Anthrax is an acute infectious zoonotic disease caused by Bacillus anthracis, a bacterium that is considered a potential biological warfare agent. Bacillus bacteriophages shape the composition and evolution of bacterial communities in nature and therefore have important roles in the ecosystem community. B. anthracis phages are not only used in etiological diagnostics but also have promising prospects in clinical therapeutics or for disinfection in anthrax outbreaks. In this study, two temperate B. anthracis phages, vB_BanS_A16R1 (A16R1) and vB_BanS_A16R4 (A16R4), were isolated and showed siphovirus-like morphological characteristics. Genome sequencing showed that the genomes of phages A16R1 and A16R4 are 36,569 bp and 40,059 bp in length, respectively. A16R1 belongs to the genus Wbetavirus, while A16R4 belongs to the genus Hubeivirus and is the first phage of that genus found to lyse B. anthracis. Because these two phages can comparatively specifically lyse B. anthracis, they could be used as alternative diagnostic tools for identification of B. anthracis infections.

What is already known about this topic? Antibiotic resistance (AR) is a serious public health threat worldwide. However, the AR and antibiotic resistance genes (ARGs) data from West Africa, especially from Sierra Leone, are limited. What is added by this report? The study revealed ARGs’ common dissemination, and multiplex antibiotic resistance genes in one sample. Genes blaNDM and blaOXA-48-like were first discovered in Sierra Leone. What are the implications for public health practice? Basic information is provided for AR research and surveillance and highlights that effective AR surveillance among diarrhea patients is necessary for Sierra Leone and West Africa.

Introduction: The epidemic of human anthrax is at a low level in China in recent years, but the reported incidence increased in 2021. In order to understand the current landscape of research and knowledge about anthrax in China, the epidemiological characteristics of anthrax in humans from 2018 to 2021 were analyzed and the prevention and control suggestions were proposed. Methods: Surveillance data of anthrax in humans and livestock, together with human outbreaks data during 2018-2021, were collected and analyzed by descriptive statistics methods. The number and proportion of outbreaks, cases and deaths by provincial-level administrative divisions (PLADs), clinical types, and contributing factors were calculated. Results: A total of 1,244 cases of human anthrax and 53 outbreaks were reported from 2018 to 2021 in China. While the incidence of anthrax declined from 2018 to 2020, it increased in 2021. The regions of anthrax were mainly located in the west and the northeast PLADs of China, though cases were reported in some central and eastern PLADs in 2021. Young and middle-aged men involved in animal husbandry were found to be at a higher risk of anthrax. All the reported outbreaks were associated with the exposure of infected livestock. A total of 296 livestock anthrax cases were reported. Conclusions: The increased incidence and wider geographical distribution of human anthrax in 2021 were found to be the result of inadequate supervision of diseased animals as well as updated diagnostic criteria. As such, the monitoring of risk factors and emergency preparation procedures should be strengthened at the national level. In addition, it is also critical to strengthen health education for high-risk occupational groups and strengthen professional training for local clinicians. Finally, more measures should be carried out to strengthen anthrax surveillance in livestock husbandry.

On 12 November 2019, one couple from the Sonid Left Qi (County) in the Inner Mongolia Autonomous Region was diagnosed with pneumonic plague in Beijing. The wife acquired the infection from her husband. Thereafter, two bubonic plague cases were identified in Inner Mongolia on November 16th and 24th. In this study, genome-wide single nucleotide polymorphism (SNP) analysis was used to identify the phylogenetic relationship of Yersinia pestis strains isolated in Inner Mongolia. Strains isolated from reservoirs in 2018 and 2019 in Inner Mongolia, together with the strain isolated from Patient C, were further clustered into 2.MED3m, and two novel lineages (2.MED3q, 2.MED3r) in the 2.MED3 population. According to the analysis of PCR-based molecular subtyping methods, such as the MLVA 14 scheme and seven SNP allele sequencing, Patients A/B and D were classified as 2.MED3m. In addition, strains from rodents living near the patients’ residences were clustered into the same lineage as patients. Such observations indicated that human plague cases originated from local reservoirs. Corresponding phylogenetic analysis also indicated that rodent plague strains in different areas in Inner Mongolia belong to different epizootics rather than being caused by spreading from the same epizootic in Meriones unguiculatus in 2019.

为解决现有内孔检测存在的无法测量、测量精度存在偏差、无法满足技术要求等问题,提供一种内孔水平度检测装置及其检测方法.此内孔水平度检测装置及其检测方法,属于装备制造领域,装置包括侨板及光学合像水平议,桥板包括桥板体、园柱支撑及水准仪,桥板体的底面对称设有2个圆柱支撑,顶面安装有水准仪,2个圆柱支撑的母线与被测内孔的母线在检测时线性接触重合,光学合像水平仪在检测时放置在处于水平的桥板体上,将桥板平放在内孔底面上,使水准仪中水银泡处在中间位置来找平桥板体,2个圆柱支撑与内孔底母线平行且完全贴合,将光学合像水平仪放在桥板体上,转动微分盘旋转钮,从窗口看到气泡两端的半边像合在一起,从微分盘上读出刻度值.此装置及检测方法满足了产品组装测量找正需求,不仅保证了产品组装质量,同时缩短了测量找正时间,提高生产效率.

The objective of this study was to construct a rapid, high-throughput, and biosafety-compatible screening method for Bacillus anthracis and Bacillus cereus based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS coupled to ClinProTools was used to discover MALDI-TOF MS biomarker peaks and generate a classification model based on a genetic algorithm (GA) to differentiate between different Bacillus anthracis and Bacillus cereus isolates. Thirty Bacillus anthracis and 19 Bacillus cereus strains were used to construct and analyze the model, and 40 Bacillus strains were used for validation. For the GA screening model, the cross-validation values, which reflect the ability of the model to handle variability among the test spectra, and the recognition capability values, which reflect the model's ability to correctly identify its component spectra, were all 100%. This model contained 10 biomarker peaks (m/z 3,339.9, 3,396.3, 3,682.4, 5,476.7, 6,610.6, 6,680.1, 7,365.3, 7,792.4, 9,475.8, and 10,934.1) used to correctly identify 28 Bacillus anthracis and 12 Bacillus cereus isolates from 40 Bacillus isolates, with a sensitivity and specificity of 100%. With the obvious advantages of being rapid, highly accurate, and highly sensitive and having a low cost and high throughput, MALDI-TOF MS ClinProTools is a powerful and reliable tool for screening Bacillus anthracis and Bacillus cereus strains.

目的 研究蜡样芽胞杆菌毒力基因在不同标本中的分布特征.方法 分离自环境监测标本(米粉、奶粉、土壤)和疾病相关标本(米饭、凉皮、眼内炎和肿瘤患者)的蜡样杆菌333株,PCR扩增蜡样杆菌11个毒力基因,包括溶血性BL基因(hblC、hblD、hblA、hblB)、非溶血性基因(nheA、nheB、nheC)、肠毒素FM基因和T基因(entFM、bceT)、细胞毒素K基因(cytK)和呕吐毒素相关基因(ces),统计不同标本菌株中毒力基因的携带数目和各毒力基因的携带率,方差分析和卡方检验比较毒力基因在不同标本中,尤其是在疾病相关标本与环境监测标本中的携带差异.结果 研究菌株携带毒力基因平均数目为5.97个,88.29％的菌株携带至少3个毒力基因,12.31％的菌株携带除ces外的所有基因.标本携带毒力基因数目从高到低依次为患者(8.75)、凉皮(8.20)、米饭(7.13)、土壤(6.22)、米粉(5.78)和奶粉(5.71).患者、凉皮分别与奶粉的基因数目两两比较有统计学差异显著性.菌株各毒力基因的携带率从高到低依次为非溶血性基因(89.19％)、entFM基因(79.88％)、bceT基因(49.85％)、溶血性BL基因(48.35％)、ctyK基因(47.75％)和ces基因(1.50％).溶血性基因在疾病相关标本中的携带率比环境监测标本高(x2 =8.230,P＜0.01),其余基因携带率则在两类标本中无统计学差异.环境监测标本中,土壤的溶血性基因携带率高于米粉和奶粉(x2=15.071,P＜0.01),非溶血性基因和entFM基因的携带率则低于后两者,检验值分别为(x2=9.603,P＜0.05)和(x2=21.634,P＜0.01).结论 蜡样杆菌毒力基因在不同标本中的分布,尤其是毒力基因数目和溶血性基因在疾病相关标本中较高的携带特点,为研究蜡样杆菌的致病性提供了一定的参考依据,具有一定的临床意义.

目的 筛选蜡样芽胞杆菌鉴定基因和快速检测毒力基因.方法 选择分离自食品和土壤的代表性蜡样芽胞杆菌共329株,聚合酶链式反应(PCR)方法检测gyrB和groEL基因的种特异性,检测毒力基因在菌株中的分布特征.基于检测结果,用多重PCR检测方案快速检测蜡样芽胞杆菌鉴定基因及其毒力因子.结果 在蜡样芽胞杆菌及其近缘芽胞杆菌中,除1株苏云金芽胞杆菌扩增阳性外,gyrB基因具有蜡样芽胞杆菌种特异性;而groEL基因在4种芽胞杆菌中均有扩增.6种毒力基因nheA、entFM、bceT、hblC、cytK和ces的携带率分别为84.19％、79.64％、49.24％、47.72％、47.11％和1.52％.选择nheA、hblC、entFM、ces、cytK和gyrB用于快速鉴定蜡样芽胞杆菌及其毒力基因,获得了双重PCR扩增体系(gryB和cytK)与4重PCR扩增体系(nheA、hblC、entFM和ces)的最佳检测方案.结论 筛选的蜡样芽胞杆菌鉴定基因和毒力基因能够全面、特异、简便、高效地检测蜡样芽胞杆菌,可为食品安全检测及快速诊断提供依据,在实际检验工作中具有良好的应用前景.

目的 对某校疑似炭疽疫苗菌株毒力恢复事件(事件)中,教学实验使用的炭疽芽孢杆菌毒力及其环境污染情况进行鉴定和调查,为合理处理该事件提供参考依据.方法 调查事件发生的情况、采集实验用培养物、冻存菌株和实验室环境样本.对实验用培养物进行噬菌体裂解和青霉素敏感试验鉴定;选取炭疽芽孢杆菌的rpoB基因和毒力相关的pagA和capC基因,应用TaqMan荧光探针法对采集样本进行检测.结果 实验用培养物的噬菌体裂解试验显示有明显的噬菌斑,青霉素敏感试验显示在青霉素纸片周围有明显的抑菌环,并且培养物和冻存菌株rpoB和pagA基因检测为阳性,capC基因检测为阴性;教学场所的环境样本rpoB、pagA和capC基因检测均为阴性.结论 该事件中实验用培养物和冻存菌株均为缺少capC基因的减毒炭疽芽孢杆菌,未发现毒力恢复情况;教学场所中无炭疽芽孢杆菌的污染.

目的 总结分析内蒙古自治区(内蒙古)炭疽暴发疫情中炭疽病例诊断方法的应用经验和问题,为炭疽预防控制工作提供参考.方法 用载玻片直接蘸取和无菌棉签擦取疑似炭疽病例皮损渗出液,同时采集疑似病例静脉血标本.涂片镜检、直接分离培养、肉汤增菌后分离培养炭疽菌、炭疽芽胞杆菌实时荧光定量PCR扩增(毒素质粒的pagA、染色体rpoB、荚膜质粒的cap)以及ELISA法检测双份血清炭疽抗体.结果 2018年8月,内蒙古通辽市疾病预防控制中心实验室共采集17例炭疽病例标本,采集时间距发病时间最短1d,最长16d,中位数为6d.15例可疑病例用过抗生素后采集标本(15/17,88.24％).从1份标本中分离到炭疽芽胞杆菌,分离阳性率为5.88％(1/17);实时荧光PCR检测17份标本,阳性率为41.18％ (7/17);ELISA检测保护性抗原抗体阳性率为83.33％ (10/12).结论 在最佳时间采集标本是疫情早期定性的关键.推荐炭疽疫情中应用快速灵敏的实时荧光PCR检测诊断方法,应采集所有可疑病例双份血清提高病例诊断率.

BackgroundLeptospirosis is one of the most important neglected tropical bacterial diseases worldwide. However, there is limited information on the genetic diversity and host selectivity of pathogenic Leptospira in wild small mammal populations.Methodology/principal findingsJiangxi Province, located in southern China, is a region highly endemic for leptospirosis. In this study, among a total of 3,531 trapped rodents dominated by Apodemus agrarius (59.7%), 330 Leptospira strains were successfully isolated from six different sites in Jiangxi between 2002 and 2015. Adding 71 local strains from humans, various kinds of livestock and wild animals in Jiangxi, a total of 401 epidemic strains were characterized using 16S rRNA gene senquencing, multilocus sequence typing (MLST) and the microscopic agglutination test (MAT). Among them, the most prevalent serogroup was Icterohaemorrhagiae (61.10%), followed by Javanica (19.20%) and Australis (9.73%); the remaining five serogroups, Canicola, Autumnalis, Grippotyphosa, Hebdomadis and Pomona, accounted for 9.97%. Species identification revealed that 325 were L. interrogans and 76 were L. borgpetersenii. Moreover, L. interrogans was the only pathogenic species in Fuliang and Shanggao and was predominant in Shangrao (95.0%); L. borgpetersenii was the most common in the remaining three sites. Twenty-one sequence types (STs) were identified. Similarly, ST1 and serogroup Icterohaemorrhagiae were most prevalent in Shangrao (86.0% and 86.4%) and Fuliang (90.4% and 90.4%), ST143 and serogroup Javanica in Shangyou (88.5% and 90.4%) and Longnan (73.1% and 73.1%), and ST105 and serogroup Australis in Shanggao (46.3% and 56.1%). Serogroup Icterohaemorhagiae primarily linked to A. agrarius (86.9%), serogroup Canicola to dogs (83.3%). There were significant differences in the distribution of leptospiral species/serogroups/STs prevalence across host species/collected locations among the 394 animal-associated strains (Fisher's exact test, p<0.001).Conclusions/significanceOur study demonstrated high genetic diversity of pathogenic Leptospira strains from wild small animals in Jiangxi from 2002 to 2015. A. agrarius was the most abundantly trapped animal reservoir, and serogroup Icterohaemorrhagiae and ST1 were the most dominant in Jiangxi. Significant geographic variation and host diversity in the distribution of dominant species, STs and serogroups were observed. Moreover, rat-to-human transmission might play a crucial role in the circulation of Leptospirosis in Jiangxi. Details of the serological and molecular characteristics circulating in this region will be essential in implementing prevention and intervention measures to reduce the risk of disease transmission in China. However, phylogenetic analysis of more Leptospira isolates should explore the impact of ecological change on leptospirosis transmission dynamics and investigate how such new knowledge might better impact environmental monitoring for disease control and prevention at a public health level.

目的 对贵州省黔西南州兴义市患者关节液中分离的疑似土拉弗朗西斯菌(土拉菌)进行鉴定.方法 将患者关节液中分离的1株革兰阴性菌(经全自动细菌鉴定及药敏分析系统检测,此菌株为土拉菌),接种到土拉菌选择性培养基上进行初步筛选.对新鲜培养的菌株进行土拉菌特异抗原乳胶凝集检测;并用16S rRNA的两对引物27f和1492r、8-27f和1500r对菌株进行菌种鉴定.采集患者血清,分别采用玻片法和试管法进行土拉菌特异抗体乳胶凝集检测.结果 该菌在土拉菌选择性培养基上不生长.土拉菌特异抗原乳胶凝集检测为阴性,采用玻片法和试管法进行土拉菌特异抗体乳胶凝集检测,均为阴性.用两对引物扩增分别得到1 379 nt和1 429nt的片段,经测序和比对分析,该菌株与阿萨姆类芽胞杆菌GPTSA 11的16S rRNA基因具有高度一致性,覆盖率为100％,E值为0.两个片段均含有类芽胞杆菌属特有的保守信号序列PAEN 515F和PAEN 862F.结论该病例排除土拉菌感染,为阿萨姆类芽胞杆菌引起感染,是首例阿萨姆类芽胞杆菌感染的临床病例.

Objective To compare the coding sequences (CDS) of Yersinia pestis D106004 strain from Yulong County in Yunnan Province and Z176003 strain from Qing-Tibet Plateau in order to find the differences between their genomes and the genetic characteristics. Methods The CDS of Yersinia pestis D106004 strain and Z176003 strain were searched and compared by BLAST. Twenty-two differential CDS were selected to design 22 pairs of primers. PCR amplification was carried out in 119 representative plague strains from different isolation sources (natural foci of Himalayan marmot plague in the Qinghai-Tibet Plateau, natural foci of Apodemus chevrieri and Eothenomys miletus plague in Yunnan), time span of about 50 years, and distribution in six ecological types including Tibet, Qinghai, Sichuan, Gansu and Yunnan, and PCR products were sequenced and verified. The strains were all from the State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention. Results In 119 representative plague strains of 6 ecological types, the cumulative sequence length of 22 differential CDS PCR amplification products was 2.13 × 106 bp. Among the 119 representative plague strains in the foci of Yulong D106004 strain and Qinghai-Tibet Plateau Z176003 strain, 22 differential CDS had high homology, there was no difference in 78.2％ (2047/2618) sequences of differential CDS, and 21.8％ (571/2618) sequences had three types of gene mutations ( deletion , missense and frameshift mutations). The characteristics of the differences were stable in the 6 ecological plague strains of the foci, and they were divided into 6 geographical distributions. Conclusion Yulong D106004 strain and Qinghai-Tibet Plateau Z176003 strain have high homology, close genetic relationship, and little difference in genome, but the genetic characteristics of different ecotype strains are stable.

On December 14, 2017, a faculty member of a university in Hunan Province reported that an anthrax vaccine strain might have recovered virulence during an undergraduate experiment and potential exposure could not be ruled out for the students involved. Upon receiving the case report, the CDC, health bureaus, and local governments at the county, prefectural, and provincial levels promptly organized experts in different fields (including epidemiologists, biosafety experts, and laboratory testing experts) for case investigation, evaluation, and response. As the investigation results showed, no virulence recovery was identified in the involved anthrax vaccine strain; and no contamination of Bacillus anthracis was detected at the involved areas. Thus, the university returned to normal functioning.

目的 原核表达炭疽杆菌PA63蛋白,以此为包被抗原建立检测相应抗体的间接ELSIA方法,用于炭疽血清免疫学诊断. 方法 选择炭疽杆菌PA63为目的基因,合成全长序列,构建pCzn1-PA63质粒,克隆至E.coli ArcticExpress表达菌株,优化IPTG表达条件,通过包涵体变性复性,Ni柱纯化获得可溶性PA63蛋白.利用棋盘滴定法确定包被PA63蛋白的最佳浓度及血清最佳稀释度建立间接ELISA方法,对炭疽阳性血清和阴性血清进行检测,计算ROC曲线下面积,确定Cut-off值,灵敏度与特异度. 结果 成功构建了pCzn1-PA63质粒,IPTG 37℃诱导表达蛋白主要以包涵体形式存在.包涵体经变性、复性,纯化后获得PA63蛋白.用5 μg/ml PA63包被酶标板,血清稀释度为1∶50建立的ELISA方法;ROC 曲线下面积为0.969(P＜0.01),Cut-off值为0.2865时的灵敏度为91.18％(95％可信区间为76.32％-98.14％),特异度为94.64％(95％可信区间为85.13％-98.88％). 结论 成功表达具有生物活性的重组炭疽杆菌PA63抗原蛋白,以此为包被抗原建立的间接ELISA检测炭疽PA抗体,具有较高的灵敏度与特异度,可用于炭疽的血清学诊断.

Paenibacillus assamensis is a bacterium usually found in warm springs. We detected P. assamensis in a man with suspected tularemia. The strain isolated from the man’s knee joint fluid was identified as P. assamensis after analysis of a homologous sequence of the 16S rRNA gene.