人胃螺旋菌的分离培养研究

我们首次从一慢性活动性胃炎病人胄粘膜活检组织中成功地分离到一株人胃螺旋菌（Ｇａｓｔｒｏｓｐｉｒｉｌｌｕｍｈｕｍｉｎｉ５,ＧＨ）,并观察到此菌与幽门螺杆菌（Ｈｅｌｉｃｏｂａｃｔｅｒｐｙｌｏｒｉ,Ｈｐ）混合感染存在。ＧＨ直径０．３μｍ（在扫描电镜下测量）,氧化酶阳性,过氧化氢酶阳性,并有很强的尿素酶活性。此菌为微需氧菌,在含５％Ｏ２、１０％ＣＯ２和８５％Ｎ２的混合气体环境中生长良好。在含６％、１０％脱纤维绵羊血布氏琼脂培养基及含１０％小牛血清布氏肉汤中表现为不同菌体形态,可呈球状、肾形及１～１４个较密螺旋状,菌体长度可达１７Ｆｔｍ。在一７０℃保存３０天后发生球形变,但在连续传代培养后可恢复典型形态。ＧＨ的分离培养成功为进一步研究其生物学特性及致病性打下了基础。     

猪胃螺杆样细菌与幽门螺杆菌的比较研究

我们检测１０例普通猪的胄组织,有８例分离到螺杆菌样细菌（ＨＬＯ）。其菌落、菌体形态和某些生化反应与幽门螺杆菌（ＨＰ）相似,但其尿素酶活性较低,ＨＬＯ全菌蛋白的ＳＤＳ一ｐＡＧＥ图谱也与ＨＰ的不同。本文就ＨＰ和ＨＬＯ及其伴发的人、猪慢性胃炎的特点,作了比较和讨论。     

幽门螺旋菌保存方法的初步研究

幽门螺旋菌(HP)过去称它为幽门弯曲菌(CP)是近年来从人体胃组织中发现的一种细菌,自1982年首次培养成功以来,已做了大量的研究工作,它与胃炎和消化性溃疡的发生有密切关系.幽门螺旋菌营养要求和培养条件都比较高,而且生物学特性,经多次传代比较容易产生变异,特别是菌种保存方面还存在不少问题,该菌的长期贮存很困难,而长期贮存对该菌的药敏,生物学特性的研究,动物模型的建立都是非常重要的。为了开展对幽门螺旋菌与慢性胃炎和消化性溃疡关系的基础研究,我们对临床胃及十二指肠患者粘膜分离出的幽门螺旋菌进行传代培养,在10％兔全血的布氏肉汤琼脂培养基上,在混合气体(10％CO_2,5％O_2,85％N_2条件下),4℃冰箱中保存于不同时间检查了存活情况,生物学特性的比较。实验表明,用这种方法保存细菌存活率,菌体形态生物学特性保存较好,取得了较满意的结果.     

ABO血型与幽门螺旋菌感染关系及其意义

本文采用细菌学和血清学方法检测了２１７例胃、十二指肠疾病患者和１６９例健康输血员幽门螺旋菌（ＨｅｌｉｃｏｂａｃｔｅｒｐｙｌｏｒｉＨＰ）感染情况和ＡＢＯ血型,以探讨ＨＰ感染与ＡＢＯ血型系统之间的关系。结果表明：消化性溃疡各组患者Ｏ型血所占的比例均较高;胃十二指肠疾病患者和健康输血员中Ｏ型血人群ＨＰ阳性率分别为８９．５８％和５１．７８％,显著高于其它血型（Ｐ＜０．００５、０．０５）;并且在同一疾病中ＨＰ阳性者Ｏ型血所占的比例显著高于ＨＰ阴性者（Ｐ＜０．０２５）。提示：Ｏ型血人群对ＨＰ易感染,这可     

动物胃内螺旋样细菌

本文对动物胃内螺旋样细菌和螺杆菌属细菌的研究历史、自然宿主、分类及致病性进行了较细致的探讨,重点讨论了这类细菌在人类Ｈｐ防治研究中的应用价值。     

原位杂交及原位PCR检测幽门螺杆菌

本研究建立了检测幽门螺杆菌的原位杂交和原位ＰＣＲ技术,采用ＰＣＲ掺入的方法标记原位杂交的生物素探针,６份ＨＰ阳性胃活检组织冰冻切片杂交均为阳性,６份阴性对照组织为阴性。９／１２份ＨＰ阳性对照组织石蜡切片杂交阳性,２份阴性对照切片为阴性。３份阳性对照猫胃粘膜冰冻切片杂交也是阳性。原位ＰＣＲ的引物来自ＨＰ的１６ｓｒＲＮＡ基因,在扩增过程掺入生物素基因。４／４例ＨＰ阳性人胃粘膜冰冻切片原位扩增阳性,２／     

狗胃内幽门螺杆菌相关菌的调查研究

本实验对２５只成年狗和１２只幼犬,通过胃镜下活检或处死后立即取胃及十二指肠球部的六个部位的粘膜标本作尿素酶试验、涂片革兰氏染色、细菌培养和组织学检查。结果显示：（１）全部成年狗均有尿素酶试验阳性、革兰氏染色阴性的人胃螺旋菌相似菌定植,而无Ｈｐ相似菌定植。（２）该螺旋菌定植于胃小凹、腺管、和腺腔的底部,且在壁细胞内常可见到螺旋菌,少数出现壁细胞破坏。（３）粘膜有弥漫性慢性胃炎改变,胃底腺壁细胞数量减少。（４）经隔离的幼犬,在生后第６０天胃粘膜均见螺旋菌定植,以胃窦多见。结果表明：（１）狗胃中的螺旋菌具有一定致病性,可破坏壁细胞。（２）狗可能是人胃螺旋菌的传染源,而不是Ｈｐ的传染源。（３）不宜选用成年狗或较大的幼犬建立Ｈｐ感染普通狗模型,所选幼犬也需隔离。     

猫胃螺杆菌小鼠感染模型在幽门螺杆菌研究中的应用

本研究应用幽门螺杆菌的近缘菌猫胃螺杆菌感染的小鼠模型,验证了一个三联治疗方案（羟氨苄青霉素＋次枸橼酸铋＋灭滴灵）的抗菌作用,发现其对Ｈｆ的清除率为９０％,与临床治疗Ｈｐ时的效果相似,说明该模型可用于活体筛检抗Ｈｐ药物,同时探讨了Ｈｆ与腺癌诱癌剂ＭＮＮＧ的协同致癌作用,发现经四个月共同作用后,单纯ＭＮＮＧ组与Ｈｆ感染在胃癌前病变发生率及反应细胞增殖的核仁形成区蛋白嗜银颗粒计数（Ａｇ－ＮＯＲ）、增殖细胞核抗原（ＰＣＮＡ）标记指数等指标均无显著差异,揭示螺杆菌属细菌感染的促癌作用可能是缓慢持久的。     

幽门螺杆菌感染的动物模型及实际应用

幽门螺杆菌已被公认为慢性胃炎、消化性溃疡、胃癌等胃癌的重要病因，但其 致病与免疫机制等多问题的深入解决尚有 待于动物模型的发展。本文好在小猪、犬、猴、裸鼠胃中建立的ＨＰＢ感染模型，或用ＨＰ近似菌如Ｈｅｌｉｃｏｂａｃｔｅｒｆｅｌｉｓ（ＨＦ）或Ｈｅｌｉｃｏｂａｃｔｅｒｍｕｓｔｅｌａｓ（ＨＭ）在小鼠、雪貂胃中建立的拟ＨＰ感染模型，并介绍了它们在观察抗ＨＰ疗法疗效、ＨＰ及其近似菌致病及免疫机制研究中的应用     

鼬鼠螺旋杆菌

1986年,美国的Fox等首先报道在雪貂(ferret)的胃内分离出一种形态和生化特性非常相似于幽门螺杆菌(HP)的细菌,称为胃弯曲菌样微生物(gastric campylobacter-like organism,GCLO),1988年,Fox建议将该菌称为幽门弯曲菌鼬鼠亚种(Campylobacter pylori subsp mustelae),其标准株为ATCC43772。1989年,Goodwin提出一个新的属—螺旋杆菌属,该菌又易名为鼬鼠     

Occurrence of Helicobacter Infection in the gastric mucosa of free-living red foxes (Vulpes vulpes)

We studied gastric Helicobacter spp. in five red foxes (Vulpes vulpes). Samples of stomach from the cardia, corpus, pyloric antrum, and duodenum were subjected to histopathologic, immunohistochemical, and transmission electron microscopy (TEM) examination for the presence of Helicobacter and gastritis. All foxes had gastric Helicobacter-like organisms (GHLOs) on examination by light microscopy and TEM. Gastric Helicobacter-like organisms were present in all areas of the stomachs. Chronic mild or moderate gastric inflammation was associated with infection by GHLOs in one or more regions of the stomach, but there was no correlation between inflammation and infection. It is not clear whether the organisms were causing the minimal histologic lesions observed, but the gastric mucosa of free-living foxes appears to be commonly colonized with GHLOs. The frequent colonization of free-living foxes with distinct GHLOs possibly reflects their special characteristic in feeding and/or social behavior or the potential commensal nature of the bacteria in free-ranging foxes.     

Use of polymerase chain reaction and enzymatic cleavage in the identification of Helicobacter spp. in gastric mucosa of human beings from North Paraná, Brazil

Helicobacter pylori is the most common gastric bacteria of human beings. Animal-borne helicobacter have been associated with gastritis, ulceration, and gastric mucosa-associated lymphoid-tissue lymphoma in people. We attempted to identify the species of Helicobacter spp. that infect human beings in north Paran , Brazil. Samples of gastric mucosa from 38 dyspeptic patients were analyzed by optic microscopy on silver stained slides, polimerase chain reaction (PCR), and enzymatic cleavage. Genus and species-specific primers to H. pylori, H. heilmannii, H. felis, and consensual primers to H. bizzozeronii or H. salomonis were used. The PCR products were submitted to enzymatic cleavage by VspI (Helicobacter spp. product) and HinfI (species products) enzymes. Thirty-two out of 38 patients evaluated had 3.2 to 5 m long bacteria that resembled H. pylori in Warthin-Starry stained slides and were positive to the genus Helicobacter by PCR. In 30 of these patients the bacteria were identified as H. pylori. Two samples positive by silver stain were negative to all species tested by PCR. None of the 38 samples was positive to animal-origin helicobacter species. These results show that PCR and enzymatic restriction are practical methods to identify the species of helicobacters present in gastric mucosa of human beings. People in north Paran appear to be infected mostly with H. pylori.     

Analysis of host responses of guinea pigs during Helicobacter pylori infection

Host responses of guinea pigs infected with Helicobacter pylori were investigated. Passaged H. pylori colonised the stomach for up to 13 weeks after infection, but after 1 month the number of bacteria fell sharply. Specific antibodies, predominantly of the IgG2 subtype, were present from week 3 onwards. Antibodies to urease A and flagella were abundant. Severe inflammation of the gastric mucosa and damage to the stomach epithelium was seen. Infiltrates of mononuclear cells and eosinophils were found near the parietal glands. As infection progressed, inflammation and tissue damage became more localised and more variable between individual animals. These parameters can be used as markers for colonisation of the stomach by H. pylori.     

Use of HP selective medium to detect Helicobacter pylori associated with other enteric bacteria in seawater and marine molluscs

AIMS: This project investigated the utility of HP selective medium to isolate H. pylori cells from seawater and from marine molluscs. METHODS AND RESULTS: Nested-PCR was performed to reveal the presence of Helicobacter genus. All samples were cultured in HP selective medium and 16 cultures were initially selected as putative Helicobacter. Helicobacter spp. DNA were detected in 9/16 cultures and three of them had 99-100% homology to H. pylori based on 16S RNA gene sequence. Helicobacter pylori isolation was unsuccessful. On the basis of 16S RNA gene sequences the contaminating organisms were shown to be Proteus mirabilis and Vibrio cholerae. CONCLUSIONS: These results indicate the coexistence of three predominant bacterial genera in the cultures and that HP selective medium can grow other enteric bacteria besides Helicobacter. Additional assays will improve the HP selective medium formulation for marine samples avoiding P. mirabilis and V. cholerae interferents. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows the effectiveness of the selective HP medium for the Helicobacter culture from marine samples.     

Role of persisting opportunistic bacteria in the pathogenesis of the gastric and duodenum ulcer

Dynamic study of the bacterial microflora of 122 patients with gastric and duodenal ulcer was carried out. Microflora examination of the bioptic samples of mucosa, obtained from the ulcerous zone of the patients, revealed that an open ulcer is like an infected wound needing sanitation. In the focus of lesion microorganisms of 32 genera and species, including Helicobacter pylori, fungi of the genus Candida, representatives of the genera Streptococcus, Staphylococcus, Corynebacterium, Bacteroides, etc., were detected. Opportunistic microorganisms were isolated in associations (up to 8 different cultures), possessing cytotoxic, hemolytic, antilysozyme, lecithinase, caseinolytic and RNAase activities. To inhibit the microflora, chitosan was used; 82-85% of the cultures of different bacteria under study proved to be sensitive to it. The inclusion of chitosan into the complex therapy suppressed the persistence of H. pylori, ensured the sanitation of mucosa affected by opportunistic bacteria and accelerated ulcers cicatrization.     

Helicobacter nemestrinae sp. nov., a spiral bacterium found in the stomach of a pigtailed macaque (Macaca nemestrina)

A new microaerophilic, spirally curved, rod-shaped bacterium was isolated from the gastric mucosa of a pigtailed macaque (Macaca nemestrina). The gram-negative cells of this bacterium are oxidase, catalase, and urease positive and strongly resemble Helicobacter pylori (Campylobacter pylori) cells. Like H. pylori, this organism does not metabolize glucose, does not reduce nitrate or produce indole, does not produce H2S from triple sugar iron agar, does not hydrolyze hippurate or esculin, and does not grow in the presence of 1% glycine, 1.5% salt, or 1% bile. Also like H. pylori, it is resistant to nalidixic acid and susceptible to cephalothin. However, unlike H. pylori, the colorless colonies are flat and have irregular edges. This organism has a unique cellular fatty acid composition, forming a new gas-liquid chromatography group, group K, and a distinctive DNA content (24 mol% guanine plus cytosine). It exhibits less than 10% DNA-DNA homology (as determined by the nylon filter blot method at 65 degrees C) with other members of the genus Helicobacter. Although the levels of DNA relatedness between previously described Helicobacter species and the new organism are low (less than 10%) and the difference in guanine-plus-cytosine content is large (24 versus 36 to 41 mol%), the genus Helicobacter is the only genus in which it is logical to include the organism at this time. We propose that our single strain represents a new species, Helicobacter nemestrinae, and we designate strain T81213-NTB (= ATCC 49396) as the type strain.     

Invasiveness of Helicobacter pylori into Human Gastric Mucosa

Background. Helicobacter pylori has generally been observed only in the gastric mucous layer or in the spaces between gastric mucus -s ecreting cells and not in the gastric epithelial cells or in the lamina propria. The purpose of this study is to determine whether H. pylori invades the gastric mucosa, using an immunoelectron microscopical examination of human gastric mucosa infected with H. pylori.
Materials and Methods. Five hundred gastric antral biopsy specimens were fixed in a periodate-lysin-paraformaldehyde solution, embedded in Lowicryl, sectioned, and examined with a light microscope. One hundred specimens moderately or severely infected with H. pylori were selected and were incubated with polyclonal rabbit anti– H. pylori antibody. The specimens were washed, incubated with 20 nm of colloidal gold–conjugated goat anti–rabbit IgG, stained with uranyl acetate and lead citrate, and observed with a transmission electron microscope.
Results. In one case, a bacterium was observed within the cytoplasm of a gastric mucus -s ecreting cell; in another case, a few bacteria were observed within the cytoplasm of a stromal cell in the lamina propria. The bacteria could be differentiated from degenerated intracellular organelles by gold particles attached to the bacteria.
Conclusion. H. pylori rarely invade the lamina propria and gastric cells.