新型甲氧苄啶耐药二氢叶酸还原酶DfrB7的生化分析与宿主菌耐药机制研究

【目的】分析新型甲氧苄啶获得性耐药蛋白DfrB7的生化性质,探究其与B家族代表性的二氢叶酸还原酶(DHFR)对甲氧苄啶获得性耐药的生化基础。【方法】构建系统进化树分析B家族DHFRs与新型DfrB7的进化关系。将dfr基因分别构建到pACYC184和pET15b(+)载体并转化到相应大肠杆菌中。使用微量肉汤稀释法确定克隆菌株对甲氧苄啶的耐药性。测定DHFRs使用NADPH作为质子供体催化二氢叶酸还原的酶活反应参数。通过等温滴定量热法测定甲氧苄啶的解离常数。【结果】克隆到大肠杆菌中的新型dfrB7基因表现出对甲氧苄啶的高耐药表型。系统进化树确定了dfrB7编码B家族的DHFRs。检测并比较DfrB7和代表性DHFRs的酶活性质、抑制剂的亲和力,与染色体上DHFR相比,DfrB7与DfrB1均表现出显著降低的催化活性,通过生化实验证实B家族二氢叶酸还原酶对甲氧苄啶结合力极差。【结论】新型dfrB7基因编码的DfrB7具有B家族二氢叶酸还原酶的普遍特征。B家族二氢叶酸还原酶赋予宿主菌对甲氧苄啶的获得性耐药与该酶对甲氧苄啶的低亲和力有关。     

Emergence of tetracycline resistance due to a multiple drug resistance plasmid in Vibrio cholerae O139

Abstract Of the 173 clinical strains of Vibrio cholerae O139 isolated from India, Bangladesh, and Thailand tested, six strains from India were resistant to tetracycline, ampicillin, chloramphenicol, kanamycin, and gentamicin. These six strains harbored a self-transmissible plasmid that mediated resistance to tetracycline, ampicillin, chloramphenicol, kanamycin, gentamicin, sulfamethoxazole, trimethoprim, and O/129. The multiple drug resistance plasmids were 200 kb in size and belonged to the incompatibility group C. Although a majority of the O139 strains (94.8%) were highly resistant to streptomycin, sulfamethoxazole, trimethoprim, and O/129, the tetracycline-susceptible strains so far tested were plasmid-negative. The data suggest the existence of two distinct multiple antimicrobial agent resistance (MAR) patterns in V. cholerae O139.     

Update on acquired tetracycline resistance genes

This mini-review summarizes the changes in the field of bacterial acquired tetracycline resistance (tet) and oxytetracycline (otr) genes identified since the last major review in 2001. Thirty-eight acquired tetracycline resistant (Tc(r)) genes are known of which nine are new and include five genes coding for energy-dependent efflux proteins, two genes coding for ribosomal protection proteins, and two genes coding for tetracycline inactivating enzymes. The number of inactivating enzymes has increased from one to three, suggesting that work needs to be done to determine the role these enzymes play in bacterial resistance to tetracycline. In the same time period, 66 new genera have been identified which carry one or more of the previously described 29 Tc(r) genes. Included in the new genera is, for the first time, an obligate intracellular pathogen suggesting that this sheltered group of bacteria is capable of DNA exchange with non-obligate intracellular bacteria. The number of genera carrying ribosomal protection genes increased dramatically with the tet(M) gene now identified in 42 genera as compared with 24 and the tet(W) gene found in 17 new genera as compared to two genera in the last major review. New conjugative transposons, carrying different ribosomal protection tet genes, have been identified and an increase in the number of antibiotic resistance genes linked to tet genes has been found. Whether these new elements may help to spread the tet genes they carry to a wider bacterial host range is discussed.     

Mutational analysis of tetracycline resistance protein transmembrane segment insertion

The tetracycline resistance proteins (TetA) of gram-negative bacteria are secondary active transport proteins that contain buried charged amino acids that are important for tetracycline transport. Earlier studies have shown that insertion of TetA proteins into the cytoplasmic membrane is mediated by helical hairpin pairs of transmembrane (TM) segments. However, whether helical hairpins direct spontaneous insertion of TetA or are required instead for its interaction with the cellular secretion (Sec) machinery is unknown. To gain insight into how TetA proteins are inserted into the membrane, we have investigated how tolerant the class C TetA protein encoded by plasmid pBR322 is to placement of charged residues in TM segments. The results show that the great majority of charge substitutions do not interfere with insertion even when placed at locations that cannot be shielded internally within helical hairpins. The only mutations that frequently block insertion are proline substitutions, which may interfere with helical hairpin folding. The ability of TetA to broadly tolerate charge substitutions indicates that the Sec machinery assists in its insertion into the membrane. The results also demonstrate that it is feasible to engineer charged residues into the interior of TetA proteins for the purpose of structure-function analysis.     

信号分子AI-2的体外合成及其对副溶血性弧菌四环素耐药性的调控作用

【背景】抗菌药的过度使用引起细菌耐药性日益严重,作为重要的食源性致病菌,副溶血性弧菌也表现出一定程度的耐药性。群体感应系统可以调控细菌的耐药性,为研究副溶血性弧菌的耐药机制和控制技术提供新的途径。【目的】探讨群体感应信号分子AI-2 (autoinducer-2)对海产品中分离的副溶血性弧菌四环素耐药性的调控作用。【方法】通过原核表达制备AI-2合成关键酶——S-核糖同型半胱氨酸酶(S-ribosylhomocysteinase, LuxS)和S-腺苷同型半胱氨酸核苷酶(S-adenosylhomocysteinenucleosidase,Pfs),体外合成AI-2,通过菌落计数法分析AI-2对副溶血性弧菌在四环素亚抑菌浓度下耐受性的影响,采用逆转录实时荧光定量PCR法测定不同浓度AI-2对副溶血性弧菌四环素耐药基因转录水平的影响。【结果】通过原核表达获得LuxS和Pfs,作用于底物S-腺苷同型半胱氨酸能合成具有生物活性的AI-2,其荧光强度约为阳性对照的6倍。在四环素亚抑菌浓度下,AI-2能显著促进副溶血性弧菌的生长,6、15、30μmol/L浓度AI-2能不同程度地提高副溶血性弧菌...     

Characterization of tetracycline resistance lactobacilli isolated from swine intestines at western area of Taiwan

To investigate the frequency of tetracycline resistance (Tet-R) lactobacilli in pig intestines, a total of 256 pig colons were analyzed and found to contain typical colonies of Tet-R lactic acid bacteria in every sample, ranging from 3.2 × 10³ to 6.6 × 10⁵ CFU/cm². From these samples, a total of 159 isolates of Tet-R lactobacilli were obtained and identified as belonging to 11 species, including Lactobacillus reuteri, Lactobacillus amylovorus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus ruminis, Lactobacillus kefiri, Lactobacillus fermentum, Lactobacillus sakei, Lactobacillus coryniformis, Lactobacillus parabuchneri and Lactobacillus letivazi. Based on the EFSA (2008) breakpoints, all isolates, after MIC analysis, were qualified as Tet-R, from which the significant high Tet-R MIC₅₀ and MIC₉₀ values indicated an ecological distribution of Tet-R lactobacilli mostly with high resistance potency in pig colons. PCR-detection identified 5 tet genes in these isolates, the most predominant one being tet (W), followed by tet (M), (L), (K), and (Q). Their detection rates were 82.0%, 22.5%, 14.4%, 8.1% and 0.9%, respectively. Noteworthily, isolates of the same species carrying identical tet gene(s) usually had a wide different MIC values. Furthermore, strain-subtyping of these isolates by REP-PCR demonstrated a notable genotypic biodiversity % (average = 62%).     

植物抗病毒病育种策略

为了得到抗病毒的寄主植物,植物育种学家进行了许多有益研究,形成了许多行之有效的抗病毒病育种策略。利用植物本身对病毒侵染所具有的一些免疫功能及其本身的一些抗性基因来获得抗性;利用来源于病毒自身基因的一些抗病性策略(PDR),如利用病毒外壳蛋白基因,病毒复制酶基因,病毒移动蛋白基因,病毒卫星RNA和反义RNA等,植物也可以获得抗性。近年来对由转录后RNA沉默引起的由RNA介导的病毒抗性策略(RMVR)也进行了深入地研究。除了PDR和RMVR以外,还有一些导致植物抗病毒的策略,包括利用美国商陆的病毒抗性蛋白(PAP),2',5’-寡腺苷酸合成酶,“植物抗体”以及病毒蛋白多肽来获得病毒抗性等。     

家养及大型养殖场鸡肠道微生物菌群及四环素耐药菌多样性的比较研究

本研究分析比较了农家家养鸡与大型养殖场鸡肠道菌群组成及抗四环素抗性基因在肠道菌群中的分布情况。通过454焦磷酸法对细菌16S rRNA V3区进行测序来分析肠道菌群的组成;用平板琼脂法筛选四环素耐药菌株,对其16S rRNA基因全长测序,并与RDP (Ribosomal Database Project)数据库比对进行菌种鉴定;聚合酶链反应(polymerase chain reaction,PCR)检测常见四环素耐药基因。家养鸡粪便中菌群香农多样性指数为5.321±0.590,养殖场鸡为4.398±0.440,前者显著大于后者(Mann-Whitney U test,P＝0.008)。从家养鸡和养殖场鸡粪便中随机分离到69株和65株四环素耐药菌株,后者四环素耐药菌的种类多于前者。家养鸡较养殖场鸡的肠道菌群更具多样性,而抗生素抗性基因在养殖场鸡的肠道菌群中分布更广泛。结果表明,不同饲养方式对鸡的肠道菌群有影响,对抗生素抗性基因的分布也有一定影响。     

Synthesis,characterization and antimicrobial activity of novel xanthene sulfonamide and carboxamide derivatives

Xanthene intermediates 4a and 4b were obtained from the reduction of nitro xanthene derivatives 3a and 3b which were synthesized via condensation of dimedone with m-nitrobenzaldehyde and p-nitrobenzaldehyde, respectively. Then xanthene sulfonamide 6a–n, and xanthene carboxamide derivatives 8a–h were synthesized by reaction of amino xanthene 4a, 4b with sulfonyl chlorides 5a–g and acyl chlorides 7a–d. Structures of the novel amino xanthene compounds and xanthene sulfonamide/carboxamide derivatives were established by their spectral data and elemental analyses. Furthermore, all the synthesized compounds were tested in vitro for their antimicrobial activity. The results were compared with reference standard antibiotics, erythromycin and nystatin. 6c, 6f, 6m and 8b Compounds were found to display most effective antimicrobial activity against a series of bacteria and fungi.     

Chromosomal studies in vivo and in vitro of trimethoprim and sulphamethoxazole (co-trimoxazole)

Because both trimethoprim (TMP) and sulphamethoxazole (SMX) could interfere with folate metabolism and the former is a pyrimidine analogue it seemed advisable to try to ascertain whether these drugs alone, or in combination, determine damage in human chromosomes.In nine patients who had been taking a standard daily dosage of co-trimoxazole for periods varying from 3 to 112 weeks no damage in chromosomes of lymphocytes was found. In vitro experiments were cultured in concentrations of TMP and SMX considerably higher than the steady state plasma levels of patients being treated with the combination of drugs, also showed no chromosomal damage.     

Identification of a truncated, but functionally active tet(H) tetracycline resistance gene in Pasteurella aerogenes and Pasteurella multocida

Molecular analysis of Pasteurella isolates of animal origin for plasmid-encoded tetracycline resistance genes identified a common tet(H)-carrying plasmid of 5.5 kbp in a single isolate of Pasteurella aerogenes and six isolates of Pasteurella multocida. This plasmid carried a truncated Tn5706 element in which one of the IS elements, IS1596, was lost completely and of the other, IS1597, only a relic of 84 bp was left. Sequencing of the resistance gene region and the flanking areas revealed the presence of a deletion in the 3' end of the tet(H) gene which shortened the tet(H) reading frame by 24 bp. The amino acid sequence of the respective TetH protein comprised only 392 amino acids. Despite this deletion, the tet(H) gene conferred high level tetracycline resistance not only to the original Pasteurella isolates but also to the respective Escherichia coli JM107 and C600 transformants as confirmed by MIC determination. The deletion was probably the result from recombinational events. Two possible recombination sites involved in the deletion of tet(H) and that of IS1597 were identified. Macrorestriction analysis of the Pasteurella isolates carrying plasmid pPAT1 confirmed horizontal and vertical transfer of this plasmid.     

Novel carbazole sulfonamide derivatives of antitumor agent: Synthesis,antiproliferative activity and aqueous solubility

The current optimization of IG-105 (3) on the carbazole-ring provided a series of new carbazole sulfonamides derivatives 13a–13m. All of the compounds have been evaluated against HepG2 cells (hepatoma cancer) for antiproliferative activity. Compounds that showed activity better or comparable to that of 3 versus HepG2 were evaluated against MCF-7 (breast cancer), MIA PaCa-2 (pancreatic cancer), and Bel-7402 (hepatoma/liver cancer) for antiproliferative activity. Of the seven compounds selected for further study five (13b, 13g, 13j, 13k and 13l) were found to give IC₅₀ values against the four cell lines comparable to those for 3. Two compounds (13f and 13i) were more active than 3 and their activity against HepG2 and MCF-7 (IC₅₀:0.01–0.07 μM) approached that of the positive controls podophyllotoxin (podo) and CA-4. Most of compounds showed aqueous solubility (0.11–19.60 μg/mL at pH 7.4 and 2.0) better than 3. These promising results warrant further development of new compounds 13f and 13i as potential potent antitumor drug candidates.     

A Dual Role for Microbial Pathogen-Derived Effector Proteins in Plant Disease and Resistance

Many proteins from plant pathogens affecting the interaction with the host plant have dual functions: they promote virulence on the host species and they function as avirulence determinants by eliciting defense reactions in host cultivars expressing the appropriate resistance genes. In viruses all proteins encoded by the small genomes can be expected to be essential for viral development in the host. However, in different plants surveillance systems have evolved that are able to recognize most of these proteins. Bacteria and fungi have specialized pathogenicity and virulence genes. Many of the latter were originally identified through the resistance gene-dependent elicitor activity of their products. Their role in virulence only became apparent when they were inactivated or transferred to different microbes or after their ectopic expression in host plants. Many microbes appear to maintain these genes despite their disadvantageous effect, introducing only few mutations to abolish the interaction of their products with the plant recognition system. This has been interpreted as been indicative of a virulence function of the gene products that is not impaired by the mutations. Alternatively, in particular in bacteria there is now evidence that pathogenicity was acquired through horizontal gene transfer. Genes supporting virulence in the donor organism's original host appear to have traveled along. Being gratuitous in the new situation, they may have been inactivated without loss of any beneficial function for the pathogen.     

小麦抗白粉病基因

到目前为止,小麦中已经鉴定出31个主效抗白杨病基因位点(Pm1-Pm31),对这些小麦抗白粉病基因位点的来源、染色体定位、遗传特点以及载体品种等方面进行了概括性综述。     

Real-time PCR methods for quantitative monitoring of streptomycin and tetracycline resistance genes in agricultural ecosystems

Antibiotic application in plant agriculture is primarily used to control fire blight caused by Erwinia amylovora in pome fruit orchards. In order to facilitate environmental impact assessment for antibiotic applications, we developed and validated culture-independent quantitative real-time PCR multiplex assays for streptomycin (strA, strB, aadA and insertion sequence IS1133) and tetracycline (tetB, tetM and tetW) resistance elements in plant and soil samples. The qPCR were reproducible and consistent whether the DNA was extracted directly from bacteria, plant and soil samples inoculated with bacteria or soil samples prior to and after manure slurry treatment. The genes most frequently identified in soils pre- and post-slurry treatment were strB, aadA, tetB and tetM. All genes tested were detected in soils pre-slurry treatment, and a decrease in relative concentrations of tetB and the streptomycin resistance genes was observed in samples taken post-slurry treatment. These multiplex qPCR assays offer a cost-effective, reliable method for simultaneous quantification of antibiotic resistance genes in complex, environmental sample matrices.