Different microbial biofilm formation on polymethylmethacrylate (PMMA) bone cement loaded with gentamicin and vancomycin

We studied the in vitro effects of gentamicin and vancomycin alone and in combination added to polymethylmethacrylate (PMMA) cement specimens on the bacterial adhesion of multiresistant clinical isolates.The PMMA specimens (discs) loaded with gentamicin (1.9%) or vancomycin (1.9%) or with a combination of the two were placed in Mueller-Hinton Broth inoculated with bacterial strains. After incubation, bacterial growth was determined by optical density (OD₅₄₀) and sub-cultures. The biofilm PMMA-associated dye (crystal violet) was measured. Antibiotic concentrations in broth were determined by fluorescence polarisation immunoassay.All antibiotic-loaded PMMA cement specimens released high, inhibitory concentrations of gentamicin and vancomycin. However, differences in strain growth and adhesion were recorded. The clinical isolates Met-R/Gent-R CoNS showed no adhesion to gentamicin-loaded specimens for 24 h; strains with Gent-Intermediate susceptibility exhibited growth after 48 h but reduced adhesion. Some Gent-R strains exhibited growth and adhesion to antibiotic-loaded specimens similar to controls (plain discs). Only the VRSA strain (Staphylococcus aureus 5/7) and Escherichia coli were able to grow and adhere to vancomycin-loaded specimens after 24 h of incubation. The specimens loaded with the gentamicin + vancomycin combination showed a synergistic inhibitory effect against all tested strains (no bacterial growth). The degree of bacterial adhesion to PMMA cement loaded with gentamicin or vancomycin may be reduced in spite of a normal growth rate and is different for the tested strains.The effect of gentamicin and vancomycin on bacterial growth and adhesion to PMMA bone cement depends on the antibiotic concentrations, on the characteristics of each specific strain and on its ability to produce biofilm and adhere to antibiotic-loaded PMMA bone cement.     

Overexpression,purification and characterization of SimL,an amide synthetase involved in simocyclinone biosynthesis

Simocyclinone D8 is a potent inhibitor of bacterial gyrase, produced by Streptomyces antibioticus Tü 6040. It contains an aminocoumarin moiety, similar to that of novobiocin, which is linked by an amide bond to a structurally complex acyl moiety, consisting of an aromatic angucycline polyketide nucleus, the deoxysugar olivose and a tetraene dicarboxylic acid. We have now investigated the enzyme SimL, responsible for the formation of the amide bond of simocyclinone. The gene was cloned, expressed in S. lividans T7, and the protein was purified to near homogeneity, and characterized. The 60 kDa protein catalyzed both the ATP-dependent activation of the acyl component as well as its transfer to the amino group of the aminocoumarin ring, with no requirement for a 4-phosphopantetheinyl cofactor. Besides its natural substrate, simocyclinone C4, SimL also accepted a range of cinnamic and benzoic acid derivatives and several other, structurally very diverse acids. These findings make SimL a possible tool for the creation of new aminocoumarin antibiotics.     

The chloramphenicol resistance gene cmlA is disseminated on transferable plasmids that confer multiple-drug resistance in swine Escherichia coli

A recent study of beta-hemolytic Escherichia coli isolated from diarrheic swine found that 53% were resistant to chloramphenicol, a drug that has been prohibited from use in food animals in the US since the mid-1980s. To identify the factors governing the persistence of chloramphenicol resistance in the absence of specific selection pressure, the location of the chloramphenicol resistance gene cmlA and its linkage to other resistance determinants were investigated. Southern blot analysis of plasmid DNA from 46 swine E. coli isolates indicated that cmlA was present on large plasmids greater than 100 kbp. Fifty-two percent of the isolates were able to transfer chloramphenicol resistance to an E. coli recipient at conjugation frequencies ranging from 10(-3) to 10(-8) per recipient. Antimicrobial susceptibility tests on transconjugant strains demonstrated that resistance to sulfamethoxazole, tetracycline, and kanamycin frequently transferred along with chloramphenicol resistance. The transconjugant strains possessed at least two distinct class 1 integrons that linked cmlA to both aminoglycoside resistance genes aadA1 and aadA2 and either to sul1 or to sul3 sulphonamide resistance genes. These results suggest that in the absence of specific chloramphenicol selection pressure, the cmlA gene is maintained by virtue of gene linkage to genes encoding resistance to antimicrobials that are currently approved for use in food animals.     

药物不良反应造成急性肾损害的临床分析

目的：研究药物不良反应（ADR）所致急性肾损伤的相关因素并探讨其诊疗思路。,方法：对56例ADR造成急性肾损害的病例进行归类统计和分析评价。结果：56例病例中,男性略多于女性,男女之比为1．24：1,平均年龄53．1岁;抗生素发生率居首位,其次为中成药;途径以静脉注射为主。结论：合理用药,早期干预治疗,预防药源性急性肾损害的发生。     

“Pseudo” γ-Butyrolactone Receptors Respond to Antibiotic Signals to Coordinate Antibiotic Biosynthesis

In actinomycetes, the onset of secondary metabolite biosynthesis is often triggered by the quorum-sensing signal γ-butyrolactones (GBLs) via specific binding to their cognate receptors. However, the presence of multiple putative GBL receptor homologues in the genome suggests the existence of an alternative regulatory mechanism. Here, in the model streptomycete Streptomyces coelicolor, ScbR2 (SCO6286, a homologue of GBL receptor) is shown not to bind the endogenous GBL molecule SCB1, hence designated “pseudo” GBL receptor. Intriguingly, it could bind the endogenous antibiotics actinorhodin and undecylprodigiosin as ligands, leading to the derepression of KasO, an activator of a cryptic type I polyketide synthase gene cluster. Likewise, JadR2 is also a putative GBL receptor homologue in Streptomyces venezuelae, the producer of chloramphenicol and cryptic antibiotic jadomycin. It is shown to coordinate their biosynthesis via direct repression of JadR1, which activates jadomycin biosynthesis while repressing chloramphenicol biosynthesis directly. Like ScbR2, JadR2 could also bind these two disparate antibiotics, and the interactions lead to the derepression of jadR1. The antibiotic responding activities of these pseudo GBL receptors were further demonstrated in vivo using the lux reporter system. Overall, these results suggest that pseudo GBL receptors play a novel role to coordinate antibiotic biosynthesis by binding and responding to antibiotics signals. Such an antibiotic-mediated regulatory mechanism could be a general strategy to coordinate antibiotic biosynthesis in the producing bacteria.     

Antimicrobial activity of steady-state cultures of Bacillus sp. CCMI 1051 against wood contaminant fungi

The effect of changing dilution rate (D) on Bacillus sp. CCMI 1051 at dilution rates between 0.1 and 0.55 h⁻¹ in a glucose-limited medium was studied. Biomass values varied between 0.88 and 1.1 g L⁻¹ at D values of 0.15–0.35 h⁻¹. Maximal biomass productivity was found to be 0.39 g L⁻¹ h⁻¹, obtained at D = 0.35 h⁻¹ and corresponding to a 54.4% conversion of the carbon into cell mass. The highest rate of glucose consumption was 4.45 mmol g⁻¹ h⁻¹ occurring at D = 0.4 h⁻¹. The glucose concentration inside the chemostat was below the detection level starting to accumulate around 0.4 h⁻¹. Growth inhibition of fifteen strains of fungi by the broth of the steady-state cell-free supernatants was assessed. Results showed that the relative inhibition differ among the target species but was not influenced by the dilution rate changing.     

Gene transfer preferentially selects MHC class I positive tumour cells and enhances tumour immunogenicity

The modulated expression of MHC class I on tumour tissue is well documented. Although the effect of MHC class I expression on the tumorigenicity and immunogenicity of MHC class I negative tumour cell lines has been rigorously studied, less is known about the validity of gene transfer and selection in cell lines with a mixed MHC class I phenotype. To address this issue we identified a C26 cell subline that consists of distinct populations of MHC class I (H-2D/K) positive and negative cells. Transient transfection experiments using liposome-based transfer showed a lower transgene expression in MHC class I negative cells. In addition, MHC class I negative cells were more sensitive to antibiotic selection. This led to the generation of fully MHC class I positive cell lines. In contrast to C26 cells, all transfectants were rejected in vivo and induced protection against the parental tumour cells in rechallenge experiments. Tumour cell specificity of the immune response was demonstrated in in vitro cytokine secretion and cytotoxicity assays. Transfectants expressing CD40 ligand and hygromycin phosphotransferase were not more immunogenic than cells expressing hygromycin resistance alone. We suggest that the MHC class I positive phenotype of the C26 transfectants had a bearing on their immunogenicity, because selected MHC class I positive cells were more immunogenic than parental C26 cells and could induce specific anti-tumour immune responses. These data demonstrate that the generation of tumour cell transfectants can lead to the selection of subpopulations that show an altered phenotype compared to the parental cell line and display altered immunogenicity independent of selection marker genes or other immune modulatory genes. Our results show the importance of monitoring gene transfer in the whole tumour cell population, especially for the evaluation of in vivo therapies targeted to heterogeneous tumour cell populations.     

Static recipient cells as reservoirs of antibiotic resistance during antibiotic therapy

How does taking the full course of antibiotics prevent antibiotic resistant bacteria establishing in patients? We address this question by testing the possibility that horizontal/lateral gene transfer (HGT) is critical for the accumulation of the antibiotic-resistance phenotype while bacteria are under antibiotic stress. Most antibiotics prevent bacterial reproduction, some by preventing de novo gene expression. Nevertheless, in some cases and at some concentrations, the effects of most antibiotics on gene expression may not be irreversible. If the stress is removed before the bacteria are cleared from the patients by normal turnover, gene expression restarts, converting the residual population to phenotypic resistance. Using mathematical models we investigate how static recipients of resistance genes carried by plasmids accumulate resistance genes, and how specifically an environment cycling between presence and absence of the antibiotic uniquely favors the evolution of horizontally mobile resistance genes. We found that the presence of static recipients can substantially increase the persistence of the plasmid and that this effect is most pronounced when the cost of carriage of the plasmid decreases the cell's growth rate by as much as a half or more. In addition, plasmid persistence can be enhanced even when conjugation rates are as low as half the rate required for the plasmid to persist as a parasite on its own.     

Nutritional requirements for the production of dithiolopyrrolone antibiotics by Saccharothrix algeriensis NRRL B-24137

The amino acid and humic acid requirements of Saccharothrix algeriensis NRRL B-24137 for growth and production of the dithiolopyrrolone antibiotics were studied in a semi-synthetic medium (SSM). Nature and concentration of amino acids and humic acid strongly influenced the growth and dithiolopyrrolone specific production.

The highest value of thiolutin (acetyl-pyrrothine) specific production was obtained in the presence of 1 g/l humic acid (336 mg/g DCW), and in the presence of 5 mM l-cystine (309 mg/g DCW) as compared to 19 mg/g DCW obtained with the control. Furthermore, thiolutin production was increased about six-fold, four-fold and three-fold in the presence of l-proline, l-glutamic acid and dl-histidine, respectively. In contrast, the production of thiolutin was reduced by addition of other amino acids such as l-glutamine, dl-ethionine, l-methionine and l-arginine. The highest value of isobutyryl-pyrrothine production was obtained in the presence of 2,6-diaminopimelic acid and l-lysine (7.8 and 1.0 mg/g DCW, respectively). However, the highest value of butanoyl-pyrrothine production was obtained in the presence of humic acid (6.6 mg/g DCW), followed by l-cysteine and l-proline (3.6 and 3.2 mg/g DCW, respectively). In addition, the maximum specific production of senecioyl-pyrrothine (29 mg/g DCW) and tigloyl-pyrrothine (21 mg/g DCW) was obtained in the presence of humic acid. We found that, except for isobutyryl-pyrrothine, production of all dithiolopyrrolones was favoured by addition of l-proline. The maximum specific production was obtained with l-proline at concentrations of 2.50 mM for thiolutin (133 mg/g DCW), 1.25 mM for senecioyl-pyrrothine, tigloyl-pyrrothine and butanoyl-pyrrothine production (29, 23 and 3.9 mg/g DCW, respectively). Production of all dithiolopyrrolones strongly decreased as the l-methionine or dl-ethionine concentration was increased in the culture medium.     

Metalloantibiotic Mn(II)-bacitracin complex mimicking manganese superoxide dismutase

Superoxide dismutase (SOD) activities of various metallobacitracin complexes were evaluated using the riboflavin-methionine-nitro blue tetrazolium assay. The radical scavenging activity of various metallobacitracin complexes was shown to be higher than those of the negative controls, e.g., free transition metal ions and metal-free bacitracin. The SOD activity of the complex was found to be in the order of Mn(II)>Cu(II)>Co(II)>Ni(II). Furthermore, the effect of bacitracin and their complexation to metals on various microorganisms was assessed by antibiotic susceptibility testing. Moreover, molecular modeling and quantum chemical calculation of the metallobacitracin complex was performed to evaluate the correlation of electrostatic charge of transition metal ions on the SOD activity.