequence-based PCR typing method to subtype a previously well-characterized collection of E. sakazakii isolates of diverse origin. While both methods successfully discriminated between the collection of isolates, repetitive sequence-based PCR identified 65 types, whereas pulsed-field gel electrophoresis identified 110 types showing > or =95% similarity. The method was quick and easy to perform, and our data demonstrated the utility and value of this approach to monitor in-process contamination, which could potentially contribute to a reduction in the transmission of E. sakazakii.     

Characterization of microorganisms at different landfill depths using carbon-utilization patterns and 16S rRNA gene based T-RFLP

A borehole core from 20 m depth of a Japanese landfill was characterized chemically and microbially. The borehole core sample was typically divided into 5 waste layers; 2.4–4.0 m, 5.7–8.5 m, 9.25–9.6 m, 9.77–14.9 m, and 15.9–17.86 m depths. The waste layers' ages spanned about 14 years between the bottom and top. Archaeal 16S rRNA gene and eubacterial 16S rRNA gene in the waste samples at their respective levels were 9.8 × 10⁵–7.2 × 10⁷ and 1.2 × 10⁷–7.2 × 10⁹ copy/g-wet. Similar to populations of viable and culturable bacteria, those populations were high at 7.0 m and 17.5 m depth, but low at 3.0 m depth. The microorganisms' phenotypes and genotypes were evaluated, respectively, using carbon-utilization tests and by eubacterial 16S rRNA gene based T-RFLP. Low dominance of the VFA-utilizing bacteria in samples and low concentrations of VFAs in all waste layers suggest that the organic decomposition in this landfill site remained. Gamma-proteobacteria dominated the microbial community at 17.5 m depth. Clostridia were detected at 7.0, 11.5, and 17.5 m depths, suggesting strict anaerobic conditions in these deep layers. The Shannon–Weaver diversity index showed lower values at 3.0 m and 11.5 m depth with a T-RF pattern. The diversity index calculated from the carbon-utilization pattern increased slightly with depth at the landfill site. The landfill-site waste layers are expected to be mutually isolated and to form unique microbial communities depending on the buried wastes' composition, temperature, moisture content, and pressure inside the landfill.     

Identification of Acetobacter strains from Thai fermented rice products based on the 16S rRNA gene sequence and 16S-23S rRNA gene internal transcribed spacer restriction analyses

BACKGROUND: Fermented rice flour (khao‐khab, a non‐glutinous rice) and related products are Thai traditional products. The types of acetic acid bacteria (AAB) microflora in khao‐khab have not been reported. In this study, Acetobacter strains were isolated and identified based on the phenotypic and chemotaxonomic characteristics and molecular aspects. RESULTS: Twenty‐five acetic acid bacteria isolated from fermented rice products and a starter for sweetened rice in Thailand by an enrichment culture approach, were assigned to the genus Acetobacter by phenotypic and chemotaxonomic characterisations. On the basis of the 16S rRNA gene sequence and 16S–23S rRNA gene ITS restriction analyses, 25 isolates were divided into six groups and identified at the specific level: (1) Group 1 included five isolates, which were identified as A. indonesiensis; (2) Group 2 included two isolates, which were identified as A. lovaniensis; (3) Group 3 included one isolate, which was identified as A. orientalis; (4) Group 4 included eleven isolates, which were identified as A. pasteurianus; (5) Group 5 included three isolates, which were identified as A. syzygii and (6) Group 6 included three isolates, which were unidentified and considered to constitute a new species. CONCLUSION: Results revealed that various Acetobacter species were distributed in Thai fermented rice flour and related products. A novel Acetobacter species was isolated from the product. Copyright © 2011 Society of Chemical Industry     

Identification of the lactic acid bacteria in Kimchi according to initial and over-ripened fermentation using PCR and 16S rRNA gene sequence analysis

This paper aimed to identify the lactic acid bacteria species involved in kimchi fermentation at different fermentation periods by using culture-independent 16S rRNA gene clone libraries, and develop polymerase chain reaction for the detection of lactic acid bacteria (LAB). We investigated 6 commercially produced kimchi samples, including kimchi at an initial stage of fermentation and kimchi that was fermented to an over-ripened stage. The results of our study show that the analysis with cultureindependent 16S rRNA gene clone libraries could successfully identify 134 clones, 11 species, including Weissella, Lactobacillus, Pediococcus, and Leuconostoc from the 6 commercial kimchi samples. Weisella koreensis and Lactobacillus brevis were the predominant LAB in the initial stage of kimchi fermentation at 4°C (pH 4.96–5.27 and acidity 0.81–0.88), and Leuconostoc gelidum and Lactobacillus sakei subsp. sakei may play an important role in kimchi fermentation at the over-ripened stage (pH 3.61–3.91 and acidity 1.70–1.79).     

Differentiation of Cronobacter sakazakii and related taxa using direct sequencing, species-specific PCR, and mini-sequencing assays

Cronobacter sakazakii and its phylogenetically closest species are considered to be an opportunistic pathogens associated with food-borne disease in neonates and infants. Clearly identifying the specific species of the C. sakazakii group using phenotypic and genotypic techniques is hard. The aim of this study was to use the tuf gene for species discrimination in the C. sakazakii and its phylogenetically closest species, as well as to develop a species-specific primer and single nucleotide polymorphism primer based on 16S rRNA and tuf gene sequence for species identification. The average sequence similarity for the tuf gene among type strains was 96.9 %, and most members of the C. sakazakii group could be distinguished. The species-specific primer was designed according to the 16S rRNA gene sequence, which was employed for PCR with the template DNA of Cronobacter strains. Single 137-bp species-specific band was found only in C. sakazakii and C. malonaticus. A mini-sequencing assay using tuf as a target gene was also developed. The specificity of the mini-sequencing assay was evaluated using 24 strains of Cronobacter species and was able to unambiguously discriminate strains belonging to the species C. sakazakii. The sequence of the tuf gene is more polymorphic than that of the 16S rRNA gene and can be used to differentiate the C. sakazakii group strains. In addition, a novel method to identify the species of the C. sakazakii and C. malonaticus was also developed by species-specific PCR combined with mini-sequencing.     

阪崎肠杆菌的生物学性状与健康危害

阪崎肠杆菌是肠杆菌科的一种,1980年由黄色阴沟肠杆菌更名为阪崎肠杆菌。阪崎肠杆菌能引起严重的新生儿脑膜炎、小肠结肠炎和菌血症,死亡率高达50%以上。目前,微生物学家尚不清楚阪崎肠杆菌的污染来源,但许多病例报告表明婴儿配方粉是目前发现的主要感染渠道。阪崎肠杆菌的生物学性状及其对人群的健康危害受到人们的关注并被报告。     

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirão Preto, São Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L. monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTE™ QPCR SYBR^® Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fast and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method.     

Real-time PCR detection of 16S rRNA genes speeds most-probable-number enumeration of foodborne Listeria monocytogenes

Quantifying foodborne pathogens at concentrations of 0.1 to 1,000 CFU/g of food generally involves most-probable-number (MPN) enumeration, which takes at least 4 days. A real-time PCR assay (RTi-PCR) was developed to accelerate MPN enumeration of foodborne Listeria monocytogenes. Foods were spiked from 70 to 110 CFU/g, and triplicate subportions from 0.0001 to 1 g were selectively enriched for 48 h at 30 degrees C. For standard MPN enumeration, the enrichments were subcultured on Oxford agar (48 h at 35 degrees C) to isolate Listeria. For RTi-PCR MPN, the L. monocytogenes cells from the same enrichments were washed and resuspended in 2 ml of sterile water. DNA was extracted by boiling for 10 min. The DNA in the extract's supernatant was targeted with published oligonucleotide primers for amplifying an Lmo-specific sequence of 16S rRNA genes. Amplification was continuously monitored with SYBR Green. The resulting amplicon was characterized by its melting temperature. The L. monocytogenes specificity of the primers was confirmed by testing L. monocytogenes (15 strains), Listeria innocua (11 strains), and Listeria welshimeri, Listeria seeligeri, Listeria ivanovii, and Listeria grayi (1 strain each). Quantitatively spiked milk, lettuce, smoked salmon, Brie cheese, ice cream, pork paté, salami, ready-to-eat shrimp, raw ground beef, and fresh soft cheese were enumerated by both the standard and the PCR MPN method. The paired results from the two MPN methods agreed well, except for the fresh cheese. For some foods, l-g samples required a decimal dilution for a positive test result, suggesting concentration-dependent food ingredient interference with the RTi-PCR. This RTi-PCR method reduced the time necessary for the MPN enumeration of foodborne L. monocytogenes from 4 to 2 days.     

基于16S rRNA基因与相关基因序列分析格氏乳球菌亲缘关系

通过PCR扩增7株分离自传统发酵乳制品中的格氏乳球菌的dnaA、rpoB、recA基因,将扩增产物测序,测得的序列构建系统发育树,进行分类鉴定.同时,比较了这三个基因位点与16S rRNA基因揭示的格氏乳球菌种内菌株间以及其与乳酸乳球菌种间的亲缘关系的远近.结果表明,dnaA、rpoB、recA基因能够有效的鉴定出格氏乳球菌,且比16S rRNA基因更清晰地揭示了格氏乳球菌菌株间以及其与乳酸乳球菌种间的亲缘关系,特别是将3个管家基因联合起来,亲缘关系更明确,验证了多个管家基因是研究细菌亲缘关系的有力工具.