Modified Live Edwardsiella ictaluri Vaccine,AQUAVAC-ESC,Lacks Multidrug Resistance Plasmids

Abstract

Plasmid-mediated antibiotic resistance was first discovered in Edwardsiella ictaluri in the early 1990s, and in 2007 an E. ictaluri isolate harboring an IncA/C plasmid was recovered from a moribund channel catfish Ictalurus punctatus infected with the bacterium. Due to the identification of multidrug resistance plasmids in aquaculture and their potential clinical importance, we sought to determine whether the modified live E. ictaluri vaccine strain in AQUAVAC-ESC harbors such plasmids, so that the use of this vaccine will not directly contribute to the pool of bacteria carrying plasmid-borne resistance. Antimicrobial sensitivity testing of the E. ictaluri parent isolate and vaccine strain demonstrated that both were sensitive to 15 of the 16 antimicrobials tested. Total DNA from each isolate was analyzed by polymerase chain reaction (PCR) using a set of 13 primer pairs specific for conserved regions of the IncA/C plasmid backbone, and no specific products were obtained. PCR-based replicon typing of the parent isolate and vaccine strain demonstrated the absence of the 18 commonly occurring plasmid incompatibility groups. These results demonstrate that the vaccine strain does not carry resistance to commonly used antimicrobials and provide strong support for the absence of IncA/C and other commonly occurring plasmid incompatibility groups. Therefore, its use should not directly contribute to the pool of bacteria carrying plasmid-borne resistance. This work highlights the importance of thoroughly investigating potential vaccine strains for the presence of plasmids or other transmissible elements that may encode resistance to antibiotics.

Received May 6, 2011; accepted July 22, 2011     

Isolation, characterization, and molecular cloning of cryptic plasmids isolated from Edwardsiella ictaluri

Fifty-five isolates of Edwardsiella ictaluri were examined for the presence of plasmid DNA by a rapid alkaline extraction procedure. All 49 isolates from channel catfish and a single isolate from Bengal danio carried 2 plasmids with molecular masses of approximately 3.2 and 3.7 megadaltons (Mdal). Five E ictaluri isolates from other fish contained 1 to 3 plasmids, which had molecular masses ranging from 2.5 to 45 Mdal. The 2 plasmids (3.2 and 3.7 Mdal) from the type strain of E ictaluri (ATCC 33202) were ligated into pUC19 cloning vectors, and restriction endonuclease maps of each insert were prepared.     

Effect of bambermycins, in vitro, on plasmid-mediated antimicrobial resistance

The potential of bambermycins (a growth-promoting antimicrobial approved for turkeys, broilers, and swine) to overcome or control plasmid-mediated antimicrobial resistance was determined in a series of in vitro experiments. Four possible modes of action of bambermycins were studied: synergistic effect with 12 other antimicrobials, elimination of resistance (R) plasmids from Escherichia coli, selective killing or inhibition of E coli carrying R plasmids, and inhibition of R plasmid transfer. Bambermycins had no synergistic activity with the other drugs tested and had little effect on eliminating plasmids from host bacteria. Dependent on plasmid type, bambermycins decreased or increased transfer frequency of R plasmids. Bambermycins also selectively inhibited growth of bacteria harboring certain R plasmids.     

Change in antimicrobial resistance pattern in Salmonella enterica serovar Typhimurium isolates detected in a beef cattle farm

Multidrug-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates with four different antimicrobial resistance patterns obtained from a beef cattle farm were characterized to determine their clonality. Macrorestriction analysis of genomic DNA revealed that these four isolates are closely related to each other and can be classified as a newly emerged pulsed-field gel electrophoresis type among cattle: cluster VII. Three of the four isolates showed resistance to extended-spectrum cephalosporins (ESCs), and this resistance was mediated by AmpC β-lactamase encoded by the bla(CMY-2) gene in a 190-kbp IncA/C plasmid. Results of restriction analysis and IncA/C backbone PCR suggest that the three 190-kbp plasmids are identical and that a 70-kbp IncA/C plasmid of the ESC-susceptible isolate is derived from the 190-kbp plasmid by a deletion event. Three isolates harboured a virulence-resistance plasmid (165 or 180 kbp), and restriction analysis revealed that these plasmids were identical or closely related to each other. These results suggest that the four S. Typhimurium cluster VII isolates originate from a common ancestor that probably invaded the farm prior to the salmonellosis outbreak. Antimicrobial resistance patterns may not necessarily reflect the relationships of the isolates.     

Selection of CMY-2 producing Escherichia coli in the faecal flora of dogs treated with cephalexin

Cephalexin is a first generation cephalosporin commonly used in dogs for treatment of pyoderma. The objective of this study was to evaluate the in vivo effects of cephalexin on selection of Escherichia coli resistant to extended-spectrum cephalosporins. A cohort study was conducted on 13 dogs presenting clinical signs of pyoderma and treated with cephalexin and 22 healthy dogs that had not been treated with antibiotics during the previous six months. Selective plating of faeces on MacConkey agar plates containing cefotaxime (CTX) yielded growth of CTX-resistant E. coli for eight of the 13 treated dogs (62%), whereas no growth was observed for any of the control dogs (Fisher exact test, P<0.001). PCR and sequence analysis identified bla(CMY-2) in all eight dogs. PCR-based replicon typing and restriction fragment length polymorphism (RFLP) of E. coli transformants revealed location of bla(CMY-2) on indistinguishable IncI1 plasmids in five of the eight dogs. One representative of these five epidemiologically related IncI1 plasmids was further characterized as sequence type (ST2) by plasmid multilocus sequence typing (pMLST). E. coli from the remaining three dogs harboured bla(CMY-2) on distinct plasmids with non-typeable replicons. A single isolate was classified as an extraintestinal pathogenic E. coli (ExPEC) due to the presence of iutA, papC and sfa/foc. The results provide a strong indication that cephalexin selects for E. coli producing plasmid-borne CMY-2 β-lactamase. The isolation of a specific IncI1 plasmid carrying bla(CMY-2) from five epidemiologically unrelated dogs suggests that cephalexin use may contribute to the spread of this plasmid lineage among Danish dogs.     

Molecular epidemiology of Salmonella Heidelberg in an equine hospital

From 1992 to 1997, multi-drug resistant (MDR) Salmonella Heidelberg isolates were cultured from a number of horses hospitalised in a veterinary hospital in Victoria, Australia. To examine the relationships between the cases, 28 isolates from the hospital were compared by pulsed field gel electrophoresis (PFGE), IS200 element profiles, antimicrobial resistance patterns, plasmid profiles and phage typing. The PFGE patterns following digestion with XbaI and BlnI restriction endonucleases showed that the isolates from the veterinary hospital originated from a common source. These isolates also had indistinguishable IS200 profiles. However, PFGE was more discriminatory than IS200 profiles. All the veterinary hospital isolates and one independent isolate had the same antimicrobial resistance pattern and had at least one plasmid in common. Localisation of antimicrobial resistance genes indicated that the veterinary hospital isolates had more than one plasmid carrying resistance genes and that the genes encoding sulphathiazole and trimethoprim resistance were not on these plasmids. Phage typing was ineffective as 22 of the 28 isolates were untypeable. In conclusion, the combination of different methods used for epidemiological studies suggested that a single strain of MDR S. Heidelberg was isolated from horses admitted to the hospital for 6 years and caused salmonellosis in susceptible horses within that period with no apparent correlation between the antimicrobials used and retention of its MDR phenotype.     

Molecular characterization of Salmonella enteritidis isolates from Maine poultry and poultry farm environments.

Eighty-six Salmonella enteritidis isolates obtained during a surveillance program of poultry farms in Maine were subjected to phage-typing, plasmid profiling and fingerprinting, outer-membrane polypeptide analysis, and antimicrobial sensitivity testing. Isolates were obtained from a variety of sources, including poultry-farm environmental samples, chicken organ samples, human stool samples, cat feces, and live-trapped rats and mice. These isolates were compared with 21 S. enteritidis isolates originating outside of Maine. Phage types isolated in Maine included 13a (60%); 14b (29%); 23 (5%); 8 (2%); and 2 (2%). All S. enteritidis isolates from Maine carried plasmid DNA, and 97% of these isolates carried a 40.3-megadalton plasmid alone (6%) or in conjunction with several smaller plasmids (91%). All 52 phage-type 13a isolates harbored 40.3- and 3.0-megadalton plasmids. All 25 phage-type 14b isolates carried 3.3- and 1.3-megadalton plasmids, and 22 isolates also carried the 40.3-megadalton plasmid. All isolates displayed highly similar outer-membrane polypeptide profiles and were sensitive to a variety of antimicrobials commonly used against gram-negative organisms. The above data suggest that phage type and plasmid content may be related in the cases of phage-type 13a and 14b isolates, and that traditional plasmid-borne antimicrobial resistance determinants were not present in Maine isolates. Results also indicate that phage-typing can be a valuable epizootiological tool for monitoring the potential spread of these strains throughout the Northeast.     

CpG寡核苷酸对IBDV VP2基因真核表达质粒免疫增效作用

以传染性法氏囊病病毒(IBDV)VP2蛋白基因表达质粒DNA为免疫原，以CpG的寡核苷酸(CpG-0DN)为免疫佐剂，肌肉注射于14日龄SPF鸡，1周后加强免疫1次，2次免疫后15d和21d分别测定血清ELISA抗体效价，并于免疫后21d用IBDV99儿强毒株攻毒和进行病理学观察。结果显示，(1)VP2基因重组质粒DNA与CpG共同免疫组的ELISA抗体水平明显高于VP2重组质粒免疫组；(2)IBD弱毒苗与VP2重组质粒免疫组抗体水平明显高于VP2重组质粒免疫组，且比VP2基因重组质粒DNA与CpG共同免疫组略高；(3)VP2基因重组质粒DNA与CpG共同免疫组及IBD弱毒苗与VP2重组质粒免疫组可明显降低IBDV强毒攻击后引起的急性发病率和死亡率。由此表明，CpG寡核苷酸对IBDV VP2蛋白基因真核表达质粒免疫具有明显增强作用，有很大的应用前景。     

鸡源大肠杆菌强毒株耐药基因的定位及耐药质粒消除

本试验对临床分离的多重耐药鸡源致病性大肠杆菌强毒株的耐药基因进行初步定位,为临床选择合适的治疗策略提供理论依据。从送检病死鸡的肝脏、心脏中分离鉴定致病菌,质粒提取试剂盒提取分离菌的耐药质粒,转化入基因工程菌JM109,通过质粒纯化、电泳和药敏试验对耐药基因进行了初步定位。并用艾叶水煮液对该菌株进行体外耐药质粒消除试验。结果分离鉴定到1株强毒力鸡源大肠杆菌,该菌呈多重耐药性,且仅对氟奇霉素和链霉素敏感;由质粒转化和药敏试验结果可初步将耐环丙沙星、青霉素、氧氟沙星、氟哌酸、林可霉素和复方新诺明的基因定位于耐药质粒上,并可随质粒的转移而使转化菌获得耐药性;用艾叶水煮液可使该菌的耐药质粒消除率达60%;质粒消除菌的药敏试验结果表明,消除耐药质粒的细菌恢复了对环丙沙星、青霉素、氧氟沙星、氟哌酸、林可霉素和复方新诺明的敏感性。本研究结果表明,分离菌的耐药基因分别位于质粒和染色体上,艾叶对耐药质粒有较强的消除作用,可作为临床治疗用药。     

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.     

河南省鸡致病性大肠杆菌耐庆大霉素质粒的检测分析

通过对12株100%耐庆大霉素的多重耐药菌株的质粒提取,电泳检测和转化试验,成功获得了2株由质粒介导的耐庆大霉素的多重耐药菌株。结果表明:同一质粒可编码一个至数个耐药性基因,不同质粒可以携带相同的耐药性基因,并且这些质粒可以传递多种耐药性。证明了菌株对庆大霉素的耐药性是由耐药性质粒介导的,细菌的抗药性与耐药性质粒的转移有很大关系。     

犬瘟热病毒小熊猫株H、F和N基因的克隆及表达

根据GenBank中发表的犬瘟热病毒（CDV）的核苷酸序列，设计并合成了扩增CDVH、F和N基因的3对引物，经RT—PCR分别扩增获得了CDV小熊猫株（LP株）H、F和N基因，并对H、F及N基因进行了克隆和序列测定。序列分析表明，CDV LP株属于强毒谱系，与CDV流行株的亲缘关系近．H基因含有较多潜在的糖基化位点．F和N基因相对比较保守。将CDV LP株H、F和N基因克隆入真核表达栽体pVAX1的CMV启动子下游，构建了CDV基因疫苗表达载体pVAXLPH、pVAXLPF、pVAXLPN，体外转染BHK-21细胞．用间接ELISA方法检测到目的蛋白的表达。用构建的3个表达质粒免疫小鼠，从小鼠血清中检测到了抗CDV抗体．初步证实用CDVH、F和N基因作为核酸疫苗免疫动物，可以激活机体的免疫应答。     

Plasmid profiles of six species of Campylobacter from human beings, swine, and sheep

Twenty-four isolates representing 6 species of Campylobacter were screened for plasmids. A large plasmid with an approximate molecular weight of 38 Mdal was detected in 5 C jejuni isolates originally recovered from diarrheic human beings, in one isolate of C coli recovered from diarrheic pigs, and in 1 isolate of C sputorum ssp mucosalis and 2 isolates of C hyointestinalis recovered from pigs with proliferative enteritis. One isolate of C coli and 1 isolate of C hyointestinalis contained an additional smaller plasmid with an approximate molecular weight of 1.6 Mdal; this plasmid was partially mapped by restriction endonuclease digestion. Fifteen Campylobacter isolates contained no detectable plasmids: 2 C coli, 2 C sputorum ssp mucosalis, 2 C fecalis, 1 C fetus ssp fetus, and 8 C hyointestinalis isolates. In summary, 37.5% of the Campylobacter isolates contained a 38-Mdal plasmid, with 8% having both 38 Mdal and 1.6-Mdal plasmids; 62.5% contained no detectable plasmids.     

3株猪源大肠埃希氏菌耐药基因的初步定位

本试验旨在对临床分离的猪源大肠埃希氏菌耐药基因进行初步定位。采用常规细菌分离培养、16S rRNA PCR扩增和序列测定方法从江西省3个规模化猪场送检的子宫脓液中分离鉴定病原菌,并通过质粒提取、转化大肠埃希氏菌DH5α感受态细胞及药敏试验对临床分离株的耐药基因进行初步定位。结果显示,分离鉴定到3株大肠埃希氏菌,其中JX-22分离株仅对氧氟沙星、大观霉素敏感,JX-26分离株仅对链霉素、氧氟沙星等4种药物敏感,JX-28分离株仅对氧氟沙星等3种药物敏感,均为多重耐药菌;3株大肠埃希氏菌均可纯化到分子质量大小不一的质粒。分离株、质粒转化菌及大肠埃希氏菌DH5α感受态细胞药敏试验对比结果显示,3株大肠埃希氏菌的耐链霉素、林可霉素、甲硝唑、氨苄西林、阿莫西林、大观霉素、丁胺卡那基因,JX-22和JX-26分离株的耐多西环素、氟苯尼考和复方新诺明基因,JX-22分离株的耐头孢曲松基因,JX-28分离株的耐头孢曲松、头孢噻肟、诺氟沙星基因均定位于细菌质粒上;JX-28分离株的耐多西环素、氟苯尼考和复方新诺明基因,JX-22分离株的耐诺氟沙星基因和JX-26分离株的耐头孢曲松、头孢噻肟基因均定位于其染色体上;3株分离株均无氧氟沙星耐药基因。本试验初步确定3株多重耐药猪源大肠埃希氏菌的大部分耐药基因定位于质粒上,为进一步研究猪源大肠埃希氏菌的耐药机理和有效控制措施奠定基础。     

TRANSFERABLE DRUG RESISTANCE IN MAN AND ANIMALS: GENETIC RELATIONSHIP BETWEEN R-PLASMIDS IN ENTERIC BACTERIA FROM MAN AND DOMESTIC PETS

SUMMARY Enteric bacteria isolated from morbid human and domestic pet populations in Canberra were examined for multiple antibiotic resistance, the transferability of such resistance, and the genetic incompatibility reactions of the presumptive R-plasmid concerned. More than one-third of the isolates obtained from domestic pets were resistant to one or more of the antibiotics tested (ampicillin, chloramphenicol, kanamycin, nalidixic acid, streptomycin, sulfathiazole, tetracycline). Ampicillin resistance was invariably a part of all multiple resistance patterns seen. Half of the resistant bacteria from pets were resistant to 5 or more of the antibiotics, with ApCmSmSuTc the most frequent combination of resistance markers. The occurrence of resistance to Km and Nd was low, probably reflecting the infrequent usage of these antibiotics in veterinary practice. Genetic similarities between identifiable R-plasmids and individual resistance markers in the 2 populations were tested by grouping on the basis of incompatibility reactions against reference plasmids for incompatibility groups Fll, ***lα, N, P and W. The results, though limited in extent by experimental conditions, suggest a close similarity between R-plasmids in domestic pets and man: the majority of assignable plasmids in each population belong to group Fll***, with smaller numbers falling into groups lα and P. Analysis of individual resistance markers reveals that apart from incompatibility group N (which contained a smaller number of markers), the distribution of resistance markers across the other incompatibility groups was similar for isolates from man and animals. These results are consistent with a common origin of R-plasmids in man and domestic pets in Canberra, or one population serving as an infection source for the other, or selection influences operating at the level of host bacteria for the plasmids which result in similarities in the bacterial population and thus of plasmid types.     

Characterization and classification of Actinobacillus (Haemophilus) pleuropneumoniae plasmids.

Actinobacillus (Haemophilus) pleuropneumoniae plasmids were characterized and classified. They were isolated from A pleuropneumoniae strains different in serotype, year isolated, or location from which isolated. Six of 8 plasmids encoded streptomycin (Sm) and sulfonamide (Su) resistance (SmSu). One of the other plasmids, pVM105, encoded ampicillin (Ap) resistance and another, pHM0, encoded no drug resistance. All SmSu plasmids were transferred to Escherichia coli strains by transformation. Among them, pABO and pMS260 were 8.1 kb and incompatible with each other; they were stable in E coli. The other SmSu plasmids, pHM1, pVM104, pVM106, and pKD25, were 4.3 kb and did not replicate stably in E coli. The former SmSu plasmids were mobilized in E coli strains by a plasmid RP4, which belonged to incompatibility (Inc) group P, but the latter plasmids were not. Further, each 8.1-kb SmSu plasmid and each 4.3-kb plasmid had the same respective restriction pattern. These results indicated that there were at least 2 types of SmSu plasmids in A pleuropneumoniae. The 2 types were classified in 2 groups: H1(pMS260 and pABO) and H2(pHM1, pVM104, pVM106, and pKD25). The H1 and H2 plasmids belonged to different Inc groups, and H2 plasmids belonged to a different Inc group from that of pHMO and pVM105.     

双表达去势DNA疫苗的构建及其稳定性研究

试验旨在构建一种更安全有效的新型免疫去势DNA疫苗,通过选取下丘脑-垂体-性腺轴（hypot halamic-pituitary-gonadal axis,HPG）的上游调控基因吻素1（KISS1）和促性腺激素释放激素（GnRH）作为靶标,借助2A肽的自剪切功能,引入平衡致死系统代替抗性基因筛选流程,成功将GnRH和KISS1转入非抗性筛选质粒pVAX-asd中,酶切验证和测序对比验证目的基因的插入方向和序列完全正确。重组质粒转染HeLa细胞,反转录后扩增目的基因结果显示,重组质粒在真核细胞内能够正常转录,保证重组质粒在导入机体后能够正常表达,从而引起特异性免疫反应。将构建成功的质粒转入减毒的猪霍乱沙门氏菌C500中,获得可直接口服免疫的活载体疫苗,酶切和测序结果表明,双表达重组质粒成功导入工程菌中。将活菌疫苗在体外连续传代50次,选取0、2、5、10、20、30、40、50代的菌株进行稳定性研究,生长曲线检测结果表明,工程菌在体外连续传代50次的过程中,其生长特性无明显变化,且与减毒猪霍乱沙门氏菌C500的生长特性一致,未因携带质粒发生明显变化;同时将各代菌液扩增沙门氏菌标志基因（invA）和毒力基因（crp）,结果表明多次传代后工程菌仍然具有沙门氏菌的特性,其减毒特性也无变化。各代菌液质粒酶切验证显示,多次传代并不影响质粒的稳定性,重组双表达质粒能够在沙门氏菌C500中维持正常拷贝功能。综上所述,该重组质粒和工程菌疫苗均具有良好的稳定性,可直接应用于动物免疫去势的研究。     

Plasmid profile analysis, phage typing, and antibiotic sensitivity of Salmonella dublin from clinical isolates in the United States.

One hundred clinical isolates of Salmonella choleraesuis subsp. choleraesuis serovar dublin (Salmonella dublin) were examined for phage sensitivity, antibiotic resistance patterns, and plasmid content. Computer analysis of the lysis patterns observed by using 27 typing phages divided the S. dublin isolates into 26 groups. One lytic pattern (Designated pattern 16) contained 52% of the isolates examined whereas 16 isolates had unique patterns, and nine patterns had fewer than ten members. Although 14 antibiotic resistance patterns were observed among the 100 isolates, 79% of the isolates grouped in three major patterns. Seven plasmid groups were identified and designated A-G based on the large plasmids found in the isolates. Of the 100 isolates, 28 contained the plasmid profile of Group A, 28 were Group B, 7 were Group C, 34 were Group D, and 1 isolate each was observed in Groups E, F, and G. The strong association between antibiotic resistance pattern and plasmid type suggest that the drug resistance genes are plasmid borne.