A bacterial reporter panel for the detection and classification of antibiotic substances

The ever‐growing use of pharmaceutical compounds, including antibacterial substances, poses a substantial pollution load on the environment. Such compounds can compromise water quality, contaminate soils, livestock and crops, enhance resistance of microorganisms to antibiotic substances, and hamper human health. We report the construction of a novel panel of genetically engineered Escherichia coli reporter strains for the detection and classification of antibiotic substances. Each of these strains harbours a plasmid that carries a fusion of a selected gene promoter to bioluminescence (luxCDABE) reporter genes and an alternative tryptophan auxotrophy‐based non‐antibiotic selection system. The bioreporter panel was tested for sensitivity and responsiveness to diverse antibiotic substances by monitoring bioluminescence as a function of time and of antibiotic concentrations. All of the tested antibiotics were detected by the panel, which displayed different response patterns for each substance. These unique responses were analysed by several algorithms that enabled clustering the compounds according to their functional properties, and allowed the classification of unknown antibiotic substances with a high degree of accuracy and confidence.     

The effect of species of Allium on soil bacteria in relation to germination of sclerotia of Sclerotium cepivorum Berk

Extracts made from Allium species were shown to exhibit marked antibiotic properties in a variety of tests. However, no evidence was obtained to suggest that intact Allium seedlings exude antibiotic compounds in sufficient quantity to cause inhibition of bacteria in the soil or in assay tests in the laboratory. Germination of sclerotia of Sclerotium cepivorum was induced by Allium extracts which were too dilute to cause antibiotic responses in laboratory tests using a number of bacteria. Synthetic methyl-methanethiolsulphinate had little effect on germination of sclerotia. The specific reversal, by Allium species and their extracts, of the mycostatic effect of unsterile soil on sclerotia of S. cepivorum does not appear to be due to antibiotic effects.     

Synergy effects of the antibiotics gentamicin and the essential oil of Croton zehntneri

The leaves of Croton zehntneri Pax et Hoffm (Euphorbiaceae) were subjected to hydrodistillation, and the essential oil extracted was examined with respect to antibacterial and antibiotic modifying activity by gaseous contact. The gaseous component of the oil inhibited the bacterial growth of Staphylococcus aureus and Pseudomonas aeruginosa with a MID of 0.5 and<1 mg/l air, respectively. The activity of the antibiotic gentamicin was increased by 42,8% against P. aeruginosa after contact with the gaseous component, showing that this oil influences the activity of the antibiotic and may be used as an adjuvant in the antibiotic therapy of respiratory tract bacterial pathogens.     

Selective inhibition of the potato scab pathogen by antagonistic bacteria and substrate influence on antibiotic production

Summary In a long-term field experiment a soybean cover crop and green manure incorporation prevented the build-up of common scab of potato whereas barley, employed in the same manner, increased disease incidence. When soil from these plots was assayed for organisms antagonistic toS. scabies, a bacterium indentified asBacillus subtilis, was found to be predominant. Laboratory tests showed thatS. scabies was more sensitive to the antibiotic produced by this bacterium than most non-pathogenicStreptomyces spp which were tested. The antibiotic was found to be similar to bacitracin and activity was expressed as units bacitracin. Water extracts of greenhouse-grown soybean and barley were compared, at different concentrations, as a substrate for growth and antibiotic production by this bacterium. It was found that on the soybean extract, antibiotic activity, as measured by the standard filter-paper-disc technique, was 2.5 to 3 times greater per unit of bacterial growth than when barley extract was used as the substrate. Similar results were obtained when extracts of partially decomposed tissue were used. It is suggested that when evaluating antagonistic organisms as a possible factor in the behavior of plant pathogens in soil, the relative sensitivity of the pathogen to the antibiotic activities of the antagonists as well as the substrates available to the antagonists should be considered.     

Susceptibility of Pseudomonas aeruginosa Urinary Tract Isolates and Influence of Urinary Tract Conditions on Antibiotic Tolerance

Pseudomonas aeruginosa is an opportunistic human pathogen, which can cause severe urinary tract infections (UTIs). Because of the high intrinsic antibiotic resistance of P. aeruginosa and its ability to develop new resistances during antibiotic treatment, these infections are difficult to eradicate. The antibiotic susceptibility of 32 P. aeruginosa isolates from acute and chronic UTIs were analysed under standardized conditions showing 19% multi-drug resistant strains. Furthermore, the antibiotic tolerance of two P. aeruginosa strains to ciprofloxacin and tobramycin was analysed under urinary tract-relevant conditions which considered nutrient composition, biofilm growth, growth phase, and oxygen concentration. These conditions significantly enhance the antibiotic tolerance of P. aeruginosa up to 6000-fold indicating an adaptation of the bacterium to the specific conditions present in the urinary tract. This reversible phenomenon is possibly due to the increased formation of persister cells and is based on iron limitation in artificial urine. The results suggest that the general high antibiotic resistance of P. aeruginosa urinary tract isolates together with the increasing tolerance of P. aeruginosa grown under urinary tract conditions decrease the efficiency of antibiotic treatment of UTIs.     

A new class of mutations altering the response of the ribosome to streptomycin

Summary Mutants were isolated from high-level streptomycin dependent strains of Escherichia coli B, which do not spontaneously revert to antibiotic independence. In these mutants the requirement for streptomycin was much reduced, but not abolished. The relieving of the antibiotic dependence was caused by qui (for quasi-independent) mutations. These were analogous to the ramA (rpsD) mutations which relieve the streptomycin requirement of other classes of streptomycin dependent mutants, but strains harboring qui mutations exhibited novel streptomycin phenotypes in conjunction with all rpsL (strA) alleles. RamA mutations increase ribosomal misreading; qui mutations either did not significantly alter misreading, or else reduced it.This work was done in partial fulfilment of the requirements for the Ph. D. degree in the Division of Medical Sciences of Harvard University     

食品中蜡样芽孢杆菌耐药性及其机制研究进展

蜡样芽孢杆菌是常见的食源性致病菌之一,其产生的毒素会引起食物中毒。蜡样芽孢杆菌主要引起2种类型的食物中毒,即呕吐和腹泻综合征,并可造成各种局部和全身感染。随着抗生素的广泛、大量使用,蜡样芽孢杆菌的耐药性不断增强,现已有报道出现多重耐药性。本文对蜡样芽孢杆菌的耐药现状及耐药性机制进行了综述,以期正确理解蜡样芽孢杆菌耐药性的特点及其规律,从而为防治蜡样芽孢杆菌耐药性的产生及合理用药提供理论依据。     

Structural and functional characterization of three Type B and C chloramphenicol acetyltransferases from Vibrio species

Chloramphenicol acetyltransferases (CATs) were among the first antibiotic resistance enzymes identified and have long been studied as model enzymes for examining plasmid‐mediated antibiotic resistance. These enzymes acetylate the antibiotic chloramphenicol, which renders it incapable of inhibiting bacterial protein synthesis. CATs can be classified into different types: Type A CATs are known to be important for antibiotic resistance to chloramphenicol and fusidic acid. Type B CATs are often called xenobiotic acetyltransferases and adopt a similar structural fold to streptogramin acetyltransferases, which are known to be critical for streptogramin antibiotic resistance. Type C CATs have recently been identified and can also acetylate chloramphenicol, but their roles in antibiotic resistance are largely unknown. Here, we structurally and kinetically characterized three Vibrio CAT proteins from a nonpathogenic species (Aliivibrio fisheri) and two important human pathogens (Vibrio cholerae and Vibrio vulnificus). We found all three proteins, including one in a superintegron (V. cholerae), acetylated chloramphenicol, but did not acetylate aminoglycosides or dalfopristin. We also determined the 3D crystal structures of these CATs alone and in complex with crystal violet and taurocholate. These compounds are known inhibitors of Type A CATs, but have not been explored in Type B and Type C CATs. Based on sequence, structure, and kinetic analysis, we concluded that the V. cholerae and V. vulnificus CATs belong to the Type B class and the A. fisheri CAT belongs to the Type C class. Ultimately, our results provide a framework for studying the evolution of antibiotic resistance gene acquisition and chloramphenicol acetylation in Vibrio and other species.     

大熊猫源细菌耐药性研究进展

耐药菌和耐药基因已成为一种新型环境污染物,引发世界公共卫生问题。细菌耐药性尤其是多重耐药菌在人医临床、畜禽养殖以及环境传播等多个方面得到越来越多的关注,而关于大熊猫等野生动物的耐药性研究相对较少。大熊猫(Ailuropoda melanoleuca)是世界公认的珍稀野生动物,其种群数量易受到各种疾病的威胁,尤其是肠道细菌性疾病。随着抗菌药物在疾病预防和控制中的普遍使用,由此带来的耐药性危害日益明显。本文总结了关于大熊猫源细菌耐药的国内外研究报道,对其耐药表型、耐药基因型、耐药机制及水平传播机制等方面内容进行了综述,旨在为大熊猫源细菌耐药性的研究和防控提供依据,为临床科学用药提供理论参考,从而助力大熊猫迁地保护。     

Streptomycin mimics the cool temperature response in rice plants

Exogenous application of streptomycin to etiolated seedlings of rice (Oryza sativa L.) during growth in darkness at moderate temperatures induced the same type of chlorosis as that elicited by cool temperatures. A comparison of sensitive (Indica) and tolerant (Japonica) cultivars indicated a close relationship between sensitivity to streptomycin and cool temperatures. Immunoblot (Rubisco LSU, SSU; CF1 complex of H⁺-ATPase; NADPH-protochlorophyllide oxidoreductase) and Northern blot analyses of plastid-encoded genes (16S rRNA; rbcL; rpoB; petB) in the streptomycin-treated sensitive cultivars revealed that the normal etioplast development was specifically inhibited by the antibiotic. Furthermore, the antibiotic did not affect the expression of mitochondrion-encoded genes (18S rRNA; atpA), which are also unaffected by cool temperatures. These result suggest that the effect of the antibiotic is quite similar to that of cool temperatures.     

Streptomyces species producing the streptovaricin complex

Morphological, cultural and physiological-biochemical properties ofStreptomyces sp. strain 1000 and its antibiotic production were investigated. Antibiotics 1011 (identical with the streptovaricin complex) and 1012 (with antibacterial action) were isolated from the cultural broth of this strain. The overproducing natural variant 1011 was isolated from the population of a strain producing antibiotic 1011 at a concentration of 1000 mg/L (activity of the parent strain represents 41 mg/L only). Comparative taxonomical characteristic ofStreptomyces sp. strain 1000 with strains fromS. spectabilis showed that the strain 1000 differed in some properties and antibiotic production being considered as a new variant ofS. spectabilis. The strain shows an expressed antibiotic activity against G+ as well as G− bacterial and yeasts.     

A simple and rapid method for the elimination of R plasmids from enteric bacteria

A rapid, simple, and effective method for the curing of a wide range ofEscherichia coli antibiotic resistance plasmids is described. Treatment with acridine orange followed by growth in sublethal concentration of antibiotics and penicillin selection under such bacteriostatic conditions resulted in a curing efficiency of more than 98% in all cases tested. The method is equally applicable, with modifications, to other enteric bacteria such asKlebsiella pneumoniae. It is also equally applicable to nutritional markers for which toxic analogues exist, and to elimination of recombinant bacteriophages containing antibiotic resistance transposons.     

Influence of <Emphasis Type="Italic">Pseudomonas aeruginosa pvdQ</Emphasis> Gene on Altering Antibiotic Susceptibility Under Swarming Conditions

In Pseudomonas aeruginosa PAO1, the pvdQ gene has been shown to have at least two functions. It encodes the acylase enzyme and hydrolyzes 3-oxo-C12-HSL, the key signaling molecule of quorum sensing system. In addition, pvdQ is involved in swarming motility. It is required for up-regulated during swarming motility, which is triggered by high cell densities. As high-density bacterial populations also display elevated antibiotic resistance, studies have demonstrated that swarm-cell differentiation in P. aeruginosa promotes increased resistance to various antibiotics. PvdQ acts as a signal during swarm-cell differentiation, and thus may play a role in P. aeruginosa antibiotic resistance. The aim of this study is to examine whether pvdQ was involved in modifying antibiotic susceptibility during swarming conditions, and to investigate the mechanism by which this occurred. We constructed the PAO1pMEpvdQ strain, which overproduced PvdQ. PAO1pMEpvdQ promotes swarming motility, while PAO1ΔpvdQ abolishes swarming motility. In addition, both PAO1 and PAO1pMEpvdQ acquired resistance to ceftazidime, ciprofloxacin, meropenem, polymyxin B, and gentamicin, though PAO1pMEpvdQ exhibited a two to eightfold increase in antibiotic resistance compared to PAO1. These results indicate that pvdQ plays an important role in elevating antibiotic resistance via swarm-cell differentiation and possibly other mechanisms as well. We analyzed outer membrane permeability. Our data also suggest that pvdQ decreases P. aeruginosa outer membrane permeability, thereby elevating antibiotic resistance under swarming conditions. Our results suggest new approaches for reducing P. aeruginosa resistance.     

A pharmacodynamic model for the action of the antibiotic imipenem onPseudomonas aeruginosa populationsin vitro

The standard method for measuringin vitro antibiotic efficacy is based on a point observation of bacterial activity 18 hours after inoculation. The method, while simple, forgoes significant information by ignoring the dynamics of the interations between antibiotic and bacteria. This paper proposes a simple dynamic model describing these interactions. The model consists of two non-linear differential equations of the S-system type. Its parameter values are estimated, through the minimization of residual errors, from data on the effect of the carbapenem antibiotic imipenem onPseudomonas aeruginosa. The model adequately describes the dynamic behavior of the bacterial populations in the presence of the antibiotic: beginning with drug administration, then through the decline of the bacterial population and possibly ending with bacterial resurgence.     

Distinct mechanisms contribute to immunity in the lantibiotic NAI‐107 producer strain Microbispora ATCC PTA‐5024

The investigation of self‐resistance in antibiotic producers is important to understand the emergence of antibiotic resistance in pathogens and to improve antibiotic production. Lantibiotics are ribosomally synthesized antibiotics that mostly target peptidoglycan biosynthesis. The actinomycete Microbispora ATCC PTA‐5024 produces the lantibiotic NAI‐107, which interferes with peptidoglycan biosynthesis by binding bactoprenol‐pyrophosphate‐coupled peptidoglycan precursors. In order to understand how Microbispora counteracts the action of its own antibiotic, its peptidoglycan composition was analysed in detail. Microbispora peptidoglycan consists of muropeptides with D‐Ala and Gly in similar proportion at the fourth position of the peptide stems and alternative 3‐3 cross‐links besides the classical 4‐3 cross‐links. In addition, the NAI‐107 biosynthetic gene cluster (mlb) was analysed for the expression of immunity proteins. We show that distinct immunity determinants are encoded in the mlb cluster: the ABC transporter MlbYZ acting cooperatively with the transmembrane protein MlbJ and the lipoprotein MlbQ. NMR structural analysis of MlbQ revealed a hydrophobic surface patch, which is proposed to bind the cognate lantibiotic. This study demonstrates that immunity in Microbispora is not only based on one determinant but on the action of the distinct immunity proteins MlbQ, MlbYZ and MlbJ.     

Molecular cloning of resistance genes and architecture of a linked gene cluster involved in biosynthesis of oxytetracycline by Streptomyces rimosus

Summary The isolation of mutants of Streptomyces rimosus which were blocked in oxytetracycline (OTC) production was described previously. The genes for the early steps of antibiotic biosynthesis mapped together. Genomic DNA fragments of S. rimosus which conferred resistance to OTC and complemented all of these non-producing mutants have been cloned. The cloned DNA is physically linked within approximately 30 kb of the genome of S. rimosus. The gene cluster is flanked at each end by a resistance gene each of which, independently, can confer resistance to the antibiotic. In OTC-sensitive strains of S. rimosus, the entire gene cluster including both resistance genes has been deleted. Complementation of blocked mutants by cloned DNA fragments in multi-copy vectors was often masked by a secondary effect of switching off antibiotic productions in strains othersise competent to produce OTC. This adverse effect on OTC production was not observed with recombinants using low copy-number vectors.     

Effect of amphotericin B on the metabolism of Aspergillus fumigatus

Action of amphotericin B on the growth and metabolism of Aspergillus fumigatus has been investigated. The fungus proved to be very sensitive to amphotericin B, showing complete inhibition of growth at 0.5 units/ml. Amphotericin B suppressed the exogenous and endogenous respiration and glycolysis of A. fumigatus as well as the assimilation of various glycolysis and TCA cycle intermediates. Addition of cations and cholesterol failed to reverse the action of amphotericin B. The treated mycelium released a variety of cellular constituents and it is inferred that the antibiotic effects the permeability of A. fumigatus cells. In experiments with ³²P labelled mycelium phosphorus compounds leached out in concentrations which were dependent on the antibiotic dose, period of contact, incubation temperature and metabolic state of the fungus.     

Proteomics-driven identification of putative AfsR2-target proteins stimulating antibiotic biosynthesis inStreptomyces lividans

AfsR2, originally identified fromStreptomyces lividans, is a global regulatory protein which stimulates antibiotic biosynthesis. Through its stable chromosomal integration, the high level of gene expression ofafsR2 significantly induced antibiotic production as well as the sporulation ofS. lividans, implying the presence of yet-uncharacterized AfsR2-target proteins. To identify and evaluate the putative AfsR2-target proteins involved in antibiotic regulation, the proteomics-driven approach was applied to the wild-typeS. lividans and theafsR2-integrated actinorhodin overproducing strain. The 2D gel-electrophoresis gave approximately 340 protein spots showing different protein expression patterns between these twoS. lividans strains. Further MALDI-TOF analysis revealed several AfsR2-target proteins, including glyceraldehyde-3-phosphate dehydrogenase, putative phosphate transport system regulator, guanosine pentaphosphate synthetase/polyribonucleotide nucleotidyltransferase, and superoxide dismutase, which suggests that the AfsR2 should be a pleiotropic regulatory protein which controls differential expressions of various kinds of genes inStreptomyces species.