Analysis and Recognition of Erythroid-Specific Gene Promoters Basing on Degenerate Oligonucleotide Motifs

We developed a method to search for degenerate oligonucleotide motifs specific for certain regions in eukaryotic gene promoters. A procedure of promoter recognition based on these motifs is presented. The methods are integrated within the program package ARGO available for Internet users (http://wwwmgs.bionet.nsc.ru/mgs/programs/argo). This method was applied to study erythroid-specific gene promoters. High efficiency of their recognition is demonstrated.     

Analysis and recognition of promoters for erythroid-specific genes based on degenerative oligonucleotide motifs]

We developed a method to search for degenerate oligonucleotide motifs specific for certain regions in eukaryotic gene promoters. A procedure of promoter recognition based on these motifs is presented. The methods are integrated within program package ARGO available for the Internet users (http://wwwmgs.bionet.nsc.ru/mgs/programs/argo). This method was applied to study erythroid-specific gene promoters. High efficiency of their recognition is demonstrated.     

Polygenes and modifier genes for tetracycline and penicillin resistance in Neisseria gonorrhoeae

The genetic basis for spontaneous resistance to tetracyline (Tet) and penicillin (Pen) in Neisseria gonorrhoeae was investigated. Tet and pen are polygenes which confer small but distinct levels of resistance to Tet and Pen, respectively. Mtr is a multiple-drug resistance polygene which increases resistance to Tet and Pen (as well as to other unrelated antibiotics). Tem is a modifier gene affecting resistance toTet and Pen. Pem is a modifier gene for Pen resistance. The following gene combinations code for resistance to five antibiotics: tet, mtr and tem for Tet; pen, mtr, pem and tem for Pen; tet, tem and mtr for doxycycline; pen and pem for ampicillin; pen, pem and mtr for nafcillin.     

畜禽养殖废物中抗生素和重金属抗性基因的产生机制和控制方法研究进展

动物饲料中常混有抗生素和重金属,导致外排的动物粪便中携带有抗生素和重金属,引发细菌产生耐药性和重金属抗性,继而产生抗生素抗性基因和重金属抗性基因。抗生素和重金属抗性基因污染已成为威胁人类身体健康及破坏生态环境的重大问题。本文从细菌进化的角度,明确了细菌的抗生素和重金属长期进化试验对抗性机制研究的重要性;抗生素抗性基因与重金属抗性基因间存在复杂的协同选择抗性,两者间相互影响,共同决定着细菌环境行为;抗性基因的水平转移增加了细菌在环境中的可变性,可移动遗传元件在抗性基因水平转移中发挥着重要作用。在抗性基因污染控制方面,高级氧化技术具有很好的抗性基因去除效果,尤其是UV/TiO₂氧化技术,能使抗生素抗性基因丰度减少4.7～5.8 log,减少率大于99.99%。其他的控制策略,如抗生素替代品博落回提取物以及噬菌体与抗生素结合使用,对于抗性基因的控制也具有重要意义。     

A DHA14 drug efflux gene from Xanthomonas albilineans confers high-level albicidin antibiotic resistance in Escherichia coli

AIMS: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. METHODS AND RESULTS: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. CONCLUSIONS: AlbF is the first apparent single-component antibiotic-specific efflux pump from a gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.     

Research progress on distribution,migration, transformation of antibiotics and antibiotic resistance genes (ARGs) in aquatic environment

Antimicrobial and antibiotics resistance caused by misuse or overuse of antibiotics exposure is a growing and significant threat to global public health. The spread and horizontal transfer of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) by the selective pressure of antibiotics in an aquatic environment is a major public health issue. To develop a better understanding of potential ecological risks die to antibiotics and ARGs, this study mainly summarizes research progress about: (i) the occurrence, concentration, fate, and potential ecological effects of antibiotics and ARGs in various aquatic environments, (ii) the threat, spread, and horizontal gene transfer (HGT) of ARGs, and (iii) the relationship between antibiotics, ARGs, and ARB. Finally, this review also proposes future research direction on antibiotics and ARGs.     

鲍曼不动杆菌耐药程度与其主动外排泵蛋白的相关性研究

目的:探讨鲍曼不动杆菌耐药程度与其主动外排泵蛋白的相关性。方法:首先用纸片扩散法检测64株临床鲍曼不动杆菌对8种抗菌药物的敏感性;将其分为A组(0～2种抗生素耐药)、B组(对3～5种抗生素耐药)和C组(对6～8种抗生素耐药);检测64株临床鲍曼不动杆菌对罗丹明6G的外排情况,筛选出罗丹明6G外排明显增加的菌株;并用逆转录-聚合酶链反应(RT-PCR)方法检测主动外排泵基因AdeABC的表达水平。结果:64株鲍曼不动杆菌中有4株对0～2种抗生素耐药(A组),对3～5种抗生素耐药的有33株(B组),对6～8种抗生素耐药的有27株(C组);多重耐药组鲍曼不动杆菌罗丹6G外排明显增高,外排程度A组相似文献   

Origins and Evolution of Antibiotic Resistance

Summary: Antibiotics have always been considered one of the wonder discoveries of the 20th century. This is true, but the real wonder is the rise of antibiotic resistance in hospitals, communities, and the environment concomitant with their use. The extraordinary genetic capacities of microbes have benefitted from man''s overuse of antibiotics to exploit every source of resistance genes and every means of horizontal gene transmission to develop multiple mechanisms of resistance for each and every antibiotic introduced into practice clinically, agriculturally, or otherwise. This review presents the salient aspects of antibiotic resistance development over the past half-century, with the oft-restated conclusion that it is time to act. To achieve complete restitution of therapeutic applications of antibiotics, there is a need for more information on the role of environmental microbiomes in the rise of antibiotic resistance. In particular, creative approaches to the discovery of novel antibiotics and their expedited and controlled introduction to therapy are obligatory.     

多重耐药鲍曼不动杆菌的耐药性及其耐药基因分析

目的:探讨多重耐药鲍曼不动杆菌(MDR-Ab)的耐药性及其耐药基因,为临床合理选择抗菌药物提供依据。方法:回顾性分析2018年1月至2018年12月鲍曼不动杆菌感染的住院患者信息。使用VITEK-32微生物分析仪/梅里埃药敏卡片GN13鉴定MDR-Ab 95株。采用聚合酶链式反应(多重PCR)检测MDR-Ab携带相关耐药基因。结果:95株MDR-Ab对头孢类抗菌药物耐药率为100%。对氨苄西林-舒巴坦和头孢哌酮-舒巴坦耐药率分别为95.79%和81.05%,对美罗培南和亚胺培南耐药率分别为56.84%和57.89%,对庆大霉素和阿米卡星耐药率均为88.42%,对环丙沙星和左氧氟沙星耐药率分别为100%和88.42%,对四环素、米诺环素、替加环素耐药率分别为87.37%、16.84%和9.47%,对多粘菌素B耐药率为1.05%。95株MDR-Ab中携带β-内酰胺酶中A类酶耐药基因TEM、PER分别95株和25株,D类酶耐药基因OXA-51、carO和adeB各95株,OXA-23基因90株。携带消毒剂耐药基因qacE 60株。携带16S r RNA甲基化酶耐药基因armA 75株。每株MDR-Ab除携带TEM+carO+adeB+OXA-51四种基因外,另同时携带四种基因20株(21.05%),三种基因38株(40.00%)。结论:MDR-Ab对多种抗菌药物的耐药率较高,携带的耐药基因型主要为TEM、carO、adeB及OXA-51。携带多种耐药基因是MDR-Ab耐药重要原因。加强医院感染防控、合理应用抗菌药物对于延缓泛鲍曼不动杆菌耐药性发展具有重要的临床意义。     

Involvement of a novel efflux system in biofilm-specific resistance to antibiotics

Bacteria growing in biofilms are more resistant to antibiotics than their planktonic counterparts. How this transition occurs is unclear, but it is likely there are multiple mechanisms of resistance that act together in order to provide an increased overall level of resistance to the biofilm. We have identified a novel efflux pump in Pseudomonas aeruginosa that is important for biofilm-specific resistance to a subset of antibiotics. Complete deletion of the genes encoding this pump, PA1874 to PA1877 (PA1874-1877) genes, in an P. aeruginosa PA14 background results in an increase in sensitivity to tobramycin, gentamicin, and ciprofloxacin, specifically when this mutant strain is growing in a biofilm. This efflux pump is more highly expressed in biofilm cells than in planktonic cells, providing an explanation for why these genes are important for biofilm but not planktonic resistance to antibiotics. Furthermore, expression of these genes in planktonic cells increases their resistance to antibiotics. We have previously shown that ndvB is important for biofilm-specific resistance (T. F. Mah, B. Pitts, B. Pellock, G. C. Walker, P. S. Stewart, and G. A. O'Toole, Nature 426:306-310, 2003). Our discovery that combining the ndvB mutation with the PA1874-1877 gene deletion results in a mutant strain that is more sensitive to antibiotics than either single mutant strain suggests that ndvB and PA1874-1877 contribute to two different mechanisms of biofilm-specific resistance to antibiotics.     

Molecular cloning of chlortetracycline resistance gene from chlortetracycline producer Streptomyces aureofaciens

The chlortetracycline (CT) resistance gene ctr was cloned from S. aureofaciens 633, a strain producing the antibiotic. The 6.6-kb DNA Bam HI fragment containing the resistance gene was cloned with the plasmid vector pIJ699. Comparison of the restriction maps of the cloned gene and the oxytetracycline (OT) resistance gene otrA from S. rimosus revealed their similarity which enabled identification of the cloned resistance gene as otrA. Investigation of the resistance determinants in S. aureofaciens 633 made it possible to identify a mtr gene(s). It was demonstrated that introduction of a ctrA gene into S. lividance provided a simultaneous increase in the resistance of the recipient strain to CT and a number of macrolide antibiotics. The CT resistance determinants in S. lividans TK64 showed properties of exogenous induction by CT and the macrolide antibiotics similar to the properties of the mtr gene(s) of S. aureofaciens. Possible adaptation properties of mtr genes are discussed.     

Abundance and Dynamics of Antibiotic Resistance Genes and Integrons in Lake Sediment Microcosms

Antibiotic resistance in bacteria causing disease is an ever growing threat to the world. Recently, environmental bacteria have become established as important both as sources of antibiotic resistance genes and in disseminating resistance genes. Low levels of antibiotics and other pharmaceuticals are regularly released into water environments via wastewater, and the concern is that such environmental contamination may serve to create hotspots for antibiotic resistance gene selection and dissemination. In this study, microcosms were created from water and sediments gathered from a lake in Sweden only lightly affected by human activities. The microcosms were exposed to a mixture of antibiotics of varying environmentally relevant concentrations (i.e., concentrations commonly encountered in wastewaters) in order to investigate the effect of low levels of antibiotics on antibiotic resistance gene abundances and dynamics in a previously uncontaminated environment. Antibiotic concentrations were measured using liquid chromatography-tandem mass spectrometry. Abundances of seven antibiotic resistance genes and the class 1 integron integrase gene, intI1, were quantified using real-time PCR. Resistance genes sulI and ermB were quantified in the microcosm sediments with mean abundances 5 and 15 gene copies/10⁶ 16S rRNA gene copies, respectively. Class 1 integrons were determined in the sediments with a mean concentration of 3.8×10⁴ copies/10⁶ 16S rRNA gene copies. The antibiotic treatment had no observable effect on antibiotic resistance gene or integron abundances.     

Determinants of resistance to chlortetracycline and other antibiotics in chlortetracycline-producing strain of Streptomyces aureofaciens

Data are presented on resistance of Streptomyces aureofaciens strain TB-633 FU--the producer of chlortetracycline (CTC) to autogenous antibiotics and a number of other antibiotics. It is demonstrated that resistance to CTC is specified by ctr genes of constitutive expression as well as by inducible genes. CTC and ethidium bromide may serve as efficient inductors of inducible ctr genes. The induction process is accompanied by increase in antibiotic biosynthesis level. Genes responsible for strain resistance to a number of macrolide antibiotics and thiostrepton are inducible and only function in the presence of appropriate antibiotics in the medium. The action of inducible mtr gene(s) is described in detail. The gene(s) simultaneously ensure increase in resistance to CTC and a number of macrolide antibiotics in the presence of exogenous inductors in media, such as both CTC and macrolide antibiotics. Mutants have been isolated which provide constitutive level of resistance to these antibiotics. A series of ctr and mtr mutants have increased CTC biosynthesis as compared to the initial level. Data on comparative analysis of the results obtained from hybridization of fragments of S. aureofaciens and S. rimosus DNAs to actI and actIII genes, responsible for polyketide synthases' synthesis, demonstrate that genes for CTC and OTC biosynthesis are situated on DNA fragments of similar size. This determines the strategy for cloning ctr and mtr genes as well as genes for CTC biosynthesis from S. aureofaciens.     

The penC mutation conferring antibiotic resistance in Neisseria gonorrhoeae arises from a mutation in the PilQ secretin that interferes with multimer stability

The penC resistance gene was previously characterized in an FA19 penA mtrR penB gonococcal strain (PR100) as a spontaneous mutation that increased resistance to penicillin and tetracycline. We show here that antibiotic resistance mediated by penC is the result of a Glu-666 to Lys missense mutation in the pilQ gene that interferes with the formation of the SDS-resistant high-molecular-mass PilQ secretin complex, disrupts piliation and decreases transformation frequency by 50-fold. Deletion of pilQ in PR100 confers the same level of antibiotic resistance as the penC mutation, but increased resistance was observed only in strains containing the mtrR and penB resistance determinants. Site-saturation mutagenesis of Glu-666 revealed that only acidic or amidated amino acids at this position preserved PilQ function. Consistent with early studies suggesting the importance of cysteine residues for stability of the PilQ multimer, mutation of either of the two cysteine residues in FA19 PilQ led to a similar phenotype as penC: increased antibiotic resistance, loss of piliation, intermediate levels of transformation competence and absence of SDS-resistant PilQ oligomers. These data show that a functional secretin complex can enhance the entry of antibiotics into the cell and suggest that the PilQ oligomer forms a pore in the outer membrane through which antibiotics diffuse into the periplasm.     

Presence of specific antibiotic (tet) resistance genes in infant faecal microbiota

The widespread use of antibiotics for medical and veterinary purposes has led to an increase of microbial resistance. The antibiotic resistance of pathogenic bacteria has been studied extensively. However, antibiotics are not only selective for pathogens: they also affect all members of the gut microbiota. These microorganisms may constitute a reservoir of genes carrying resistance to specific antibiotics. This study was designed to characterize the gut microbiota with regard to the presence of genes encoding tetracycline resistance proteins (tet) in the gut of healthy exclusively breast-fed infants and their mothers. For this purpose we determined the prevalence of genes encoding ribosomal protection proteins (tet M, tet W, tet O, tet S, tet T and tet B) by PCR and characterized the gut microbiota by FISH in stools of infants and their mothers. The gene tet M was found in all the breast-fed infants and their mothers. tet O was found in all of the mothers' samples, whilst only 35% of the infants harboured this gene. tet W was less frequently found (85% of the mothers and 13% of the infants). None of the other genes analysed was found in any sample. Our results suggest that genes carrying antibiotic resistance are common in the environment, as even healthy breast-fed infants with no direct or indirect previous exposure to antibiotics harbour these genes.     

The YompC protein of<Emphasis Type="Italic">Yersinia enterocolitica</Emphasis>: Molecular and physiological characterization

The structural gene coding for YompC has been identified in the genome of a pathogenic strain of Yersinia enterocolitica O:9, and was subsequently cloned and sequenced. Detailed alignment of the deduced amino acid sequence showed that YompC is a member of the OmpC porin family with the highest degree of homology to Klebsiella pneumoniae. The mutant lacking YompC porin was constructed by insertional inactivation of the yompC gene which resulted from the integration of suicide vector at the yompC locus. In intact cells of Y. enterocolitica, loss of the YompC protein reduced the outer membrane permeability for beta-lactam antibiotics and tetracycline and resulted in a 2-5-fold increase in resistance to these compounds, depending on their chemical properties. Mutation in the ompR regulatory gene resulted in the loss of both YompC and YompF porins, which led to a greater increase of resistance to antibiotics, as compared with the YompC mutant strain. Moreover, the binding assay with HEp-2 cells suggests that YompC may play a role in the adhesion properties of Y. enterocolitica strains.     

Identification of the CysB-regulated gene,hslJ, related to the Escherichia coli novobiocin resistance phenotype

The cysB gene product is a LysR-type regulatory protein required for expression of the cys regulon. cysB mutants of Escherichia coli and Salmonella, along with being auxotrophs for the cysteine, exhibit increased resistance to the antibiotics novobiocin (Nov) and mecillinam. In this work, by using lambdaplacMu9 insertions creating random lacZ fusions, we identify a gene, hslJ, whose expression appeared to be increased in cysB mutants and needed for Nov resistance. Measurements of the HSLJ::lacZ gene fusion expression demonstrated that the hslJ gene is negatively regulated by CysB. In addition we observe the negative autogenous control of HslJ. When the control imposed by CysB is lifted in the cysB mutant, the elevation of Nov resistance can be achieved only in the presence of wild-type hslJ allele. A double cysB hslJ mutant restores the sensitivity to Nov. Overexpression of the wild-type HslJ protein either in a cysB(+) or a cysB(-) background increases the level of Nov resistance indicating that hslJ product is indeed involved in accomplishing this phenotype. The HSLJ::OmegaKan allele encodes the C-terminaly truncated mutant protein HslJ Q121Ter which is not functional in achieving the Nov resistance but when overexpressed induces the psp operon. Finally, we found that inactivation of hslJ does not affect the increased resistance to mecillinam in cysB mutants.     

肠球菌万古霉素耐药基因簇遗传特性

万古霉素耐药肠球菌自20世纪80年代后期被发现以来,已逐渐发展成为重要的医院感染病原菌。此类耐药肠球菌携带的万古霉素耐药基因簇编码产物可催化合成与万古霉素、替考拉宁等糖肽类抗生素亲和力极低的细胞壁前体导致耐药。目前已在肠球菌中发现的万古霉素耐药基因簇根据基因序列及构成不同分为9个型别;依据它们编码的连接酶合成产物不同又可分为D-Ala:D-Lac连接酶基因簇(VanA、VanB、VanD及VanM型)和D-Ala:D-Ser连接酶基因簇(VanC、VanE、VanG、VanL和VanN型)。这些耐药基因簇介导的耐药水平及其传播模式各有特点。文章综述了肠球菌中万古霉素耐药基因簇的类型、基因构成及传播特性。     

Inactivation of chloramphenicol and florfenicol by a novel chloramphenicol hydrolase

Chloramphenicol and florfenicol are broad-spectrum antibiotics. Although the bacterial resistance mechanisms to these antibiotics have been well documented, hydrolysis of these antibiotics has not been reported in detail. This study reports the hydrolysis of these two antibiotics by a specific hydrolase that is encoded by a gene identified from a soil metagenome. Hydrolysis of chloramphenicol has been recognized in cell extracts of Escherichia coli expressing a chloramphenicol acetate esterase gene, estDL136. A hydrolysate of chloramphenicol was identified as p-nitrophenylserinol by liquid chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. The hydrolysis of these antibiotics suggested a promiscuous amidase activity of EstDL136. When estDL136 was expressed in E. coli, EstDL136 conferred resistance to both chloramphenicol and florfenicol on E. coli, due to their inactivation. In addition, E. coli carrying estDL136 deactivated florfenicol faster than it deactivated chloramphenicol, suggesting that EstDL136 hydrolyzes florfenicol more efficiently than it hydrolyzes chloramphenicol. The nucleotide sequences flanking estDL136 encode proteins such as amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxidase. The most closely related genes are found in the bacterial family Sphingomonadaceae, which contains many bioremediation-related strains. Whether the gene cluster with estDL136 in E. coli is involved in further chloramphenicol degradation was not clear in this study. While acetyltransferases for chloramphenicol resistance and drug exporters for chloramphenicol or florfenicol resistance are often detected in numerous microbes, this is the first report of enzymatic hydrolysis of florfenicol resulting in inactivation of the antibiotic.