Investigations into the antiadhesive activity of herbal extracts against Campylobacter jejuni

Campylobacter jejuni is one of the most common bacterial causes of diarrhoea in the industrialized world, being associated with the occurrence of Guillain‐Barré Syndrome, and inducing diseases partially through intestinal adherence. With increasing reports of C. jejuni drug resistance against standard antibiotics, investigations into antiadhesive agents for the prevention of bacterial infection are highly significant. Given the consumer‐driven development towards holistic and integrative healthcare, research into additional anti‐Campylobacter effects of herbal medicines that are already used for their beneficial effects on bowel and digestive functions is important. Twenty‐one herbal extracts were screened for antiadhesive activity against C. jejuni using modifications of previously published antiadhesion assays. Antiadhesion effects with IC₅₀ values <3 mg/mL were obtained for seven ethanol plant extracts, with Zingiber officinale (ginger), Capsicum annum (cayenne) and Glycyrrhiza glabra (licorice) displaying the highest antiadhesion activity against C. jejuni (IC₅₀: <0.1 mg/mL, 0.29 mg/mL and 0.65 mg/mL, respectively). Differences in antiadhesion activity were found for two different Echinacea species, with E. purpurea displaying significantly higher and dose dependent antiadhesion activity than E. angustifolia. No significant antiadhesion activity (IC₅₀ values >35 mg/mL) was found for Agrimonia eupatoria (agrimony), Andrographis paniculata (andrographis), Matricaria recutita (chamomile), Foeniculum vulgare (fennel), Filipendula ulmaria (meadowsweet) and Artemisia absinthium (wormwood) extracts. This study provides evidence for additional beneficial effects of marketed herbal medicines in gastrointestinal disorders. Copyright © 2011 John Wiley & Sons, Ltd.     

吉兰-巴雷综合征相关空肠弯曲菌cstⅡ基因序列对比研究

目的 研究华北地区3株吉兰-巴雷综合征(Guillain-Barré syndrome,GBS)相关空肠弯曲菌(Campylobacter jejuni,C.jejuni)的cstⅡ基因序列并寻找GBS相关及GBS无关的两类空肠弯曲菌菌株间可能与GBS致病性相关的基因突变位点和蛋白高级结构变化,分析菌株间遗传进化关系.方法 选取分离自GBS患者粪便并经动物模型证实为致GBS的3株空肠弯曲菌菌株进行培养并提取基因组DNA测序,与GenBank中GBS无关空肠弯曲菌基因序列进行配对对比,寻找两类菌株间可能导致空肠弯曲菌致病能力差异的cstⅡ基因碱基突变位点及蛋白高级结构改变,观察这种变化是否为GBS相关菌株的共性,并构建遗传进化树.结果 与GBS无关菌株相比,3株致GBS菌株cstⅡ基因的氨基酸序列二级结构的第7个α螺旋的165～180区段均被打开形成了折叠或转角,该变化在其他GBS相关菌株的二级结构变化中同样得到体现.实验中75%的GBS相关菌株cstⅡ基因为双功能,25%GBS相关菌株及所有GBS无关菌株cstⅡ基因为单功能.LL株与实验中涉及的GBS相关菌株在进化上高度类聚.结论 双功能cstⅡ可能与空肠弯曲菌的致GBS特性相关.LL株cstⅡ基因在一定程度上反映了亚洲地区的GBS相关菌株cstⅡ基因的序列特征,为进一步探索GBS发病的地域性特点并对GBS菌株进行监测提供了资料.

Abstract：

Objective To investigate the pathogenic mechanism of Campylobacter jejuni(C.jejuni) associated with Guillain-Barré syndrome(GBS) and provide strategy for gene modification, the cstⅡ gene from 8 GBS-associated C.jejuni strains were compared with that from 3 GBS-unrelated C.jejuni strains, getting the base and amino acid mutations, the changes of secondary structures and finding the region which may be responsible for the pathogenicity of C.jejuni inducing GBS. Methods Three GBS-associated C.jejuni strains isolated from stools of GBS patients in north China were selected and cultured, which has been confirmed as GBS-associated by animal model. After sequencing the genome of them, the nucleotide sequences of cstⅡ gene were got through sequence alignment. The nucleotide sequences and deduced amini acid sequences of 3 GBS-associated cstⅡ genes were compared with that from 3 GBS-unrelated C.jejuni strains through bioinformatics software, getting the base and amino acid mutations, the changes of secondary structures. Other 5 GBS-associated cstⅡ genes were also aligned to know whether the differences we got above makes sense. In this way the genetic differences between two kinds of C.jejuni strains may be found and speculating the gene region related to the pathogenicity of GBS became possible. Results The cstⅡgene of 3 GBS-associated C.jejuni strains were all composed of 876 base pairs. Compared with GBS-unrelated C.jejuni strains, there were 9 consistent mutation sites in cstⅡ gene of LL and QYT stains, leading to 3 consistent amino acid mutation. The amino acid mutation of 114 and 182 sites in LL and QYT stains existed in other 5 GBS-associated C.jejuni strains. The sole amino acid mutation of ZHX strain -169 site, located near the 182 site. The seventh α-helix(165-180 region)of the secondary structure of the amino acid sequence from GBS-associated strains were shorter than that from GBS-unrelated strains, and the shorter regions were opened to form β-sheet or coli, which also existed in other GBS-related strains in this study.75% of the GBS-associated cstⅡ genes were Asn-51, while 25% of the GBS-associated and all of the GBS-unrelated cstⅡ genes were Thr-51.LL strain showed highly identity to other GBS-unrelated strains in this study. Conclusion The 165-180 segment of secondary structures in cstⅡ gene from local 3 GBS-associated C.jejuni strains are probably the responsible region involved in inducing GBS. The senior structure changes in this region may affect the activity of sialyltransferase and the structures of ganglioside epitope, so that the C.jejuni can acquire the pathogenicity of GBS. This finding may give a clue to genetic modified site. The bi-functional cstⅡ of C.jejuni may be related to the pathogenicity of GBS. The cstⅡ of LL strain to some extent represents the characteristics of Asian strains, which may directs strains monitoring.     

Accuracy of the API Campy system,the Vitek 2 Neisseria–Haemophilus card and matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the identification of Campylobacter and related organisms

Biochemical identification of Campylobacter and related organisms is not always specific, and may lead to diagnostic errors. The API Campy, the Vitek 2 system and matrix-assisted desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are commercially available methods that are routinely used for the identification of these microorganisms. In the present study, we used 224 clinical isolates and ten reference strains previously identified by multiple PCR assays, whole cell protein profiling and either DNA–DNA hybridization or sequencing analysis to compare the reliability of these three methods for the identification of Campylobacter and related pathogens. The API Campy accurately identified 94.4% of Campylobacter jejuni ssp. jejuni and 73.8% of Campylobacter coli, but failed to correctly identify 52.3% of other Epsilobacteria. The Vitek 2 Neisseria–Haemophilus card correctly identified most C. jejuni ssp. jejuni (89.6%) and C. coli (87.7%) strains, which account for the majority of campylobacterioses reported in humans, but it failed in the identification of all of the other species. Despite a good identification rate for both C. jejuni ssp. jejuni and C. coli, both methods showed poor sensitivity in the identification of related organisms, and additional tests were frequently needed. In contrast to API Campy and Vitek, MALDI-TOF MS correctly identified 100% of C. coli and C. jejuni strains tested. With an overall sensitivity of 98.3% and a short response time, this technology appears to be a reliable and promising method for the routine identification of Campylobacter and other Epsilobacteria.     

急性感染性多发性神经根炎患儿空肠弯曲菌感染与抗神经节苷脂GM_1抗体关系

目的：急性感染性多发性神经根炎(GBS)病因尚不清楚,目前认为与感染,尤其与空肠弯曲菌(CJ)感染有关,本文研究CJ感染与神经节苷脂(GM1)损伤的关系,探讨GBS的发病机理。方法：采用酶联免疫吸附试验测定31例GBS患儿(经典型急性感染性多发性神经根炎AIDP 23例,急性运动性轴素神经病AMAN 8例)急性期、恢复期血清和急性期脑脊液CJ-IgG抗体及GM1-IgG、GM1-IgM抗体的变化;并与非GBS神经系统疾病患儿(NGBS组)和10例正常儿童(正常组)对比。结果：AMAN急性期、恢复期血清CJ-IgG抗体水平高于NGBS组(P0.05)。GBS组脑脊液GM1-IgM水平高于NGBS组(P<0.05)。CJ-IgG与GM1-IgG、GM1-IgM具有明显的相关性(R=0.722,P=0.05)。结论：空肠弯曲菌感染是GBS发病的重要病因。神经节苷脂GM1的免疫损伤在GBS发病中起重要作用。     

石家庄地区格林-巴利综合征抗空肠弯曲菌抗体研究

目的：本文在严格对照的背景下,研究石家庄地区GBS病人的空弯感染的情况。方法：分别采集了与GBS病人发病同期,相同居住环境的同一村庄及邻村健康人群做背景,对石家庄地区GBS病人的空弯感染的血清学情况做厂研究。共同时采集CSF及血清标本,对比两者抗体水平,以探讨CSF中Cj抗体的可能来源。且对病后不同病程的患者Cj空弯抗体滴度的变化规律进行了研究。结果GBS患者Cj近期感染率达80％左右,GBS病人发病同期,相同居住环境的同一村庄健康人群Cj感染达25．1％,GBS患者Cj抗体滴度自病后2周内逐渐增高,1月后呈下降趋势。患者血脑屏障通透性增高,CSF中检出Cj抗体,且CSF中CjIgG水平与血清抗体水平密切相关（r=O．88,P＜0．01）。结论：石家庄地区GBS病人的发病与Cj感染密切相关。     

200株弯曲菌对30种抗生素敏感性研究

为了解近几年弯曲菌对抗生素的敏感性,我们采用琼脂扩散法进行了200株弯曲菌对30种抗生素的敏感实验,结果 1994－1998氨基糖苷类、硫霉素、多粘菌素B、克拉霉素、交沙霉素、麦迪霉素均无耐药菌。红霉素耐药率为3％;氟喹诺酮类为44．5％。另外,氟喹诺酮类有完全性交叉耐药。红霉素和庆大霉素耐药菌五年来相对稳定,仍可用来治疗弯曲菌肠炎。我们这一地区氟喹诺酮类严重耐药,将来应严格控制使用。     

空肠弯曲菌共同抗原的提取及应用:SPA协同凝集试验快速检测空肠弯曲菌的实验研究

本文报道一株空肠弯曲菌用甘氨酸提取的共同抗原,能和52种空肠弯曲菌分型血清起交叉反应。用这种共同抗原免疫家兔制备的抗血清致敏SPA后,协同凝集法检测了23株不同血清型的空肠弯曲菌标准株,结果均阳性,而对18株其它肠道菌检测结果均阴性。协同凝集试验与培养法平行检测57份鸡标本,两者均为阳性31份,均阴性14份,总符合率78.9％。无显著性差异。本法具有特异、敏感、快速、简单的特点,便于推广。对临床快速诊断空肠弯曲菌肠炎具有一定价值。     

用Lior玻片凝集法作空肠弯曲菌血清学分型与流行病学分析

本文用Lior氏玻片凝集法,对从福建省人与动物中检出的379株空肠弯曲菌进行血清学分型,有212株被分成18个型14个亚型。其中人源菌分布14型,以29、21、8型较多见,占可分型菌的62.5％,鸡菌分布15型,以2、21、44、11和29型较为多见,占65.7％,鸭菌分布9个型,猪菌分布10型,以29、20、2和9型较多见,占66.7％。鸡、鸭、猪检出菌与人源菌同血清型菌株数分别为91.7％、83.3％和95.8％。因而这些禽、畜属于人类空肠弯曲菌的主要传染来源。     

广州地区儿童空肠弯曲菌感染的病原学研究

目的：了解广州地区腹泻儿童空肠弯曲菌（CJ）的感染及耐药情况,为临床及时、合理诊断和治疗提供实验室依据。方法：对2005年7月至2008年6月该院门诊3 351份腹泻患儿的粪便标本进行CJ的分离培养,并对分离菌株进行Lior分型及耐药试验。结果：3 351份腹泻患儿粪便标本共检出267株CJ,分离率为8.0%;婴幼儿（1个月至1岁）是CJ的易感人群,占91.0%（243/267）;夏秋季是高发季节,占76.8%（205/267）。该菌对亚胺培南、阿米卡星、头孢哌酮/舒巴坦和氯霉素敏感,对大环内酯类和林可霉素类耐药率<5%,对氨苄西林、喹诺酮类和四环素耐药率在20%~40%之间,对复方新诺明和头孢哌酮均耐药。Lior分型以Ⅰ型为主,占 75.3%（201/267）。结论：CJ是婴幼儿腹泻的重要病原菌,其耐药形势日趋严重,应引起重视。临床应结合药敏试验结果选择抗菌药物治疗CJ感染。［中国当代儿科杂志,2009,11（6）：422-424］