Surveillance of Antibiotic Resistant Staphylococcus aureus in Agricultural Production Chain of Mongolia

Monitoring of food borne pathogens in food is the primary tool for the implementation of food safety systems. It is necessary to monitor the prevalence of food borne pathogens for effective food safety planning and targeted interventions. Staphylococcus aureus is considered as the third largest cause of food related illness in worldwide. The present study aimed at surveillance of S. aureus contamination of meat on meat supply chain stages, which is a common benchmark of meat market in Mongolia, and characterization of isolated and collected strains from other agricultural sources. The cultural and polymerase chain reaction （PCR） methods were used for isolation, identification and characterization of S. aureus. In 216 cultures of S. aureus among 634 Staphylococci isolates obtained from different sources throughout the agricultural production chain in this study, common gene for S. aureus （98.74%）, and nuc （97.47%）, mecA （44.12%）, msrA （9.66%）, gyrA （32.77%） and ermC （29.41%） genes were identified. As seen in the surveillance result, the prevalence of methicillin-resistance S. aureus （MRSA） is 44% among S. aureus isolates from agricultural production chain. Confirmed cases of food-borne infections and intoxications caused by S. aureus should be considered as one of mean criteria of food safety issues in Mongolia, and special attentions should be paid on antibiotic resistant bacteria, such as S. aureus.

Potentiating activity of rhein in targeting of resistance genes in methicillin-resistant Staphylococcus aureus

Objective: To investigate the synergistic effect between rhein(RHE) and oxacillin against Staphylococcus aureus(MRSA) at the gene level. Method: A minimum inhibitory concentration and checkerboard dilution test were conducted to evaluate antibacterial activity. Reverse transcriptase polymerase chain reaction was conducted to investigate the gene expressions. Results: RHE exhibited a minimum inhibitory concentration of 62.5-250.0 μg/mL against various MRSA strains and the reference strain, respectively. As revealed by the checkerboard assay, a combination of RHE and oxacillin exhibited synergistic or partially synergistic effects against MRSA strains. RHE decreased the expressions of mecA/blaZ in a dose-dependent manner. RHE also decreased the expressions of the regulator genes mecI/blaI and mecR1/blaR1. Conclusions: We suggest that RHE affects the activity of mecR1/blaR1, which is located in the cell membrane of MRSA and results in the suppression of mecA/mecI/mecR1 and blaZ/blaI/blaR1 gene expressions.

Tuf m RNA rather than 16S r RNA is associated with culturable Staphylococcus aureus

AIM: To study the presence of various nucleic acids targets of Staphylococcus aureus(S. aureus) during bacterial growth and antibiotic induced killing in relation to viability.METHODS: S. aureus was cultured to log phase and spiked in Todd Hewitt(TH) broth and whole blood of healthy human volunteers. Viability of S. aureus after flucloxacillin treatment(0, 1, 3 and 6 d) was assessed by culture on bloodagar plates. DNA and RNA were isolated from 200 μL. c DNA synthesis was performed by using random primers. The presence of S. aureus DNA, r RNA, and m RNA were determined by real-time polymerase chain reaction of the 16 S r DNA and tuf gene(elongation factor Tu).RESULTS: S. aureus spiked in TH broth without antibiotics grew from day 0-6 and DNA(tuf and 16S), and 16 S r RNA remained detectable during this whole period. During flucloxacillin treatment S. aureus lost viability from day 3 onwards, while the 16 S r RNA-gene and its RNA transcripts remained detectable. DNA andr RNA can be detected in flucloxacillin treated S. aureus cultures that do not further contain culturable bacteria.However, tuf m RNA became undetectable from day 3onwards. Tuf m RNA can only be detected from samples with culturable bacteria. When spiking S. aureus in whole blood instead of broth no bacterial growth was seen, neither in the absence nor in the presence of flucloxacillin. Accordingly, no increase in DNA and RNA levels of both 16 S r DNA and the tuf gene were detected. CONCLUSION: Tuf m RNA expression is associated with culturable S. aureus and might be used to monitor antibiotic effects.

Detection of bacterial DNA from cholesterol gallstones by nested primers polymerase chain reaction

AIM To search for bacterial DNA sequences in cholesterol gallstones with negative bacterial culture. METHODS DNA was extracted from cholesterol gallstones in gallbladders and nested primers polymerase chain reaction (NP PCR) was used to amplify bacterial gene fragments for identifying the existence of bacteria. The samples of bacterial DNA extracted from potentially causative or unrelated living bacteria were amplified in vitro as the standard markers and comparative 16S ribosomal RNA sequence analysis was made for bacterial identification. RESULTS The gallbladder gallstones of 30 patients were analyzed and bacterial DNA was found in 26 patients. Among them, gallstones with cholesterol content between 30%-69% were seen in 5 (5/5) patients, 70%-90% in 11 (11/14) patients, and more than 90% in 10 (10/11) patients. There was no difference either in cholesterol and water content of gallstones or in harboring bacterial DNA of gallstones. E.coli related DNA fragments appeared in the stones of 8 (26 67%) patients; propionibacteria type DNA in 7 (23 33%); and harbored bacterial gene fragments in 2 patients, similar to Streptococcus pyogenes . A more heterogeneous sequence collection was found in 7 (23 33%) patients, which could belong to multiple bacterial infections. Two (6 67%) patients had bacterial DNA with low molecular weight which might be related to some unidentified bacteria. CONCLUSION Most cholesterol gallstones harbor bacterial DNA. It is important to determine whether these microorganisms are innocent bystanders or active participants in cholesterol gallstone formation.

Risk factors affecting nasal colonization of methicillin-resistant Staphylococcus aureus when admitted in intensive care unit

Background Colonization with methicillin-resistant Staphylococcus aureus （MRSA） is a risk factor for subsequent invasive MRSA infection,particularly in patients admitted for critical care.The purpose of this study was to investigate the risk factors affecting nasal colonization of MRSA in patients admitted to intensive care units （ICU）.Methods Between August 1,2011 and June 30,2012,we screened for MRSA nasal colonization in 350 patients by Real-time PCR within 24 hours of admission by means of swab samples taken from the anterior nares.According to the results of PCR,the patients were divided into 2 groups：the positive group with nasal MRSA colonization and the negative group without nasal MRSA colonization.The 31 （8.86％） patients were MRSA positive.The risk factors evaluated included thirteen variables,which were analyzed by t test for continuous variables and X2 test for discrete variables.The variables with significance （P ＜0.05） were analyzed with stepwise Logistic regression.Results There were differences （P ＜0.05） in four variables between two groups.The duration of stay in hospital prior to ICU admission in the positive group was （35.7±16.1） days,vs.（4.5±3.1） days in the negative group.The average blood albumin level was （28.4±2.9） g/L in the positive group,vs.（30.5±4.3） g/L in the negative group.Of 31 patients in the positive group,seven had been treated with antibiotics longer than seven days vs.34 of 319 patients in the negative group.In the positive group,four of 31 patients received treatment with more than two classes of antibiotics prior to admission in ICU,contrasted to 13 of 319 patients in the negative group.Furthermore,stepwise Logistic regression analysis for these four variables indicates that the duration of stay in hospital prior to ICU admission may be an independent risk factor.Conclusions MRSA colonization in ICU admission may be related to many factors.The duration of stay in hospital prior to ICU admission is an independent risk factor.

Community-acquired pneumonia:The importance of the early detection of drug-resistant organisms

Pneumonia is a disease associated with significant healthcare burden with over 1.5 million hospitalizations annually and is the eighth leading cause of death in the United States.While community-acquired pneumonia(CAP)is generally considered an acute time-limited illness,it is associated with high long-term mortality,with nearly one-third of patients requiring hospitalization dying within one year.An increasing trend of detecting multidrug-resistant(MDR)organisms causing CAP has been observed,especially in the Western world.In this editorial,we discuss about a publication by Jatteppanavar et al which reported that a case of a MDR organism was the culprit in developing pneumonia,bacteremia,and infective endocarditis that led to the patient’s death.The early detection of these resistant organisms helps improve patient outcomes.Significant advances have been made in the biotechnological and research space,but preventive measures,diagnostic techniques,and treatment strategies need to be developed.

TaqMan探针双重荧光聚合酶链式反应技术检测食源性沙门氏菌和志贺氏菌

目的建立TaqMan探针双重荧光聚合酶链式反应(polymerase chain reaction,PCR)技术检测食源性沙门氏菌和志贺氏菌的方法。方法通过比对沙门氏菌和志贺氏菌的基因组序列,选择同源性高、保守性好的区域设计特异性引物和探针,经过引探筛选、浓度调试等一系列优化,建立了食源性沙门氏菌和志贺氏菌双重荧光PCR核酸检测方法,并对其特异性、质粒最低检出限、菌液敏感性、重复性、稳定性以及实际样品进行了验证。结果该方法与大部分食源性致病菌无交叉反应,特异性强;沙门氏菌和志贺氏菌质粒的最低检出限可达5 copies/μL,沙门氏菌菌液敏感性为1.0×10^(2) cfu/mL,志贺氏菌菌液敏感性10 cfu/mL;质粒和菌液核酸梯度的批内和批间变异系数均在0.177%~1.958%之间,小于5.0%,具有较强的稳定性;标准曲线相关系数(r^(2))均大于0.999。结论本研究成功建立了一种TaqMan探针双重荧光PCR技术同时检测食源性沙门氏菌和志贺氏菌的方法,该方法扩增时间短,只需要30 min即可,而且特异性强、稳定性好,可用于疑似沙门氏菌和志贺氏菌污染样品的检测,为食品安全的快速检测提供技术支撑。

数字聚合酶链式反应技术在食品安全核酸检测领域中的研究进展及标准化现状

数字聚合酶链式反应(digital polymerase chain reaction,dPCR)是一种新型核酸扩增技术,无需借助内参基因和标准曲线,通过极限稀释和泊松分布统计即可实现核酸单分子层面的绝对定量分析,具有高灵敏度、高精确度和高耐受性等技术优势,在食品安全核酸检测领域具有极大的发展潜力。本文介绍了dPCR的技术原理、优缺点及商业化平台,综述了dPCR在食源性致病菌检测、动物和植物源性成分检测、转基因成分检测和食源性病毒检测等领域的研究进展及标准化现状,并分析了该技术存在的技术难题和未来发展方向,以期为dPCR在食品安全核酸检测领域中的应用推广和标准制定提供研究思路。

叠氮丙啶-实时荧光聚合酶链式反应-高通量测序技术鉴定冷链食品中多种病原菌

目的 应用叠氮丙啶-实时荧光聚合酶链式反应-高通量测序技术鉴定冷链食品中多种病原菌,构建病原菌分子进化树。方法 以冷链食品中的沙门氏菌、金黄色葡萄球菌、蜡样芽胞杆菌、副溶血性弧菌、单核细胞增生李斯特氏菌5种病原菌作为研究对象,应用叠氮丙啶-实时荧光聚合酶链式反应技术作为冷链食品中病原菌检测初筛手段,应用微生物培养法以及生化鉴定仪器法进行方法比对与结果验证,运用高通量测序以及分子进化树构建作为冷链食品中所分离病原菌的种属地位确证方法。结果 叠氮丙啶-实时荧光聚合酶链式反应技术成功扩增了冷链食品中生活状态病原菌的特征性核酸片段,排除了死亡细菌以及阴性对照菌的干扰,病原菌检出限可达到1×10^(3)CFU/mL,一次反应可检测42份试样,可以在18 h内完成检测工作。在冷链食品中病原菌抽样检测调查中,随机采集的751份冷链食品,共检出62株病原菌,病原菌总体检出率为8.3%(62/751)。通过后续的16S rRNA测序以及葡萄球菌属、弧菌属以及李斯特菌属分子进化树的构建,成功溯源了金黄色葡萄球菌的污染来源并完成病原菌种属定位。结论 本方法特异性好、灵敏度高、检测通量高,为冷链食品及相关食品中病原菌的精确检测与溯源分析提供新的思路与方法。

RAPID MOLECULAR DETECTION OF METHICILLIN -RESISTANT S.AUREUS IN THE INTENSIVE CARE UNIT

［1］Daniel JG, James RU, Cynthia A G, et al. Multiplex PCR for identification of methicillin-resistant staphylo-cocci in the clinical laboratory [J]. J Clin Microbiol,1994,32(7):1768［2］Chambers HF ,Sachdeva M. Binding of β-lactam antibi-otics to penicillin-binding proteins in methicillin-resis-tant staphylococcus aureus [J]. J Infect Dis, 1990,161:1170［3］Towner KJ,Talbot DC,Curran R, et al. Development and evaluation of a PCR-based immunoassay for the rapid detection of methicillin-resistant staphylococcus aureus[J]. J Med Microbiol, 1998,47 :607［4］Murakami KW, Minamide KW,Nakamura E, et al. I-dentification of methicillin-resistant strains of staphylo-cocci by polymerase chain reaction[J]. J Clin Microbiol,1991,29:2240［5］Hulimann-Dalel RL,Ryffel C, Kayser F H, et al. Sur-vey of the methicillin resistant -associated genes mecA,mecRI-mecI,and femA-femB in clinical isolates of me-thicillin-resistant Staphylococcus aureus[J]. Antimicrob Agents Chemother, 1992,36: 2617［6］Kopp U,Roos M ,Weck J, et al. Staphylococcal peptido-glycan interpeptide bridge biosynthesis: a novel anti-staphylococcal target[J]? Microb Drug Resist, 1996,2:29［7］Rychlik W, Spencer WJ,Rhoads R E. Optimization of the annealing temperature for DNA amplification in vitro [J]. Nucleic Acids Res, 1990,18 :

食品检测中快速检测技术的应用

本文全面介绍了食品安全问题的重要性以及常规食品检测技术和快速检测技术的应用。常规食品检测技术包括化学分析法、仪器分析法和微生物检测法,而快速检测技术包括聚合酶链式反应技术、免疫学检测技术、生物传感技术和基于成像的检测技术。此外,针对快速检测技术常见的问题,本文提出了相应的解决方案,为食品安全监测提供借鉴和参考。

生物检测技术在食品检验中的应用分析

本文通过对酶联免疫吸附技术、聚合酶链式反应技术、生物传感器技术和生物芯片技术等主流生物检测技术的原理和特点进行介绍,并结合这些技术在食品农药残留检测、食品中微生物检测、食品过敏原检测和食品营养物质检测方面的具体应用实例进行分析,揭示了生物检测技术凭借其高效、灵敏、特异、快速等优点在食品安全检测领域的广阔应用前景。研究表明,生物检测技术的应用极大地提高了食品安全检测的效率和准确性,为保障食品安全、维护消费者健康提供了有力的技术支撑。

数字聚合酶链式反应技术在食品安全核酸检测中的研究进展

数字聚合酶链式反应(Digital Polymerase Chain Reaction,dPCR)技术是一种新兴的核酸绝对定量分析技术,无须标准品和绘制标准曲线,通过极限稀释样品和泊松概率分布原理就可以精准检测样本初始的DNA拷贝数,具有特异性强、灵敏度高和重复性好的特性,在食品安全核酸检测领域具有较好的发展潜力。本文介绍了dPCR技术的基本原理和特点,分析了dPCR技术在食品安全核酸检测领域的研究现状,并对其在食品安全中的应用前景进行展望,以期为促进dPCR在食品安全核酸检测领域中的应用提供一定参考。

核酸分子检测技术在食品药品检测中的应用

核酸分子检测技术在食品药品检测领域发挥着至关重要的作用。本文综述了聚合酶链式反应(Polymerase Chain Reaction,PCR)技术、实时荧光定量PCR技术以及DNA芯片技术等核酸分子检测技术在食品药品检测中的应用,探讨了核酸分子检测技术的发展前景。

红曲醋酿造过程中菌群动态分析

红曲醋是在多菌种共同作用下发酵酿造而成,其酿造周期与微生物有着重要关系。研究通过聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)和高通量测序对红曲醋中的微生物菌群结构进行分析,并借助热图分析醋中的优势菌群,为揭示红曲醋中的微生物演替规律提供依据,并对缩短永春老醋酿造周期奠定基础。高通量测序研究结果发现,红曲醋中的优势菌群是乳酸菌(Lactobacillus)、醋酸菌(Acetobacter)、红球菌(Rhodococcus)、苍白杆菌(Ochrohactrum)、芽孢杆菌(Bacillus)、沉积物杆状菌(Sediminibacterium)、不动杆菌(Acinetobacter)、甲基杆菌(Methylobacterium)、酵母菌(Saccharomycetales sp.)、解脂耶氏酵母(Yarrowia lipolytica)、子囊菌(Ascomycota sp.)、假丝酵母菌(Candida xylopsoci)和毛孢壳属(Coniochaetaceae sp.)。芽孢杆菌(Bacillus)与酵母菌(Saccharomycetales sp.)有拮抗作用,红曲霉菌(Monascus purpureus)与克拉菌(Cryocola)、无氧芽孢菌(Anoxybacillus)、沙雷氏菌(Serratia)、微杆菌(Microbacterium)、甲基杆菌(Methylobacterium)有较强的促进作用。

核酸扩增技术在动物源性食品掺假中的研究进展

核酸扩增技术是一种在体外快速扩增特定DNA片段的分子生物学技术,目前已广泛应用于传染病检测、生物勘测、食品安全检测、临床诊断和公共卫生监测等研究领域。其中,食品安全领域的问题日渐成为人们关注的焦点,尤其是动物源性食品掺假的现象屡禁不止。在过去的科学研究中,动物源性食品掺假的核酸扩增技术发展迅速,取得了很大进展。该文就核酸扩增技术中的凝胶电泳PCR、实时荧光定量PCR、数字液滴PCR、环介导等温扩增、交叉引物扩增、滚环扩增、重组酶聚合酶扩增等技术的原理及在动物源性食品掺假检测中的应用进行综述。讨论了各类核酸扩增技术的关键优势和局限性,简要介绍了现有的挑战和进一步的研究进展,旨在为动物源性食品掺假核酸扩增技术的发展指明方向。

眼表定植菌群分布特征及常见菌耐药性分析

目的:探讨眼表定植菌群分布及抗菌药物耐药性,为临床经验性用药提供指导。方法:收集健康体检人群眼结膜囊标本共93例,进行细菌培养、鉴定及体外药物敏感试验,统计、分析细菌的检出和分布及主要革兰阳性球菌的耐药情况。结果:93例健康受试者结膜囊标本经培养,87例为阳性,共分离出168株细菌。凝固酶阴性葡萄球菌对多种抗生素呈现出耐药性,耐甲氧西林凝固酶阴性葡萄球菌(MRCoNS)组对抗生素的耐药率明显高于甲氧西林敏感凝固酶阴性葡萄球菌(MSCoNS)组,其中青霉素、环丙沙星、左氧氟沙星、克林霉素及复方新诺明五种药物耐药性比较差异有统计学意义(均P<0.05),PCR检测mecA基因与VITEK 2 Compact仪器法对MRCoNS苯唑西林耐药性预测的结果符合率是97.5%。结论:G+球菌为眼表的主要定植菌,其中凝固酶阴性葡萄球菌为优势菌,我们应加强对葡萄球菌特别是MRS耐药性的监测,指导临床根据药敏结果合理规范使用抗生素。

基因扩增技术在食品检测中的质量控制要点

随着食品安全监管需求的不断提升,相应的检测技术向着更加准确、快捷的方向发展已成为必然趋势。聚合酶链式反应是基因扩增技术最重要的应用方向,具有省时高效的特点,现已被食品安全检测领域普遍采用。本文对聚合酶链式反应主要的应用途径进行了论述,并结合实践经验,对检测过程的质量控制要点及内、外源影响因素进行了总结,为相关实验室建设提供了参考依据。

食品微生物检测技术应用探讨

食品微生物检测对保障食品安全具有重要意义。本文概述了食品微生物检测技术的发展现状,重点阐述了聚合酶链式反应、酶联免疫吸附测定、生物传感器等常用检测技术的原理、步骤、应用情况,分析了各技术的优势和局限性,为食品微生物检测技术的选择提供参考。

微生物检测技术在食品检验中的应用探讨

随着人们对食品安全关注度的不断提高,微生物检测技术在食品检验中的应用越来越广泛。本文介绍了常见的微生物检测方法,探讨了其在食品检验中的应用情况和优势,并分析了当前微生物检测技术面临的挑战和未来发展方向。