四环素耐药基因在鸡源沙门氏菌中的分布和传播

试验分析了84株鸡源沙门氏菌分离株的四环素耐药性,用PCR方法检测四环素耐药基因在分离株中的分布情况。结果显示,四环素耐药率为49%(41/84),鸡白痢和鸡伤寒沙门氏菌仅携带tet(A)基因(23/23),肠炎沙门氏菌和德尔卑沙门氏菌携带tet(A)(8/18)、tet(B)(17/18)或tet(G)(10/18)三种基因,tetC基因在这些沙门氏菌中都没有检测到。该类基因多数位于结合性质粒上,但是不在整合子范围内。     

鼠伤寒沙门菌cya基因的克隆表达及生物信息学分析

研究旨在克隆鼠伤寒沙门菌cya基因并对其进行生物信息学分析。以鼠伤寒沙门菌SL1344株为模板PCR扩增并克隆了鼠伤寒沙门菌cya基因,构建原核表达质粒pET-32a-cya,转化大肠杆菌BL21（DE3）感受态细胞,用1 mmol/L IPTG对其进行诱导表达、纯化,同时利用相关的生物信息学软件对其进行生物信息学分析。结果显示,鼠伤寒沙门菌cya基因大小为1 185 bp。经SDS-PAGE和Western blotting分析显示,在原核表达系统中主要以包涵体形式存在,蛋白分子质量大小约为58 ku。生物信息学分析表明,Cya蛋白由394个氨基酸组成,理论分子质量为44.97 ku,分子式为C₂₀₂₆H₃₁₄₆N₅₆₂O₅₇₈S₁₁,等电点（pI）为7.01,其中带负电荷残基总数（Asp+Glu）43个,带正电荷残基总数（Arg+Lys）42个。Cya蛋白无信号肽与跨膜区,为膜内蛋白,含有4个N-糖基化位点、1个O-糖基化位点、27个磷酸化位点、13个线性B细胞结合位点、11个T细胞结合位点。二级结构中α-螺旋、延伸链、β-转角、无规则卷曲所占比例分别为38.07%、19.80%、5.58%和36.55%。本试验结果为进一步研究鼠伤寒沙门菌Cya蛋白的功能及建立以Cya蛋白为抗原的免疫鉴别检测方法奠定了基础。     

Prevalence,Characterization and Antibiotic Resistance of Salmonella Isolates in Large Corvid Species of Europe and North America Between 2010 and 2013

It is well understood that Salmonella is carried by animals and in majority of cases as asymptomatic hosts. Surveillance efforts have focused on the role of agriculture and contamination points along the food chain as the main source of human infection; however, very little attention has been paid to the contribution of wildlife in the dissemination of Salmonella and what effect anthropogenic sources have on the circulation of antibiotic resistant Salmonella serovars in wildlife species. A purposive survey was taken of large corvids roosting yearly between November and March in Europe and North America. Two thousand and seven hundred and seventy‐eight corvid faecal specimens from 11 countries were submitted for Salmonella spp. culture testing. Presumptive positive isolates were further serotyped, susceptibility tested and analysed for antibiotic resistance genes. Overall, 1.40% (39/2778) (CI = 1.01, 1.90) of samples were positive for Salmonella spp. Salmonella Enteritidis was the most prevalent serovar followed by S. Infantis, S. Montevideo and S. Typhimurium. No significant difference (P > 0.05) was found in the proportion of Salmonella recovered in Europe versus North America. The most variability of serovars within a site was in Kansas, USA with five different serovars recovered. European sites were significantly more likely to yield Salmonella resistant to more than one antibiotic (OR 71.5, P < 0.001, CI = 3.77, 1358) than North American sites, where no resistance was found. Resistance to nalidixic acid, a quinolone, was recovered in nine isolates from four serovars in four different sites across Europe. Large corvids contribute to the transmission and dissemination of Salmonella and resistance genes between human and animal populations and across great distances. This information adds to the knowledge base of zoonotic pathogen prevalence and antibiotic resistance ecology in wild birds.     

Prevalence and Antimicrobial Resistance Pattern of Salmonella Isolates from Apparently Healthy Slaughtered Cattle in Ethiopia

The prevalence and antimicrobial resistance pattern of Salmonella isolates was determined from apparently healthy slaughtered cattle at Debre Zeit (Ethiopia). A total of 323 cattle were examined for the presence of Salmonella in faeces, mesenteric lymph nodes, abdominal and diaphragmatic muscles. Salmonellae were cultured from 23 (7.1%) of the animals. Salmonellae were isolated from 2 (3.1%) and 3 (4.5%) of 65 pooled faecal and mesenteric lymph node samples, respectively. Nine (2.8%) abdominal muscle and 10 (3.1%) diaphragmatic muscle samples (n = 323 of each) were contaminated by Salmonella. About 60% of the serovars identified in the abdominal and diaphragmatic muscles were also detected from faeces and mesenteric lymph node samples. The five different serovars isolated were Salmonella mishmarhaemek (48%), S. typhimurium (20%), S. enteritidis (12%), S. guildford (12%) and S. dublin (48%). The antimicrobial resistance profiles of 25 of the Salmonella isolates with 17 antimicrobials showed that 52% (13/25) of them were resistant to three or more antimicrobials. Both strains of Salmonella (S. mishmarhaemek and S. typhimurium) showed multiple resistance to ampicillin, sulfamethoxazole and ticarcillin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Whole‐genome sequencing reveals resistome of highly drug‐resistant retail meat and human Salmonella Dublin

Non‐typhoidal Salmonella (NTS) are a significant source of foodborne illness worldwide, with disease symptoms most often presenting as self‐limiting gastroenteritis; however, occasionally the infection spreads and becomes invasive, frequently requiring anti‐microbial treatment. The cattle‐adapted Dublin serovar of NTS has commonly been associated with invasive illness and anti‐microbial resistance (AMR). Here, the enhanced resolution conferred by whole‐genome sequencing was utilized to elucidate and compare the resistome and genetic relatedness of 14 multidrug‐resistant (MDR) and one pan‐susceptible S. Dublin, isolated primarily in Pennsylvania, from fresh retail meat (one isolate) and humans (14 isolates). Twelve different genetic AMR determinants, including both acquired and chromosomal, were identified. Furthermore, comparative plasmid analysis indicated that AMR was primarily conferred by a putative IncA/C2 plasmid. A single pan‐susceptible S. Dublin isolate, collected from the same timeframe and geographical region as the MDR isolates, did not carry an IncA/C2 replicon sequence within its genome. Moreover, the pan‐susceptible isolate was genetically distinct from its MDR counterparts, as it was separated by ≥267 single nucleotide polymorphisms (SNPs), whereas there was a ≤38 SNP distance between the MDR isolates. Collectively, this data set advances our understanding of the genetic basis of the highly drug‐resistant nature of S. Dublin, a serovar with significant public health implications.     

动物源大肠杆菌及沙门氏菌抗药性的快速检测

利用本室自主研制的新型96点阵药敏检测盒检测山东地区动物源大肠杆菌、沙门氏菌的药物敏感性,试验结果表明,大肠杆菌对氨苄西林和喹诺酮类药物的抗药率达98%以上,禽源大肠杆菌的抗药率高于猪源大肠杆菌,猪源大肠杆菌的抗药谱广于禽源大肠杆菌;沙门氏菌抗药率略低于大肠杆菌,但同样呈现多重抗药,主要集中在5抗-7抗和13抗-14抗两个范围。96点阵抗生素药敏检测盒是适合于试验室进行大规模抗药性监测的新工具,具有简易、准确、快速、大通量、节约的优点,为临床治疗中抗生素的使用、细菌病的治疗提供了技术保障,从而达到避免盲目使用抗生素,降低细菌抗药率的目的。     

Identification of a Plasmid‐Mediated Quinolone Resistance Gene in Salmonella Isolates from Texas Dairy Farm Environmental Samples

A recent increase in plasmid‐mediated quinolone resistance (PMQR) has been detected among Salmonella isolated from humans in the United States, and it is necessary to determine the sources of human infection. We had previously isolated Salmonella from dairy farm environmental samples collected in Texas, and isolates were tested for anti‐microbial susceptibility. Two isolates, serotyped as Salmonella Muenster, showed the discordant pattern of nalidixic acid susceptibility and intermediate susceptibility to ciprofloxacin. For this project, whole‐genome sequencing of both isolates was performed to detect genes associated with quinolone resistance. The plasmid‐mediated qnrB19 gene and IncR plasmid type were identified in both isolates. To our knowledge, this is the first report of PMQR in Salmonella isolated from food animals or agricultural environments in the United States.     

规模化猪场猪源沙门氏菌的耐药性及相关耐药基因检测

为了解贵州省某规模化猪场猪源沙门氏菌分离株的药物敏感性和耐药基因的存在情况,也为进一步研究细菌耐药的分子机制和临床用药提供资料,利用K-B法检测了6株猪源沙门氏菌对15种药物的敏感性,采用PCR方法检测了9种常见耐药基因在分离株中的分布情况。结果显示,所有菌株对青霉素、氨苄西林和吡哌酸的耐药率最高均为100%,对头孢克洛、链霉素和庆大霉素的耐药率最低。从9种常见耐药基因中扩增到3种β-内酰胺类耐药、3种氨基糖苷类、2种氟喹诺酮耐药基因。研究表明,沙门氏菌极易产生耐药性,耐药基因广泛存在于耐药菌株中,但耐药表型与耐药基因之间无绝对相关性。     

Salmonella Serovars and Antimicrobial Resistance Profiles in Beef Cattle,Slaughterhouse Personnel and Slaughterhouse Environment in Ethiopia

The present study was undertaken to determine the occurrence, distribution and antimicrobial resistance profiles of Salmonella serovars in slaughter beef cattle, slaughterhouse environment and personnel engaged in flaying and evisceration during slaughtering process. A total of 800 samples (each sample type, n = 100) consisting of swabs from hides, slaughterhouse personnel hands at flaying and evisceration, rumen and caecal contents, mesenteric lymph nodes, carcasses and holding pens were collected. Of the total 100 beef cattle examined, 14% were Salmonella positive in caecal content and/or mesenteric lymph nodes. Of the various samples analysed, Salmonella was detected in 31% of hides, 19% of rumen contents, 8% of mesenteric lymph nodes, 6% of caecal contents, 2% of carcass swabs, 9% of palm swabs taken from the hands of personnel in the slaughterhouse during flaying (7%) and evisceration (2%), and in 12% of holding pen swabs. The Salmonella isolates (n = 87) belonged to eight different serovars of which S. Anatum (n = 54) and S. Newport (19) were the major serovars and both serovars were detected in all sample sources except in carcass swabs. Eighteen of the 87 (20.7%) Salmonella serovars consisting of Newport (n = 14), Anatum (n = 3) and Eastbourne (n = 1) were resistant to one or more antimicrobials. Among the antimicrobial resistant Salmonella serovars, S. Newport was multidrug resistant (15.6%) and exhibited resistance to streptomycin, sulphisoxazole and tetracycline.     

Transduction of bla(CMY-2), tet(A), and tet(B) from Salmonella enterica subspecies enterica serovar Heidelberg to S. Typhimurium

Antimicrobial resistance of Salmonella spp., especially resistance mediated by extended spectrum β-lactamases (ESBL), is a growing public health concern. Understanding the mechanisms through which Salmomella spp. acquire the resistance genes can lead to the development of intervention and mitigation strategies. Thirty-one Salmonella isolates of bovine origin were analyzed by serotyping, antimicrobial susceptibility testing, phage induction and bacterial host range determination, and phage transduction of antimicrobial resistance. The Salmonella isolates consisted of 12 serovars. Resistance to 1 or more antibiotics was detected in 12 isolates. Inducible phages were recovered from 19 Salmonella (61%) by spot lysis assay. Of the 19 phage samples, 13 were able to multiply in 2 or more Salmonella of various serovars. A cross-serovar transduction of antimicrobial resistance was observed from S. Heidelberg to S. Typhimurium. Transfection of an antimicrobial-susceptible strain of S. Typhimurium with a phage propagated in a S. Heidelberg resistant to multiple β-lactam antibiotics and tetracycline resulted in independent acquisition of bla_CMY-2, tet(A), and tet(B). These data indicate that inducible phages are common in Salmonella of bovine origin, many of them demonstrate a broad host range, and wild-type phage mediated transduction may contribute to the dissemination of antimicrobial resistance, including the resistance mediated by ESBL.     

独龙牛和云岭牛初乳的理化特性比较

【目的】 通过对独龙牛（Bos frontalis）和云岭牛初乳成分测定,揭示两牛种初乳成分的差异性。【方法】 采集独龙牛和云岭牛（年龄、胎次和饲养管理基本一致）分娩第1天初乳样品共45份,测定其乳蛋白、乳脂肪、乳糖、总固形物（TS）、非脂固形物（SNF）和乳尿素氮（MUN）含量及体细胞数（SCC）和冰点（FP）,采用独立样本t检验比较独龙牛和云岭牛初乳成分差异。【结果】 独龙牛初乳中乳蛋白、乳脂肪、TS、SNF含量分别为16.77%、4.70%、27.82%和22.39%,比云岭牛的分别高28.11%（P<0.01）、5.38%（P>0.05）、22.45%（P<0.01）和26.93%（P<0.01）;乳糖、MUN含量和SCC分别为2.98%、354.2 mg/L和35.73×10⁴/mL,比云岭牛的分别低10.38%（P<0.05）、39.63%（P<0.01）和78.60%（P<0.01）;FP为－0.5337 ℃,比云岭牛的高0.1383 ℃（P<0.01）;乳脂蛋白比（FPR）为0.266,比云岭牛的低0.131（P>0.05）。【结论】 独龙牛和云岭牛分娩第1天的初乳成分存在较大差异,独龙牛初乳营养价值较高。     

Analysis of Salmonella enterica serotype Enteritidis isolated from human and chickens by repetitive sequence-PCR fingerprinting, antibiotic resistance and plasmid profiles

A total of 22 Salmonella enterica serotype Enteritidis (S. Enteritidis) strains isolated from human and chicken were subjected to DNA fingerprinting by repetitive sequence PCR using ERIC and BOX primers, antibiotic resistance and plasmid patterns. Both ERIC and BOX PCR amplification data revealed a highly genetic homogeneity between isolates from human and chicken except one isolate, which originated from chicken and showed a different DNA band pattern from others. Eleven of 22 S. Enteritidis isolates (50%) were resistant to more than one antibiotics and characterized by 5 resistance patterns. The most common pattern was penicillin resistant (63.6%). Only one isolate from chicken showed a multiple drug resistance patterns to 4 antibiotics. All 22 S. Enteritidis isolates harbored more than two plasmids with eight different plasmid profiles including two to six plasmids with approximate molecular size ranging from 1.9 to 21 kb. A band of 15 kb size was detected in all isolates tested, however, the band sizes smaller than 15 kb were found only in isolates from chicken.     

Characterization of Salmonella isolates from captive lizards

Reptile-associated salmonellosis in humans is an increasing public health issue. This study aimed at characterizing Salmonella isolates from captive lizards and to compare them to human isolates. Salmonella was isolated from 25 of 33 cloacal and 47 of 79 faecal samples from captive lizards (75.8 and 59.5%, respectively). The strains belonged to 44 serotypes of subspecies I (27 serotypes), II (9), IIIb (3) and IV (5). Two strains, one of serotype Enteritidis and one of serotype Amsterdam, were resistant to nitrofurantoin. Invasion assays in Caco-2 cells were performed with 40 saurian isolates of subspecies I, 15 isolates of subspecies II, 4 strains of subspecies IIIb, 6 subspecies IV isolates and 17 human isolates of corresponding serotypes of subspecies I. Saurian isolates belonging to subspecies I invaded the Caco-2 cells to a higher extent than those from the other subspecies. The human isolates invaded the Caco-2 cells to a lesser degree compared to their saurian counterparts. In the same strains, the presence of virulence genes agfA, shdA, spvR, pefA and sopE was determined using PCR. Whereas agfA was detected in all strains, pefA was only detected in one saurian and in the human serotype Enteritidis strains. The spvR gene was detected in the same serotype Enteritidis strains and in 33% of the subspecies IV strains. The shdA gene was present in all the human isolates and in 86% of subspecies I saurian isolates. SopE was found in 17% of the human isolates, in 24% of the saurian subspecies I strains and in all of the subspecies IV strains.     

食品动物源沙门氏菌质粒介导喹诺酮类耐药基因的检测与分析

为了解食品动物源沙门氏菌质粒介导喹诺酮类耐药性(Plasmid-mediated quinolone resistance,PMQR),采用微量肉汤稀释法和PCR方法,检测了316株食品动物源沙门氏菌对20种抗菌药物的敏感性,以及菌株中PMQR基因的携带率.结果显示:316株沙门氏菌对20种抗菌药物呈不同程度的耐药性,95.57％菌株为多重耐药菌;316株菌中未检出qnrA、qnrC、qnrD、qnrS和qepA基因,7.91％菌株检出qnrB基因,15.19％菌株检出aac(6 ′ )-Ib-cr基因,7.91％菌株检出oqxA基因,8.86％菌株检出oqxB基因,这是首次在沙门氏菌中发现oqxAB基因;98.11％PMQR阳性菌同时携带2种及以上的耐药基因,呈8～17耐的多重耐药性,其中以qnrB和aac(6′)-Ibcr基因型为主;53株PMQR阳性菌分属于5种不同的基因型,耐药表型或耐药基因型不同的菌株却有相同的PFGE谱型.本次检测的316株食品动物源沙门氏菌耐药较为严重;菌株主要携带qnrB、aac(6 ′ )-Ib-cr及oqxAB基因;不同来源菌株存在同一耐药克隆株的流行.     

沙门菌肠毒素基因克隆及序列分析

研究常见的不同血清型沙门菌肠毒素(stn)基因核苷酸序列之间的差异及其分布情况。根据沙门菌的stn核苷酸序列设计一对引物,应用PCR技术,分别对肠炎沙门菌、鼠伤寒沙门菌和鸡白痢沙门菌进行PCR扩增,对扩增产物进行克隆及序列分析,并用所设计的引物检测7种血清型沙门菌(42株)。结果显示,3种沙门菌经PCR均扩增出749 bp的特异条带,DNA序列分析证实,沙门菌的stn核苷酸序列比较保守,42株沙门菌stn的检出率为100%。本试验成功克隆出沙门菌的stn,调查其在不同血清型沙门菌中的分布及序列分析,为进一步研究stn致病机理及研制减毒沙门菌活菌疫苗奠定了基础。     

鼠伤寒沙门菌毒力基因SptP的原核表达及生物信息学分析

【目的】试验旨在获得高效表达的鼠伤寒沙门菌SptP蛋白并进行生物信息学分析,为其功能研究和互作蛋白的筛选提供理论依据。【方法】通过PCR技术扩增SptP基因,并将该序列连接至pET-32a （+）载体,构建鼠伤寒沙门菌SptP基因原核表达载体pET32a-SptP。通过热激法将重组质粒导入大肠杆菌BL21（DE3）感受态细胞后经IPTG诱导表达、纯化重组蛋白,并经过SDS-PAGE和Western blotting验证;应用在线软件对SptP蛋白进行生物信息学分析。【结果】PCR成功扩增出大小为1 632 bp的SptP基因。SptP重组蛋白在大肠杆菌BL21（DE3）感受态细胞中成功诱导表达、纯化,得到分子质量为79.7 ku的蛋白。SptP蛋白分子式为C₂₆₂₅H₄₂₅₇N₇₄₅O₈₁₂S₂₅,分子质量为60 047.68 u,无跨膜结构,无信号肽存在,理论等电点为8.75,有57个潜在的磷酸化位点,主要定位于细胞核、细胞质、高尔基体、细胞骨架、分泌系统的囊泡、质膜,占比分别为43.5%、34.8%、8.7%、4.3%、4.3%和4.3%。SptP蛋白二级结构由α-螺旋、延伸链、β-转角及无规则卷曲组成,占比分别为43.65%、14.92%、4.42%和37.02%。【结论】本研究构建了表达SptP蛋白的重组质粒pET32a-SptP,获得分子质量为79.7 ku的SptP重组蛋白,阐明了SptP蛋白的基本理化性质和生物学功能,为后续SptP蛋白与宿主细胞互作的作用机制及鼠伤寒沙门菌新疫苗的制备提供理论基础和试验依据。     

Deletions in the pyruvate pathway of Salmonella Typhimurium alter SPIl-mediated gene expression and infectivity

Background： Salmonella enter/ca serovar Typhimurium is a major foodborne pathogen worldwide. S. Typhimurium encodes type III secretion systems via Salmonella pathogenicity islands （SPI）, producing the major effector proteins of virulence. Previously, we identified two genes of Salmonella pyruvate metabolism that were up-regulated during chicken cell infection： pyruvate formate lyase I （pf/B） and b/functional acetaldehyde-CoA/alcohol dehydrogenase （adhE）. We were therefore interested in examining the role these genes may play in the transmission of Salmonella to humans. Methods： Mutant strains of Salmonella with single gene deletions for pflB and adhE were created. Invasion and growth in human HCT-8 intestinal epithelial cells and THP-1 macrophages was examined. Quantitative PCR was performed on 19 SPI-1 genes. Results： In HCT-8 cells, both mutant strains had significantly higher intracellular counts than the wild-type from 4 to 48 h post-infection. Various SPI-1 genes in the mutants were up-regulated over the wild-type as early as 1 h and lasting until 24 h post-infection. In THP-1 cells, no significant difference in internal Salmonella counts was observed; however, SPI-1 genes were largely down-regulated in the mutants during the time-course of infection. We also found five SPI-1 genes - hilA, hiIC hill）, sicP and rtsA - which were up-regulated in at least one of the mutant strains in log-phase broth cultures alone. We have therefore identified a set of SPI-1 virulence genes whose regulation is effected by the central metabolism of Salmonella.     

鸡白痢沙门菌的分离鉴定与耐药性分析

本研究采集某鸡场疑似感染鸡白痢的病死鸡组织,进行了沙门菌的分离、鉴定,以及对10种常见抗生素的药物敏感性试验.结果显示:分离株均为鸡白痢沙门菌,对阿米卡星(0％)、庆大霉素(0％)、头孢噻肟(3.70％)的耐药率较低,对链霉素(88.89％)、四环素(62.96％)、阿莫西林(51.85％)的耐药率较高;分离株至少可对...     

鱼塘生态体系中大肠埃希菌和沙门菌的耐药性研究

为了解鱼塘生态体系大肠埃希菌和沙门菌的耐药情况,从广东省佛山某鱼塘随机采集草鱼及生活圈的水土样品中(包括鱼肠内容物、鱼塘底泥、鱼塘水)分离出47株大肠埃希菌和23株沙门菌.采用纸片扩散法(Kirby-bauer,KB)对分离菌株进行了15种抗生素的敏感性试验,结果显示100%的菌株均耐1种及1种以上的抗生素多表现出对氨...