肠炎血清型沙门菌多重PCR检测方法的建立及应用

摘 要：目的 为临床确诊及基层实验室快速准确鉴定肠炎血清型沙门菌提供实验室支持,同时为沙门菌传统血清学鉴定方法提供技术补充。方法 根据沙门菌菌体抗原A/D1群基因、鞭毛抗原基因（fliC-HG）及肠炎沙门菌特异性基因片段（sdf I）设计引物,建立多重PCR体系并进行反应条件优化。对所建立的体系的特异性进行检测,并将该体系应用于浙江省2009-2010年分离的共计53株样本的检测。结果 建立并优化了肠炎沙门菌检测的多重PCR体系,该体系具有高特异性,可准确快速鉴定肠炎血清型沙门菌,也可区分A群及D群血清群的沙门菌,并能检测鞭毛抗原为HG的沙门菌,对实际样本检测符合率达100%。结论 该体系能正确鉴定肠炎血清型沙门菌,可作为沙门菌传统血清学分型的辅助方法,可弥补商品化血清质量层次不齐的缺陷。并为肠炎沙门菌的监测与实验室诊断提供了一种简单快速、重复性好的新方法。     

安徽地区腹泻病人粪便标本中沙门菌分离及其血清型鉴定

目的 了解安徽地区近期临床分离沙门菌血清型、药物敏感性、毒力基因携带及血清型相同菌株的基因溯源情况。方法 应用血清凝集法检测沙门菌的血清型,微量肉汤稀释法检测药物敏感性,PCR法扩增毒力基因,PFGE法检测基因同源性并进行溯源。结果 43株沙门菌中鼠伤寒沙门菌和肠炎沙门菌分别占(39.5%/29.6%)。43株菌对四环素、甲氧苄啶/磺胺甲噁唑、萘啶酸和氯霉素的耐药率分别为(60.5%/39.5%/37.2%/30.2%),43株菌均携带InvA和InvE毒力基因,鼠伤寒沙门菌基因同源性较低,肠炎沙门菌基因同源性较高。结论 本次安徽地区临床分离的沙门菌以鼠伤寒沙门菌和肠炎沙门菌为主,其他血清型散在分布。对常规抗生素呈较高的耐药性,分离菌株均携带毒力基因对人体具有致病性。本地区流行的鼠伤寒沙门菌大部分来源于不同的基因克隆株,亲缘关系较远。而流行的肠炎沙门菌大部分来源于同一克隆株或亲缘关系较近的克隆群体。     

30株水禽沙门菌的分离鉴定与血清型分析

目的 通过分析佛山及其周边地区水禽沙门菌感染的血清型分布特征,为防控水禽沙门菌病与制定公共卫生安全措施提供参考信息。方法 本试验对发病水禽采用细菌分离纯化、生化特性鉴定、PCR方法鉴定获得30株水禽沙门菌,以玻片凝集反应法作血清型鉴定。结果 30株水禽沙门菌的血清型含4(B、D、C₁、E₁ )个群别7种血清型,B群有25株沙门菌分离株,包括19株鼠伤寒沙门菌(S.Typhimurium)、3株乙型副伤寒沙门菌(S.Paratyhi)、2株印第安纳沙门菌(S.Indiana)、1株斯坦利沙门菌(S.Stanley);D群有肠炎沙门菌(S.Enteritidis)1株;C1群有2株波茨坦沙门菌(S.Potsdam);E₁群有2株明斯特沙门菌(S.Muenster)。结论 30株水禽沙门菌分离株均为人兽共患血清型,其中B群鼠伤寒沙门菌为优势血清型,对于水禽感染沙门菌病的防控,应优先考虑鼠伤寒沙门菌感染。     

腹泻儿童非伤寒沙门菌的耐药性及毒力基因分析

目的 探讨腹泻儿童非伤寒沙门菌血清型和耐药性及毒力基因的情况。方法 收集苏州大学附属儿童医院2020年1月至2022年1月腹泻患儿粪培养标本中分离培养的71株非伤寒沙门菌作为研究对象，采用全自动质谱仪鉴定菌株，采用玻片凝集法进行血清学分型，纸片扩散法和全自动细菌鉴定药敏分析仪进行抗生素耐药分析。聚合酶链反应(PCR)检测，非伤寒沙门菌株的21种毒力基因和3种耐药基因。结果 从腹泻患儿粪培养分离71株NTS血清型，以鼠伤寒沙门菌为主(52.11%),其次为肠炎沙门菌(14.08%)。NTS对氨苄西林的耐药率最高为88.73%。其次是哌拉西林(54.93%)和复方新诺明(40.85%),对头孢曲松和头孢哌酮的耐药率均为32.39%。亚胺培南耐药和中介各1株。鼠伤寒沙门菌对毒力基因sitC、sseL、sifA、siiE及phoP的携带率为100%。肠炎沙门菌对毒力基因sifA、siiE、phoP、pagN及pagC携带率高达90%。毒力基因spvB和prot6E常成对出现，主要分布在肠炎沙门菌。bapA和pefA的检测存在互补关系，bapA基因阳性则pefA阴性。结论 苏州地区腹泻儿童非伤寒...     

福氏志贺菌4av和Yv血清型PCR鉴定方法的建立及应用

目的 建立福氏志贺菌4av和Yv血清型PCR鉴定方法。方法 根据福氏志贺菌4av和Yv血清型O抗结构,针对O抗合成基因wzx、IV型抗原决定基因gtrIV和MASF IV-1抗原决定基因opt,建立血清型4av和Yv 的PCR鉴定方法。并应用该方法对126株福氏志贺菌临床分离株进行血清型检测。结果 建立了一种福氏志贺菌4av和Yv血清型PCR鉴定方法,在一个反应中包括4对引物,Yv血清型PCR扩增为wzx及opt阳性;4av血清型PCR扩增为wzx、opt及gtrIV阳性。该方法可将4av和Yv血清型与目前已知的其他福氏志贺菌血清型完全区分。对126株不同血清型福氏志贺菌临床分离株的鉴定结果显示,该方法具有很好的特异性。结论 本研究建立的福氏志贺菌4av和Yv血清型PCR鉴定方法,可以用于志贺菌检测和监测。     

深圳市腹泻患者沙门菌感染状况和耐药性分析

目的 分析2014-2017年深圳地区腹泻患者沙门菌的感染特征和耐药性情况,为沙门菌感染的防控提供科学依据。方法 从深圳市3家哨点医院腹泻门诊收集患者粪便标本,进行沙门菌的分离鉴定、血清分型及药敏试验;对7家医院上送的住院病人分离的沙门菌进行血清分型和药敏试验。结果 4 847份门诊患者的粪便标本中,分离出192株沙门菌,检出率为4.0%。5-10月是沙门菌检出的高峰, 5岁以下儿童和6-17岁是感染的主要人群,检出率分别为6.2%和5.3%。192株沙门菌中鉴定出18种血清型,其中鼠伤寒沙门氏菌、鼠伤寒沙门氏菌变种和肠炎沙门氏菌是最常见的3种血清型,另有19株未能分型。77株住院病人分离的沙门菌中鉴定出20种血清型,其中肠炎沙门氏菌、鼠伤寒沙门氏菌变种、甲型副伤寒沙门氏菌占比分别为33.8%、13.0%和11.7%,另有3株未能分型。45株沙门菌对氨苄西林、环丙沙星和左旋氧氟沙星的耐药最为严重,自2015年开始出现对喹诺酮类药物的耐药和多重耐药菌株,10株鼠伤寒沙门氏菌中多重耐药率为50.0%。结论 深圳市腹泻患者的沙门菌感染以17岁以下儿童为主,5-10月份为感染的高峰期,沙门菌的血清型分散,其中肠炎沙门氏菌和鼠伤寒沙门氏菌、鼠伤寒沙门氏菌变种是主要的血清型,多重耐药自2015年开始日趋严重,临床上应合理规范用药并且加强耐药监测。     

泰安市感染性腹泻非伤寒沙门菌耐药监测及分型研究

目的分离鉴定泰安市腹泻病沙门菌的血清型分布、耐药性及PFGE分子分型和同源性特点。方法对分离自泰安市腹泻病人的粪便标本中的31株沙门菌进行生化鉴定和血清分型,采用微量肉汤稀释法对其进行16种抗生素敏感试验,利用PFGE进行分子分型及聚类分析。结果 31株沙门菌分属于10种血清型,肠炎沙门菌占35.5%(11/31)为优势血清型;31株沙门菌对头孢西丁和亚胺培南100%敏感,对磺胺异唑的耐药率为77.42%(24/31);31株沙门菌按照100%的相似度可分为17个PFGE型。结论药敏显示所检测样本存在较为严重的多重耐药性,肠炎沙门菌遗传变异在该地区相对较为保守,且存在散发流行的可能,提示应做好肠炎沙门菌传染病的早期预警及防控。     

无锡市腹泻病人沙门菌的病原学特征及分子分型研究

目的分析无锡市腹泻人群中分离的沙门菌的流行特征;同时比较主要血清型菌株间的脉冲场凝胶电泳(PFGE)带型差异,为沙门菌感染的防控提供实验数据。方法收集2015年无锡市腹泻病人的粪便标本,分别进行沙门菌的分离鉴定、药敏试验、血清型分型以及PFGE分型分析。结果 756份粪便标本中共分离到32株沙门菌,阳性率为4.23%;检出时间主要集中在5~10月份,感染人群以老年人为主。药敏试验说明无锡地区的沙门菌对氨苄西林的耐药率最高,达56.25%;对环丙沙星和头孢他啶的耐药率最低,均为6.25%。32株沙门菌共鉴定出11种血清型,以肠炎沙门菌和鼠伤寒沙门菌为主,分别占31.25%和21.88%。对这2种血清型的沙门菌进行PFGE分析,显示肠炎沙门菌所有带型的相似度均在85%以上,鼠伤寒沙门菌的带型各不相同。结论无锡市沙门菌的感染具有明显季节和年龄分布差异性,流行的优势血清型为肠炎沙门菌。无锡市需同时加强对食品和环境中沙门菌的监测。     

成都市人源沙门菌基因组特征分析

目的 基于全基因组测序技术,探究成都市人源沙门菌的基因组特征, 为监测与预防沙门菌感染提供资料。方法 收集35株成都市人源沙门菌(分离自腹泻患者粪便)进行全基因组测序。根据测序数据,进行血清型预测、耐药基因及可移动遗传元件预测、毒力因子注释及分布分析。结果 35株沙门菌分离株经全基因组测序,共预测到5种血清型,包括15株I 4,[5],12:i:-沙门菌(13株为ST34、2株为新ST型)、12株鼠伤寒沙门菌(ST19)、5株肠炎沙门菌(ST11)、2株德比沙门菌(ST40)与1株火鸡沙门菌(ST463)。35株沙门菌分离株共预测到10类42种不同的耐药基因,其中氨基糖苷类aac(6')-Iaa携带率为100%(35/35)。I 4,[5],12:i:-沙门菌的耐药基因、质粒及插入序列数目及种类多于其他血清型菌株。I 4,[5],12:i:-、鼠伤寒沙门菌和肠炎沙门菌的毒力因子数目大于其他血清型菌株。部分鼠伤寒沙门菌有更高的毒力质粒、质粒编码菌毛和血清抗性毒力因子分布。结论 成都市人源沙门菌不同血清型菌株之间耐药基因、可移动遗传元件、毒力因子分布差异明显,值得在转录组、蛋白组进一步探究其耐药与毒力机制。     

上海市沙门菌血清型流行特征

目的建立本市沙门菌数据库,初步了解本地区部分沙门菌血清型的流行特征。方法以1999-2007年于从业人员体检、食品、腹泻病人质控(QC)和沙门菌监测对象分离的1078株沙门菌的血清型进行回顾性分析;其中2006年和2007年按照全球沙门菌监测(GSS)方案进行连续性腹泻病例监测。结果血清型谱显示本市不同来源的沙门菌型在分布上存在优势交叉,GSS分离菌株中有7个型为国内首次报告;通过分子分型证明本市2006年的山夫登堡和2007年的汤卜逊病例分别属于2个和1个克隆型。结论肠炎、鼠伤寒仍是本市优势致泻性血清型,山夫登堡和汤卜逊病例符合沙门菌性相对散发和集中暴发的分子流行特征。加强对沙门菌综合监测和准确及时的血清、分子分型能力有助于检索和追溯流行菌型。     

From the Cover: Patterns of genome evolution that have accompanied host adaptation in Salmonella

Many bacterial pathogens are specialized, infecting one or few hosts, and this is often associated with more acute disease presentation. Specific genomes show markers of this specialization, which often reflect a balance between gene acquisition and functional gene loss. Within Salmonella enterica subspecies enterica, a single lineage exists that includes human and animal pathogens adapted to cause infection in different hosts, including S. enterica serovar Enteritidis (multiple hosts), S. Gallinarum (birds), and S. Dublin (cattle). This provides an excellent evolutionary context in which differences between these pathogen genomes can be related to host range. Genome sequences were obtained from ∼60 isolates selected to represent the known diversity of this lineage. Examination and comparison of the clades within the phylogeny of this lineage revealed signs of host restriction as well as evolutionary events that mark a path to host generalism. We have identified the nature and order of events for both evolutionary trajectories. The impact of functional gene loss was predicted based upon position within metabolic pathways and confirmed with phenotyping assays. The structure of S. Enteritidis is more complex than previously known, as a second clade of S. Enteritidis was revealed that is distinct from those commonly seen to cause disease in humans or animals, and that is more closely related to S. Gallinarum. Isolates from this second clade were tested in a chick model of infection and exhibited a reduced colonization phenotype, which we postulate represents an intermediate stage in pathogen–host adaptation.The central importance of horizontal acquisition of mobile genetic elements in the development of virulence in bacteria has been well described. It has frequently been observed that, as pathogens acquire virulence determinants, they become increasingly adapted to a specific host (1, 2). Exquisitely host-restricted pathogens also often exhibit extensive genome decay, through insertion sequence element proliferation, genomic rearrangement, and/or pseudogene formation (1, 3, 4). Investigating mechanisms involved in host adaptation is key to an understanding of pathogen evolution and has directly translatable relevance to the epidemiology and potentially the control of human and zoonotic infectious disease.By concentrating upon individual pathogenic clades, insights have been obtained into specific adaptations relating to specific hosts; however, comparative analysis is relatively rare. By broadening this approach to examine multiple human and animal pathogens, derived from a single closely related lineage but with differing host specializations, there is an opportunity to understand the fundamental evolutionary processes involved in host adaptation. Lineage-specific changes that have become fixed can then be distinguished from those stochastic changes that differentiate individual isolates.A single lineage within Salmonella enterica presents such an opportunity. S. enterica is a leading cause of foodborne gastroenteritis, globally responsible for 80 million cases annually (5). Differentiation of S. enterica is largely based upon somatic (O) and flagellar (H) antigens, but it is increasingly being typed by genomic methods, such as multilocus sequence typing (MLST). Somatic serogrouping and MLST have identified a single lineage with closely related members that exhibit a range of different host specializations (6–9). These include two of the most important Salmonella pathogens: S. Enteritidis and S. Gallinarum. In addition to the contribution of S. Enteritidis to human disease, in many countries both of these pathogens are notifiable diseases in poultry farming and egg production. Despite their close phylogenetic relatedness, they exhibit strikingly different host ranges, with S. Enteritidis capable of infecting multiple host species, whereas S. Gallinarum is restricted to infection in galliforme birds. This lineage also includes S. Gallinarum biovar Pullorum (hereafter S. Pullorum), also restricted to galliformes, and S. Dublin, which is strongly associated with infection of cattle and more rarely that of humans (10). The Salmonella pathogens adapted to particular hosts are associated with a much more invasive disease than generalists like S. Enteritidis, which tend to cause enteritis.Fifty-nine isolates of this Salmonella lineage were selected to capture the diversity of sequence types, phage types, and geographical and temporal spread available at the time. Through genome sequencing, we generated a phylogeny to act as a framework with which to reconstruct the evolutionary history of the lineage, onto which observed gene loss and acquisition could be plotted.This dataset provides a compelling record of the degradation of common metabolic pathways during host specialization to date. We have documented the order of events during the evolution of an entire Salmonella lineage. To link this specifically to host adaptation, we have tested isolates occupying key positions in the phylogeny in their cognate host to assess the effects of gene degradation on the manner and severity of disease caused.     

Salmonella serovars isolated from humans in São Paulo State, Brazil, 1996-2003

Salmonellosis remains an important cause of diarrheal illness in humans in S?o Paulo State, Brazil. In this study were identified 3554 Salmonella isolates from human infections, during the period 1996-2003. Among 68 different serovars determined, S. Enteritidis was the most frequent one in gastrointestinal and extra-intestinal infections accounting for 67.4% of all isolates. S. Typhimurium and S. enterica subsp. enterica (4,5,12:i:-) were most frequently isolated from children aged < 1-4 year-old, in contrast, people with S. Enteritidis infections were most likely to be 20-50 year-old. In our geographic area the continued laboratorial surveillance of salmonellosis, including serotyping, has showed the trends in Salmonella serovars causing infections in humans throughout the time.     

Dna probes for identification of leptospires and disease diagnosis

A newly identified 1 kb DNA fragment amplified by PCR using (AG)8T inter-simple sequence repeats (ISSR) primer and a 631 bp segment of 16S rRNA ribosomal gene amplified by PCR using reported primers were labeled with a alpha32P dCTP for use as DNA probes. These probes were hybridized with DNA extracted from 19 standard pathogenic serovars, 3 standard saprophytic serovars, 33 pathogenic isolates (12 from patients, 1 from a tapwater source, and 20 from rodents), and 22 saprophytic isolates from environmental sources. The pathogen-specific 16S rRNA DNA probe specifically hybridized all 33 standard pathogenic serovars, to 13 pathogenic isolates. Similarly, the saprophyte specific 1 kb ISSR DNA probe specifically hybridized the 3 standard saprophytic serovars and the 22 saprophytic Leptospira isolates. The sensitivity of the 1 kb labeled saprophytic Leptospira specific DNA probe was 1.95 ng, and for the 16S rRNA pathogen specific probe 3.90 ng. The 16S rRNA gene segment DNA probe could also identify the leptospiremic stage in mice or guinea pigs infected experimentally with the pathogenic serovars australis, autumnalis or icterohaemorrhagiae. DNA probes therefore, owing to their high specificity and sensitivity, appear useful for easy, rapid, and reliable differentiation of pathogenic Leptospira strains and also hold promise for direct identification of organisms in blood samples to diagnose leptopsirosis.     

A regional outbreak of S. Enteritidis phage type 5, traced back to the flocks of an egg producer, Austria

In the spring and summer of 2002, the Nationale Referenzzentrale für Salmonellen (National Reference Centre for Salmonella - NRCS) in Austria noticed a cluster of human Salmonella enterica subsp. enterica ser. Enteritidis phage type 5 (S. Enteritidis PT5) infections in two neighbouring districts of Austria. Another small outbreak of S. Enteritidis PT5 infections that occurred in the same region in 1999 had been traced back to the flocks of a local egg producer (approximately 6 000 hens). Attention was therefore again directed at this farm. The results of voluntary bacteriological examinations from the farm and further epidemiological investigations identified the same egg producer as the source of the second outbreak. The 70 human isolates of S. Enteritidis PT5 ascertained in 2002 represented a minority of all infections. It is realistic to estimate that several hundred infections occurred in the course of the 2002 outbreak. The farmer had not vaccinated new flocks against Salmonella since August 2001. It is likely that the change in vaccination policy resulted in the reappearance of the S. Enteritidis PT5 infections. By the end of September 2002 the farmer had stopped selling untreated table eggs. In October 2002 only one isolate of S. Enteritidis PT5 was ascertained in the region.     

Recovery and its evaluation of Shigella bacilli or Salmonella from healthy food handlers in Tokyo (1961-1997)]

Since 1961, recovery of Shigella bachilli from healthy food handlers in Tokyo has been carrying out, and detection of Salmonella carriers has also been adding from 1980. Recovery rate of Shigella has decreased from 0.28% (589 cases) in 1961 to 0.01% (9 cases) in 1969, and 7 cases between 1971 and 1975 and only 3 carriers since 1976 have been detected. On the other hand, Salmonella has been detected from about 9,000 cases (0.07%) during 18 years. The isolates were typed into 150 serovars, in which the most frequent one was S. Enteritidis, following S. Litchfield, S. Thompson, S. Hadar, S. Typhimurium, S. Infantis, S. Tennessee, S. Montevideo, S. Agona and S. Braenderup. These serovars except S. Agona caused in 90% of 1,650 Salmonella food poisoning outbreaks which had occurred between 1980 and 1996. Recovery of S. Enteritidis from healthy subjects increased year by year since 1989, and this tendency was well consistent with the increase of food poisoning outbreak caused by this serovar. These results indicate that the recovery of carrier with enteropathogen from food handlars is significant as preventive measures or food hygiene.     

Characterization of Salmonella isolated in Okinawa, Japan

Salmonella enterica strains isolated in Okinawa between 1995 and 2005 were analyzed with respect to their serovars and antimicrobial susceptibility, and pulsed-field gel electrophoresis (PFGE) was used to examine their digestion patterns. A total of 1,071 isolates, including 610 from humans, 358 from animal rectal swabs and 103 from meat obtained at grocery stores, were examined. The first 3 most frequent serovars in human isolates were Enteritidis, Weltevreden and Bareilly, together accounting for 65% of the isolates. In isolates from the rectal swabs of laying hens, the predominant serovars were Albany, Saintpaul and Aarhus, accounting for 82% of the isolates. In broilers, 123 of 124 isolates belonged to serovar Infantis, which reflected the high ratio of this serovar in the chicken sold at grocery stores. An antibiogram of human isolates was different from that of broilers and chicken. Chromosomal DNAs of S. Infantis isolated from humans and from the rectal swab of broilers and chickens were examined by PFGE using the restriction enzymes XbaI and BlnI. The digestion patterns of human isolates were not coincident with those of the isolates from the rectal swab of broilers and chicken-meat samples.     

Multiplex polymerase chain reaction assay for selective detection of Salmonella enterica serovar typhimurium

A multiplex polymerase chain reaction (PCR) assay was developed for the identification of Salmonella enterica serovar Typhimurium. Three sets of primers were designed for detecting O4, H:i, and H:1,2 antigen genes from the antigen-specific genes rfbJ, fliC, and fljB, respectively. These were evaluated in a multiplex PCR assay by using DNAs from S. enterica serovar Typhimurium, 15 other Salmonella serovars, and 8 non-Salmonella enteric pathogens. Multiplex PCR proved to be capable of identifying S. enterica serovar Typhimurium specifically and differentiating it from other Salmonella serovars in addition to non-Salmonella enteric pathogens. Thus, this multiplex PCR assay can be practically applied to the identification of S. enterica serovar Typhimurium.     

Serovar-distribution and drug-resistance of Salmonella strains isolated from domestic and imported cases during 1995-1999 in Tokyo

A total of 2,277 non-typhoidal Salmonella strains consisting of 1,807 domestic strains and 470 imported strains isolated from sporadic cases during 1995-1999 in Tokyo, were examined regarding their serovar-distibution and their drug-resistance. The serological typing results showed that the domestic strains were classified into 17 O-groups and 99 serovars, and the imported strains were classified into 12 O-groups and 58 serovars. Among the serovars identified, Salmonella serovar Enteritidis (S. Enteritidis), S. Thompson, S. Hadar, S. Infantis, S. Typhimurium, and S. Litchfield were predominant in the domestic strains, whereas S. Enteritidis, S. Anatum, S. Hadar, and S. Weltevreden were predominant in the imported strains. The drug-resistance test using 9 drugs (CP, TC, SM, KM, ABPC, ST, NA, FOM, and NFLX) showed that 34.0% of the domestic strains and 33.0% of the imported strains were resistant to any of the drugs examined. The serovars of a high resistant rate during this period were S. Blockley (100%), S. Hadar (96.6%), S. Typhimurium (63.6%), and S. Enteritidis (62.2%) in the domestic strains and S. Blockley (100%), S. Hadar (97.1%), S. Rissen (88.9%), S. Emek (83.3%), S. Panama (83.3%), and S. Typhimurium (77.8%) in the imported strains. Drug-resistance patterns of the resistant isolates varied to 60 types. Prevalent patterns recognized were SM, TC.SM, TC, TC.SM.KM.ST, TC.SM.KM, and CP.TC.SM.ABPC in the domestic strains and TC.SM, TC, NA, TC.SM.KM.NA, and TC.SM.NA in the imported strains.     

肠炎沙门菌sifA基因缺失株的无痕构建及其在巨噬细胞内的增殖特性研究

目的 本研究旨在探究sifA基因对肠炎沙门菌胞内寄生和增殖的影响,以探索肠炎沙门菌的致病机理。方法 根据同源重组原理,无痕构建肠炎沙门菌C50041的sifA基因缺失株,通过蓝白斑、抗生素耐药性、PCR和基因测序方法对基因缺失株进行鉴定,并从生物学特性、胞内沙门菌增殖、sifA基因在胞内外菌中表达等方面,分析sifA基因对肠炎沙门菌的影响。结果 成功构建肠炎沙门菌C50041ΔsifA,蓝白斑、抗生素耐药性、PCR和基因测序方法证明该菌株是正确的。其生长速度和生化特性没有发生改变。ΔsifA株感染巨噬细胞后,其胞内沙门菌量显著少于其野生株。sifA基因在spiC基因缺失株中表达量增加。结论 肠炎沙门菌C50041ΔsifA被成功构建,其在巨噬细胞内的增殖量显著降低,sifA基因表达可能受spiC基因调控,本研究为深入探究肠炎沙门菌在细胞内增殖及sifA基因的功能奠定基础。     

Class 1 integrons and resistance gene cassettes among multidrug resistant Salmonella serovars isolated from slaughter animals and foods of animal origin in Ethiopia

The present study was undertaken to identify and characterize integrons and integrated resistance gene cassettes among multidrug resistant (MDR) Salmonella isolates from slaughter animals and food products of animal origin in Ethiopia. A total of 98 epidemiologically unrelated Salmonella isolates comprising 13 serovars were characterized using serotyping, phage typing, antimicrobial resistance testing and the pulsed-field gel electrophoresis (PFGE) method. Integron-PCR was used to detect the presence of class 1 and class 2 integrons in the MDR strains. The associated individual resistance gene cassettes were identified using specific PCRs and DNA sequencing. The location of the integrons was determined by Southern blot hybridization analysis. Among the Salmonella serovars, a high level of antimicrobial resistance was found to streptomycin (82.6%), tetracycline (75.5%), sulfamethoxazole (60.2%), spectinomycin (53.1%), ampicillin (42.8%), nalidixic acid (34.7%), nitrofurantoin (30.6%), trimethoprim (27.5%), gentamicin (20.4%) and ciprofloxacin (19.4%). Class 1 integrons were detected in 53.1% of the MDR isolates comprising serovars Anatum, Braenderup, Kentucky, Saintpaul and Typhimurium. Of the class 1 integron positive isolates 61.5% harboured the integron-associated gene cassettes: aadA2, aadA2+bla(PSE-1), dfrA1-aadA1 and dfrA12-orf-aadA2 (amplicon sizes 1000 bp, 1000+1200 bp, 1600 bp and 1900 bp, respectively). The chromosomally located aadA2 and aadA2+bla(PSE-1) resistance gene cassettes occurred exclusively in S. Typhimurium DT104 isolates, the other cassettes were found on large plasmids in several serovars. An aacCA5-aadA7 gene cassette array (amplicon size 1600 bp) was exclusively found in all MDR S. Kentucky strains of R type Str/SpeSmxGenNalAmpTetCipCef and this integron was shown to be chromosomally located. Results of the present study indicate that class 1 integrons carrying gene cassettes, which confer resistance to different classes of antimicrobials such as aminoglycosides, beta-lactams and trimethoprim are widespread among the MDR Salmonella serovars isolated from slaughter animals and food products of animal origin in Ethiopia indicating the important role of these genetic elements in the dissemination of multidrug resistance.