Action of quinolones against Staphylococcus aureus topoisomerase IV: basis for DNA cleavage enhancement

Topoisomerase IV is the primary cellular target for most quinolones in Gram-positive bacteria; however, its interaction with these agents is poorly understood. Therefore, the effects of four clinically relevant antibacterial quinolones (ciprofloxacin, and three new generation quinolones: trovafloxacin, levofloxacin, and sparfloxacin) on the DNA cleavage/religation reaction of Staphylococcus aureus topoisomerase IV were characterized. These quinolones stimulated enzyme-mediated DNA scission to a similar extent, but their potencies varied significantly. Drug order in the absence of ATP was trovafloxacin > ciprofloxacin > levofloxacin > sparfloxacin. Potency was enhanced by ATP, but to a different extent for each drug. Under all conditions examined, trovafloxacin was the most potent quinolone and sparfloxacin was the least. The enhanced potency of trovafloxacin correlated with several properties. Trovafloxacin induced topoisomerase IV-mediated DNA scission more rapidly than other quinolones and generated more cleavage at some sites. The most striking correlation, however, was between quinolone potency and inhibition of enzyme-mediated DNA religation: the greater the potency, the stronger the inhibition. Dose-response experiments with two topoisomerase IV mutants that confer clinical resistance to quinolones (GrlA(Ser80Phe) and GrlA(Glu84Lys)) indicate that resistance is caused by a decrease in both drug affinity and efficacy. Trovafloxacin is more active against these enzymes than ciprofloxacin because it partially overcomes the effect on affinity. Finally, comparative studies on DNA cleavage and decatenation suggest that the antibacterial properties of trovafloxacin result from increased S. aureus topoisomerase IV-mediated DNA cleavage rather than inhibition of enzyme catalysis.     

A staphylococcal plasmid that replicates and expresses ampicillin, gentamicin and amikacin resistance in Escherichia coli

Plasmid pPG1 from Staphylococcus aureus coding for ampicillin (Apr), gentamicin (Gmr) and amikacin (Akr) resistance was transformed into Escherichia coli. Transformation efficiency was about 2 x 10(3) transformants/micrograms of plasmid DNA. The plasmids present in the E. coli transformants were identical to pPG1 according to their restriction patterns. The copy number of pPG1 was estimated to be at least 20-times less in E. coli than in S. aureus. The minimal inhibitory concentrations (MICs) for Ap and Gm were lower in E. coli than in S. aureus. However, the MIC for Ak was higher in E. coli transformants than in S. aureus. pPG1 was maintained in the E. coli transformants for at least 80 generations at 37 degrees C without antibiotic selection pressure.     

琼脂糖凝胶的合适浓度对于回收酶切后质粒载体的重要性

目的：探讨琼脂糖凝胶的不同浓度对回收不同大小的酶切后质粒载体纯度的影响。方法：将2种质粒载体酶切,并经不同浓度的琼脂糖凝胶电泳分析,通过比较酶切前和酶切后载体的相对位置,研究胶浓度对回收酶切后载体纯度的影响。结果：不同浓度的琼脂糖凝胶中,酶切前和酶切后载体的相对位置会发生变化,对能否成功回收到纯度高的酶切后DNA片段有重要影响;质粒大小不同,胶浓度的影响也不同。结论：合适的胶浓度对于回收酶切后质粒载体具有重要意义,应选择合适的胶浓度回收酶切后质粒载体。     

mRNA stability and plasmid copy number effects on gene expression from an inducible promoter system

The effects of mRNA stability and plasmid copy number on gene expression in Escherichia coli were evaluated by constructing multicopy (pMB1-based) and low-copy (F-based) plasmids containing an arabinose-inducible promoter system, the lacZ reporter gene, and mRNA-stabilizing 5' hairpin structures. Product formation and cell growth were evaluated under a number of inducer concentrations. The introduction of a 5' hairpin into the untranslated region of the mRNA resulted in significantly higher gene expression from the multicopy plasmids at low inducer concentrations and increased gene expression from the low-copy plasmids across all inducer concentrations investigated. With high inducer concentrations, expression from high-copy plasmids significantly slowed cell growth, whereas expression from the low-copy plasmids had little effect on growth rate. At inducer concentrations between 1 x 10(-4) and 4 x 10(-4)%, the productivity of low-copy plasmids containing the 5'-hairpin was equal to or greater than that from multicopy plasmids. Together, these two gene expression strategies may find important use in metabolic engineering and heterologous gene expression.     

亚胺培南对铜绿假单胞菌敏感和耐药菌株DNA释放的影响

目的：观察亚胺培南对铜绿假单胞菌标准菌株和耐药菌株体外培养释放DNA的影响。方法：选择铜绿假单胞菌的标准菌株和临床分离耐药菌株作为实验菌株,检测其亚胺培南的最低抑菌浓度（MIC）,琼脂糖凝胶电泳法观察不同浓度亚胺培南作用下铜绿假单胞菌的DNA释放情况,并用Quantity One软件分析所释放DNA片段的分子量和含量。结果：亚胺培南作用下铜绿假单胞菌从1／2MIC开始释放小片段DNA,大于1／2MIC仅释放l条大片段DNA,且含量少,等于1／2MIC可释放3条DNA,但无小片段DNA,小于1／2MIC可释放4．5条DNA,且含量多。1／16MIC时,标准菌株释放的第1、5条DNA片段含量偏少,3、4条DNA片段含量偏多,而耐药菌株相反。耐药菌株与标准菌株相比释放的DNA片段有所不同,耐药菌株比标准菌株多释放一条DNA片段（第2条）,分子量为2784bp,耐药菌株的第1、3、4、5条DNA片段分子量均比标准菌株小。耐药菌株释放的第3、4、5条DNA含量明显高于标准菌株,P〈0．01,其统计有显著学意义。结论：不同亚胺培南浓度作用下,铜绿假单胞菌会释放不同片段大小和含量的DNA分子,耐药菌株与标准菌株释放的DNA片段数量和含量有明显差异,其与耐药机制的关系值得进一步研究。     

Mitochondrial plasmids in related cultivars of crookneck and stable and unstable butternut squash

Summary Two series of two to four plasmids are contained in mitochondria of butternut squash (Cucurbita moschatd). The plasmids are composed of DNA, and are consistent in number within a cultivar but vary in number among cultivars. Plasmid patterns and homology suggest that the faster migrating series of plasmids are the supercoiled form of the slower running plasmids. Plasmid 1 was present in all cultivars except New Hampshire Butternut. Plasmid 2 was only observed in the original crookneck cultivar, Canada Crookneck. Plasmids 3 and 4 were present in all accessions tested. The presence of plasmid 1 in a cultivar does not follow a maternal pattern of inheritance; this may be due to the ability of the plasmid to insert into and excise from the main mitochondrial DNA, or the recombination of some of the smaller plasmids to create the larger plasmid 1. There is some homology between plasmid 1 and plasmids 3 and 4, and between plasmid 1 and main band mitochondrial DNA. The equal presence of plasmid 1 in mitochondria of F₁ seedlings from the pollination of New Hampshire Butternut by Ponca Butternut to that in F₁ seedlings of the reciprocal cross indicates that there is probably a dominant nuclear effect on the ability to produce plasmid 1 either by release from the mitochondrial genome or by recombination of the smaller plasmids. There is no obvious relationship between the presence or number of the mitochondrial plasmids and the butternut fruit shape or stability of the butternut trait of the cultivars.     

Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains

Abstract To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics. Total DNA, i.e. both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation. Arbitrarily primed polymerase chain reaction was carried out for all of these preparations. Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present. Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target. These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA. Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present.     

Variations in R-plasmid DNA concentrations of Escherichia coli during starvation in sewage and brackish waters

Cell culturability and plasmid stability in Escherichia coli containing plasmids RP1, R388 and pUB824 were studied in raw and treated wastewater, and in brackish water. The E. coli strain survived well in the three samples of water employed. Moreover, the three plasmids were maintained under all conditions studied. Interestingly, plasmid DNA concentration of individual plasmids followed the same evolution as the culturable bacteria in the corresponding selective medium when the bacteria grew in raw or treated wastewater. In contrast, in brackish water, the stress due to the oligotrophic and salinity conditions of the medium produced an initial paradoxical increase in plasmid DNA concentration, followed by a decrease in the number of culturable bacteria in the corresponding selective medium. Maintenance of RP1 (56 kbp) and R388 (33 kbp) was markedly influenced by nutritive conditions, which caused a segregation of the plasmids from cells. The results of the present study suggest that variations in plasmid DNA concentrations in an aquatic environment depend on the quality of the water and also on the molecular weight of the plasmid considered.     

The insertion of large pieces of foreign genetic material reduces the stability of bacterial plasmids

The stability of genetically engineered bacterial plasmids under continuous culture fermentation is of crucial importance in the application of microbiology to many processes of potential industrial importance. In order to determine the effect of inserting large pieces of foreign DNA on the stability of bacterial plasmids we have studied the behavior of pAT153 with DNA inserts of various sizes derived from cytomegalovirus. Foreign DNA up to 2 kb in length had no effect on stability, whereas the insertion of an 8-kb fragment resulted in a transient instability. This instability was overcome by the spontaneous appearance of leu+ cells in the culture. Insertion of a 21-kb DNA fragment resulted in a rapid loss of plasmid, which was not prevented by the appearance of leu+ cells. In all cases copy number analyses indicated that plasmid loss was due to segregational instability, probably because the plasmid placed an unacceptable metabolic load on the cell.     

Linear- and circular-plasmid copy numbers in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease agent, and other members of the spirochetal genus Borrelia have double-stranded linear plasmids in addition to supercoiled circular plasmids. The copy number relative to the chromosome was determined for 49- and 16-kb linear plasmids and a 27-kb circular plasmid of the type strain, B31, of B. burgdorferi. All three plasmids were present in low copy number, about one per chromosome equivalent, as determined by relative hybridizations of replicon-specific DNA probes. The low copy number of Borrelia plasmids suggests that initiation of DNA replication and partitioning are carefully controlled during the cell division cycle. The copy numbers of these three plasmids of strain B31 were unchanged after approximately 7,000 generations in continuous in vitro culture. A clone of B. burgdorferi B31 that did not contain the 16-kb linear plasmid was obtained after exposure of a culture to novobiocin, a DNA gyrase inhibitor. The plasmid-cured strain contains only one linear plasmid, the 49-kb plasmid, and thus has the smallest genome reported to date for B. burgdorferi.     

Application of agarose gel electrophoresis to the characterization of plasmid DNA in drug-resistant enterobacteria.

A simple gel electrophoresis method has been described for the detection of plasmid DNA in bacteria (Meyers et al., 1976). We investigated further the problems encountered in using this method for the analysis of plasmids in wild enterobacterial strains. The migration of open circular and linear plasmid DNA was examined, since these forms sometimes caused difficulty in the interpretation of the plasmid content of uncharacterized strains. Electrophoresis at different agarose concentrations was employed to resolve clearly plasmid DNA from the chromosomal DNA fragments in the crude preparations. Dissociation of some plasmids occurs in Salmonella typhimurium, and this was detected by electrophoresis. The technique was applied to the study of drug-resistant strains of S. typhimurium phage type 208 from several Middle Eastern countries. The cultures carry a drug resistance plasmid of the FIme compatibility group, and at least two other plasmids which were detected and identified by gel electrophoresis. The studies supported and extended the genetic findings and provided information on the distribution of particular plasmids.     

Effect of suprainhibitory concentrations of quinolones on hydrophobicity and motility of Serratia marcescens

The effect of suprainhibitory concentrations of quinolones (ciprofloxacin, enoxacin and norfloxacin) on the growth, hydrophobicity and motility of a nosocomial pathogen Serratia marcescens was studied. A postantibiotic effect (PAE) was induced by 2x of 4x MIC concentrations for 0.5 h. By using the 2x MIC concentrations all three quinolones induced equally long PAE approximately 1 h. The longest PAE of 5.4 h at 4x MIC concentration was induced by enoxacin. The results obtained showed that suprainhibitory concentrations of quinolones significantly stimulated the adhesion of S. marcescens to xylene, with the exception of enoxacin, which inhibited the adhesion to 61.2% at 4x MIC concentration. These results correlated with those in the salt aggregation test. The adhesion of strains to nitrocellulose filters did not influence the aftereffect of suprainhibitory concentrations of quinolones. Exposure of bacterial cells to suprainhibitory concentrations of ciprofloxacin and norfloxacin caused a reduction in motility, while this effect was more distinct at 4x MIC concentration. The results suggest that any consideration of postantibiotic effects should include the residual antibiotic effects on virulence factors, in addition to the defined suppression of bacterial regrowth.     

Involvement of the cell envelope in plasmid maintenance: Plasmid curing during the regeneration of protoplasts

Observations are described that demonstrate elimination of certain plasmids in up to 80% of Staphylococcus aureus cells during the formation and regeneration of lysostaphin-induced protoplasts of these organisms. All of nine small (≤3 megadaltons (Mdal)) plasmids studied showed the protoplast-dependent elimination to a greater or lesser extent; none of three larger (≤15 Mdal) plasmids showed the effect. This difference in behavior was not due to molecular weight per se, as curing was not shown by one of the large plasmids with a deletion of two-thirds of its genome but was shown by a chimera consisting of a 3-Mdal plasmid with a 5.7-Mdal DNA insertion. The curing effect was not related to copy number, as all of the curable plasmids have substantially greater copy numbers than the noncurable ones. Physical loss of plasmid DNA from the protoplasts could not be demonstrated; replication of plasmids in protoplasts appeared normal; but most of the plasmid-positive regenerant colonies consisted of mixed populations of plasmid-positive and negative organisms with a very wide range of composition. On the basis of these observations, we conclude that plasmid elimination occurs during the several protoplast divisions that occur before cell wall regeneration is completed and that it is due to a disruption of the plasmid partition system as a consequence of removal of the cell wall. If so, then the noncurable plasmids must be partitioned by a mechanism that is different from that by which the curable ones are normally partitioned.     

Effects of subinhibitory concentrations of antibiotics on biological properties ofSalmonella typhimurium

We followed the effects of subinhibitory concentrations (sub-MICs) of 7 antibiotics (ticarcilin, cefotaxim, streptomycin, gentamicin, ciprofloxacin, pefloxacin, mitomycin C) on the sensitivity of aSalmonella typhimurium strain to standard bacteriophages, on the phage DNA as well as on the factors of virulence (permeability and cytotoxic activity). The phage type was not changed by the sub-MICs of the tested antibiotics. However, differences were found in culture filtrates prepared from the bacterial suspensions of the strain cultivated with the sub-MICs. Marked inducing effects on phage DNA were exhibited by mitomycin C (1/2, 1/4, 1/8 of the MIC), pefloxacin (1/2, 1/4, 1/8 of the MIC) and ciprofloxacin (1/2, 1/4, weakly also 1/8 of the MIC). Ticarcilin (1/2 of the MIC), like the aminoglycosides streptomycin and gentamicin (1/2, 1/4, 1/8 of the MIC), had a weak effect. Sub-MICs of the studied antibiotics (with the exception of 1/8 of the MIC of ciprofloxacin and 1/4 of the MIC of ticarcilin) decreased the permeability reaction in rabbit skin. Most effective was streptomycin (1/2 of the MIC). Sub-MICs of the tested antibiotics (with the exception of 1/4 and 1/8 of the MIC of ciprofloxacin and 1/4 of the MIC of pefloxacin) caused also an inhibition of the factor responsible for morphological changes on Vero cells. Gentamicin and streptomycin were effective at all the sub-MICs tested.     

Relationship between Nif plasmids of fast-growing Rhizobium species and Ti plasmids of Agrobacterium tumefaciens.

By use of the Southern blot hybridization technique the extent of DNA homology was determined between the Nif plasmid of a number of fast-growing Rhizobium species and Ti plasmids of the octopine (pTiAch5) and nopaline (pTiC58) type. DNA sequences common to these plasmids were located on functional maps of the Ti plasmids. No homology between Nif plasmids and the T region of Ti plasmids was detected.     

Synergistic effect of cefepime on the phagocytic killing of Staphylococcus aureus by human polymorphonuclear leucocytes and the determination of this effect by means of nitrite production

In this study the effect of cefepime on the phagocytosis and intracellular killing of Staphylococcus aureus by human polymorphonuclear leucocytes (PMNL) was determined. The opsonophagocytic killing of S. aureus was synergistically enhanced by cefepime at concentrations below 0.5 times the minimal inhibitory concentration (MIC), and four times the MIC at higher concentrations. The effect of cefepime on phagocytosis and the bactericidal activity of PMNL was also investigated by the measurement of nitrite levels using a Sievers analyser. According to the nitrite levels, cefepime enhanced not only the phagocytosis by PMNL 2.1-fold in the 0.5 MIC and 2.8-fold in the four MIC values but also the bactericidal activity of neutrophils 2.5-fold in the 0.5 MIC and 2.8-fold in the four MIC values, respectively. The beneficial cefepime-leucocyte interaction may explain the efficacy of cefepime against intracellular pathogens.     

Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacteria

The minimum inhibitory concentrations (MICs) of 6 different antibiotics (chloramphenicol, clindamycin, erythromycin, streptomycin, tetracycline and vancomycin) were determined for 143 strains of lactic acid bacteria and bifidobacteria using the Etest. Different MICs were found for different species and strains. Based on the distribution of these MIC values, most of the strains were either susceptible or intrinsically resistant to these antibiotics. However, the MIC range of some of these antibiotics showed a bimodal distribution, which suggested that some of the tested strains possess acquired antibiotic resistance. Screening for resistance genes was performed by PCR using specific primers, or using a DNA microarray with around 300 nucleotide probes representing 7 classes of antibiotic resistance genes. The genes identified encoded resistance to tetracycline [tet(M), tet(W), tet(O) and tet(O/W)], erythromycin and clindamycin [erm(B)] and streptomycin [aph(E) and sat(3)]. Internal portions of some of these determinants were sequenced and found to be identical to genes described in other bacteria. All resistance determinants were located on the bacterial chromosome, except for tet(M), which was identified on plasmids in Lactococcus lactis. The contribution of intrinsic multidrug transporters to the antibiotic resistance was investigated by cloning and measuring the expression of Bifidobacterium breve genes in L. lactis.     

Co-transfected SV40 origin of replication activates expression from SV40 promoterless constructs.

Co-transfection with expression plasmids is widely used to control DNA uptake efficiency in transient transfection experiments. However, a number of problems have been associated with their use. Here, we describe the activation of expression of constructs not containing the simian virus 40 (SV40) origin of replication (ori) by co-transfection in COS-7 cells with plasmids containing the SV40 ori. This effect has consequences for the use of such plasmids to control transfection efficiency.