Fusidic acid resistance in Rhodococcus erythropolis due to an inducible extracellular inactivating enzyme

Fusidic acid is a steroid antibiotic inhibiting Gram-positive microorganisms. The mechanism of resistance previously characterised involves mutation alteration of the target moiety, protein synthesis elongation factor EFG. In the nocardioform bacterium Rhodococcus erythropolis, a novel resistance mechanism to this antibiotic was found, i.e., an inducible extracellular fusidic acid-in-activating enzyme. The presence of this enzyme increased roughly 30-fold the antibiotic concentration at which the organism could grow.     

Light inhibits rifampicin inactivation and reduces rifampicin resistance due to a cloned mycobacterial ADP-ribosylation gene

Rifampicin is a principal drug used to combat infections by mycobacteria and related organisms. Most strains of Mycobacterium are able to inactivate this antibiotic by ribosylation via an ADP-ribosylated intermediate. We found that this inactivation was inhibited by light at levels similar to those prevailing in laboratory environments. Rifampicin resistance arising from the cloned ADP-ribosyl transferase was also greatly diminished at these light levels. The cloned Rhodococcus equi monooxygenase which inactivates this antibiotic by a different mechanism was, in contrast, not inhibited by light.     

Cloning of nocardioform DNA conferring the ability to inactivate rifampicin

A novel mechanism of resistance to rifampicin has recently been reported in which this antibiotic is inactivated. Here we describe the cloning of DNA from a nocardiofrom strain conferring this ability to inactivate rifampicin. Cloning was on the basis of complementation, as an increased resistance to the antibiotic. The genetic information was on a 8.3-kb BglII fragment.     

Specific preadaptations of Rhodococcus equi cooperate with its Virulence-associated protein A during macrophage infection

Gram-positive Rhodococcus equi (Prescotella equi) is a lung pathogen of foals and immunocompromised humans. Intra-macrophage multiplication requires production of the bacterial Virulence-associated protein A (VapA) which is released into the phagosome lumen. VapA pH-neutralizes intracellular compartments allowing R. equi to multiply in an atypical macrophage phagolysosome. Here, we show that VapA does not support intra-macrophage growth of several other bacterial species demonstrating that only few bacteria have the specific preadaptations needed to profit from VapA. We show that the closest relative of R. equi, environmental Rhodococcus defluvii (Prescotella defluvii), does not multiply in macrophages at 37°C even when VapA is present because of its thermosensitivity but it does so once the infection temperature is lowered providing rare experimental evidence for ‘thermal restriction’. Using growth experiments with isolated macrophage lysosomes and modified infection schemes we provide evidence that R. equi resists the attack by phagolysosome contents at low pH for several hours. During this time, R. equi produces and secretes VapA which enables it to grow at the expense of lysosome constituents. We present arguments that, under natural infection conditions, R. equi is VapA-less during the initial encounter with the host. This has important implications for vaccine development.     

Mycobacterium massiliense is differentiated from Mycobacterium abscessus and Mycobacterium bolletii by erythromycin ribosome methyltransferase gene (erm) and clarithromycin susceptibility patterns

Erythromycin ribosome methyltransferase gene (erm) sequences of Mycobacterium massiliense and Mycobacterium bolletii isolates were newly investigated. Forty nine strains of M. massiliense that were analyzed in the present study had a deleted erm(41). Due to a frame‐shift mutation, large deletion, and truncated C‐terminal region, the Erm(41) of M. massiliense had only 81 amino acids encoded by 246 nucleotides. Corresponding to these findings, most of the M. massiliense isolates (89.8%) were markedly clarithromycin susceptible, but resistant strains invariably had a point mutation at the adenine (A₂₀₅₈ or A₂₀₅₉) in the peptidyltransferase region of the 23S rRNA gene, which is quite different from Mycobacterium abscessus and M. bolletii. In addition, erm(41) sequences of M. massiliense were more conserved than those of M. abscessus and M. bolletii. The results of species identification using erm(41) showed concordant results with those of multi‐locus sequence analysis (rpoB, hsp65, sodA and 16S‐23S ITS) where there were originally inconsistent results between rpoB and hsp65 sequence analysis in previous research. Therefore, erm(41) PCR that was used in the present study can be efficiently used to simply differentiate M. massiliense from M. abscessus and M. bolletii.     

Molecular and infection biology of the horse pathogen Rhodococcus equi

The soil actinomycete Rhodococcus equi is a pulmonary pathogen of young horses and AIDS patients. As a facultative intracellular bacterium, R. equi survives and multiplies in macrophages and establishes its specific niche inside the host cell. Recent research into chromosomal virulence factors and into the role of virulence plasmids in infection and host tropism has presented novel aspects of R. equi infection biology and pathogenicity. This review will focus on new findings in R. equi biology, the trafficking of R. equi -containing vacuoles inside host cells, factors involved in virulence and host resistance and on host–pathogen interaction on organismal and cellular levels.     

牛分支杆菌与肺结核分支杆菌基因组的比较

通过比较基因组学的方法研究发现,牛分支杆菌与肺结核杆菌基因组的同源性为99．95％,但在牛分枝杆菌基因组中有11个缺失区,大小从1kb到12．7kb,遗传信息的缺失引起牛分枝杆菌的基因组减小;牛分枝杆菌与肺结核分枝杆菌H37Rv间存在着2437个单核苷酸多态性（SNPs）,与肺结核分枝杆菌CDC1551间存在着2423个单核苷酸多态性（SNPs）,牛分支杆菌与肺结核分枝杆菌在编码细胞壁和分泌蛋白上变异程度也是巨大的。研究结果揭示了牛分支杆菌与肺结核分枝杆菌的遗传关系,为研究分支杆菌疫苗和诊断试剂提供理论依据,对牛肺结核病的防治有着非常重要的意义。     

N-acylhomoserine lactonase producing Rhodococcus spp. with different AHL-degrading activities

N-acylhomoserine lactones (AHLs) are conserved signal molecules that control diverse biological activities in quorum sensing system of Gram-negative bacteria. Recently, several soil bacteria were found to degrade AHLs, thereby interfering with the quorum sensing system. Previously, Rhodococcus erythropolis W2 was reported to degrade AHLs by both oxido-reductase and AHL-acylase. In the present study, two AHL-utilizing bacteria, strains LS31 and PI33, were isolated and identified as the genus Rhodococcus. They exhibited different AHL-utilization abilities: Rhodococcus sp. strain LS31 rapidly degraded a wide range of AHLs, including N-3-oxo-hexanoyl-l-homoserine lactone (OHHL), whereas Rhodococcus sp. strain PI33 showed relatively less activity towards 3-oxo substituents. Coculture of strain LS31 with Erwinia carotovora effectively reduced the amount of OHHL and pectate lyase activity, compared with coculture of strain PI33 with E. carotovora. A mass spectrometry analysis indicated that both strains hydrolyzed the lactone ring of AHL to generate acylhomoserine, suggesting that AHL-lactonases (AHLases) from the two Rhodococcus strains are involved in the degradation of AHL, in contrast to R. erythropolis W2. To the best of our knowledge, this is the first report on AHLases of Rhodococcus spp.     

A self-compartmentalizing protease in Rhodococcus: the 20S proteasome

The 26S proteasome represents a major, energy-dependent and self- compartmentalizing protease system in eukaryotes. The proteolytic core of this complex, the 20S proteasome, is also ubiquitous in archaea. Although absent from most eubacteria, this multi- subunit protease was recently discovered in Rhodococcus and appears to be confined to actinomycetes. The eubacterial 20S proteasome represents an attractive complementary system to study proteasome assembly, quaternary structure, and catalytic mechanism. In addition, it is likely to contribute substantially to our understanding of the role of various self-compartmentalizing proteases in bacterial cells.     

19F NMR study on the biodegradation of fluorophenols by various Rhodococcus species

Of all NMR observable isotopes 19F is the one perhaps most convenient for studies on biodegradation of environmental pollutants. The reasons underlying this potential of 19F NMR are discussed and illustrated on the basis of a study on the biodegradation of fluorophenols by four Rhodococcus strains. The results indicate marked differences between the biodegradation pathways of fluorophenols among the various Rhodococcus species. This holds not only for the level and nature of the fluorinated biodegradation pathway intermediates that accumulate, but also for the regioselectivity of the initial hydroxylation step. Several of the Rhodococcus species contain a phenol hydroxylase that catalyses the oxidative defluorination of ortho-fluorinated di- and trifluorophenols. Furthermore, it is illustrated how the 19F NMR technique can be used as a tool in the process of identification of an accumulated unknown metabolite, in this case most likely 5-fluoromaleylacetate. Altogether, the 19F NMR technique proved valid to obtain detailed information on the microbial biodegradation pathways of fluorinated organics, but also to provide information on the specificity of enzymes generally considered unstable and, for this reason, not much studied so far.     

快速方法检测结核分枝杆菌耐药基因突变

快速准确地鉴定结核分枝杆菌与结核分枝杆菌对利福平和异烟肼耐药基因突变的快速检测,对结核病人的诊断与治疗具有重要指导意义。本次根据结核分枝杆菌标准株H37RV序列,利用覆盖rpoB、katG、inhA基因突变区的系列寡核苷酸探针,并检测临床样品中结核分枝杆菌的基因突变情况,以此来判断耐药结果,并对其进行方法学评价。     

Oil desorption from mineral and organic materials using biosurfactant complexes produced by Rhodococcus species

Rhodococcus strains from the culture collection at the Institute of Ecology and Genetics of Microorganisms, Perm, Russia were examined for biosurfactant production during growth on n-alkanes and the ability to remove oil associated with contaminated sands and oil shale cuttings. Members of the genus, particularly R. ruber, were shown to produce low toxicity surfactants effective in removing oil from surfaces. The extent of desorption was inversely related to the concentration of high molecular weight hydrocarbons, namely asphaltenes and resins. In addition, crude surfactant complexes enhanced the degradation of crude oil, in the short term, when added to contaminated agricultural soil during bioremediation studies utilizing biopiling technology.     

Dithiothreitol reactivates desacetoxycephalosporin C synthetase after inactivation

Dithiothreitol (DTT) has been found to stimulate the desacetoxycephalosporin C synthetase (expandase) activity of a frozen crude extract of Streptomyces clavuligerus, even in the presence of an optimum concentration of ascorbate. Catalase, both native and heat-denatured, and bovine serum albumin (BSA) also stimulated the enzyme activity but were less active than DTT. Although fresh extracts were sporadically stimulated, frozen extracts or extracts inactivated by shaking at 37°C in air were consistently activated. It is felt that DTT functions as a reactivating, rather than an activaing, agent. Similar effects were observed with extracts of Cephalosporium acremonium.     

痰涂片标本用于结核分枝杆菌耐药性检测的研究

目的 研究抗酸染色结核分枝杆菌(简称结核杆菌)阳性痰涂片标本直接用于耐药性检测的方法。方法 对18株临床分离培养的结核杆菌用利福平进行药敏试验。分别提取菌株DNA和与之对应的痰涂片标本的菌体DNA,用聚合酶链反应(PcR)扩增ropB基因后进行固相杂交和核酸测序检测结核杆菌的耐药性。结果 18株结核杆菌中有12株对利福平耐药。经PCR扩增的ropB片段与探针杂交后,敏感菌株未发现rpoB基因的突变,自耐药菌株提取的DNA中rpoB突变体的检出率为100％(12／12),痰涂片提取DNA的检出率为91．7％(11／12)。所有耐药菌株DNA与痰涂片DNA核酸测序结果相吻合,都有rpoB基因核心区域碱基突变。结论 抗酸染色痰涂片阳性标本可直接用于检测结核杆菌利福平耐药基因rpoB突变体,是一种值得临床实验室推广使用的耐药菌诊断方法。     

红球菌DS—3脱除二苯并噻吩中有机硫的性能初探

从孤岛油田分离到一株红球菌(Rhodococcus sp.)DS—3,能专一地切断二苯并噻吩(DBT)中的C－S键,沿4S途径代谢,生成二羟联苯。实验证明,以2％的接种量脱除50μｇ／mL DBT底物中的硫效果最佳。在此条件下,适宜菌株生长和脱硫的碳源为葡萄糖,氯源为硝酸铵,初始PH为8．2,生长温度为30℃,15mmol/L的硫酸根离子能使其丧失脱硫能力。在上述适宜条件下,培养72h后DBT中34．04％的硫被脱除。     

Degradation of 1,3-dichloropropene by a soil bacterial consortium and Rhodococcus sp. AS2C isolated from the consortium

A bacterial consortium capable of degrading the fumigant 1,3-D ((Z)- and (E)-1,3-dichloropropene) was enriched from an enhanced soil. This mixedculture degraded (Z)- and (E)-1,3-D only in the presence of a suitable biodegradable organic substrate, such as tryptone, tryptophan, or alanine. After 8 months of subculturing at 2- to 3-week intervals, a strain of Rhodococcus sp. (AS2C) that was capable of degrading 1,3-D cometabolically in the presenceof a suitable second substrate was isolated. (Z)-3-chloroallyl alcohol (3-CAA) and (Z)-3-chloroacrylic acid (3-CAAC), and (E)-3-CAA and (E)-3-CAAC were the metabolites of (Z)- and (E)-1,3-D, respectively. (E)-1,3-D was degraded faster than (Z)-1,3-D by the strain AS2C and the consortium. AS2C also degraded (E)-3-CAA faster than (Z)-3-CAA. Isomerization of (E)-1,3-D to (Z)-1,3-D orthe (Z) form to the (E) form did not occur.     

Photodynamic inactivation of Aeromonas hydrophila by cationic phthalocyanines with different hydrophobicity

Antibacterial photodynamic therapy is a pioneering method for the inactivation of pathogenic bacteria. Four tetra alkyl-substituted cationic phthalocyanines with different hydrocarbon chains attached to the pyridyloxy group were synthesized. These photodynamic sensitizers were studied for antibacterial inactivation of a multidrug-resistant strain of Gram-negative bacterium Aeromonas hydrophila. Aeromonas species are recognized as etiological agents of a wide spectrum of diseases in humans and animals. The uptake of phthalocyanines by the bacterial cells decreased with an increase in cell density. Following the phthalocyanine solubility from hydrophilic to hydrophobic complexes, the accumulation capacity increased. Full inactivation was achieved with phthalocyanine with (methoxy) pyridyloxy substitution following a short exposure time, low drug concentration and mild irradiation. Although the phthalocyanine with the longest hydrocarbon chain (C12) has some toxic effect in the absence of light, substantial phototoxic effect was obtained with the optimal combination of drug-irradiation parameters.     

衣壳蛋白靶向灭活 一种新型抗病毒策略

衣壳蛋白靶向灭活（CTVI）是一种新型抗病毒策略,它是通过将病毒衣壳蛋白与核酸酶（金黄色葡萄球菌和酸梅、核糖核酸酶Barnase、大肠杆菌RNase HI等）的融合蛋白装配到病毒粒子中,使核酸酶接触并降解病毒核酸,从而达到抑制病毒复制的目的。该策略已经在人免疫缺陷病毒、鼠白血病病毒、乙肝病毒、登革病毒等病毒的抗病毒研究中取得良好效果,展示了广阔的应用前景。     

Chelating agents protect hydrogenase against oxygen inactivation

The effect of chelation on rate of air inactivation of hydrogenase from Clostridium pasteurianum has been investigated. All chelating agents used, whether water-soluble or water-insoluble, afforded protection against oxygen inactivation. EDTA appeared to be the most effective. Thus, in the absence of EDTA, hydrogenase in aqueous solution was nearly totally inactivated after 1 hour incubation in air, whereas 0.5 M EDTA (which did not affect significantly catalytic activity) allowed 41% retention of the initial activity even after 3 days incubation.     

Radiation inactivation of (Na+ + K+)-ATPase a small target size for the K+-occluding mechanism

Radiation inactivation of partially purified (Na⁺ + K⁺)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pig kidney outer medulla shows that the target size for Rb⁺ occlusion by the enzyme (in the absence of phosphorylation) is much smaller than the target size for $\text{p-}\text{nitrophenyl phosphatase activity}$, which is itself smaller than the reported target size for (Na⁺ + K⁺)-ATPase activity.