Environmental survey for four pathogenic bacteria and closely related species using phylogenetic and functional genes

Bacterial species with high DNA sequence similarity to pathogens could affect the specificity of assays designed to detect biological threat agents in environmental samples. The natural presence of four pathogenic bacteria, Bacillus anthracis, Clostridium perfringens, Francisella tularensis, and Yersinia pestis and their closely related species, was determined for a large collection of soil and aerosol samples. Polymerase chain reaction (PCR) and gene sequencing were used using group-specific 16S rRNA primers to identify pathogens and related species, and pathogen-specific virulence genes. Close relatives of B. anthracis (B. cereus group species) were detected in 37% of the soils and 25% of the aerosol samples. The B. anthracis protective antigen (pag) gene or a close homolog was detected in 16 of these samples. For the other three pathogen groups, the frequency of detection was much lower, and none of the samples were positive with both the phylogenetic and virulence gene primer sets.     

广东省发病猪禽大肠杆菌中耶尔森菌强毒力岛的分布

为了解广东省发病猪、禽大肠杆菌中耶尔森菌强毒力岛(HPI)的分布和HPI阳性菌的血清型,从广东省发病的猪、禽养殖场共分离鉴定了608株大肠杆菌,针对耶尔森菌强毒力岛的标志基因irp2基因设计1对引物,对这些大肠杆菌进行了irp2基因的PCR检测;并对HPI阳性菌进行O血清型鉴定。结果显示,有105株菌含有irp2基因,即HPI阳性率为17.27%(105/608)。猪源、鹅源、鸽源、鸡源、鸭源、鹧鸪源大肠杆菌的HPI检出率分别是16.51%、17.81%、21.05%、17.74%、19.01%及0;105株HPI阳性菌的血清型分布较为分散,没有明显规律;主要的血清型有O1、O26、O45、O114、O136、O18等。研究表明,广东省猪、禽养殖场大肠杆菌的HPI携带率较高,血清型复杂。     

禽致病性大肠杆菌中耶尔森菌强毒力岛的分子流行病学调查

为了解耶尔森菌强毒力岛(HPI)在禽致病性大肠杆菌(APEC)中的流行情况,根据HPI结构基因irp2和fyuA参考序列设计了引物,用PCR方法和斑点杂交法对从江苏等地分离的APEC基因组进行了扩增和检测,并对E.coliNTJC040406菌株相关基因进行了克隆和序列分析。结果表明,216株APEC中有44.9%的菌株携带有HPI,序列分析表明相关基因与GenBank中参考序列的同源性高达98%以上。提示HPI在APEC中广泛存在,经进一步分析,发现分离菌株是否携带HPI与O78等特定血清型有一定的相关性。     

Detection of Anthrax and Other Pathogens Using a Unique Liquid Array Technology

A bead‐based liquid hybridization assay, Luminex^® 100?, was used to identify four pathogenic bacteria, Bacillus anthracis, Clostridium botulinum, Francisella tularensis subsp. tularensis, and Yersinia pestis, and several close relatives. Hybridization between PCR‐amplified target sequences and probe sequences (located within the 23S ribosomal RNA gene rrl and the genes related to the toxicity of each bacterium) was detected in single‐probe or multiple‐probe assays, depending on the organism. The lower limits of detection (LLDs) for the probes ranged from 0.1 to 10 ng. Sensitivity was improved using lambda exonuclease to digest the noncomplementary target strand. All contributors in 33 binary, ternary, and quaternary mixtures in which all components were present in a 1:1 ratio were identified with an 80% success rate. Twenty‐eight binary mixtures in which the two components were combined in various ratios were further studied. All target sequences were detected, even when the minor component was overshadowed by a tenfold excess of the major component.     

Investigations into Enhancing Yersinia pestis Cells Viability following Environmental Sampling for Forensic Analysis

Following an intentional or accidental bio-warfare agent (BWA) release, environmental sample analysis is absolutely critical to determine the extent of contamination. When dealing with nonspore forming BWA (e.g., Yersinia pestis), retention of cell viability is central to such analyses. Even though significant advances have been achieved in DNA sequencing technologies, a positive identification of BWAs in environmental samples must be made through the ability of cells to form colony-forming units upon culturing. Inability to revive the cells between collection and analysis renders such studies inconclusive. Commercial kits designed to preserve the viability of pathogens contained within clinical samples are available, but many of them have not been examined for their ability to preserve samples containing suspected BWAs. The study was initiated to examine the applicability of commercial solutions aiding in retention of Y. pestis viability in samples stored under nonpermissive temperatures, that is, 40 and 37°C. While none of the tested solutions sustained cell viability at 40°C, the results show five out of 17 tested preservatives were capable of supporting viability of Y. pestis at 37°C.