创伤弧菌基因组学研究进展

创伤弧菌是人类三大致病弧菌之一,能够通过食源性传播或者伤口感染导致发病,近年来全球发病率逐步升高。随着新一代测序技术的发展,已有上百株创伤弧菌全基因组序列在国际公共数据库公布。这些数据为创伤弧菌基因组学研究提供了重要基础,并促进了对该病原菌遗传多样性、传播和致病机制的认识。本文从基因分型、种群结构和重要毒力因子3个方面对创伤弧菌基因组学研究进展进行归纳总结,以期为创伤弧菌感染、进化和防控研究提供借鉴。     

创伤弧菌FJ03-X2株培养条件的优化研究

目的探讨创伤弧菌毒株FJ03 X2的优化培养。方法以创伤弧菌毒株FJ03 X2进行实验,研究摇瓶培养温度、接种量、培养基、初始pH值、溶解氧等因素对菌株生长的影响,确定最适培养条件为：TSB培养基初始pH值为7.0,适宜接种量10%,培养温度28℃,装液量25mL/250mL,转速180r/min。培养菌数可达8×109cfu/mL,生物量为25mg/mL。以TSB为基础培养基,采用单因子和正交实验相结合的方法,确定最佳培养基主要成份为：葡萄糖0.2%、胰蛋白胨1.8%、大豆胨0.4%和玉米浆0.4%。以最佳培养基配方给菌体提供营养,在最适培养条件下培养,可以使培养液中的细菌生物量高达50mg/mL以上。结果得出创伤弧菌FJ03 X2的优化培养条件和培养基配方。结论国内首次优化鳗源创伤弧菌培养条件,并给出相关依据。     

创伤弧菌的药物敏感性

目的创伤弧菌(Vibrio vulnificus)是"人鱼共患病"的重要致病菌,从患"腐皮病"卵形鲳鲹(Trachinotus ovatus)鱼中分离到创伤弧菌TO-3,以阿莫西林等44种药物进行敏感性试验。结果对青霉素类抗菌药物不敏感,对头孢菌素类等抗菌药物很强的耐药性,而对氟哌酸、头孢氯氨苄、氟嗪酸、米诺环素、呋喃妥因、复达欣、萘啶酸、四环素、庆大霉素等抗菌药物高度敏感。在17味中草药中对五倍子、诃子、黄连、石榴皮等中草药极为敏感。     

黄姑鱼创伤弧菌的分离和鉴定

目的从患病黄姑鱼体内分离出致病力较强的菌株L1,人工感染实验证实该菌株为黄姑鱼的病原菌。对该细菌进行了形态、生理生化特性测定和16SrRNA分子鉴定,测定16SrRNA基因序列,分析相关细菌相应序列的同源性,构建了系统进化树。结果表明菌株L1与创伤弧菌(Vibrio vulnificus)的亲缘关系最近,具有98.7%的同源性。结合该菌株的生理生化特性,可鉴定菌株L1为创伤弧菌。     

创伤弧菌溶细胞毒素单克隆抗体制备及其鉴定

目的制备创伤弧菌(Vibrio vulnificus)溶细胞素vvhA基因产物鼠源性单克隆抗体并鉴定其特异性和免疫性,为进一步研制创伤弧菌检测试剂盒奠定基础。方法采用IPTG诱导目的重组蛋白rvvhA表达,Ni-NTA亲和层析法提纯rvvhA,SDS-PAGE检测表达和提纯效果。采用杂交瘤技术和rvvhA-ELISA制备并筛选分泌rvvhA单克隆抗体的细胞株,有限稀释法进行细胞克隆。采用免疫双扩散法鉴定单克隆抗体类型。采用ELISA、免疫双扩散法和Western Blot鉴定单克隆抗体的效价和特异性。结果在0.5mmol/L IPTG诱导下,rvvhA产量可占细菌总蛋白的18%。提纯的rvvhA经SDS-PAGE后仅显示单一的蛋白条带。共获得9株rvvhA抗体阳性的杂交瘤细胞株,其中A5E8和C3B6株可持续分泌高效价特异性单克隆抗体,其抗体类型分别为IgG1和IgG2a。A5E8和C3B6单克隆抗体有较高特异性,与多种其它细菌蛋白不发生免疫反应,对rvvhA及创伤弧菌GTC333株和WZ01株蛋白的ELISA检测阳性的效价可达1∶4000~1∶8000、免疫双扩散效价为1∶4~1∶8,Western Blot结果显示此等单克隆抗体均能有效识别rvvhA。结论本研究成功地获得了2株稳定分泌rvvhA特异性单克隆抗体的鼠源性杂交瘤细胞株,rvvhA单克隆抗体可用于检测自然表达的创伤弧菌溶细胞素。     

创伤弧菌溶细胞素基因在大肠杆菌中的表达及溶血活性鉴定

目的用大肠杆菌表达系统表达创伤弧菌溶细胞素,并对其溶血活性进行评价。为今后的免疫学活性研究和单克隆检测试剂盒的研发奠定基础。方法构建pET28a( )-vvhA表达载体,对包涵体进行三步洗涤后,用金属亲合层析(HisTag)纯化重组蛋白,并用溶血试验验证重组蛋白活性。结果用基因工程的方法成功获得高表达、高纯度(纯度≥96%)重组蛋白VVC。利用兔红细胞溶血试验检测表明,重组蛋白具有溶血活性,其活性为0.2μg/HU。结论成功用大肠杆菌表达系统表达创伤弧菌溶细胞素并对其纯化、复性条件进行优化。     

植物乳杆菌DOMLa对创伤弧菌诱导炎症反应的抑制作用

目的 观察植物乳杆菌DOMLa对创伤弧菌诱导小鼠腹腔巨噬细胞产生细胞因子和细胞毒作用的影响,探讨其在协助机体抵抗细菌感染以及对过强炎性反应的影响。方法 选择合适剂量的创伤弧菌感染小鼠腹腔巨噬细胞,同时加入不同剂量的植物乳杆菌DOMLa共培养。作用3 h后,采用乳酸脱氢酶释放法检测细胞毒作用,酶联免疫吸附试验检测相关细胞因子的释放量。结果 在MOI=100时,植物乳杆菌DOMLa显著降低创伤弧菌的细胞毒作用,明显抑制了创伤弧菌诱导的三种促炎细胞因子IL-6、IL-1β、TNF-α的产量。结论 植物乳杆菌DOMLa可降低创伤弧菌对小鼠腹腔巨噬细胞的细胞毒作用,并可减轻病原菌诱导巨噬细胞的过强炎症反应。该植物乳杆菌对宿主免疫细胞的其他作用和影响还有待进一步研究。     

日本鳗鲡混合感染迟缓爱德华氏菌与创伤弧菌的分离与鉴定

目的对日本鳗鲡混合感染迟缓爱德华氏菌(Edwardsiella tarda)和创伤弧菌(Vibrio vulnificus)的病原进行了分类鉴定。方法对两株病原菌进行了形态、API-ID32E鉴定系统和分子生物学鉴定,分别测定了菌株AnGH080301和An-GH080302的16SrRNA和HSP60基因序列,构建了系统进化树。结果按形态特征和API-ID32E鉴定系统分别初步鉴定菌株AnGH080301和AnGH080302为迟缓爱德华氏菌和创伤弧菌。菌株AnGH080301的16SrRNA基因部分序列(登录号FJ646618)与迟缓爱德华氏菌的16S rRNA基因(登录号AB050832)同源性最高,达99.7%;菌株AnGH080302的HSP60基因部分序列(登录号FJ646619)与创伤弧菌HSP60基因(登录号BA000037)的同源性最高,达99.8%。结论综合菌株的生理生化特性和分子生物学鉴定结果,可将菌株AnGH080301和AnGH080302分别鉴定为迟缓爱德华氏菌和创伤弧菌,迟缓爱德华氏菌与创伤弧菌均为人兽共患病病原,其混合感染日本鳗鲡致病的报道尚属首次。     

鳗鲡创伤弧菌的分子鉴定

目的对分离自发病欧洲鳗鲡的创伤弧菌疑似菌株进行准确的鉴定。方法首先尝试利用一对16SrRNA基因特异性通用引物PCR扩增一株鳗源创伤弧菌疑似株的基因组DNA，得到一个约500bp的DNA片段，将该DNA片段亚克隆至pMDl8-T载体，鉴定克隆化成功之后，送专业公司进行测序，得到一个502bp的DNA产物，NCBI上同源性比较表明，该片段与GeneBank上注册的创伤弧菌的16SrDNA序列同源性最高（100％），同时排除了哈维氏弧菌的可能。设计一对创伤弧菌溶血素特异性引物，实现了对鳗鲡创伤弧菌的分子鉴定。结果建立一种简洁的鳗鲡创伤弧菌的分子鉴定方法。结论国内首次自发病欧鳗分离到创伤弧菌，并给出分子鉴定证据。     

Tracking the establishment of local endemic populations of an emergent enteric pathogen

Shigella sonnei is a human-adapted pathogen that is emerging globally as the dominant agent of bacterial dysentery. To investigate local establishment, we sequenced the genomes of 263 Vietnamese S. sonnei isolated over 15 y. Our data show that S. sonnei was introduced into Vietnam in the 1980s and has undergone localized clonal expansion, punctuated by genomic fixation events through periodic selective sweeps. We uncover geographical spread, spatially restricted frontier populations, and convergent evolution through local gene pool sampling. This work provides a unique, high-resolution insight into the microevolution of a pioneering human pathogen during its establishment in a new host population.The bacterium Shigella sonnei is a human-adapted bacterial pathogen that accounts for approximately one-sixth of the global dysentery burden of >160 million infections and 1 million deaths annually (1, 2). We have recently shown that S. sonnei emerged in Europe ∼500 y ago and spread intercontinentally in the last few decades to establish new and locally evolving populations in countries where it is now considered endemic (3). Most of these newly disseminated S. sonnei populations belonged to a single, globally distributed, multidrug-resistant (MDR) clade of S. sonnei lineage III, which we refer to as Global III. Members of this clade are biotype g and carry a class II integron insertion within the chromosome bearing MDR genes (3). Recent shifts have been reported in the dominant agents of bacterial dysentery, with S. sonnei replacing Shigella flexneri in Vietnam, Thailand, Malaysia, China, and several other countries undergoing economic development (4–8). Here, we have sequenced the genomes of >250 S. sonnei isolated in Vietnam over a 15-y period to investigate the microevolution of this pathogen during its establishment in a naïve human population.     

Multiclonal human origin and global expansion of an endemic bacterial pathogen of livestock

Most new pathogens of humans and animals arise via switching events from distinct host species. However, our understanding of the evolutionary and ecological drivers of successful host adaptation, expansion, and dissemination are limited. Staphylococcus aureus is a major bacterial pathogen of humans and a leading cause of mastitis in dairy cows worldwide. Here we trace the evolutionary history of bovine S. aureus using a global dataset of 10,254 S. aureus genomes including 1,896 bovine isolates from 32 countries in 6 continents. We identified 7 major contemporary endemic clones of S. aureus causing bovine mastitis around the world and traced them back to 4 independent host-jump events from humans that occurred up to 2,500 y ago. Individual clones emerged and underwent clonal expansion from the mid-19th to late 20th century coinciding with the commercialization and industrialization of dairy farming, and older lineages have become globally distributed via established cattle trade links. Importantly, we identified lineage-dependent differences in the frequency of host transmission events between humans and cows in both directions revealing high risk clones threatening veterinary and human health. Finally, pangenome network analysis revealed that some bovine S. aureus lineages contained distinct sets of bovine-associated genes, consistent with multiple trajectories to host adaptation via gene acquisition. Taken together, we have dissected the evolutionary history of a major endemic pathogen of livestock providing a comprehensive temporal, geographic, and gene-level perspective of its remarkable success.

The emergence of new pathogens typically arises through host-jump events and is a major threat to public health and food security (1). The domestication of animals and the expansion of agriculture in the Neolithic era increased the opportunities for the zoonotic and anthroponotic transmission of pathogens (2). Subsequent intensification of farming, industrialization, and globalization have increased the likelihood of successful expansion and dissemination of new pathogenic clones. However, our understanding of the evolutionary history of the major bacterial pathogens affecting livestock is very limited. In order to mitigate the emergence of new pathogens, or design novel interventions to limit spread, it is imperative that we understand the evolutionary and ecological drivers for the success of existing pathogens that have originated via host-switching events.S. aureus is a multihost bacterial species and a major pathogen of humans and livestock. In particular, S. aureus is a leading cause of bovine mastitis resulting in huge economic losses to the global dairy industry (3). In addition, bovine S. aureus is recognized as an emergent zoonotic threat (4), but the relative frequency of human infections caused by different clones of bovine S. aureus has not been examined to date. Previously, we demonstrated that the evolution of S. aureus has involved host-switching events between humans and domesticated animals in both directions leading to the emergence of clones circulating in human and livestock populations (5). In addition, we identified gene acquisition as a major driver for the adaptation of S. aureus to a new host species after a host-switch event. However, the limited number of isolates from livestock sources included in the study was insufficient to support analysis of the origin, clonal expansion, and global spread of contemporary livestock clones.Numerous studies have employed whole genome sequencing to explore the diversity of S. aureus from dairy cattle but they have tended to include a limited number of isolates from geographically restricted regions (6–15). In order to address this gap in understanding, we established a genome sequence dataset of 10,254 S. aureus genomes including 1,896 bovine isolates from 32 countries in 6 continents to carry out a comprehensive phylodynamic and accessory genome network analysis. We provide broad insights into the evolutionary origins of bovine S. aureus, including the key impact of human activities, and reveal the adaptive and geographical trajectories that have driven its global success.     

Fusion of two divergent fungal individuals led to the recent emergence of a unique widespread pathogen species

In a genome alignment of five individuals of the ascomycete fungus Zymoseptoria pseudotritici, a close relative of the wheat pathogen Z. tritici (synonym Mycosphaerella graminicola), we observed peculiar diversity patterns. Long regions up to 100 kb without variation alternate with similarly long regions of high variability. The variable segments in the genome alignment are organized into two main haplotype groups that have diverged ～3% from each other. The genome patterns in Z. pseudotritici are consistent with a hybrid speciation event resulting from a cross between two divergent haploid individuals. The resulting hybrids formed the new species without backcrossing to the parents. We observe no variation in 54% of the genome in the five individuals and estimate a complete loss of variation for at least 30% of the genome in the entire species. A strong population bottleneck following the hybridization event caused this loss of variation. Variable segments in the Z. pseudotritici genome exhibit the two haplotypes contributed by the parental individuals. From our previously estimated recombination map of Z. tritici and the size distribution of variable chromosome blocks untouched by recombination we estimate that the hybridization occurred ～380 sexual generations ago. We show that the amount of lost variation is explained by genetic drift during the bottleneck and by natural selection, as evidenced by the correlation of presence/absence of variation with gene density and recombination rate. The successful spread of this unique reproductively isolated pathogen highlights the strong potential of hybridization in the emergence of pathogen species with sexual reproduction.     

Contact between bird species of different lifespans can promote the emergence of highly pathogenic avian influenza strains

Outbreaks of highly pathogenic strains of avian influenza viruses (AIVs) cause considerable economic losses to the poultry industry and also pose a threat to human life. The possibility that one of these strains will evolve to become transmissible between humans, sparking a major influenza pandemic, is a matter of great concern. Most studies so far have focused on assessing these odds from the perspective of the intrinsic mutability of AIV rather than the ecological constraints to invasion faced by the virus population. Here we present an alternative multihost model for the evolution of AIV in which the mode and tempo of mutation play a limited role, with the emergence of strains being determined instead principally by the prevailing profile of population-level immunity. We show that (i) many of the observed differences in influenza virus dynamics among species can be captured by our model by simply varying host lifespan and (ii) increased contact between species of different lifespans can promote the emergence of potentially more virulent strains that were hitherto suppressed in one of the species.Avian influenza viruses (AIVs) exist within a complex ecology that includes common interspecies transmission among birds (1, 2). Like all influenza viruses, AIV can be divided into subtypes (e.g., H5N1) on the basis of variation in the hemagglutinin (HA) and neuraminidase (NA) surface proteins and also exhibit extensive antigenic diversity within a particular subtype (e.g., refs. 3 and 4). Most AIV cocirculating between domestic and wild birds are classified as being of low pathogenicity (LPAIV). Occasionally, however, highly pathogenic (HPAIV) forms arise, causing high mortality in poultry. Severe illness and death also occur in humans infected by HPAIV: There have been around 650 human cases of subtype H5N1 HPAIV, with 384 deaths, since 2003. However, a recent epidemic of subtype H7N9 LPAIV, which has to date claimed more than 100 lives from ∼400 confirmed cases, illustrates that inducing severe disease in humans is not the sole preserve of HPAIV.In avian species other than domestic poultry, AIV infection is largely asymptomatic (5, 6) with the notable exception of HP H5N1, which has caused recorded deaths in domestic and exotic waterfowl. Among ducks, the outcome of HP H5N1 infection is variable (7–11) and several factors suggest that domestic ducks may act as an asymptomatic reservoir (or “Trojan horse”) (12–14). Since 2002, HP H5N1 viruses have been regularly found among wild birds, including various species of migratory ducks and geese (15) in Asia, but other HPAIV have been isolated only sporadically from wildfowl (e.g., ref. 7).HP H5N1 was first isolated in 1997 from chickens, ducks, and geese but the direction of transmission among these species remains unclear (16, 17). By contrast, genetic analyses of the virus responsible for the 2013 H7N9 outbreak show that virus to have moved first from wild birds to geese and ducks and then to chickens (18). Such studies offer insights into the dispersal history of AIV but they do not explain why new viral lineages or strains associated with phenotypes of interest (including, but not limited to, pathogenicity) arise and spread. The current understanding is that their emergence is limited by the occurrence of mutational events that are assumed to be rare (4, 19), but this is not wholly consistent with both the high observed rates of AIV nucleotide substitution (20) and the fact that many viral phenotypes are defined by a small number of genetic changes (21): Changes at as few as seven amino acid sites can explain recent patterns of antigenic evolution in humans (22). Here, we show many of these questions can be answered by recognizing that the virus population comprises a large pool of interchangeable gene segments (2) circulating within a variety of wild and domestic species of widely different lifespans. We demonstrate that host lifespan is a key determinant of the population dynamics of the virus and that the emergence of particular phenotypes may be driven by the immunodynamics of multispecies transmission rather than by the generation of those phenotypes by mutation.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Evolutionary diversification,coevolution between populations and their antagonists,and the filling of niche space

The population component of a species’ niche corresponds to the distribution of individuals across environments within a region. As evolutionary clades of species diversify, they presumably fill niche space, and, consequently, the rate of increase in species numbers slows. Total niche space and species numbers appear to be relatively stable over long periods, and so an increase in the species richness of one clade must be balanced by decrease in others. However, in several analyses, the total population niche space occupied per clade is independent of the number of species, suggesting that species in more diverse clades overlap more in niche space. This overlap appears to be accommodated by variation in the populations of each species, including their absence, within suitable niche space. I suggest that the uneven filling of niche space results from localized outcomes of the dynamic coevolutionary interactions of populations with their pathogens or other antagonists. Furthermore, I speculate that relationships with pathogens might constrain diversification if pathogen diversity increased with host diversity and resulted in more frequent host switching and emergent disease. Many indirect observations are consistent with these scenarios. However, the postulated influence of pathogens on the filling of niche space and diversification of clades primarily highlights our lack of knowledge concerning the space and time dimensions of coevolutionary interactions and their influence on population distribution and species diversification.     

Gene evolutionary trajectories in Mycobacterium tuberculosis reveal temporal signs of selection

Genetic differences between different Mycobacterium tuberculosis complex (MTBC) strains determine their ability to transmit within different host populations, their latency times, and their drug resistance profiles. Said differences usually emerge through de novo mutations and are maintained or discarded by the balance of evolutionary forces. Using a dataset of ∼5,000 strains representing global MTBC diversity, we determined the past and present selective forces that have shaped the current variability observed in the pathogen population. We identified regions that have evolved under changing types of selection since the time of the MTBC common ancestor. Our approach highlighted striking differences in the genome regions relevant for host–pathogen interaction and, in particular, suggested an adaptive role for the sensor protein of two-component systems. In addition, we applied our approach to successfully identify potential determinants of resistance to drugs administered as second-line tuberculosis treatments.

The Mycobacterium tuberculosis complex (MTBC) is a genetically monomorphic group of bacteria (1, 2) whose members cause tuberculosis in humans and animals. The MTBC comprises both human-associated (L1, L2, L3, L4, L5, L6, L7, L8, and L9) and animal-associated (A1, A2, A3, and A4) clades (3–7). Due to the absence of horizontal gene transfer, plasmids, and measurable recombination among strains and other species (8–10), chromosomal mutations represent the source of MTBC genetic diversity. The maximum genetic distance between any two MTBC strains is around 2,500 single-nucleotide polymorphisms (SNPs). Strikingly, studies have highlighted large phenotypic differences between strains involving traits like gene expression, drug resistance, transmissibility, and immune response, despite this limited variation. In some cases, the mutations driving phenotypic differences have been identified—for example, nonsynonymous variants in genes, such as rpoB, katG, or gyrA, cause drug-resistant phenotypes (11–13). Furthermore, single mutations in regulatory elements can induce alterations to downstream gene expression, which can foster differential virulence characteristics (14, 15). Finally, specific gene mutations may affect transmission (9), host tropism within the complex (16), and the host immune response (17). However, many of the genomic determinants of these phenotypes remain elusive, despite robust evidence that they are driven by genetic differences between strains (18, 19).Several types of evolutionary forces play crucial roles in the fixation of mutations in bacterial populations. Previous research has provided evidence for the ongoing positive selection of specific genes and regions (9, 20–23), while other studies have reported ongoing purifying selection of specific genomic regions, especially in epitopes and essential genes (24). Additionally, there exists some evidence that genetic drift may have significant functional and evolutionary consequences (25).Detecting selection in MTBC at the genome-wide level remains a challenging task due to limited genetic diversity. The significant accumulation of nonsynonymous substitutions has been previously used to characterize patterns of mutation accumulation in large categories of genes (24, 26); however, these studies employed a limited number of strains. Of note, the number of MTBC sequences has undergone a recent and rapid expansion, with studies involving hundreds to thousands of strains. The large number of available sequences has allowed, for example, the estimation of the ratio of nonsynonymous to synonymous substitutions (dN/dS) signatures in more than 10,000 strains (27), thereby allowing the identification of targets of selection with some probably related to host–pathogen interactions. Host–pathogen interaction signals are specially challenging as they are likely obscured by the force exerted by antimicrobial therapies. Weaker signals are also expected in genes related to second-line drugs related to the relative underuse of related treatments and the low abundance of associated resistant strains in genome databases (28).We reasoned that to detect signs of selection, we should focus on when and/or where they occurred in the phylogenetic tree instead of averaging signs across the phylogeny. In this study, we developed a methodology to study temporal signs of selection in MTBC genes and identified positive selection in a larger number of genes than previously described. This allowed the identification of past and currently unknown players in the MTBC evolution, particularly two-component systems (2CSs), related to host adaptation and second-line drug resistance. This methodology can be applied to other tuberculosis settings to explore signs of selection associated with changing selective pressures and could be extremely useful to unravel hidden details in the evolution of other human pathogens.     

上海市市售水产品中副溶血性弧菌的分离、鉴定及耐药性研究

目的了解上海市市售水产品中副溶血性弧菌(Vibrio parahaemolyticus,Vp)污染状况和耐药性,为防治Vp引起的食源性疾病提供依据。方法采集上海市各大农贸市场3类水产品共273份检测Vp,用统计学处理分析结果;K-B法进行药敏试验。结果水产品中Vp平均检出率为38.46%,其中甲壳类的检出率为50.96%,贝类为27.12%,鱼类为15.79%,三者间有极显著差异(P<0.01)。药敏试验结果显示,105株分离菌株对头孢曲松、萘啶酸和诺氟沙星100%敏感,对氨苄西林的耐药率达69.52%,有20株对多种抗生素耐药。结论上海市售水产品中Vp污染比较严重,药敏结果对多种抗生素耐药。     

宁波地区海产品及环境中副溶血弧菌主要毒力及耐药性分析

目的了解浙江省宁波地区海产品和环境中副溶血弧菌主要毒力和耐药性。方法采用生化反应、抗菌药物敏感性试验及PCR方法对分离的宁波地方副溶血弧菌进行毒力及耐药性测定。结果 90株分离株中,耐热直接溶血素基因（tdh）阳性率为2.2%,与神奈川溶血表型（KP,Kanagawa phenomenon）一致,但在tdh＋和KP阳性的分离株中未检测到Ⅲ型分泌系统2（T3SS2）;trh与ureC基因携带率分别为20.0%和12.2%,而在11株trh＋-ureC＋菌株中未显示尿素酶表型阳性,2株尿素酶阳性的菌株未携trh或ureC基因;Ⅵ型分泌系统的携带率为17.8%。所有的分离株对氟喹诺酮类、氯霉素类、四环素类药物敏感;72%以上分离株对青霉素类、磺胺类及氨基糖苷类中链霉素和卡那霉素耐药;分离株中至少耐2种药物,最多耐10种药物,超过82%的分离株对6种以上药物耐药。耐药基因tetB的检出率为0,bla TEM、sul2和strB的携带率分别为91.7%、16.7%和43.3%。结论宁波地区相当比例的菌株携带毒力,并呈现不同程度耐药。     

江苏省部分地区淡水产品中弧菌菌群及其致病性分析

目的 了解江苏省市场上淡水产品中弧菌的菌群组成及其致病性。方法 对采集的江苏省部分地区淡水产品样本经TCBS培养基分离、纯化弧菌分离株,应用特异性基因tlh、HSP60以及16S rDNA PCR方法鉴定;并检测致病基因、溶血性和引起小鼠肠积水能力分析其致病性。结果 江苏省部分地区55份淡水产品中分离出细菌分离株256株,分别是副溶血弧菌占74.55%、溶藻弧菌占85.45%、哈维氏弧菌占9.1%、河口弧菌占3.6%、麦奇尼科夫弧菌占5.5%、天蓝色弧菌占3.6%株、产钠弧菌占1.8%和重氮养弧菌占1.8%。所有分离株均未检出致病相关基因tdh、trh;15.02%分离株有溶血性(KP⁺);大部分KP⁺分离株能引起小鼠肠积水。结论 江苏省淡水产品中弧菌菌群呈现多样性分布,副溶血弧菌和溶藻弧菌是主要菌群;不同地区和不同样品中的弧菌菌群和检出率有较大差异,沿海地区的检出率明显高于内陆地区,鱼、虾中弧菌的检出率和弧菌种类明显高于贝、蟹。致病性分析说明小部分分离菌株可能具有潜在的致病性