Detection of Cronobacter species in powdered infant formula by probe-magnetic separation PCR

Cronobacter species are opportunistic foodborne pathogens associated with serious infections in preterm neonates and infants. Based on the epidemiological research, infant formula products are considered to be the main source of infections from this organism. Therefore, accurate methods are required for detection of Cronobacter species. In this study, the specific probe and primers for detection of this organism were designed and verified. The probe-magnetic beads were prepared for sequence capture, followed by PCR assay to detect the target gene. This probe-magnetic separation PCR assay could detect as few as 10³ cfu/mL of Cronobacter in artificially contaminated infant formulas in less than 4 h. The combination of magnetic beads and PCR showed the potential for the detection of Cronobacter in infant formulas and may have applications in the dairy industry.     

Direct application of supercritical carbon dioxide for the reduction of Cronobacter spp. (Enterobacter sakazakii) in end products of dehydrated powdered infant formula

The objective of this study was to develop a viable new method for inactivation of Cronobacter spp. that could be applied directly to dehydrated powdered infant formula (PIF) using supercritical carbon dioxide (SC-CO₂). Samples inoculated with Cronobacter spp. were subjected to SC-CO₂ treatment under various conditions (temperature: 63, 68, and 73°C; pressure: 15, 20, and 25 MPa; time: 10, 20, and 30 min). The survival of Cronobacter spp. was assayed, as were any changes in the quality of the treated PIF. Inactivation of Cronobacter spp. by SC-CO₂ was enhanced as temperature and pressure conditions increased (>6.32 log₁₀ cfu/g). In a validation assay using low-level inoculation (3.21 log₁₀ cfu/g), treatment at 73°C and 15 MPa for 30 min, 20 MPa for 20 and 30 min, or 25 MPa for 20 and 30 min reduced Cronobacter spp. to undetectable levels, with no recovery of cell viability. There was no significant change in water activity, pH, and color of the treated PIF. Overall, the optimum conditions for elimination of Cronobacter spp. were determined to be 73°C and 20 MPa for 20 min. These parameters for effective SC-CO₂ treatment are feasibly applicable to end product of dehydrated PIF. The results of our study may contribute to the development of an efficient method for improving the microbiological safety of PIF.     

枯草芽孢杆菌木聚糖酶的克隆表达及其酶解两种木聚糖的产物分析

通过基因克隆方法获得枯草芽孢杆菌木聚糖酶XynA,考察经镍离子亲和柱纯化后的XynA分别在桦木木聚糖和毛榉木木聚糖中的酶解情况,利用薄层色谱法（thin layer chromatography,TLC）及基质辅助激光解吸/电离飞行时间质谱（matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry,MALDI-TOF/MS）法鉴定木聚糖酶XynA的酶解产物。运用MALDI-TOF/MS分析枯草芽孢杆菌木聚糖酶XynA酶解桦木木聚糖和毛榉木木聚糖产物不同的聚合度寡糖的分布情况。结果表明：在桦木木聚糖酶解液中,产生的中性木寡糖主要为木二糖（X2）和木三糖（X3）,酸性木寡糖聚合度为4～12,并且每一个酸性木寡糖上仅连接着一个甲基葡萄糖醛酸侧链（MeG）。在毛榉木木聚糖酶解液中,产生的中性木寡糖与在桦木木聚糖酶解液中相同,酸性木寡糖的结构相似,聚合度为4～16。因此木聚糖酶XynA具有生产木二糖（X2）和木三糖（X3）以及酸性木寡糖（MeGXn）的功能。     

Short communication: Identification of subclinical cow mastitis pathogens in milk by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Subclinical mastitis is a common and easily disseminated disease in dairy herds. Its routine diagnosis via bacterial culture and biochemical identification is a difficult and time-consuming process. In this work, we show that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows bacterial identification with high confidence and speed (1 d for bacterial growth and analysis). With the use of MALDI-TOF MS, 33 bacterial culture isolates from milk of different dairy cows from several farms were analyzed, and the results were compared with those obtained by classical biochemical methods. This proof-of-concept case demonstrates the reliability of MALDI-TOF MS bacterial identification, and its increased selectivity as illustrated by the additional identification of coagulase-negative Staphylococcus species and mixed bacterial cultures. Matrix-assisted laser desorption-ionization mass spectrometry considerably accelerates the diagnosis of mastitis pathogens, especially in cases of subclinical mastitis. More immediate and efficient animal management strategies for mastitis and milk quality control in the dairy industry can therefore be applied.     

基质辅助激光解吸电离飞行时间质谱与VITEK和rpoB基因测序三种方法对食品中葡萄球菌鉴定效果的比较

目的 比较基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flightmass spectrometry,MALDI-TOF MS)、rpoB基因测序和VITEK 2 Compact三种方法对葡萄球菌的鉴定效果.方法 同时采用三种...     

基质辅助激光解吸电离飞行时间质谱在食源性致病菌鉴定中的研究进展

;基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)具有高效、准确、检测速度快等优点。作为近年来发展的新型食源性致病菌鉴定技术,MALDI-TOF MS为食品病原微生物靶标性监测和食品安全事件应急检验提供了一种高效的鉴别技术参考。本文检索了近年来国内外MALDI-TOF MS技术在食源性致病菌检测中的相关研究,综述基质辅助激光解吸电离飞行时间质谱检测原理及具体案例,在此基础上分别从参考菌株数据库建设、标准化程序规范等方面对MALDI-TOF MS在食源性致病菌检测领域的研究方向进行展望,以期为后续食品安全检测及快速监管提供技术支持。     

Growth inhibition of Cronobacter spp. strains in reconstituted powdered infant formula acidified with organic acids supported by natural stomach acidity

Cronobacter is associated with outbreaks of rare, but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in newborns. This study was conducted to determine the effect of organic acids on growth of Cronobacter in laboratory medium and reconstituted powdered infant formula (PIF) as well as the bacteriostatic effect of slightly acidified infant formula when combined with neonatal gastric acidity. Inhibitory effect of seven organic acids on four acid sensitive Cronobacter strains was determined in laboratory medium with broth dilution method at pH 5.0, 5.5 and 6.0. Acetic, butyric and propionic acids were most inhibitive against Cronobacter in the laboratory medium. The killing effect of these three acids was partially buffered in reconstituted PIF. Under neonatal gastric acid condition of pH 5.0, the slightly acidified formula which did not exert inhibition effect solely reduced significantly the Cronobacter populations. A synergistic effect of formula moderately acidified with organic acid combined with the physiological infant gastric acid was visible in preventing the rapid growth of Cronobacter in neonatal stomach. The study contributed to a better understanding of the inhibitory effect of organic acids on Cronobacter growth in different matrixes and provided new ideas in terms of controlling bacteria colonization and translocation by acidified formula.     

婴幼儿配方乳粉菌落总数检测与细菌污染种属分析

目的考察婴幼儿配方乳粉的微生物污染情况,分析婴幼儿配方乳粉的菌相构成。方法采用国标方法测定30批次1段、2段、3段婴幼儿配方乳粉菌落总数,使用基质辅助激光解吸电离飞行时间质谱对检出的优势菌落进行鉴定。结果 30批次婴幼儿配方乳粉的菌落总数均符合国家限量要求,其中1段乳粉菌落总数显著性低于2段和3段乳粉(P0.05)。婴幼儿配方乳粉中检出的细菌主要为芽孢杆菌属、类芽孢杆菌属、球形芽孢杆菌属、肠球菌属、葡萄球菌属和链球菌属,其中芽孢杆菌属检出率最高。此外, 9批次样品中检出蜡样芽胞杆菌。结论我国婴幼儿配方乳粉质量仍存在一定的风险,监管部门应加强日常监管工作。     

对位芳纶纤维及其打浆粉末端基结构的MALDI-TOF MS分析

利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对两种对位芳纶短切纤维(A1-PPTA、A2-PPTA)及其打浆粉末的端基结构进行分析,通过谱图特征峰与端基结构的对应关系,分析端基结构与反应机理的关联性及打浆过程产生粉末的原因。结果显示,A1-PPTA和A2-PPTA的聚合过程均采用了近似等摩尔的反应物来控制聚合过程;A2-PPTA的聚合过程发生溶剂链转移的几率更高;A2-PPTA中存在较多不利于纤维强度的端基结构,导致其打浆过程中产生更多粉末。     

Two novel analytical methods based on polyclonal and monoclonal antibodies for the rapid detection of Cronobacter spp.: Development and application in powdered infant formula

Cronobacter spp. are opportunistic pathogens, and infections are associated with a high mortality rate. In the current study, monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were generated using heat-inactivated C. sakazakii strain ATCC29544 as the immunogen. Following assay optimization, an indirect enzyme-linked immunosorbent assay (ELISA) based on pAbs and a sandwich ELISA based on mAbs and pAbs were established for the detection of Cronobacter spp. The indirect ELISA detected all species of Cronobacter assayed, and the limit of detection (LOD) was established as 10⁵ cfu/mL. In contrast, the sandwich ELISA was specific for C. sakazakii and had greater sensitivity than the indirect ELISA (LOD of 2 × 10⁴ cfu/mL). Following 10 h of enrichment, Cronobacter spp. were detected using either of the two analytical methods in samples inoculated with 1 cfu/100 g powdered infant formula (PIF). The results from this study demonstrated that both of these novel ELISAs were specific, sensitive, and rapid assays for the screening of pathogenic Cronobacter spp. in PIF.     

Isolation and molecular identification of Cronobacter spp. from powdered infant formula (PIF) in Bangladesh

Cronobacter spp. formerly known as Enterobacter sakazakii is an occasional contaminant of powdered infant formula (PIF). This pathogen has been associated with out-breaks of a rare form of infant meningitis, necrotizing enterocolitis (NEC), bacteremia and neonate deaths. The organism is ranked by the International Commission for Microbiological Specifications for Foods (ICMSF) as a ‘Severe hazard for restricted populations, life threatening or substantial chronic sequelae or long duration’. Present study aimed to isolate Cronobacter spp. from PIF and clinical samples, such as blood, stool and CSF collected from 93 neonates and child patients, age ranged from 0 to 24 months. We did not detect Cronobacter spp. in any of these samples. Later 32 PIF samples collected from retail markets in Bangladesh were tested for the presence of Cronobacter spp. Of these only one was found to be contaminated with Cronobacter sp. This is the first case of Cronobacter contaminated PIF found in Bangladesh to be reported. The organism was successfully identified based on its typical culture characteristics, producing blue-green colonies on chromogenic DFI agar and also by a standardized conventional PCR assay targeting the alpha glucosidase and 16 S rRNA gene sequence of Cronobacter sp. The 16 S rRNA gene was partially sequenced to provide for the phylogenetic analysis of this isolate (DA01) and found to cluster with some other Cronobacter isolates in the phylogram.     

唐菖蒲伯克霍尔德氏菌三种鉴定方法的比较

以食品及环境样品中初步分离的13株疑似唐菖蒲伯克霍尔德氏菌（Burkholderia gladioli）为研究对象,通过比较生理生化检 测法、基质辅助激光解析电离飞行时间质谱（MALDI-TOF-MS）法和recA序列分析法,得到快速准确鉴定唐菖蒲伯克霍尔德氏菌的方 法。 结果表明,13株疑似菌株经MALDI-TOF-MS法和recA序列分析法鉴定为唐菖蒲伯克霍尔德氏菌,而VITEK 2 COMPACT生理生 化法鉴定8株（62%）为唐菖蒲伯克霍尔德氏菌,其他5株为栖稻假单胞菌（Pseudomonas oryzihabitans）。 在唐菖蒲伯克霍尔德氏菌的鉴 定方法中,MALDI-TOF-MS法和recA序列分析法鉴定结果一致且准确,而VITEK 2 COMPACT生化检测法存在缺陷。 相比传统生理 生化鉴定方法,MALDI-TOF-MS法和recA序列分析法在唐菖蒲伯克霍尔德氏菌鉴定中更为快速、准确。     

Identification of bovine-associated coagulase-negative staphylococci by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a direct transfer protocol

This study evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) for the identification of bovine-associated coagulase-negative staphylococci (CNS), a heterogeneous group of different species. Additionally, we aimed to expand the MALDI-ToF MS database with new reference spectra as required to fill the gaps within the existing commercial spectral library. A total of 258 isolates of CNS were used in the study, covering 16 different CNS species. The majority of the isolates were previously identified by rpoB gene sequencing (n = 219), and the remainder were identified by sequencing of 16S rRNA, hsp60, or both rpoB and hsp60. The genotypic identification was considered the gold standard identification. All MALDI-ToF MS identifications were carried out using the direct transfer method. In a preliminary evaluation (n = 32 isolates; 2 of each species) with the existing commercial database, MALDI-ToF MS showed a typeability of 81% (26/32) and an accuracy of 96% (25/26). In the main evaluation (n = 226 isolates), MALDI-ToF MS with the existing commercial Biotyper (Bruker Daltonics Inc., Billerica, MA) database achieved a typeability of 92.0% (208/226) and an accuracy of 99.5% (207/208). Based on the assessment of the existing commercial database and prior knowledge of the species, a total of 13 custom reference spectra, covering 8 species, were created and added to the commercial database. Using the custom reference spectra expanded database, isolates were identified by MALDI-ToF MS with 100% typeability and 100% accuracy. Whereas the MALDI-ToF MS manufacturer's cutoff for species-level identification is 2.000, the reduction of the species level cutpoint to ≥1.700 improved the species-level identification rates (from 64 to 92% for the existing commercial database) when classifying CNS isolates. Overall, MALDI-ToF MS using the direct transfer method was shown to be a highly reliable tool for the identification of bovine-associated CNS.     

Bovine mastitis bacteria resolved by MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method to identify the most common pathogenic bacteria in humans and animals. The goals of this study were to amend a commercial database with additional species, evaluate the amended database for identification of bacterial genera and species causing bovine mastitis, and describe the plethora of species involved. In total, 500 udder pathogenic isolates were subjected to MALDI-TOF MS using bacterial or fungal colony material; 93.5% could be identified to the species level, and 6.5% were identified only to the genus level. Isolates identified to the genus level required further identification to the species level by conventional methods or 16S rDNA sequencing. Mass spectra from verified species were used to expand the MALDI-TOF MS database to improve future identification ability. A total of 24 genera and 61 species were identified in this study. Identified isolates were mainly staphylococci, streptococci, Enterobacteriaceae, and coryneforme bacteria. In conclusion, MALDI-TOF MS is a powerful, rapid, and reliable technique to identify the most common microorganisms causing bovine mastitis, and the database can be continuously expanded and improved with additional species.     

3种方法鉴定鸡肉粉中沙门氏菌的比对研究

目的采用3种方法对鸡肉粉中沙门氏菌进行检测。方法3份样品的前处理依据组织方提供的《作业指导书》进行,每瓶样品直接加入4.5 mL灭菌去离子水复溶,作为初始样本,后续实验依据GB4789.4-2016《食品安全国家标准食品微生物学检验沙门氏菌检验》操作。鉴定分离出疑似菌后,加入了实时荧光定量PCR法、基质辅助激光解吸电离飞行时间质谱法2种方法作为辅助检测,并用全自动微生物鉴定系统进行生化鉴定,再结合生化鉴定与血清学实验综合评判检测结果。结果使用3种鉴定方法在JS015、JS110、JS140样本中均检出沙门氏菌。结论本研究采用的实时荧光定量PCR法、基质辅助激光解吸电离飞行时间质谱法2种方法均能快速准确鉴定出沙门菌,特别是基质辅助激光解吸电离飞行时间质谱法和血清学分型配合使用,对沙门菌的快速鉴定有重要意义。     

基于MALDI-TOF MS技术对李斯特氏菌属两种细菌的快速鉴定

为提高基质辅助激光解析电离飞行时间质谱技术(MALDI-TOF MS)在李斯特氏菌属鉴定中的分辨能力,建立快速准确鉴定单增和英诺克李斯特氏菌的质谱学方法。通过采集79株单增和57株英诺克李斯特氏菌的指纹图谱,利用Clin Pro tools软件对数据进行统计学分析,建立数学判别模型并验证其准确性。峰统计结果显示,两组数据峰强度差异显著的特征峰有16个,推测出单增李斯特氏菌生物标志物6个,英诺克李斯特氏菌10个,发现在单增李斯特氏菌中质量峰3985/7970 u和3972/7942 u是独立且连锁存在。基于遗传算法的判别模型交叉验证率和检测识别能力最强,分别为99.44%和100.00%,经验证准确率达到96%以上,可实现对单增和英诺克李斯特氏菌的快速准确鉴定。同时,利用Bruker Biotyper软件将以上菌株建库形成了实验室内部李斯特氏菌谱库,对8株未测李斯特氏菌进行搜库鉴定,匹配分数均高于商品化数据库,提升了MALDI-TOF MS对李斯特菌属的自动鉴定能力。     

基质辅助激光解吸电离飞行时间质谱分析技术在微生物鉴定与分型中的应用

微生物的鉴定方法有很多种, 这些方法基于表型、免疫学、遗传学等, 目前普遍采用16S rRNA和18S rRNA基因测序为标准, 但这些方法也有一些局限性, 费时费力、操作复杂、劳动力和试剂成本都很高, 限制了在常规微生物检测实验室的普及与推广。近年来, 基质辅助激光解吸电离飞行时间质谱法(matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS)在微生物学领域的应用越来越重要, 与应用于微生物鉴定的其他技术不同, MALDI-TOF MS更快速、准确、经济且易于操作。随着样品制备、数据库丰富和算法优化的发展, 使用MALDI-TOF MS进行鉴定的准确性和速度不断提高。虽然大多数研究都集中在细菌上, 但这项技术也应用于真菌和病毒。本文对MALDI-TOF MS技术对细菌、真菌、病毒鉴定与分型的研究进展以及目前该技术的局限性进行综述, 旨为该技术在临床诊断、食品检测等领域的多种应用奠定基础。     

响应面优化蒲公英橡胶草菊糖提取工艺及其MALDI-TOF MS分析

以蒲公英橡胶草根为原料,采用超声提取的方式,通过单因素实验和响应面试验探究了超声功率、提取时间、超声温度、液料比对蒲公英橡胶草菊糖提取率的影响,得到蒲公英橡胶草菊糖提取的最佳工艺为:超声功率230 W、超声时间34 min、超声温度61 ℃、液料比30:1（mL:g）,菊糖提取率为20.14%±0.19%。采用氢氧化钙-磷酸法、三氯乙酸法、Sevage法进行脱蛋白纯化,得出氢氧化钙-磷酸法的效果最好,其蛋白清除率达90.78%,菊糖损失率为26.44%。将菊糖粗提液经蛋白纯化、脱色、旋蒸、醇沉及真空冷冻干燥后得到纯度为80.8%的菊糖,为白色固体粉末。经MALDI-TOF MS进行表征分析,确定其单体的分子量为162,端基的分子量和为179,能检测到的聚合度范围为2~30。此研究为蒲公英橡胶草的综合利用研究提供了理论依据。     

Molecular characterization of non-aureus Staphylococcus spp. from heifer intramammary infections and body sites

The purpose of this study was to investigate non-aureus Staphylococcus spp. intramammary infections (IMI) in periparturient heifers and determine the relationship of precalving body site isolation with precalving IMI and postcalving IMI using molecular speciation and strain-typing methods. Primiparous heifers were enrolled at approximately 14 d before expected calving date. Precalving mammary quarter secretions and body site swabbing samples (teat skin, inguinal skin, muzzle, and perineum) were collected. Postcalving, mammary quarter milk samples were collected for culture and somatic cell counting. Precalving body site samples were cultured, and up to 10 staphylococcal colonies were saved for characterization. Staphylococcal isolates were speciated using matrix-assisted laser/desorption ionization time-of-flight mass spectrometry or sequencing of rpoB or tuf. Pulsed-field gel electrophoresis was used to strain type a subset of isolates. Overall, Staphylococcus chromogenes, Staphylococcus agnetis, and Staphylococcus simulans were the most common species identified in precalving mammary secretions, whereas S. chromogenes, Staphylococcus xylosus, and S. agnetis were the most common species found in postcalving milk samples. The most common species identified from body site samples were S. chromogenes, S. xylosus, and Staphylococcus haemolyticus. Mammary quarters that had a precalving mammary secretion that was culture positive for S. agnetis, S. chromogenes, or Staphylococcus devriesei had increased odds of having an IMI with the same species postcalving. A S. chromogenes IMI postcalving was associated with higher somatic cell count when compared with postcalving culture-negative quarters. Among heifers identified with a non-aureus Staphylococcus spp. IMI either precalving or postcalving, heifers that had S. agnetis or S. chromogenes isolated from their teat skin had increased odds of having the same species found in their precalving mammary secretions, and heifers with S. chromogenes, S. simulans, and S. xylosus isolated from their teat skin precalving were at increased odds of having an IMI with the same species postcalving. Overall, 44% of all heifers with a S. chromogenes IMI around the time of parturition had the same strain isolated from a body site. Based on pulsed-field gel electrophoresis, a high level of strain diversity was found.     

Short communication: Evaluation of MALDI-TOF mass spectrometry and a custom reference spectra expanded database for the identification of bovine-associated coagulase-negative staphylococci

This study evaluated MALDI-TOF mass spectrometry and a custom reference spectra expanded database for the identification of bovine-associated coagulase-negative staphylococci (CNS). A total of 861 CNS isolates were used in the study, covering 21 different CNS species. The majority of the isolates were previously identified by rpoB gene sequencing (n = 804) and the remainder were identified by sequencing of hsp60 (n = 56) and tuf (n = 1). The genotypic identification was considered the gold standard identification. Using a direct transfer protocol and the existing commercial database, MALDI-TOF mass spectrometry showed a typeability of 96.5% (831/861) and an accuracy of 99.2% (824/831). Using a custom reference spectra expanded database, which included an additional 13 in-house created reference spectra, isolates were identified by MALDI-TOF mass spectrometry with 99.2% (854/861) typeability and 99.4% (849/854) accuracy. Overall, MALDI-TOF mass spectrometry using the direct transfer method was shown to be a highly reliable tool for the identification of bovine-associated CNS.