Macrolide resistance genotypes and phenotypes among erythromycin-resistant clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, Italy

One hundred macrolide-resistant staphylococcal isolates from clinically relevant infections in Italy during a 19-month period were studied. Four distinct resistance phenotypes were observed using the triple-disk induction test (erythromycin, clindamycin, telithromycin): the cMLS_B phenotype (24 isolates); the iMLS_B phenotype (41 isolates); the MS phenotype (three isolates); and the iMTS phenotype (erythromycin-induced telithromycin resistance) (32 isolates). ermC and ermA genes predominated within erythromycin-resistant Staphylococcus aureus isolates with iMLS_B phenotype and cMLS_B phenotype, respectively. Among erythromycin-resistant CoNS isolates, half of the strains showed the iMTS or MS/ msrA association, and ermC gene predominated among isolates with MLS_B phenotype. By pulsed-field gel electrophoresis, high genetic heterogeneity was observed among the isolates studied. Both independent acquisition of macrolide resistance genes and spread of specific resistant clones were observed. Association between certain clonal types and specific types of infection could be detected. To our knowledge, this is the first report on characterization of erythromycin-resistant staphylococci in Italy.     

The role of copper in drug-resistant murine and human tumors

Multidrug resistance (MDR) is still a major threat to successful clinical application of cancer chemotherapy. Copper plays an important role in biological systems, and copper is also involved in carcinogenesis. In the present investigation, we addressed the question whether metal copper might be involved in drug resistance of murine and human tumors. By means of atomic absorption spectroscopy, we determined serum copper concentrations. We found that the blood serum of tumor-bearing mice contained higher amounts of copper than healthy mice with tumors. Secondly, mice bearing doxorubicin-resistant Ehrlich ascites carcinoma- or cyclophosphamide-resistant Lewis lung carcinoma contained more copper in their serum than mice bearing the corresponding drug-sensitive parental tumors. Furthermore, the analysis of patients with breast cancer, colon carcinoma or lung cancer showed that the serum copper contents were higher in patients not responding to chemotherapy when compared to patients whose tumors responded to treatment. The copper levels in serum of healthy volunteers were lower than in cancer patients irrespective of their response to chemotherapy. Our results imply that the level of serum copper may be considered as a biomarker for treatment response.     

Bacterial resistances to mercury and copper.

Heavy metals are toxic to living organisms. Some have no known beneficial biological function, while others have essential roles in physiological reactions. Mechanisms which deal with heavy metal stress must protect against the deleterious effects of heavy metals, yet avoid depleting the cell of a heavy metal which is also an essential nutrient. We describe the mechanisms of resistance in Escherichia coli to two different heavy metals, mercury and copper. Resistance of E. coli to mercury is reasonably well understood and is known to occur by transport of mercuric ions into the cytoplasmic compartment of the bacterial cell and subsequent reductive detoxification of mercuric ions. Recent mutational analysis has started to uncover the mechanistic detail of the mercuric ion transport processes, and has shown the essential nature of cysteine residues in transport of Hg(II). Resistance to copper is much less well understood, but is known to involve the increased export of copper from the bacterial cell and modification of the copper; the details of the process are still being elucidated. Expression of both metal resistance determinants is regulated by the corresponding cation. In each case the response enables the maintenance of cellular homeostasis for the metal. The conclusions drawn allow us to make testable predictions about the regulation of expression of resistance to other heavy metals.     

Contact killing and antimicrobial properties of copper

With the emergence of antibiotic resistance, the interest for antimicrobial agents has recently increased again in public health. Copper was recognized in 2008 by the United States Environmental Protection Agency (EPA) as the first metallic antimicrobial agent. This led to many investigations of the various properties of copper as an antibacterial, antifungal and antiviral agent. This review summarizes the latest findings about ‘contact killing’, the mechanism of action of copper nanoparticles and the different ways micro‐organisms develop resistance to copper.     

Chromosome-encoded inducible copper resistance inPseudomonas strains

NinePseudomonas strains were selected by their high copper tolerance from a population of bacteria isolated from heavy-metal polluted zones. Copper resistance (Cu ^r ) was inducible by previous exposure of cultures to subinhibitory amounts of copper sulfate. All nine strains possessed large plasmids, but transformation and curing results suggest that Cu ^r is conferred by chromosomal genes. Plasmid-lessPseudomonas aeruginosa PAO-derived strains showed the same level of Cu ^r as environmental isolates and their resistance to copper was also inducible. Total DNA from the environmentalPseudomonas, as well as fromP. aeruginosa PAO strains, showed homology to a Cu ^r P. syringae cop probe at low-stringency conditions but failed to hybridize at high-stringency conditions.     

弯曲菌对大环内酯类抗生素耐药机制研究进展

弯曲菌是一种全球普遍关注的引起人兽共患病的病原菌。由于临床治疗和养殖业的不合理使用抗生素,导致弯曲菌的耐药性日益严重。核糖体靶位点突变、核糖体蛋白变构和细胞膜上外排系统改变是弯曲菌对大环内酯类抗生素耐药的主要原因。就弯曲菌对大环内酯类抗生素的耐药现状和耐药机制进行综述。     

Differential expression of pathogenicity- and virulence-related genes of Xanthomonas axonopodis pv. citri under copper stress

In this study, we used real-time quantitative PCR (RT-qPCR) to evaluate the expression of 32 genes of Xanthomonas axonopodis pv. citri related to pathogenicity and virulence that are also involved in copper detoxification. Nearly all of the genes were up-regulated, including copA and copB. Two genes homologous to members of the type II secretion system (xcsH and xcsC) and two involved in the degradation of plant cell wall components (pglA and pel) were the most expressed in response to an elevated copper concentration. The type II secretion system (xcs operon) and a few homologues of proteins putatively secreted by this system showed enhanced expression when the bacteria were exposed to a high concentration of copper sulfate. The enhanced expression of the genes of secretion II system during copper stress suggests that this pathway may have an important role in the adaptative response of X. axonopodis pv. citri to toxic compounds. These findings highlight the potential role of these genes in attenuating the toxicity of certain metals and could represent an important means of bacterial resistance against chemicals used to control diseases.     

Comparison of genotypes and antibiotic resistance of Campylobacter jejuni isolated from humans and slaughtered chickens in Switzerland

Aims: To get an overview of genotypes and antibiotic resistances in Swiss Campylobacter jejuni implicated in human gastroenteritis and to examine the association with isolates from chickens. Methods and Results: Multilocus sequence typing (MLST) and flaB typing were applied to 136 human clinical isolates. Phenotypic resistance to 12 antimicrobials and genotypic resistance to macrolides and quinolones were determined. MLST resulted in 35 known and six new sequence types (ST). The flaB analysis revealed 35 different types, which – in combination with MLST – increased the resolution of the typing approach. Resistance to quinolones, tetracycline and ampicillin was found in 37·5, 33·1 and 8·1% of the isolates, respectively, whereas macrolide resistance was found only once. Genotypic and phenotypic resistance correlated in all cases. A comparison to Camp. jejuni isolated from slaughtered chickens was performed. While 86% of the quinolone‐sensitive human isolates showed overlapping MLST‐flaB types with those of chicken origin, resistant strains showed only 39% of matching types. Conclusion: Mainly quinolone‐sensitive Camp. jejuni strains implicated in human campylobacteriosis showed matching genotypes with isolates originating from chickens. Significance and Impact of the Study: A large proportion of human cases in Switzerland are likely to originate from domestic chickens, confirming that prevention measures in the poultry production are important.     

西瓜食酸菌RND蛋白家族外排转运体cusB基因抗铜功能研究

【目的】研究RND外排泵中cus B基因突变对西瓜食酸菌抗铜性的影响。【方法】采用Tn5转座子随机插入基因组制备筛选得到突变体,通过双亲杂交的方法构建功能互补菌株,并从西瓜食酸菌抗铜性、胞外纤维素酶和胞外蛋白酶分泌、胞外多糖产生、生物膜形成、致病性及过敏性反应等方面阐明RND外排泵中MFP蛋白亚基对西瓜食酸菌的影响。【结果】突变体Δcus B在含有1.25 mmol/L或2.5 mmol/L Cu SO4的KMB平板上不能生长,cus B基因的突变导致西瓜食酸菌的胞外多糖分泌和生物膜形成与野生型有差异,但不影响胞外纤维素酶、胞外蛋白酶、致病性及过敏性反应。【结论】RND外排泵相关基因cus B的突变会影响西瓜食酸菌的某些生物学特性,并导致病菌对铜十分敏感。研究以RND外排泵转运重金属为导向初步解析了西瓜食酸菌的抗铜机制。     

Effect of medium copper concentration on the growth,uptake and intracellular balance of copper and zinc in Menkes' and normal control cells

The precise nature of the variation in cellular copper load against medium copper concentration is defined using a comprehensive logarithmically incremented series of medium copper concentrations ranging from low levels (4.8 p.p.b.) through normal to toxic levels (40 p.p.m.) in which fibroblasts were grown followed by determination of intracellular content. Menkes' fibroblasts showed an unexpected plateau region of stable intracellular copper content against a change in medium concentration of over 100-fold, albeit only when sufficient copper was present in the medium (0.08–8.0 p.p.m.). Thus, Menkes' cells are clearly capable of balancing uptake/efflux providing copper availability allows. Simultaneous analysis of cellular copper and zinc load at various medium copper concentrations shows an indistinguishable intracellular copper:zinc ratio between the two cell lines. The nature of non-labeled copper uptake by fibroblasts over a 40 min and 7 day period is reported. During the 40 min period copper uptake (20 p.p.m.) was essentially the same in both cell lines. However, copper absorbed was superimposed upon large pre-existing copper pools in the case of Menkes' cells only. Advantages of techniques determining non-labeled copper in copper uptake/efflux experiments are discussed in the light of these results. Fibroblast growth studies showed that, compared with normal cells, Menkes' cells are significantly (P < 0.01) more growth sensitive to extended exposure to low copper concentrations. Thus, Menkes' disease appears to be not only a result of copper maldistribution but also a direct result of an inability of Menkes' cells to function normally in low copper environments.     

生物耐铜的分子机理及铜污染环境的生物联合修复

铜是动植物和人类必需的微量元素,缺乏或过多都将产生不良影响。随着社会经济的发展,人类活动对环境的干扰日益加剧,工业和农业生产活动常可导致土壤铜污染,铜已成为土壤重金属污染的主要元素之一。总结了铜在植物体内的自发内稳态调节机制,在细菌和真菌体内的吸收、分布、解毒和调节因子,同时以蚯蚓为例简要阐述了土壤动物对铜的解毒机理;从分子生物学角度对重金属铜在生物体内的代谢机理及生物对环境中过量铜的联合修复研究进展进行了综述,以期为铜污染环境的植物、微生物和动物联合修复的分子机理研究提供借鉴。     

Genes involved in copper resistance influence survival of Pseudomonas aeruginosa on copper surfaces

Aims:  To evaluate the killing of Pseudomonas aeruginosa PAO1 on copper cast alloys and the influence of genes on survival on copper containing medium and surfaces.
Methods and Results:  Different strains of P. aeruginosa were inoculated on copper containing medium or different copper cast alloys and the survival rate determined. The survival rates were compared with rates on copper-free medium and stainless steel as control. In addition, the effect of temperature on survival was examined.
Conclusions:  Copper cast alloys had been previously shown to be bactericidal to various bacteria, but the mechanism of copper-mediated killing is still not known. In this report, we demonstrate that P. aeruginosa PAO1 is rapidly killed on different copper cast alloys and that genes involved in conferring copper resistance in copper-containing medium also influenced survival on copper cast alloys. We also show that the rate of killing is influenced by temperature.
Significance and Impact of the Study:  To use copper surfaces more widely as bactericidal agents in various settings, it is important to understand how genes influence survival on these surfaces. Here we show that genes shown to be involved in copper resistance in P. aeruginosa PAO1 can have an impact on the length of survival time on copper cast alloys under certain conditions. This is an important first step for evaluation of future use of copper surfaces as bactericidal agents.     

植物铜转运相关蛋白研究进展

环境中过量的铜会损害植物细胞的功能、降低酶的活性并且破坏蛋白质的结构。植物中有一个复杂的金属转运网络,对维持植物体内铜的动态平衡发挥着重要作用。综述了重金属铜对植物的毒害,详细介绍了铜转运相关蛋白及其对铜的转运和调控机制。     

Genes regulating copper metabolism

The metabolism of Cu is intimately linked with its nutrition. From gut to enzymes, Cu bioavailability to key enzymes and other components operates through a complex mechanism that uses transport proteins as well as small molecular weight ligands. Steps in Cu transport through the blood, absorption by cells, and incorporation into enzymes are slowly being understood. Cloning and sequencing of the genes for Menkes disease and Wilson disease has shown that membrane-bound enzymes analogous to Cu-ATPases in prokaryotes are equally important to Cu transport and homeostasis in mammalian cells. The primary structure of the mammalian Cu-ATPases has been deduced from cDNAs from tissues and organs. It now appears that mammalian Cu-ATPase have tissue and developmental specificity. In this review, we will focus on the Cu-ATPase that has been identified with Menkes disease. An emphasis will be placed on the existence of multiple forms of the ATPase and some indication as to how the different isoforms befit their role in the normal physiology of copper, specifically transmembrane transport and maintenance of a favorable internal cellular environment.     

Copper exposure and potential biomarkers of copper metabolism

Relevant biological effects associated with mild to moderate copper deficiency and copper excess are unknown. It is difficult to identify markers of these early changes because limits of the homeostatic range are still undefined and early changes may represent adaptive responses that do not imply necessarily risk of damage. We report here a series of studies carried out to shed light on the responses within the homeostatic range, by assessing classic parameters of copper status in humans at different copper exposure. In adult healthy volunteers that had an estimated daily intake of 0.9 mg Cu/day (approximately 15 g/kg/d), exposure to additional 50–60 g of copper/kg/day for three months or up to 150 g/kg/d for two months resulted in no significant changes of SOD activity in erythrocytes, of copper concentration (in serum, erythrocytes and mononuclear cells) and of serum ceruloplasmin (ANOVA). Neither were found differences by gender or age. As in previous studies in infants, the non-ceruloplasmin copper fraction was positively correlated to serum copper (r=0.58). Assessing variations on copper absorption, infants supplemented/not supplemented with oral copper (80 ug/kg/14 days), at age 1 and 3 months, showed copper absorption close to 80% at both ages; no effect was observed for age or supplementation, suggesting that either these concentrations do not elicit regulatory mechanisms or that at this age down regulation for copper absorption is not efficient. These studies indicate that in the range of the copper homeostasis area the markers tested are not suitable to detect mild changes (within the homeostatic range) of copper metabolism.     

Growth competition of macrolide-resistant and -susceptible Helicobacter pylori strains

We examined population dynamics in a mixed culture of clonally related macrolide-resistant and -susceptible Helicobacter pylori strains isolated from a single patient. The resistant strain had a macrolide resistance-conferring A2143G mutation in the 23S rRNA gene. The growth rate of these two strains did not apparently differ when cultured separately. On the other hand, by conducting sequential passage of a mixed culture of the resistant and the susceptible strains, the ratio of the resistant strain to the susceptible strain in the culture typically decreased per passage, indicating that the resistance imposed a significant disadvantage on bacterial fitness in the population.     

狮子山铜尾矿植物对铜的吸收及土壤特的影响

我国铜矿储藏丰富,铜矿开采带来巨大经济利益的同时,也对生态环境造成极大的破坏,这种恶劣的环境严重阻碍了植物的定居,但是自然界物种繁多,总有一些植物能适应这种环境而生存下来.本文通过对狮子山优势植物吸收和积累铜的分析,发现这些植物均能富集较多的铜,在土壤铜含量很高的情况下,依然生长旺盛,没有出现受害症状,成为尾矿上的优势种,并形成了单优群落或多优小群落.这些植物的存在改变了土壤的理化特性,降低了土壤中的重金属的含量,提高了土壤的全N、全P、全K和有机质含量,一定程度上改善了土壤的不良环境,在尾矿的植被恢复和土壤修复中起着非常重要的作用.     

Polypropylene with embedded copper metal or copper oxide nanoparticles as a novel plastic antimicrobial agent

Aims: To develop novel polypropylene composite materials with antimicrobial activity by adding different types of copper nanoparticles. Methods and Results: Copper metal (CuP) and copper oxide nanoparticles (CuOP) were embedded in a polypropylene (PP) matrix. These composites present strong antimicrobial behaviour against E. coli that depends on the contact time between the sample and the bacteria. After just 4 h of contact, these samples are able to kill more than 95% of the bacteria. CuOP fillers are much more effective eliminating bacteria than CuP fillers, showing that the antimicrobial property further depends on the type of copper particle. Cu²⁺ released from the bulk of the composite is responsible for this behaviour. Moreover, PP/CuOP composites present a higher release rate than PP/CuP composites in a short time, explaining the antimicrobial tendency. Conclusions: Polypropylene composites based on copper nanoparticles can kill E. coli bacteria depending on the release rate of Cu²⁺ from the bulk of the material. CuOP are more effective as antimicrobial filler than CuP. Significance and Impact of the Study: Our findings open up novel applications of these ion‐copper‐delivery plastic materials based on PP with embedded copper nanoparticles with great potential as antimicrobial agents.     

Mycoplasma bovis isolates from dairy calves in Japan have less susceptibility than a reference strain to all approved macrolides associated with a point mutation (G748A) combined with multiple species‐specific nucleotide alterations in 23S rRNA

Erythromycin, tylosin and tilmicosin are approved for use in cattle in Japan, the latter two being used to treat Mycoplasma bovis infection. In this study, 58 M. bovis isolates obtained from Japanese dairy calves all exhibited reduced susceptibility to these macrolides, this widespread reduced susceptibility being attributable to a few dominant lineages. All 58 isolates contained the G748A variant in both the rrl3 and rrl4 alleles of 23S rRNA, whereas a reference strain (PG45) did not. G748 localizes in the central loop of domain II (from C744 to A753) of 23S rRNA, which participates in binding to mycinose, a sugar residue present in both tylosin and tilmicosin. A number of in vitro‐ selected mutants derived from M. bovis PG45 showed reduced susceptibility to tylosin and tilmicosin and contained a nucleotide insertion within the central loop of domain II of rrl3 (U747–G748Ins_CU/GU or A743–U744Ins_UA), suggesting that mutations around G748 confer this reduced susceptibility phenotype. However, other Mycoplasma species containing G748A were susceptible to tylosin and tilmicosin. Sequence comparison with Escherichia coli revealed that M. bovis PG45 and isolates harbored five nucleotide alterations (U744C, G745A, U746C, A752C and A753G) in the central loop of domain II of 23S rRNA, whereas other Mycoplasma species lacked at least two of these five nucleotide alterations. It was therefore concluded that G748 mutations in combination with species‐specific nucleotide alterations in the central loop of domain II of 23S rRNA are likely sufficient to reduce susceptibility of M. bovis to tylosin and tilmicosin.