Correlation between the distribution pattern of virulence genes and virulence of Aeromonas hydrophila strains

Nine strains of Aeromonas hydrophila isolated from diseased fish or soft-shelled tortoise were tested for the presence of three virulence genes including the genes encoding aerolysin,hemolysin,and extracellular serine protease (i.e.,aerA,hlyA,and ahpA,respectively).These genes were investigated using polymerase chain reaction (PCR)with specific primers for each gene.And the pathogenicities to Carrassius auratus ibebio of these strains were also assayed.PCR results demonstrated that the distribution patterns of aerA,hlyA,and ahpA were different in these strains.6/9 of A.hydrophila strains were aer A positive,8/9 of strains hly A positive,7/9 of strains ahp A positive,respectively.However,the assay for pathogenesis showed that two strains (A.hydrophila XS91-4-1 and C2)were strong virulent,two strains (A.hydrophila ST78-3-3 and 58-20-9)avirulent and the rest middle virulent was to the fish.In conclusion,there are significant correlation between the distribution pattern of the three virulence genes and the pathogenicity to Carrassius auratus ibebio.All strong virulent A.hydrophila strains were aerA+hlyA+ahpA+genotype,and all aerA+hlyA+ahpA+strains were virulent.Strains with the genotype of aerA-hlyA-ahpA+have middle pathogenicity.In the present study,we found for the first time that all A.hydrophila isolated from the ahpA positive were virulent to Carrassius auratus ibebio.Additionally,there was a positive correlation between the virulence of A.hydrophila and the presence of aerA and ahpA.     

多重聚合酶链反应检测猪链球菌7种主要毒力因子

目的 建立2个分开的多重聚合酶链反应(PCR)体系,以及对猪链球菌7种主要毒力因子的检测。方法 根据猪链球菌7种主要毒力因子mrp、epf、sly、gdh、gapdh、orf2和fbps的基因序列,设计和合成7对特异性引物,通过对它们在2个分开反应体系PCRⅠ和PCRⅡ中的组合和优化,建立猪链球菌7种主要毒力因子的2组多重PCR检测方法,并对实验室的29株背景明确的猪链球菌保存菌株进行检测。结果 29株猪链球菌菌株的检测结果和菌株的背景情况一致,阳性、阴性对照均成立。结论 此猪链球菌7种主要毒力因子的2组分开体系的多重PCR检测方法,特异性和敏感性均好,可用于快速诊断以及猪链球菌毒力因子的分子流行病调查。     

Identity and virulence properties of Aeromonas isolates from diseased fish, healthy controls and water environment in China

Aims: To investigate the species distribution in Aeromonas isolates from diseased fish, healthy controls and water environment in China; to evaluate the frequency of the aerolysin (aer), cytotonic enterotoxin (alt), cytotoxic enterotoxin (act), temperature‐sensitive protease (eprCAI) and serine protease (ahp) genes in Aeromonas isolates; and to determine the potential pathogenicity of these isolates. Methods and Results: Two hundred and two Aeromonas isolates from diseased fish (n = 42), healthy fish (n = 120) and water environment (n = 40) in China were identified to species levels based on sequencing of the housekeeping gene gyrB, while the distribution of five virulence factors, including aer, alt, act, eprCAI and ahp, was investigated by PCR. Aeromonas veronii (25/42; 60%) and Aeromonas hydrophila (14/42; 33%) were the species most commonly isolated from diseased fish, while Aer. veronii was the most common species in healthy fish (90/120; 75%) and water samples (25/40; 62·5%). All the five virulence genes were present in 9% (19/202), among which 10 strains were from diseased fish and nine were identified as Aer. hydrophila. For the strains carrying five virulence genes, the average 50% lethal doses (LD_50s) of strains from diseased fish were lower when compared with the strains from healthy fish and water environment. Conclusions: Aeromonas veronii is the most common species, but no significant difference exists in the isolates obtained from diseased fish and from healthy fish. However, Aer. hydrophila isolates were significantly more frequent from diseased fish than from healthy fish. aer⁺alt⁺act⁺eprCAI⁺ahp⁺ was more frequent virulence genotype in Aeromonas isolates from diseased fish than from healthy fish and water environment, and the aer⁺alt⁺act⁺eprCAI⁺ahp⁺ isolates were more virulent to zebrafish comparing to the other genetic profiles. Significant and Impact of the Study: Aeromonas species in aquatic environments are various and have considerable virulence potential, and therefore, there is a need for more careful and intensive epidemiology studies.     

Co-migration of RAPD-PCR amplicons from Aeromonas hydrophila

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) uses arbitrary primers and low stringency annealing conditions to amplify anonymous DNA fragments which are then depicted in agarose gels. RAPD-PCR fingerprints have been used for typing and differentiation of bacteria and, increasingly, for the study of genetic relationships between strains and species of microorganisms, plants and animals. The analysis of such fingerprints is based upon the assumption that co-migration of amplicons does not occur and that any given band contains a single amplicon. This report shows that co-migration of fragments of nearly identical size, but different nucleotide sequences, occurs between different isolates and within single RAPD-PCR bands from Aeromonas hydrophila. The possibility of the same phenomenon occurring for other prokaryotic or eukaryotic genomes argues for caution in the interpretation of RAPD-PCR fingerprints.     

Correlation between the distribution pattern of virulence genes and virulence of Aeromonas hydrophila strains

Nine strains of Aeromonas hydrophila isolated from diseased fish or soft-shelled tortoise were tested for the presence of three virulence genes including the genes encoding aerolysin, hemolysin, and extracellular serine protease (i.e., aerA, hlyA, and ahpA, respectively). These genes were investigated using polymerase chain reaction (PCR) with specific primers for each gene. And the pathogenicities to Carrassius auratus ibebio of these strains were also assayed. PCR results demonstrated that the distribution patterns of aerA, hlyA, and ahpA were different in these strains. 6/9 of A. hydrophila strains were aerA positive, 8/9 of strains hlyA positive, 7/9 of strains ahpA positive, respectively. However, the assay for pathogenesis showed that two strains (A. hydrophila XS91-4-1 and C2) were strong virulent, two strains (A. hydrophila ST78-3-3 and 58-20-9) avirulent and the rest middle virulent was to the fish. In conclusion, there are significant correlation between the distribution pattern of the three virulence genes and the pathogenicity to Carrassius auratus ibebio. All strong virulent A. hydrophila strains were aerA ⁺ hlyA ⁺ ahpA ⁺ genotype, and all aerA ⁺ hlyA ⁺ ahpA ⁺ strains were virulent. Strains with the genotype of aerA ⁻ hlyA ⁻ ahpA ⁺ have middle pathogenicity. In the present study, we found for the first time that all A. hydrophila isolated from the ahpA positive were virulent to Carrassius auratus ibebio. Additionally, there was a positive correlation between the virulence of A. hydrophila and the presence of aerA and ahpA. __________ Translated from Acta Scientiarum Naturalium Universitatis Sunyatseni, 2006, 45(1): 82–85 [译自: 中山大学学报 (自然科学版)]     

Characterization of Aeromonas and Vibrio species isolated from a drinking water reservoir

AIMS: To study the phenotypic and chemotaxonomic (i.e. phospholipid and cellular fatty acid composition) characteristics of environmental Aeromonas spp. and Vibrio spp. isolated from a drinking water reservoir near Vladivostok City, and the application of some chemotaxonomic markers for discrimination of the two genera and species. METHODS AND RESULTS: Presumptive Aeromonas species were dominant in surface water samples (up to 25% of the total number of bacteria recovered). These strains were consistent with respect to the cultural and biochemical properties used to define the species Aeromonas sobria (seven strains) and Aer. popoffii (three strains). Vibrio mimicus (two strains) and Vibrio metschnikovii (one strain) were identified according to phenotypic features and cellular fatty acid composition. CONCLUSION: Environmental Aer. sobria isolates were atypical in their ability to grow at 42 degrees C, and were haemolytic, proteolytic and cytotoxic. Although it was present in a high proportion in the water samples, atypical Aer. sobria is not an indicator of polluted water. SIGNIFICANCE AND IMPACT OF THE STUDY: The incidence of Aeromonas in the drinking water reservoirs in the Far East of Russia is reported for the first time.     

饮用水系统硝化微生物分布规律、环境影响因素及调控应用的研究进展

硝化微生物广泛存在于饮用水系统中。水处理过程中,硝化微生物对含氮污染物的去除有突出贡献;而输配水过程中,硝化微生物会加剧消毒剂氯胺的降解,造成一系列饮用水微生物安全问题。本文介绍了常用硝化微生物检测方法,综述了硝化微生物在滤池、市政主管网、二次供水系统中的分布特征和规律,分析了环境因子及工程条件对硝化微生物的影响机制,探讨了硝化微生物强化应用及管控的实际工程措施,展望了未来饮用水系统中硝化微生物的研究重点与应用前景。     

The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water

Current models to study Legionella pathogenesis include the use of primary macrophages and monocyte cell lines, various free-living protozoan species and murine models of pneumonia. However, there are very few studies of Legionella spp. pathogenesis aimed at associating the role of biofilm colonization and parasitization of biofilm microbiota and release of virulent bacterial cell/vacuoles in drinking water distribution systems. Moreover, the implications of these environmental niches for drinking water exposure to pathogenic legionellae are poorly understood. This review summarizes the known mechanisms of Legionella spp. proliferation within Acanthamoeba and mammalian cells and advocates the use of the amoeba model to study Legionella pathogenicity because of their close association with Legionella spp. in the aquatic environment. The putative role of biofilms and amoebae in the proliferation, development and dissemination of potentially pathogenic Legionella spp. is also discussed. Elucidating the mechanisms of Legionella pathogenicity development in our drinking water systems will aid in elimination strategies and procedural designs for drinking water systems and in controlling exposure to Legionella spp. and similar pathogens.     

Detection of virulence associated genes in clinical strains of vibrio mimicus

A total of 42 clinical strains of Vibrio mimicus were examined for the presence of virulence associated genes toxR, toxS, toxT, tcpP, ctx and tcpA by PCR assay. Almost all strains were shown to have the toxR gene, while the toxS gene was found in 27 strains. On the other hand, five strains possessed both toxT and tcpP genes, but others had neither. Only two strains were positive for amplification of the ctx gene, whereas no PCR product with tcpA primers was detected. The results indicate the incomplete copies of virulence cascade in V mimicus strains. The pathogenesis and epidemic potential of this species is also discussed.     

Sequences from the aspergillopepsin PEP gene of Aspergillus fumigatus: evidence on their use in selective PCR identification of Aspergillus species in infected clinical samples

In immunodeficient patients, Aspergillus species emerge as circumstantial pathogens. Aspergillus fumigatus is a distant first among the pathogenic aspergilli, which cause deep-seated mycoses. Sequences of the pep gene of A. fumigatus as potential PCR primers, which have not been tested before, were used to identify this species and if possible, differentiate it from other, co-identified, clinically important species of the genus. We present results of the three most promising primer pairs, pep-1/pep-22, pep-15/pep-22 and pep-21/pep32. The second pair was of better specificity when tested with DNA extracted from pure cultures of a multitude of aspergilli, whereas the first co-amplified four clinically significant Aspergillus species. The compatibility of the PCR method with the CTAB DNA extraction protocol varied according to the biological fluid tested and the primer pair used. The first two pairs showed moderate adaptability to the different commercial DNA extraction kits, which were tested in whole blood, spiked with Aspergillus fumigatus hyphae and conidia - as were all the biological fluids used. Restriction of the amplification products with MspI produced distinct patterns for different Aspergillus spp. This approach, as a potential diagnostic tool, seems reliable and sensitive due to its flexibility, speed, low cost, ease of application and selectable breadth of detection.     

The occurrence of Campylobacter in river water and waterfowl within a watershed in southern Ontario,Canada

Aims: Quantitative PCR and a culture method were used to investigate Campylobacter occurrence over 3 years in a watershed located in southern Ontario, Canada that is used as a source of drinking water. Methods and Results: Direct DNA extraction from river water followed by quantitative PCR analysis detected thermophilic campylobacters at low concentrations (<130 cells 100 ml^?1) in 57–79% of samples taken from five locations. By comparison, a culture‐based method detected Campylobacter in 0–23% of samples. Water quality parameters such as total Escherichia coli were not highly correlated with Campylobacter levels, although higher pathogen concentrations were observed at colder water temperatures (<10°C). Strains isolated from river water were primarily nalidixic acid‐susceptible Campylobacter lari, and selected isolates were identified as Campylobacter lari ssp. concheus. Campylobacter from wild birds (seagulls, ducks and geese) were detected at a similar rate using PCR (32%) and culture‐based (29%) methods, and although Campylobacter jejuni was isolated most frequently, C. lari ssp. concheus was also detected. Conclusions: Campylobacter were frequently detected at low concentrations in the watershed. Higher prevalence rates using quantitative PCR was likely because of the formation of viable but nonculturable cells and low recovery of the culture method. In addition to animal and human waste, waterfowl can be an important contributor of Campylobacter in the environment. Significance and Impact of the Study: Results of this study show that Campylobacter in surface water can be an important vector for human disease transmission and that method selection is important in determining pathogen occurrence in a water environment.     

供水系统非结核分枝杆菌生长因素及控制措施研究进展

近年来,非结核分枝杆菌感染在世界范围内日益普遍,严重威胁公众健康。供水系统是非结核分枝杆菌重要环境来源和主要传播途径,但目前对供水系统非结核分枝杆菌生长因素及控制措施的认识仍有较多不足。本文介绍了供水系统非结核分枝杆菌的生长传播特征,探讨了多个工程环境因素(如消毒剂、有机碳、管材和温度)和生物因子(如生物膜、阿米巴原虫和细菌)对非结核分枝杆菌丰度和物种多样性特征的影响,分析了供水全流程不同阶段控制措施对非结核分枝杆菌的控制效用,提出了深化认识供水系统非结核分枝杆菌的研究需求。