气单胞菌的表型特性与毒素原性研究

１９９０－１９９４年，自山东省９市（地）从临床和外环境等６种不同性质标本中检出２６０株气单菌，对其表型特性进行了研究。结果表明：山东省以温和气单胞菌为主，嗜水气单胞菌、豚鼠气单胞菌次之。自淡水鱼中检出维隆气单胞菌和易损气单胞菌各１株。随机抽取１６３株应用溶血试验、ＣＨＯ细胞测毒试验、兔肠结扎及ＣＴ基因探针杂交试验进行毒素原性研究。结果显示：温和、嗜水及豚鼠气单胞菌均可产生溶血素、肠毒素、某些菌株的     

基于rpoB基因分析方法鉴定艾伯特埃希菌

目的探讨一种合理的方法对既往分离到的鉴定为致病性大肠杆菌(EPEC)的菌株进行重新鉴定,发现可能存在的艾伯特埃希菌。方法以2013—2017年上海市浦东地区腹泻监测中分离到的491株EPEC为分析对象,首先用基于表型的方法进行筛检,然后用聚合酶链式反应(PCR)检测毒力基因,并对可疑株rpoB基因进行扩增和测序,获得的序列在genbank数据库中进行匹配,以艾伯特埃希菌参考株以及其他密切相关的大肠埃希菌和志贺菌的rpoB基因序列作为参考序列,使用MEGA8.0的最邻接法构建进化树。结果经筛选,33株生化表型符合艾伯特埃希菌株中,10株clpX、lysP、mdh以及ctdB基因阳性,符合艾伯特埃希菌定义,其rpoB基因序列与艾伯特埃希菌参考菌株的序列高度相似,并与其聚类为一簇,与其他23株菌株以及志贺菌、大肠埃希菌等参考菌株有显著区别。结论表型鉴定方法结合rpoB基因测序分析可用于艾伯特埃希菌的准确鉴定。     

黏液玫瑰单胞菌16S rRNA基因序列及耐药性分析

目的对常规方法不易鉴定的一株临床细菌进行16S rRNA基因序列分析,并探讨该细菌的耐药性。方法菌株分离培养后进行常规的鉴定及药物敏感性分析,同时选择细菌的16S rRNA基因为靶序列,进行PCR扩增并测序,测序结果与GenBank比对。结果 API 20NE板条(法国生物梅里埃公司)将该菌鉴定为嗜中温甲基杆菌;细菌的16S rRNA基因序列与黏液玫瑰单胞菌相似性为99.5%;细菌的药敏试验显示对三代头孢耐药性高。结论黏液玫瑰单胞菌引起的感染比较罕见,菌株对三代头孢的高耐药应引起临床重视;16S rRNA基因序列分析方法可以较好地鉴定常规方法难以鉴定的不典型及罕见菌株。     

从田螺中分离出四种气单胞菌

本文报告了从28份田螺肠内容物中,分离出13株气单胞菌,分离率为46.4％,同时选用10项关键性生化试验,结合血清疑集试验,将13株阳性菌鉴定为4个种,分别是混和气单胞(8株),嗜水气单胞菌(2株)豚鼠气单胞菌(2株)和达气单胞菌(1株)。     

甲鱼,河蟹致病性气单胞菌的分离鉴定及其卫生评价研究

从江苏扬州5个人工养殖甲鱼场10只濒死甲鱼肝脏中分离到10株具有β溶血的气单胞菌,其中4株为温和气单胞菌、5株为嗜水气单胞菌、1株为易损气单胞菌,从5个人工养殖河蟹场10只频死河蟹肌肉中分离到10株具有β溶血的气单胞菌,其中5林为温和气单胞菌,4株为嗜水气单胞菌,1株为豚鼠气单胞菌。检菌株生长特性试验表明,4～37℃均能生长,最适生长温度为25℃～30℃。加热试验表明,65℃100min、90℃min能将细菌灭活。调味品杀菌试验表明,50％食醋5min能将细菌灭活,而33％加饭酒24h内对细菌无作用,提示应重视醉制生食水产品的卫生。     

1126例腹泻病患者气单胞菌的检测及分析

本文报道１１２６例腹泻病患者气单胞菌的检测结果。从１１２６份粪便标本中检出４种７８株气单胞菌,其中豚鼠气单胞菌４７株（６０．２６％）,温和气单胞菌２１株（２６．９２％）,嗜水气单胞菌８株（１０．２６％）,易损气单胞菌２株（２．５６％）,检出率为６．９３％。嗜水、温和及豚鼠气单胞菌均能产生β溶血,以温和气单胞菌溶血阳性率最高（８０．９５％）,２株易损气单胞菌不溶血。药敏试验结果：菌株对红霉素、痢特灵敏感性差,对先锋、四环素、氨苄青霉素的耐药率达３３．３３％～８６．６６％,对庆大霉毒、氟哌酸等敏感。     

自贡市44株临床非结核分枝杆菌的鉴定

目的对自贡市2012—2016年临床分离44株疑似非结核分枝杆菌进行菌种鉴定。方法使用PCR-荧光探针法对自贡市临床44份疑似非结核患者的菌株进行初步筛查,并对44份菌株的hsp65及rpoB基因进行扩增并测序,通过序列比对获得菌株型别。结果 44株菌株分枝杆菌经PCR-荧光探针法检测结果显示均为非结核分枝杆菌.对44株分枝杆菌进行hsp65及rpoB基因测序比对,进一步菌种鉴定结果显示,44株分枝杆菌分别为11种非结核分枝杆菌。结论自贡市非结核分枝杆菌种类较多,44株菌即分为11个种。PCR-荧光探针法及基因测序法可对临床非结核分枝杆菌进行快速、准确的鉴定。     

河北省3株携带blaNDM-1基因阴沟肠杆菌基因及耐药性检测

目的了解河北省3株携带bla_NDM-1基因阴沟肠杆菌的基因序列及其耐药性。方法通过生化鉴定、基因测序和药敏试验对河北省保定市和承德市肝癌晚期、输尿管结石和慢性肾衰竭3例住院患者分离的3株菌株进行基因序列及其耐药性检测。结果生化鉴定结果表明,3株菌株为携带bla_NDM-1基因的阴沟肠杆菌;bla_NDM-1基因测序结果与国外基因序列比对,同源性为100%;药敏试验结果显示,3株菌株均对氨基糖苷类抗生素敏感,其中12280010P1、12280010P2两菌株对氟喹诺酮类抗生素敏感。结论河北省存在携带bla_NDM-1基因的阴沟肠杆菌,耐药问题形势严峻,需要加强监测力度。     

游泳池水中嗜水气单胞菌的毒力因子的研究

目的:对游泳池水检测出的19株嗜水气单胞菌进行毒力因子的研究.方法:通过蛋白酶试验检测嗜水气单胞菌的蛋白酶,采用血平板法观察溶血素,采用PCR方法检测hlyA和aerA两种毒素基因,并进行小白鼠毒力试验.结果:19株嗜水气单胞菌菌株含hlyA基因的占78.95%(15/19),含aerA基因占73.68%(14/19),18株嗜水气单胞菌菌株产生蛋白酶,溶血试验显示的结果与hlyA基因检测的结果相符.菌株对小白鼠的平均致死率达85.96%(49/57).结论:游泳池水中分离出的嗜水气单胞菌大多携带毒力基因.采用多种方法检测其毒力因子能客观地对其毒力进行判定.     

幽门螺杆菌分离株iceA基因克隆及序列分析

目的 克隆幽门螺杆菌中国郑州市慢性萎缩性胃炎患者分离株MEL-Hp27iceA基因,测序后对其进行序列分析。方法 根据iceA基因上下游基因序列设计引物,PCR扩增iceA基因,并将其连接至pMD19-T载体,测序后采用相关数据库和生物信息学软件对基因序列进行分析。结果 成功克隆幽门螺杆菌Hp27菌株iceA基因序列,扩增产物大小约为790 bp;重组质粒pMD19-T-iceA单酶切产生3 820 bp片段,双酶切产生3 000和820 bp的2个片段;Hp27iceA基因与大多数美国来源菌株同源性>85%,与日本、印度等地区来源菌株同源性<70%。序列分析结果表明,所克隆到的基因为iceA2亚型,与美国株Alaska strain 219、Alaska strain 213关系最近,同源性分别为87%和95%,与芬兰株Finland strain 9496等菌株存在聚类关系,同源性88%～93%,与CR9等其他菌株距离较远。结论 成功克隆幽门螺杆菌Hp27菌株iceA基因序列;不同地区幽门螺杆菌分离株的iceA基因间存在着一定差异。     

Phylogenetic diversity of Aeromonas from "alheira," a traditional portuguese meat product

Abstract "Alheira" is a traditional smoked meat sausage produced in the north of Portugal, representing an important economic resource for the region. This meat product has been subjected to research studies with the aim of detecting the presence of common foodborne pathogens, but, to our knowledge, isolation of emerging foodborne Aeromonas from alheira has never been previously described. Present work attempts to evaluate the Aeromonas species diversity of 84 isolates of Aeromonas spp. collected from 32 alheira samples. All presumptive Aeromonas isolates were subjected to genotyping by enterobacterial repetitive intergenic consensus-polymerase chain reaction analysis. The isolates presenting a different pattern were subjected to gyrB gene sequencing for species classification, and the species A. hydrophila, A. salmonicida, A. caviae, A. media, and A. allosaccharophila were identified. The Aeromonas species diversity found has not been previously described in any other meat product evaluated in previous studies. It is also important to highlight the presence of A. hydrophila and A. caviae because they were previously associated with illness in humans, including gastroenteritis.     

Distribution of Aeromonas phenospecies and genospecies among strains isolated from water, foods or from human clinical samples.

A total of 332 Aeromonas spp. originating from drinking water (n = 75), fresh water (n = 57), chicken and ground beef (107), human faecal samples in association with travelling (n = 49), human faecal samples not associated with travelling (n = 38), and six strains from human blood cultures were studied by phenotypic methods and by using analysis of ribopatterns as a molecular method for the identification of the 13 known hybridization groups (HGs). Also included were the reference strains of each HG. A. hydrophila HG 1, A. caviae HG 4 and A. veronii biotype sobria HG 8/10 were the most important genospecies identified in human faecal samples. A. hydrophila HG 2 and A. media HG 5B predominated in drinking water and A. hydrophila HG 2 and HG 3, A. media HG 5A and HG 5B predominated in fresh water. In drinking water only one isolate was A. hydrophila HG 1 and two isolates were A. caviae HG 4. Clinically important Aeromonas spp. HG 1 (A. hydrophila), HG 4 (A. caviae) and HG 8/10 (A. veronii biotype sobria) were common in chicken and ground beef. In contrast to the drinking water samples, HG 5A was common in chicken and ground beef samples. Atypical, unidentified isolates were most often found in fresh water samples (12/57 strains). Although water has been suspected of being an important source of human aeromonas infections, clinically important HGs were found to be in the minority among Aeromonas spp. identified in drinking water or fresh water. The distribution of Aeromonas spp. HGs among drinking water, chicken and ground beef samples was also different, suggesting that contamination of meat or chicken may not originate from water.     

产毒串珠镰刀菌rDNA及其间区序列的比较研究

目的研究中国产毒串珠镰刀菌与水稻枯萎病赤霉群的系统发育关系。方法对4株ATCC典型菌株和12株从中国不同地域、不同样品中分离的串珠镰刀菌进行了rDNA及其间区序列ITS1和ITS2的测定分析,绘制了系统发育进化树及同源树,并在DNA数据库中注册。通过与美国、南非等地区的菌株进行序列分析比较,阐述不同国家、地区产毒串珠镰刀菌的分布特点及rDNA间区序列与产毒基因的相关性。结果中国伏马菌素产毒基因阳性株的rDNA间区ITS2可变区为I型,与南非分离的串珠镰刀菌伏马菌素产毒株结果一致。不产伏马菌素的串珠镰刀菌分离株,ITS2可变区为II型。2株胶孢镰刀菌SD197-fmv047和SD207-fmv049为非伏马菌素产毒株,ITS2可变区即非I型,也非II型,且其序列与尖孢镰刀菌高度同源。结论结果对进一步研究水稻枯萎病赤霉群的系统发育关系提供新的、可靠的理论依据和技术支撑。     

Prevalence, pathogenesis, antibiotic susceptibility profiles, and in-vitro activity of selected medicinal plants against Aeromonas isolates from stool samples of patients in the Venda region of South Africa

The prevalence, pathogenic indices, such as haemolytic and haemagglutinating activities, antibiograms, and in-vitro activities of local medicinal plants against Aeromonas isolates in Vhembe district of Limpopo province, South Africa, were studied using standard microbiological methods. In total, 309 diarrhoeic stool samples were collected from patients attending five health centres in the region during December 2004-May 2005. Aeromonas species were identified using the API 20E system. The haemagglutinating and haemolytic activities of isolates on human, sheep, pig and chicken red blood cells were investigated. Antibiotic susceptibility profiles of the isolates to several antibiotics and in-vitro activity of local medicinal plants were also ascertained using previously-reported schemes. Results showed that 104 (33.6%) of the 309 samples were positive for Aeromonas species, of which 89 (85.6%) were Aeromonas hydrophila, 12 (11.5%) A. sobria, and three (2.9%) A. caviae. All strains of A. hydrophila and A. caviae produced haemolysis on sheep blood, while eight of the 12 A. sobria strains were haemolytic on sheep blood. The haemolytic activities of the isolates were variable on other red blood cells tested. High level of resistance was observed to amoxicillin and ampicillin, followed by cefuroxime (79%), chloramphenicol (74%), and erythromycin (65%). The carbapenems were the most active drugs with only 7% resistance to meropenem and 11% to imipenem. About 12% of the isolates were resistant to ciprofloxacin. The extracts of three of seven medicinal plants tested showed inhibitory activity against all Aeromonas isolates; these included acetone and hexane extracts of Pterocarpus angolensis, Syzygium cordatum, and Zornia milneana. The results suggest a high prevalence of Aeromonas species in the region. The isolates demonstrated multiple resistant profiles to different antibiotics tested. Some local medicinal plants were inhibitory to Aeromonas isolates, indicating a potential role in the management of Aeromonas-related infections. Structural elucidation of the active components may pave the way for the discovery of candidate templates for eventual drug design. Most isolates possessed important virulence characteristics based on their haemolytic and haemagglutinating ability. However, the genetic characterization of the isolates will further confirm their pathogenicity and the origin of multiple antibiotic resistance.     

肠杆菌及临近种属的多位点序列分析

目的 为建立适宜的分子分型方法,以弥补16S rDNA在肠杆菌种属聚类分析上的不足,为后续类似菌株鉴定提供参考。方法 以肠杆菌属内各个种的模式菌株为研究对象,选择16S rDNA及rpoB、infB、gyrB和atpD基因序列,采用CLUSTALX软件和BioEdit软件进行序列比对和拼接;16S rDNA的相似性通过EzBioCloud数据库在线比对,拼接后的序列经MegAlign软件分析相似性;采用MEGA 7.0软件对序列多态性及系统发育关系进行分析。结果 肠杆菌属部分种间的16S rDNA序列高度相似(>99%),且与Lelliottia、Klebsiella及Kosakonia等属的部分种也有98%以上的相似性,其系统发育树的聚类分析显示属内成员并没有完全聚于一簇;16S rDNA序列的变异位点占比为6.44%(74/1149),rpoB、infB、gyrB和atpD基因串联后的变异位点占比为14.44%(380/2630)。结论 在16S rDNA高度同源的部分种属间,多位点序列分析较清晰地将肠杆菌属与其临近种属区分开,更能真实、准确地反映其生物分类学地位。     

Adherence to HEp-2 cells and enteropathogenic potential of Aeromonas spp.

Aeromonas strains (total = 60) of clinical, water and food origin were tested for adherence to HEp-2 cells. Environmental strains were selected (except for A. caviae) to include primarily those expressing other virulence-associated properties. Adhesion was markedly species-dependent (A. veronii biotype sobria, 15 of 26 [58%]. A caviae, 4 of 12 [33%] and A. hydrophila, 2 of 8 [11%]). A. veronii biotype sobria were adhesive, irrespective of source (62 and 54% for clinical and environmental strains, respectively). Adherent strains of this species were enterotoxin-positive and most (13 of 15) grew at 43 degrees C. A. caviae isolated from clinical specimens contained a higher proportion (75%) of adherent strains than environmental strains (13%). Virulent subsets of A. veronii biotype sobria and A. caviae are adherent to HEp-2 cells. The HEp-2 assay is a useful model for investigating mechanisms of adherence and enteropathogenicity of virulent Aeromonas species.     

Detection of Aeromonas caviae in the common housefly Musca domestica by culture and polymerase chain reaction.

Aeromonas caviae has been implicated in diarrhoeal disease of livestock and humans. The potential role of houseflies in the epidemiology of this pathogen was investigated by examining the prevalence of A. caviae in houseflies collected from two South Carolina farms and one restaurant. Isolation was accomplished by culture of flies in alkaline peptone water followed by identification with Aeromonas-specific PCR using novel primers (APW-PCR). All isolates cultured from houseflies were identified as A. caviae by biochemical characteristics and direct sequencing approximately 800 bp of the 16S rRNA gene. Aeromonas caviae was detected in 78% (272/349) dairy farm flies, 55% (54/99) pig farm flies and 39% (77/200) restaurant flies. Faeces from cows and pigs at the farms also were positive for A. caviae (58% and 100%, respectively). The APW PCR method provided a rapid, convenient way to identify A. caviae from faeces and houseflies that contained hundreds of bacterial species.     

浦东新区腹泻样本中气单胞菌的分离鉴定及耐药性分析

目的:了解上海浦东新区感染性腹泻病人气单胞菌的感染及耐药情况,为相关防控策略的制定提供科学依据。方法:对2010年上海市浦东新区2个监测点,1245件急性腹泻样本进行气单胞菌检测,阳性菌进行药物敏感试验。结果:从1245件标本中检出气单胞菌93株,阳性率为7.47%,其中嗜水气单胞63株,温和气单胞26株,豚鼠气单胞4株。6月-8月份为高峰期。药敏试验结果:80%以上菌株对庆大,环丙沙星,阿米卡星、头孢噻肟敏感,对氨苄西林,复方新诺明、四环素、头孢噻吩、萘啶酸不同程度耐药。结论:气单胞菌感染存在季节性差异,与夏秋季腹泻关系密切。持续对气单胞菌及其耐药性的监测对细菌性腹泻病的预防与治疗具有指导意义。     

Aeromonas detection and their toxins from drinking water from reservoirs and drinking fountains

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. A total of 200 drinking water samples from domestic and public reservoirs and drinking fountains located in São Paulo (Brazil), were analyzed for the presence of Aeromonas. Samples were concentrated by membrane filtration and enriched in APW. ADA medium was used for Aeromonas isolation and colonies were confirmed by biochemical characterization. Strains isolated were tested for hemolysin and toxin production. Aeromonas was detected in 12 samples (6.0%). Aeromonas strains (96) were isolated and identified as: A. caviae (41.7%), A. hydrophila (15.7%), A.allosacharophila (10.4%), A. schubertii (1.0%) and Aeromonas spp. (31.2%).The results revealed that 70% of A. caviae, 66.7% of A. hydrophila, 80% of A. allosacharophila and 46.6% of Aeromonas spp. were hemolytic. The assay for checking production of toxins showed that 17.5% of A. caviae, 73.3% of A. hydrophila, 60% of A. allosacharophila, 100% of A. schubertii, and 33.3% of Aeromonas spp. were able to produce toxins. The results demonstrated the pathogenic potential of Aeromonas, indicating that the presence of this emerging pathogen in water systems is a public health concern.     

Aeromonas detection and their toxins from drinking water from reservoirs and drinking fountains

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. A total of 200 drinking water samples from domestic and public reservoirs and drinking fountains located in S?o Paulo (Brazil), were analyzed for the presence of Aeromonas. Samples were concentrated by membrane filtration and enriched in APW. ADA medium was used for Aeromonas isolation and colonies were confirmed by biochemical characterization. Strains isolated were tested for hemolysin and toxin production. Aeromonas was detected in 12 samples (6.0%). Aeromonas strains (96) were isolated and identified as: A. caviae (41.7%), A. hydrophila (15.7%), A.allosacharophila (10.4%), A. schubertii (1.0%) and Aeromonas spp. (31.2%). The results revealed that 70% of A. caviae, 66.7% of A. hydrophila, 80% of A. allosacharophila and 46.6% of Aeromonas spp. were hemolytic. The assay for checking production of toxins showed that 17.5% of A. caviae, 73.3% of A. hydrophila, 60% of A. allosacharophila, 100% of A. schubertii, and 33.3% of Aeromonas spp. were able to produce toxins. The results demonstrated the pathogenic potential of Aeromonas, indicating that the presence of this emerging pathogen in water systems is a public health concern.