To determine whether Yersinia pestis (Y. pestis) signals were present in the soil, 78 soil samples were collected from 10 counties identified as Rattus tanezumi plague foci and another six counties in non-foci areas in Yunnan, China, from August 2024 to May 2025. Nucleic acids were extracted using the DNeasy PowerSoil Pro Kit (Qiagen) and detected with a Bio-Rad QX200 droplet digital PCR (ddPCR) system targeting two Y. pestis genes (caf1 and ypo0392). Our sampling design also considered two additional dimensions: nine soil types and three habitat types. The raw data obtained from ddPCR were copy numbers. A gene was considered positive when its copy number exceeded the limit of detection (LoD). A sample was considered positive if the copy number of ypo0392 exceeded its LoD (regardless of caf1), or if both genes exceeded their respective LoDs. No correlation was detected between the positivity rate of Y. pestis and the division of sampling sites into plague focus and non-focus regions (p = 0.758). Similarly, the Mann-Whitney U test revealed non-significant differences in pathogen copy number across the two site categories, with p = 0.603 for the caf1 gene and p = 0.372 for the ypo0392 gene. No statistical difference in positivity rate was found for either soil types or habitat types. However, for both the grouping variables, a statistical difference in copy number was observed. Our results indicated that quantifying nucleic acid abundance by copy number provided richer information than a simple positive/negative determination. The detection of Y. pestis signals was associated with soil type, rather than with whether an area was classified as a focus or non-focus region. Accordingly, future research on the preservation mechanism of Y. pestis should not be restricted to the concept of natural foci but should adopt a broader perspective.

Plague, one of the most devastating infectious diseases in human history, is caused by the bacterium Yersinia pestis. Since the 1950s, the Dehong Dai–Jingpo Autonomous Prefecture (DH) in Yunnan Province, China, has recorded plague outbreaks that have resulted in 1,153 human cases and 379 deaths. The genetic diversity and transmission characteristics of Y. pestis strains in this region remain unknown. Here, we performed high-resolution genomic epidemiological analysis of 175 Y. pestis strains isolated from five counties and 19 towns in DH between 1953 and 2007. Phylogenetic analysis revealed that most DH strains were located in lineage 1.ORI2, which could be further subdivided into seven sub-phylogroups (SPG1-SPG7). The dominant sub-phylogroups of Y. pestis in DH varied during different periods and presented a population shift. Genomic evidence showed that plague might have emerged from the southwest of DH (e.g., Longchuan or Ruili counties) or its bordering countries, and subsequently spread to the northeast in multiple waves between 1982 and 2007. Our study infers a fine-scale phylogeny and spread pattern of the DH Y. pestis population, which extends our knowledge regarding its genetic diversity and provides clues for the future prevention and control of plague in this region.

目的 了解云南某大型铅锌矿企附近儿童铅污染的情况及其影响因素.方法 于2016年8月7-18日,选择居住在云南省某大型铅锌矿企附近24个村小组的1 380名儿童进行血铅、尿铅测定.结果 血铅中位数为66.0μg/L(1.0～450 μg/L),血铅异常儿童占24.3％,其中高铅血症占18.1％,铅中毒率为6.2％.多因素logistic回归分析显示,男性、大龄、居住地离核心矿区500 m范围内是儿童患高铅血症和铅中毒的危险因素(P＜0.01).男童尿铅中位数为0.5μg/L(0.5～174μg/L),检出率为4.4％,超标率为0.4％.尿铅用于诊断儿童铅中毒其ROC(AUC)为0.513,P＞0.05.饮用水均未检出铅.结论 云南某大型铅锌矿企附近居住儿童已存在不同程度的血铅超标,血铅超标率与性别、年龄、距离有关.

目的 调查云南省普洱市家鼠鼠疫疫源地宿主动物中携带鼠疫噬菌体的情况,并进行鉴定.方法 2015年11月-2016年6月在普洱市的澜沧县、墨江县及思茅区选取9个疫点,采用鼠铗法捕获老鼠,取其肠道标本;以鼠疫疫苗株EV76为饲养菌,采用双层平板法分离鼠疫噬菌体,并对噬菌体进行电镜扫描等鉴定.同时对肠道标本进行鼠疫菌特异基因caf1检测.结果 共采集286份样本,从中分离到4株鼠疫噬菌体,总分离率1.40％;4株鼠疫噬菌体分离自3个疫点,其中澜沧县勐朗镇勐宾村自226份标本中分离到2株,墨江县龙潭乡大沙坝村自4份标本中分离到1株,思茅区云仙乡大石头村自7份标本中分离到1株;4株鼠疫噬菌体中,有3株分离自黄胸鼠,1株分离自斯氏家鼠;4株鼠疫噬菌体初次分离时其噬斑表现出多态性,选择其中2株噬菌体进行电镜扫描,皆为肌尾病毒科噬菌体;所有肠道标本cafl检测阴性.结论 首次在普洱市家鼠鼠疫疫源地中分离到鼠疫噬菌体,所分鼠疫噬菌体为肌尾病毒科噬菌体且具有多态性,值得进一步研究.

目的 阐明云南剑川野鼠疫源地鼠疫菌种群的演化及其与云南省其他疫源地菌株之间的遗传进化关系.方法 按不同时间、不同疫点、不同分离源,随机选取24株剑川分离鼠疫菌进行研究;采用差异区段(DFR)、规律成簇的间隔短回文重复序列(CRISPRs)及多位点可变数目串联重复序列分析(MLVA)进行基因组多态性分析;以DFR+ CRISPRs聚类分析划分簇,再以MLVA26对簇进行聚类分析.结果 24株剑川菌株中,20株菌聚为一个簇(剑川野鼠鼠疫簇);2017年疫情分离3株菌位于丽江野鼠鼠疫簇,与丽江菌株(2017LZ)存在2个位点的差异(N2577,M23);剩余1株为一个独立株;50年代菌株与其邻近的80年代菌株间位点差异未超过3.结论 剑川原野鼠疫源地的发现时间可往前推至上世纪50年代,同时疫情还波及过家鼠和人群;剑川县境内存在2种类型的野鼠疫源地,其鼠疫防控具有复杂性,应继续加强鼠间鼠疫的动态监测.

目的 探讨云南家鼠疫源地鼠疫菌在云南鼠疫菌遗传进化上所处的位置,及其种群流行演化规律. 方法 按照不同地点、时间及分离源随机选取186株家鼠疫源地鼠疫菌,采用差异区段(DFR)、规律成簇的间隔短回文重复序列(CRISPRs)及多位点可变数目串联重复序列分析(MLVA)3种方法进行分子分型分析;以DFR+ CRISPRs双重方法聚类分析划分基因簇,以MLVA26对基因簇再次聚类分析划分亚簇. 结果 186株家鼠疫源地鼠疫菌中的184株聚为一个簇(家鼠鼠疫簇),其余2株为独立株;近史流行期、复燃流行期和2016年疫情分离株分别处于不同的亚簇,它们之间的位点差异至少有5个;复燃流行期菌株共有5个亚簇和6个独立株,其中2个亚簇为主要的基因亚簇,即滇西亚簇和滇西南-滇东-广西-贵州亚簇,它们之间有4个位点差异. 结论 家鼠鼠疫菌是云南鼠疫菌中最晚出现的菌株,近史流行期菌株与复燃流行期菌株菌株存在较大差异,而复燃流行期的鼠疫流行是复燃和扩散并存的结果.

目的 测定分离自云南省澜沧县室外捕获黄胸鼠的1株野生型鼠疫噬菌体(命名为Lc-241)的生物特性,初步探讨其在家鼠鼠疫疫源地微生态的作用. 方法 用透射电镜观察Lc-241形态,双层平板法观察其噬菌斑大小、状态及效价,单层平板法观察鼠疫噬菌体Lc-241对130株菌在28℃、37℃及室温(18℃)条件下的裂解能力. 结果 Lc-241为肌尾噬菌体在28℃时Lc-241较鼠疫诊断噬菌体裂解性低,而在37℃时较鼠疫诊断噬菌体裂解性高;在3种温度下,对6株鼠疫耶尔森菌、1株福氏志贺菌2a型、1株福氏志贺菌X变种完全裂解,对宋内志贺菌I相菌半裂解,对其余菌株均不裂解. 结论 Lc-241鼠疫噬菌体为1株宽裂解谱噬菌体,其对鼠疫菌的裂解作用随温度升高而增强.

Objective To investigate the correlation between the frequency of antibiotics and the resistance rate of Pseudomonas aeruginosa and Acinetobacter baumannii, and to provide guidance for the rational use of antibiotics and the reduction of bacterial resistance. Methods The frequency of antibiotics used in the Third People’s Hospital of Yunnan Province from January 2013 to December 2017, and the drug resistance of Pseudomonas aeruginosa and Acinetobacter baumannii isolated at the same time was investigated and analyzed, retrospectively. Results The frequency of antibiotics used of antibacterial drugs in our hospital was 28 809 in 2014, which increased significantly compared with that of 2013 (16 259). The resistance rates of Pseudomonas aeruginosa to ceftriaxone, cefepxime, aztreonam, gentamicin, levofloxacin, imipenem, meropenem, cefoperazone/sulbactam and piperacillin/tazobactam were significantly increased; and the resistance rates of Acinetobacter baumannii to ceftriaxone, cefepxime, gentamicin, imipenem and meropenem cefoperazone/sulbactam and piperacillin/tazobactam were also increased. Acinetobacter baumannii was more sensitive to cefoperazone/sulbactam, which remained high resistance rates to other commonly used antimicrobial agents, especially to cephalosporins (mean resistance rate was 78.23%) and carbapenems (mean resistance rate was 72.69%). The analysis showed that the resistance rate of Pseudomonas aeruginosa was positively correlated with the frequency of levofloxacin using from 2013 to 2017 (r = 0.70, P = 0.20); and Acinetobacter baumannii was positively correlated with the frequency of meropenem using(r = 0.94, P = 0.02). Conclusions The drug resistance rate of Pseudomonas aeruginosa isolated in our hospital was positively correlated with the frequency of levofloxacin using, and the resistance rate of Acinetobacter baumannii was positively correlated with the frequency of meropenem using. In order to avoid the occurrence of common infectious bacteria and colonized bacteria, the supervision of antibiotics application should be strengthened. Key words: Frequency of antibiotics used; Pseudomonas aeruginosa; Acinetobacter baumannii; Drug resistance

Plague, caused by Yersinia pestis, was classified as a reemerging infectious disease by the World Health Organization. The five human pneumonic plague cases in Yulong County in 2005 gave rise to the discovery of a Yulong plague focus in Yunnan province, China. Thereafter, continuous wild rodent plague (sylvatic plague) was identified as the main plague reservoir of this focus. In this study, the epizootics in Yulong focus were described, and three molecular typing methods, including the different region (DFR) analysis, clustered regularly interspaced short palindromic repeats (CRISPRs), and the multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) (14+12), were used for the molecular typing and source tracing of Y. pestis isolates in the Yulong plague focus. Simultaneously, several isolates from the vicinity of Yunnan were used as controls. The results showed that during the 10-year period from 2006 to 2016, an animal plague epidemic occurred in 6 of those years, and 5 villages underwent an animal plague epidemic within a 30-km2 area of the Yulong plague focus. Searching for dead mice was the most effective monitoring method in this plague focus. No positive sample has been found in 6937 captured live rodents thus far, suggesting that the virulence of strains in the Yulong plague focus is stronger and the survival time of mice is shorter after infection. Strains from Lijiang, Sichuan and Tibet were of the same complex based on a typing analysis of DFR and CRISPR. The genetic relationship of Y. pestis illustrated by MLVA “14+12” demonstrates that Tibet and Sichuan strains evolved from the strains 1.IN2 (Qinghai, 1970 and Tibet, 1976), and Lijiang strains are closer to Batang strains (Batang County in Sichuan province, 2011, Himalaya marmot plague foci) in terms of genetic or phylogenic relationships. In conclusion, we have a deeper understanding of this new plague focus throughout this study, which provides a basis for effective prevention and control.

Objective To investigate whether the host animals of Yulong plague foci carry Yersiniapestis phage,and to identify isolated plague phage.Methods Rodent specimens were collected in 5 villages of Yulong plague foci in spring and autumn of 2016,respectively.Vaccine strain EV76 was used as breeding bacteria.Phage was isolated from the specimens by double-layer plate method and plaque morphology was identified.Results ① Totally 409 samples collected in spring failed in phage isolation.A total of 40 of Yersinia pestis phages were isolated from 444 samples in autumn,and the total isolation rate was 9.01％ (40/444).② The Yersinia pestis phages were isolated in all of 5 villages,and the isolation rate was of no significant difference (x2 =5.055,P ＞ 0.05).③ Of the 40 strains of phage,37 strains were isolated from Apodemus chevrieri,2 strains from Eothenomys Miletus and 1 strain from Crocidura Dracula.④Based on the appearance,the plaque of the phage was divided into three:large (diameter 1.5-2.5 mm),middle (0.5-＜ 1.5 mm) and small (＜ 0.5 mm).Conclusion There is a higher number of plague phage in the host animals of the plague foci in Yulong County of Yunnan Province,the plaques are diverse in morphology,and their biological characteristics may be polymorphic.

目的 调查剑川野鼠鼠疫疫源地宿主动物中是否携带鼠疫噬菌体,并探讨其流行病学意义.方法 2017年1-6月采集剑川野鼠鼠疫疫源地4个曾流行过鼠疫乡镇6个自然村的鼠类标本,以鼠疫疫苗株EV76为饲养菌,采用双层平板法分离鼠疫噬菌体,并对分离结果进行流行病学分析,同时挑取部分噬菌体进行电镜扫描.结果 共获得641份标本(齐氏姬鼠334只,大绒鼠236只,其余71只),分离到9株鼠疫噬菌体,总分离率1.40％;有4个疫点(白山母村、石龙村、新松村、大庆村)分离到了鼠疫噬菌体,另外2个点(长乐村、西门社区)未分到鼠疫噬菌体;9株鼠疫噬菌体中,8株分离自齐氏姬鼠,1株自大绒鼠;初次分离这些鼠疫噬菌体时,其噬斑在双层平板上表现为大(直径～2.0 mm)、中(～1.0 mm)及小＜0.5 mm)3种噬斑;4株有代表性噬菌体皆为肌尾病毒科噬菌体.结论 剑川野鼠鼠疫疫源地中普遍存在鼠疫噬菌体,齐氏姬鼠是主要的携带宿主,所分鼠疫噬菌体为肌尾病毒科噬菌体且具有多态性,值得进一步研究.

目的 测定一株分离自丽江野鼠鼠疫疫源地鼠巢土的野生型鼠疫噬菌体(命名为LJ-7)的裂解谱,分析LJ-7的裂解能力及特点. 方法 采用双层平板法观察LJ-7对248株菌(包括10株云南鼠疫耶尔森菌、1株鼠疫疫苗株EV76、6株假结核耶尔森菌、37株致病小肠结肠炎耶尔森菌、17株非致病小肠结肠炎耶尔森菌、100株大肠埃希菌和77株其他菌株)在28℃、37℃及18℃3种温度时的裂解能力. 结果 LJ-7对10株云南鼠疫耶尔森菌、1株鼠疫疫苗株EV76及1株弗氏志贺菌有裂解作用;对其余236株菌均不裂解.在37℃时LJ-7较鼠疫诊断噬菌体裂解性高,而在28℃时LJ-7较鼠疫诊断噬菌体裂解性低. 结论 J-7在28℃、37℃及18℃时均能裂解鼠疫耶尔森菌,对弗氏志贺菌也有裂解性作用.表明LJ-7裂解细菌谱较宽,在不依赖鼠疫菌的情况下可由其他细菌提供营养而存在,它的发现对我国鼠疫疫源地环境微生态学和流行病学研究具有重要意义.

The third plague pandemic originated from Yunnan Province, China in the middle of the 19th century. The last human plague epidemic in Yunnan occurred from 1986-2005. On June 6, 2016, a case of human plague was reported in the Xishuangbanna Prefecture, Yunnan. The patient suffered from primary septicemic plague after exposure to a dead house rat (Rattus flavipectus), which has been identified as the main plague reservoir in the local epizootic area. Moreover, a retrospective investigation identified another bubonic plague case in this area. Based on these data, human plague reemerged after a silent period of ten years. In this study, three molecular typing methods, including a clustered regularly interspaced short palindromic repeats (CRISPR) analysis, different region analysis (DFR), and multiple-locus variable number of tandem repeats analysis (MLVA), were used to illustrate the molecular characteristics of Yersinia pestis (Y. pestis) strains isolated in Yunnan. The DFR profiles of the strains isolated in Yunnan in 2016 were the same as the strains that had previously been isolated in this Rattus flavipectus plague focus. The c3 spacer present in the previously isolated strains was absent in the spacer arrays of the Ypc CRISPR loci of the strains isolated in 2016. The MLVA analysis using MLVA (14+12) showed that the strains isolated from the human plague case and host animal plague infection in 2016 in Yunnan displayed different molecular patterns than the strains that had previously been isolated from Yunnan and adjacent provinces.

Objective To analyze the antibiotic use in recent years in our hospital, analyze its influence on the prevalence of drug resistance of Escherichia coli and Klebsiella pneumoniae and provide guidance for the reasonable clinical use of antibiotics and the control and decrease of bacterial drug-resistance. Methods The use frequency and use dose of antibiotics, and the drug resistance level of E. coli and K. pneumoniae isolated in our hospital from 2013 to 2017 were investigated. Results The antibiotic use dose (AUD) increased year by year from 2013 to 2015 and decreased in 2016 in our hospital. According to the analysis of defined daily dose (DDD), the drug resistant rate of E. coli was positively correlated with the use frequencies of ceftazidime and cefuroxime, but negatively correlated with the use frequencies of other antibiotics. While the drug resistance rate of K. pneumoniae was obviously positively correlated with the use frequencies of meropenem and levofloxacin (r＝0.980, P＝0.003 and r＝0.902, P＝0.036), and positively correlated with use frequencies of ceftazidime, cefepime, gentamicin and cefoperazone/sulbactam, but negatively correlated with use frequencies of cefuroxime, imipenem and piperacillin/tazobactam. Conclusion The use frequencies of commonly used antibiotics had certain relationship with the drug resistance of E. coli and K. pneumoniae, especially the relationship between the high frequency uses of meropenem and levofloxacin and the drug resistance of K. pneumoniae, to which close attention should be paid.

目的 查明鹤庆新发野鼠鼠疫疫源地内是否存在鼠疫噬菌体,并对所离分鼠疫噬菌体进行形态鉴定及噬菌谱分析.方法 以鹤庆县马厂村为核心,选择5 km范围内的自然村为采样点,采用鼠铗法进行捕鼠,实验室中取鼠盲肠置入改良PBS中,采用双层平皿对样本进行筛选、纯化得到噬菌体,观察噬菌斑形态,电镜检查噬菌体形态,并在22℃、24℃、28℃及37℃对21株鼠疫菌与115株非鼠疫菌进行裂解特性分析.结果 采集的354份标本,分离到2株鼠疫噬菌体;2株噬菌体在高于24℃温度下可裂解鼠疫菌,低于24℃不能裂解鼠疫菌,而非鼠疫菌在4个温度下均不裂解;电镜形态检测2株噬菌体均属于肌尾噬菌体.结论 鹤庆新发野鼠疫疫源地内存在着鼠疫噬菌体,且所分鼠疫噬菌体只有在高于24℃时才裂解鼠疫菌,此特性与鼠疫菌在鹤庆县的长期存在相关,且两株噬菌体存在裂解谱较窄,特异性良好,可用于备用诊断噬菌体的筛选株.

Objective Using quantitative real-time PCR to establish a rapid specific genetic diagnostic technique for Yersinia pestis.Methods ①Four sets of specific probes and primers were designed,which targeted to chromosome genes of YPO0392,YPO1094,YPO2087 and YPO2090,respectively.②The probes and primers were tested for stability and specificity with 40 strains of Yersinia pestis and 47 strains of non-Yersinia pestis of different sources in Yunnan.③Eight positive DNA in Yulong,Yunnan,were tested with the screened probes and primers.Results ①Two sets probes and primers were selected,they were targeting YPO0392 and YPO1094,respectively.②The results were all positive of the eight positive DNA samples tested.Conclusion Two sets of primers and probes are selected for rapid specific diagnosis of Yersinia pestis.

目的 对剑川县野鼠鼠疫疫源地小肠结肠炎耶尔森菌分布情况调查分析.方法 从剑川采集鼠盲结肠标本进行小肠结肠炎耶尔森菌分离培养、表型鉴定和特异基因分析.结果 从采集的645份鼠类标本中,分离到34株小肠结肠炎耶尔森菌,分离率为5.27％ (34/645);其中致病株1株、非致病株33株.鼠类标本分别是大绒鼠、贝氏树嗣、褐家鼠、齐氏姬鼠、小林姬鼠等,其中大绒鼠采集率和检出率最高(2.79％),其次为齐氏姬鼠(1.55％).结论 剑川县野鼠鼠疫疫源地中,小肠结肠炎耶尔森菌的主要宿主与鼠疫菌主要宿主基本一致,推测小肠结肠炎耶尔森菌的存在与鼠疫的相对静息相关.

目的 对云南省玉龙县一起鼠间鼠疫进行实验室判定.方法 取6份干燥自毙鼠标本的肌肉及骨骼,用生理盐水进行浸泡,然后取浸泡液进行免疫胶体金(GICA)及鼠疫反相间接血凝抑制试验(RIHA)检测;采用DNA提取试剂盒对以上6份标本的骨骼及肌肉进行了全DNA的提取;采用聚合酶链式反应(PCR)检测caf1及pla基因;采用实时荧光定量PCR(real-time PCR)检测caf1、pla及YPO0392基因;采用LB平板对肌肉及骨髓标本进行细菌分离培养.结果 经GICA初筛,6份标本中4份为阳性;RIHA检测4份阳性标本的效价分别是1∶320、1∶640、1∶1 280及1∶640;4份免疫学阳性标本的普通PCR检测,其中3份标本为阳性,1份为阴性;实时荧光PCR检测4份免疫学阳性标本DNA,其中3份标本为阳性,1份为阴性;所有6份标本细菌培养皆阴性.结论 判定玉龙县此次事件为鼠间鼠疫疫情,同时建立了一套对干燥标本进行鼠疫实验室诊断的技术流程.

To determine a rat plague epidemic in ancient city administrative region of Lijiang,China,one of drying self-dead Eothenomys miletus were detected with methods of reverse indirect hemoagglutination inhibition test(RIHA),Polymerase Chain Reaction(PCR)and real-time PCR(RT-PCR).Results showed that the immunological detection,PCR and RT-PCR detection to this sample were positive.It was confirmed that a rat plague epidemic occurred in ancient city administrative region of Lijiang.

The emergence and spread of multidrug-resistant (MDR) Klebsiella pneumoniae has been regarded as one of the major challenges among health care-associated infections worldwide. Here, we report the draft genome sequence of an extensively drug-resistant (XDR) K. pneumoniae strain isolated in 2013 from Yunnan Province, China.