In vivo transfer of pAMβ1 from Lactobacillus reuteri to Enterococcus faecalis

Trials were conducted to determine the in vivo transferability of plasmid-mediated antibiotic resistance between two strains of enteric Gram-positive bacteria. Germfree mice were associated with the donor Lactobacillus reuteri DSM 20016 strain, carrying the broad host range pAMβ1 plasmid, and with the Enterococcus faecalis JH2SS recipient strain.
Analysis of faecal content of associated mice demonstrated that the in vivo transfer of this plasmid did occur and that frequencies of conjugation were affected by the presence of subtherapeutic levels of antibiotic in the diet.     

Plasmid- and strain-specific factors drive variation in ESBL-plasmid spread in vitro and in vivo

Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global rise of antibiotic resistance. However, the relative contributions of factors that underlie the spread of plasmids and their roles in conjugation in vivo are unclear. To address this, we investigated the spread of clinical Extended Spectrum Beta-Lactamase (ESBL)-producing plasmids in the absence of antibiotics in vitro and in the mouse intestine. We hypothesised that plasmid properties would be the primary determinants of plasmid spread and that bacterial strain identity would also contribute. We found clinical Escherichia coli strains natively associated with ESBL-plasmids conjugated to three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain. Final transconjugant frequencies varied across plasmid, donor, and recipient combinations, with qualitative consistency when comparing transfer in vitro and in vivo in mice. In both environments, transconjugant frequencies for these natural strains and plasmids covaried with the presence/absence of transfer genes on ESBL-plasmids and were affected by plasmid incompatibility. By moving ESBL-plasmids out of their native hosts, we showed that donor and recipient strains also modulated transconjugant frequencies. This suggests that plasmid spread in the complex gut environment of animals and humans can be predicted based on in vitro testing and genetic data.Subject terms: Antibiotics, Microbial ecology, Phylogenomics     

Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S, sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey     

黄芩对去污染小鼠体内痢疾杆菌R质粒消除试验研究

本文以去污染的瑞士小白鼠为模型用黄苓对福氏痢疾杆菌F13株的R质粒进行了体内消除试验。选用红霉素、四环素和利福平对小鼠肠道进行去污染,降低其定居阻力,然后用福氏痢疾杆菌F13株经口感染,同时利用四环素造成一种选择压力,以利于对四环素耐药的F13株菌生长繁殖。利用小鼠肠道菌群重建的间隙,进行R质粒的消除试验。结果表明,黄芩对福氏痢疾杆菌F13菌株R质粒在体内具有一定的消除作用。     

cosmid克隆筛选的体内同源重组法——一个含有小鼠t复合体连锁DNA顺序的cosmid克隆的分离

一个从cosmid分子克隆库中筛选特别基因顺序的遗传学方法——体内同源重组(invlvo homologous recombination)法。即使探针DNA与分子克隆库中带有与探针同源顺序的克隆发生体内重组,然后以遗传学方法进行筛选。cosmid分子克隆库构建在rec宿主细胞内,经体内包装(in vivo Packaging)成λ噬菌体颗粒,把该噬菌体颗粒转入带有探针DNA的rec~+细胞内,探针是已被克隆在与cosmid载体没有同源顺序的质粒(如PUC8或PUC9)内的。经过一段时间(1—3小时),待重组发生后,把cosmid进行体内包装。此时探针DNA连同质粒已整合入cosmid基因组内,因此它带有原为两个载体所分别带有的双重抗性——Amp~r(氨苄青霉素,PUC8或PUC9)和Kan~r(卡那霉素,cosmid)。这种双重抗性菌落可在含有这2种抗菌素的培养平皿上选出,该重组cosmid借助于λ切除酶的作用将已被整合的探针质粒重新切除,再经体内包装后,该cosmid被还原并纯化,然后可用一含有Xgal的培皿识别和选出。本文用此法以有关DNA探针从cosmid分子克隆库中分离得到含有与小鼠t复合体连锁的基因组顺序的克隆,并对该克隆作了物理图谱分析。     

CS3菌毛抗原和霍乱毒素B亚基在人伤寒菌中的表达

通过体内外重组的方法,构建了人伤寒菌流行株Ｔｙ２的△ａｒｏＡ,△ａｒｏＣ和ａｓｄ＾－基因缺失突变体（ＲＳ４１７）作为抗原载体菌：同时,构建包含ａｓｄ基因的表达质粒ｐＹＸ１０２,与ＲＳ４１７一起,构成宿主－载全平衡致死系统,用于在没有抗生素条件选择的情况下,稳定表达克隆在表达质粒上的外源抗原基因,将肠毒素性大肠杆菌的菌毛抗原ＩＩＩ（ＣＳ３）基因和霍乱弧菌毒素Ｂ亚基（ＣＴＢ）基因分别克隆至ｐＹＸ１０２     

肠毒素性大肠杆菌CS3纤毛抗原在痢疾菌中的表达及其免疫效果研究

首先通过体内外重组的方法,构建了福氏2a痢疾菌T32asd基因缺陷的突变体FaD,作为抗原载体菌;同时,构建包含asd基因的表达质粒pYX102,与FaD一起,构成宿主-载体平衡致死系统,用于在没有抗生素条件选择的情况下,稳定表达克隆在表达质粒上的外源抗原基因.将肠毒素性大肠杆菌的CS3菌毛抗原基因克隆至pYX102,构建成重组表达质粒pYX103,ELISA检测结果证实CS3在痢疾菌中可以很好地表达.免疫小鼠后可诱生相应的抗体,虽然口服免疫和注射免疫产生的CS3抗体效价有一定差别,但对痢疾菌的毒株攻击均可提供较好保护.该结果为细菌性腹泻疫苗的研制提供了候选株.     

The primary screening of bifidobacteria and lactobacilli strains to develop effective probiotic preparations based on them

10 Bifidobacterium strains and 10 Lactobacillus strains were studied for their antagonistic activity with respect to Escherichia coli, Klebsiella ozaenae, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and for their sensitivity to antibiotics, widely used in clinical practice. L. acidophilus strain 5/4, L. acidophilus strain 18/4, B. adolescentis strain UX, B. longum strain 44 exhibited the highest antagonistic activity and the highest degree of antibiotic resistance. The restriction analysis of the chromosomal DNA of these strains was then made and their plasmid content was studied, making it possible to recognize these strains in future in the course of in vivo experiments.     

Mutations in Salmonella typhimurium recovered from livers and spleens of mice

Balb/c mice were inoculated intraperitoneally with TA2662, a smooth derivative of the Salmonella typhimurium Ames tester strain (TA102) which carries the mutable hisG locus on a multicopy plasmid, or TA103, which carries the same hisG gene on the chromosome. The bacteria were recovered at various times from the livers and spleens of the infected mice. Total numbers of bacteria were determined and the mutant frequency was estimated. The frequency of occurrence of histidine prototrophs in experiments using TA2662 was substantially above the frequency found with this strain grown in vitro. The mutant frequencies in experiments using TA103 recovered from mice were also highly significantly increased above background. We did not identify factors which might suggest selection in vivo for histidine prototrophs. There is sufficient histidine in body fluids of the host for the growth of His- bacteria. The His- and His+ derivatives were found to grow equally well in vitro in the presence of amounts of histidine approximating concentrations known to exist in vivo. It is probable that mutations in TA2662 are greatly underestimated, since the hisG-containing plasmid is lost at relatively high frequency during incubation in a variety of conditions.     

Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo

A method has been developed that allows the isolation of genomic clones from a cosmid library by homologous recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene. The genomic cosmid library was packaged in vivo into lambda phage particles. A recombination-proficient host strain carrying IL2 cDNA sequences in a non-homologous plasmid vector was infected by the packaged cosmid library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both vectors were selected. From a recombinant cosmid clone the chromosomal IL2 gene was restored. After DNA mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively.     

Effects of yoghurt intake on plasmid transfer and colonisation with transconjugants in the digestive tract of mice associated with human faecal flora

This study deals with the effects of yoghurt intake on wild-type and recombinant plasmid transfer from an exogenous Escherichia coli K12-derivative donor strain to an endogenous recipient strain in the digestive tract of mice associated with human faecal flora. We showed that the self-transmissible plasmid R388 was efficiently transferred to recipient strain PG1 in mice associated with human faecal flora (HFF-PG1) and that the resulting transconjugants (PG1-R388) became established at a high and maximal population level without any selective pressure. Using HFF-PG1 mice made it possible to determine whether yoghurt consumption decreases R388 transfer efficiency and the ability of transconjugant PG1-R388 to successfully colonise the digestive tract. Results indicated that yoghurt consumption had two effects: it reduced the efficacy of plasmid transfer 10-fold and decreased the transconjugant PG1-R388 population density 100-fold, compared to the control group. We also describe another HFF mouse model in which recipient PG1 was replaced by EM0 with which no plasmid transfer was observed. This model made it possible to demonstrate the potential promoting effect of yoghurt intake on transconjugant formation and establishment. Our results indicated no yoghurt effect; no transconjugants appeared in the digestive tract of HFF-EM0 mice fed on yoghurt or on standard food. In both mouse models, HFF-PG1 and HFF-EM0, yoghurt intake did not promote the mobilisation of either the non-self-transmissible plasmid pUB2380 or the recombinant plasmid pCE325.     

Effect of in situ expression of human interleukin-6 on antibody responses against Salmonella typhimurium antigens

In an attempt to trigger increased mucosal secretory immune responses against bacterial surface antigens, we constructed an optimized human interleukin (hIL)-6-secreting Salmonella typhimurium strain (X4064(pCH1A+pYL3E)), utilizing the hemolysin (Hly) exporter for secretory delivery of a functional hIL-6-hemolysin fusion protein (hIL-6-HlyA(s)). Through stable introduction of a second hIL-6-HlyA(s) expression plasmid (pYL3E) in the previously described X4064(pCH1A) strain, hIL-6-HlyA(s) secretion efficiencies were increased by at least 10-fold. As pCH1A in the parental strain, pYL3E was stable in vitro in the absence of antibiotic selection and in vivo neither did plasmids interfere in their stabilities. Increased hIL-6-HlyA(s) expression did not adversely interfere with bacterial growth. Comparative immunization experiments in mice with oral application of the different hIL-6-secreting strains revealed that increased in situ hIL-6-production influenced systemic antibody responses against Salmonella antigens but had no marked effect on mucosal responses. In mice immunized with X4064(pCH1A+pYL3E) significantly higher sera IgG and IgA titers for lipopolysaccharide (LPS) were found compared to mice immunized with X4064(pCH1A) and a hIL-6-negative control strain. Higher sera antibody titers were accompanied by increased numbers of IgG- and IgA-specific antibody-secreting cells in spleens and Peyer's patches, respectively. These data suggest that systemic antibody responses against Salmonella LPS are largely effected by IL-6 and, moreover, the amount and the cellular location of recombinantly expressed IL-6 appears to be crucial for enhancement of immune responses.     

Construction of a non-antibiotic expression system in a marine cyanobacterium Synechococcus sp. PCC 7002 and its application in production of oral vaccine against enterotoxin of Escherichia coli

Synechococcus sp. PCC 7002 is a marine cyanobacterium that is rich in thylakoid membranes and performs oxygenic photosynthesis. In this study, we inactivated its psbEF genes encoding cytochrome b-559 of photosystem II and the mutant strain EF211 could not grow photoautotrophically. A non-antibiotic plasmid (pAQEF559) was constructed in such a way that it has a psbEF operon and all antibiotic genes are deleted. The plasmid could complement EF211 and restore photoautotrophical growth. This non-antibiotic plasmid was used to express the gene encoding heat-labile enterotoxin B-subunit, LT-B. The overproduced LT-B in the cyanobacterium could form pentamers and was associated with thylakoid membranes. Oral immunization of mice with the cyanobacterium overproducing LT-B showed that it was very effective in stimulating production of both IgG in serum and IgA in intestine. Our results demonstrate that the non-antibiotic vector in combination with the constructed host cyanobacterium is useful in production of recombinant proteins in cyanobacteria and the cyanobacterial cells producing LT-B can be an effective oral vaccine against enterotoxins.     

Bacteriocin plasmids ofPediococcus acidilactici

Summary Pediococcus acidilactici strains E, F and H isolated from fermented sausages produced bacteriocins which were protein in nature and inhibitory to a variety of spoilage and pathogenic microorganisms often encountered in foods. These strains harbored two to three plasmids ranging in size from 7.4 to 40.2 megadaltons. Curing experiments and plasmid profile analysis indicated the involvement of plasmid DNA with bacteriocin activity in all three strains. Carbohydrate fermentation and antibiotic resistance phenotypes did not appear to be associated with bacteriocin plasmids. Both bacteriocin activity and resistance determinants were linked in strain H and mediated by a 7.4-megadalton plasmid, whereas in strains E and F these two traits were not linked.     

Strain identification in Rhizobium trifolii using DNA restriction analysis, plasmid DNA profiles and intrinsic antibiotic resistances

Abstract Indigenous strains of R. trifolii , originating from different geographical regions, were compared using total DNA restriction analysis, plasmid DNA profiles and intrinsic antibiotic resistance patterns. The results of strain relatedness using 2 of these techniques (DNA restriction and antibiotic resistance patterns) were similar, and indicated that these strains formed a heterogeneous group. A large variation in plasmid DNA profiles was observed among the R. trifolii isolates investigated, indicating heterogeneity of plasmid DNA pools. This variation occurred even in some strains that were shown to be related on the basis of their antibiotic resistance patterns. The application of these techniques to differentiate indigenous R. trifolii strains is discussed in this report.     

Mechanism of additive genetic transformation in Haemophilus influenzae.

Transforming deoxyribonucleic acid (DNA) preparations from Haemophilus influenzae Rd strains carrying a chromosomally integrated, conjugative, antibiotic resistance transfer (R) plasmid were exposed to ultraviolet radiation and then assayed for antibiotic resistance transfer on sensitive wild-type Rd competent suspensions and on similar suspensions of a uvr-1 mutant unable to excise pyrimidine dimers. No host cell reactivation of resistance transfer (DNA repair) was observed. Parallel experiments with ethanol-precipitated, heated, free R plasmid DNA preparations gave much higher survival when assayed on the wild-type strain compared to the survival on the uvr-1 strain. These observations indicate that additive genetic transformation (in this case, the addition of the integrated R plasmid to the recipient genome) involves single-strand insertion.     

Photonic detection of bacterial pathogens in living hosts

The study of pathogenic is often limited to ex vivo assays and cell-culture correlates. A greater understanding of infectious diseases would be facilitated by in vivo analyses. Therefore, we have developed a method for detecting bacterial pathogens in a living host and used this method to evaluate disease processes for strains of Salmonella typhimurium that differ in their virulence for mice. Three strains of Salmonella were marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescent Salmonella allowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive. We observed patterns of bio-luminescence that suggested the caecum may play a pivotal role in Salmonella pathogenesis. In vivo efficacy of an antibiotic was monitored using this optical method. This study demonstrates that the real time non-invasive analyses of pathogenic events and pharmacological monitoring can be performed in vivo .     

A second large plasmid encodes conjugative transfer and antimicrobial resistance in O119:H2 and some typical O111 enteropathogenic Escherichia coli strains

A novel and functional conjugative transfer system identified in O119:H2 enteropathogenic Escherichia coli (EPEC) strain MB80 by subtractive hybridization is encoded on a large multidrug resistance plasmid, distinct from the well-described EPEC adherence factor (EAF) plasmid. Variants of the MB80 conjugative resistance plasmid were identified in other EPEC strains, including the prototypical O111:NM strain B171, from which the EAF plasmid has been sequenced. This separate large plasmid and the selective advantage that it confers in the antibiotic era have been overlooked because it comigrates with the virulence plasmid on conventional gels.     

Plasmids in clinical isolates ofStreptococcus pneumoniae

Forty clinical isolates ofStreptococcus pneumoniae with various antibiotic resistance profiles were screened for the presence of plasmids. Plasmids were demonstrated in five isolates. Three procedures for plasmid isolation were evaluated. A 10.8-kb plasmid was demonstrated by all three methods, but a further four plasmids were detected with one method only. The sizes of these plasmids were 11.5 kb, 10.8 kb, and 3.0 kb (two strains). Curing experiments were performed, but no plasmid/antibiotic correlation was observed. Tetracycline, erythromycin, and clindamycin resistance was lost in one strain, although the plasmid was still present.