Metal-Resistant Heterotrophic Bacteria in Coastal Waters of Primorye

Colony-forming heterotrophic bacteria were isolated from several locations in coastal waters of Primorye that differ in the extent and character of heavy metal pollution. Statistically significant differences in the minimum inhibitory concentrations of some metals were found for bacteria isolated from the environments with different levels of pollution. Bacterial strains that were highly tolerant of several heavy metals also showed resistance to a wide spectrum of antibiotics, and some strains bore plasmids of varying numbers and sizes. Bacteria of the family Enterobacteriaceae and the genus Pseudomonas were the dominant metal-resistant bacterial forms in the polluted coastal wasters of Primorye. No strict correlation was found between metal resistance and taxonomic identity of bacteria.Original Russian Text Copyright © 2005 by Biologiya Morya, Bezverbnaya, Buzoleva, Khristoforova.     

环境抗生素耐药性风险评价中最小抑菌浓度的研究进展

环境中抗生素耐药性(antimicrobial resistance, AMR)的产生和传播对人类健康构成了严重威胁,最小抑菌浓度(minimum inhibitory concentration, MIC)是评价环境中抗生素耐药性风险的关键指标。本研究基于文献梳理,发现常用的MIC测试方法中大部分采用肉汤微稀释法,其次是琼脂稀释法和E-test法,测试方法的不同对MIC值的影响不明显。基于EUCAST数据库,梳理了目前针对不同菌种和抗生素的MIC测试数据,发现革兰氏阴性菌(G⁻)的AMR问题得到了更多关注,其MIC数据量远大于革兰氏阳性菌(G⁺),然而,G⁺对抗生素的耐药性比G⁻更强。鲍曼不动杆菌(Acinetobacter baumannii)和屎肠球菌(Enterococcus faecium)分别是G⁻和G⁺中耐药性最强的细菌。有关AMR的研究主要集中于β-内酰胺类抗生素,而磺胺类和多肽类研究相对较少。在所研究的抗生素中,细菌对氨苄西林钠、链霉素和夫西地酸的耐药性最强。本研究梳理和总结了MIC测试方法与数据研究现状,指出目前数据远远不足,建议持续扩大MIC研究的覆盖面,并促进耐药信息共享。     

分离自母婴肠道的假小链双歧杆菌的耐药性分析

【背景】由于滥用抗生素导致细菌耐药性日益严重。对于双歧杆菌,人们往往注重其益生功能的挖掘而忽视了对其耐药性的研究,存在一定的安全隐患。【目的】检测母婴肠道中假小链双歧杆菌的耐药性,探究婴儿肠道中假小链双歧杆菌耐药性的来源。【方法】利用微量肉汤稀释法测定48株分离自母婴肠道的假小链双歧杆菌对14种抗生素的耐药性,比较分离自不同家庭母婴肠道中假小链双歧杆菌的耐药性。【结果】48株母婴肠道分离株对四环素、氯霉素、新霉素、环丙沙星100%耐药,对其余10种抗生素耐药率依次为：卡那霉素98%、利福平80%、克林霉素78%、甲氧苄啶63%、红霉素59%、庆大霉素43%、链霉素16%、万古霉素14%、氨苄西林6%、利奈唑胺2%。母婴肠道分离株的耐药性无显著差异,分离自同一家庭母婴肠道的菌株具有相似的耐药表型。【结论】分离自母婴肠道的假小链双歧杆菌对多种抗生素具有耐药性,婴儿肠道中假小链双歧杆菌的耐药性可能是由母亲肠道垂直传递而来。     

Antifungal activity of essential oils and their combinations in in vitro and in vivo conditions

Natural additives are in demand for the control of microbial growth in foods. Several natural compounds including essential oils (EOs) are being explored for food uses. In the present investigation, the antifungal activity of cinnamaldehyde, eugenol, peppermint and clove EOs and their combinations was evaluated against 12 species of Aspergillus, Fusarium, Penicillium and Rhizopus in in vitro and tomato fruit system (in-vivo). The EOs were able to inhibit complete growth of tested fungi at or below 0.6% level and 80?μL of EOs (except peppermint oil) in in vitro condition and tomato system, respectively. The fractional inhibitory studies showed either additive or indifferent effect by combining eugenol and peppermint, and indifferent or antagonist effect by combining the cinnamaldehyde and clove in both in vitro and in vivo studies. The findings may be useful for application of these EOs in foods, but their effects on organoleptic quality of foods need to be investigated.     

棘白菌素对氟康唑耐药的念珠菌体外药物敏感性的研究

目的评价3种棘白菌素类药物（卡泊芬净、米卡芬净、阿尼多芬净）体外对氟康唑耐药念珠菌的药物敏感性。方法采用微量液体稀释法和琼脂稀释法测定最小抑制浓度（MIC）。结果微量液体稀释法：59株耐药白念珠菌3种药物MIC50均为0.06μg/mL,米卡芬净、阿尼多芬净的MIC范围均为0.015～0.125μg/mL,卡泊芬净为0.015～0.25μg/mL;8株耐药光滑念珠菌MIC值均为0.063μg/mL。琼脂稀释法：59株耐药白念珠菌和8株耐药光滑念珠菌3种药物MIC值均为0.063μg/mL。结论3种棘白菌素类药物可能具有治疗氟康唑耐药的念珠菌感染的临床价值。     

链霉菌H03发酵液提取物的抗菌活性研究

以常见的病原细菌和植物病原真菌为指示菌研究了链霉菌发酵液提取物的抗菌活性,测定了发酵液提取物的抗菌谱和最小抑菌浓度(MIC)。结果表明:与青霉素、链霉素相比,该菌发酵液提取物的抗菌范围更广,对植物病原真菌棉花黄萎病菌、苹果轮纹病菌、小麦赤霉病菌、油菜菌核病菌、黄瓜炭疽病菌、甜菜褐斑病菌、稻瘟病菌,以及常见病原细菌大肠杆菌、枯草杆菌、绿脓杆菌和金黄色葡萄球菌都具有明显的抑制作用。利用试管二倍稀释法测定发酵液提取物对上述诸菌的最小抑菌浓度(MIC),结果分别为0.125、0.062 5、0.125、0.25、0.015、0.03、0.015、0.015、0.03、0.250、.015 mg/mL。其中对植物病原真菌中的黄瓜炭疽病菌、甜菜褐斑病菌、稻瘟病菌,以及病原细菌中的大肠杆菌、枯草杆菌、金黄色葡萄球菌抑制效果最佳。将发酵液提取物置于100℃下加热处理10 min后,其对金黄色葡萄球菌的抑菌活性不变,说明该发酵液提取物具有较好的热稳定性。     

Template synthesis of macrocyclic complexes and their spectroscopic and antibacterial studies

A new series of macrocyclic complexes of type [M(TML)X]X₂, where M = Cr(III), Fe(III), TML is tetradentate macrocyclic ligand, and X = Cl^?, NO₃^?, CH₃COO^?, have been synthesized by condensation of isatin and ethylenediamine in the presence of metal salt. The complexes were synthesized by both conventional and microwave methods. The complexes have been characterized with the help of elemental analysis, conductance measurement, magnetic measurement, and infrared, far infrared, and electronic spectral studies. Molar conductance values indicate them to be 1:2 electrolytes. Electronic spectra along with magnetic moments suggest five-coordinate square pyramidal geometry for these complexes. The complexes were also tested for their in vitro antibacterial activity. Some of the complexes showed satisfactory antibacterial activitiy.     

山东某规模化鸡场鸡毒支原体的分离鉴定、基因分型、耐药性和致病性分析

【背景】 鸡毒支原体(Mycoplasma galliscepticum, MG)主要引起家禽慢性呼吸道疾病,不同地区MG菌株的耐药性和致病性差异大,给临床防控增加了难度。【目的】 明确山东省烟台市某规模化鸡场MG分离株的基因分型、耐药情况及致病力,为该地区MG防控提供科学依据。【方法】 从MG阳性鸡群上腭裂拭子分离、纯化获得MG分离株,利用atpG、plsC、mraW、ugpA、DUF3196、lgT和dppC共7个管家基因进行多位点序列分型(multilocus sequence typing, MLST)并构建系统发育树。测定分离株对金霉素、大观霉素、林可霉素、泰乐菌素、泰万菌素、泰妙菌素、沃尼妙林和多西环素的最小抑菌浓度(minimum inhibitory concentration, MIC),并对相关耐药基因23S rRNA基因V结构域、L4及L22蛋白基因进行测序分析。通过点眼攻毒和气管攻毒两种方式感染28日龄SPF鸡,评价感染10、20和30 d后气囊炎的发病率、气管组织MG载量和病理损伤。【结果】 分离纯化获得9株MG,并首次发现菌株YTX2、YTX5.6、YTX11、YTX12、YTX13、YTX14和YTX15的管家基因plsC和mraW为新的等位基因,ID号分别为33和31,这7株MG分离株属于ST97型,而菌株YTX9和YTX10属于ST16型。所有MG分离株对沃尼妙林和泰妙菌素敏感(MIC≤0.25 μg/mL),对泰万菌素和多西环素次之(MIC≤4 μg/mL),对金霉素和林可霉素有一定程度耐药(MIC≥16μg/mL);属于ST97的7个MG分离株对泰乐菌素存在中等程度耐药(MIC：4-8 μg/mL);选取菌株YTX5.6、YTX2和YTX10对耐药性相关的23S rRNA基因V结构域、L4和L22蛋白基因测序分析,菌株YTX5.6和YTX2在23S rRNA基因V结构域发生A2069G突变;菌株YTX5.6和YTX2的L4蛋白基因出现G568T突变,在L22蛋白基因上出现T102C、T129C、T252C、C336T和G404A突变;对泰万菌素、泰乐菌素、泰妙菌素、沃尼妙林和多西环素敏感的YTX10株与对照菌株MG R_low株序列一致,未发生突变。在感染后10、20和30 d,菌株YTX5.6气管攻毒组气囊炎发病率分别为80%、60%和80%;菌株YTX10气管攻毒组仅在感染后20 d和30 d出现气囊炎,发病率分别为40%和20%,其余组在整个实验过程中均未发现明显气囊炎。气管攻毒组的鸡在整个感染过程中气管黏膜增厚情况均比点眼攻毒组严重(P<0.05),菌株YTX5.6气管攻毒组在感染后第20天气管黏膜增厚最严重,平均厚度为300.6 μm。菌株YTX5.6和YTX10气管攻毒方式在感染10、20和30 d后,气管中的MG载量均比菌株YTX5.6和YTX10点眼攻毒方式高。【结论】 本研究从山东省烟台市某规模化鸡群共分离出9株MG菌株,分别属于ST97和ST16基因型,分离株对目前常用抗生素存在不同程度的耐药性,动物感染试验表明不同基因型菌株存在不同致病力。     

Antimicrobial activity of honey from the stingless bee Trigona carbonaria determined by agar diffusion,agar dilution,broth microdilution and time‐kill methodology

Aims: The aim of this study was to determine the spectrum of antimicrobial activity of 11 samples of stingless bee honey compared to medicinal, table and artificial honeys. Methods and Results: Activity was assessed by agar diffusion, agar dilution, broth microdilution and time‐kill viability assays. By agar dilution, minimum inhibitory concentration (MIC) ranges were 4% to >10% (w/v) for Gram‐positive bacteria, 6% to >16% (w/v) for Gram‐negative bacteria and 6% to >10% (w/v) for Candida spp. By broth microdilution, all organisms with the exception of Candida albicans and Candida glabrata were inhibited at ≤32% (w/v). Geometric MIC (w/v) means for stingless bee honeys ranged from 7·1% to 16·0% and were 11·7% for medicinal honey and 26·5% for table honey. Treatment of organisms with 20% (w/v) stingless bee honey for 60 min resulted in decreases of 1–3 log for Staphylococcus aureus, >3 log for Pseudomonas aeruginosa and <1 log for C. albicans. Similar treatment with each control honey resulted in decreases of <1 log for all organisms. Conclusions: Stingless bee honey has broad‐spectrum antibacterial activity although activity against Candida was limited. Stingless bee honey samples varied in activity and the basis for this remains to be determined. Significance and Impact of the Study: Stingless bee honey had similar activity to medicinal honey and may therefore have a role as a medicinal agent.     

Experimental and statistical methods to evaluate antibacterial activity of a quaternary pyridinium salt on planktonic,biofilm-forming,and biofilm states

Robust evaluation and comparison of antimicrobial technologies are critical to improving biofilm prevention and treatment. Herein, a multi-pronged experimental framework and statistical models were applied to determine the effects of quaternary pyridinium salt, 4-acetyl-1-hexadecylpyridin-1-ium iodide (QPS-1), on Streptococcus mutans in the planktonic, biofilm-forming and biofilm cell states. Minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) were determined via common methods with novel application of statistical approaches combining random effects models and interval censored data to estimate uncertainties. The MICs and MBCs for planktonic and biofilm-forming states ranged from 3.12 to 12.5 μg ml^?1, with biofilm values only ≈ 8 times higher. Potent anti-biofilm activity and reactive structural features make QPS-1 a promising antibacterial additive for dental and potentially other biomedical devices. Together, the experimental framework and statistical models provide estimates and uncertainties for effective antimicrobial concentrations in multiple cell states, enabling statistical comparisons and improved characterization of antibacterial agents.     

Antimicrobial activity of Coniothyrium minitans and its macrolide antibiotic macrosphelide A

Aims: Assessment of antimicrobial activity of the mycoparasite Coniothyrium minitans and its macrolide antibiotic macrosphelide A. Methods and Results: Thirteen isolates of C. minitans were tested for ability to inhibit a number of filamentous fungi, yeasts, oomycetes and bacteria in agar based tests. Activity was found against some ascomycetes, basidiomycetes, oomycetes and Gram‐positive bacteria, but not against zygomycetes, yeasts or Gram‐negative bacteria tested. Six C. minitans isolates (Conio, Contans, IVT1, CM/AP/3118, B279/1, A1/327/1) were found to produce macrosphelide A in liquid culture and no other antibiotics were detected. On agar, macrosphelide A inhibited growth of some ascomycetes, basidiomycetes, oomycetes and all four Gram‐positive bacteria tested, including the medically important Staphylococcus aureus with a minimum inhibitory concentration of ≤500 μg ml^?1. There was no inhibition observed against the yeasts and Gram‐negative bacteria when macrosphelide A was tested at 700 μg ml^?1. Conclusions: The spectrum and level of activity of macrosphelide A produced by C. minitans against micro‐organisms are extended markedly compared to previous reports. Significance and Impact of the Study: Macrosphelide A was effective against Staph. aureus. Further study on the control of this bacterium is merited in view of the development of antibiotic resistance.     

An adaptable HPLC method for the analysis of frequently used antibiotics in ocular samples

Four different antibiotics, delivered individually to rabbit eyes via hydrophilic intraocular lenses soaked in the drug solution prior to implantation, were measured in aqueous and vitreous humor samples from the eyes. To meet this analytical need, we developed a sensitive, high performance liquid chromatographic (HPLC) method for measuring the concentrations of moxifloxacin, gatifloxacin, linezolid, and cefuroxime in the ocular tissue. Separations were carried out on a LichroSpher RP-18 column, maintained at room temperature. The fluoroquinolones were eluted with a mobile phase consisting of 20% acetonitrile, in 0.1% trifluoroacetic acid (pH 3.0) with 30 mM tetrabutylammonium chloride. Linezolid and cefuroxime were eluted with 25% acetonitrile in 25 mM Na acetate buffer, pH 5.0. All elutions were isocratic. With ultraviolet detection, the lower limit of quantitation (LLOQ) for these compounds approached 1 ng (on-column injection). By using fluorescence detection, the LLOQ for the fluoroquinolones improved to 200 pg. The overall accuracy of the method was ≥90%. With minor modifications, the method was optimized for each of the agents, and the resulting analytical sensitivity made the method suitable for clinical investigations of the ocular penetration of these drugs.     

Recombineering Reveals a Diverse Collection of Ribosomal Proteins L4 and L22 that Confer Resistance to Macrolide Antibiotics

Mutations in ribosomal proteins L4 and L22 confer resistance to erythromycin and other macrolide antibiotics in a variety of bacteria. L4 and L22 have elongated loops whose tips converge in the peptide exit tunnel near the macrolide-binding site, and resistance mutations typically affect residues within these loops. Here, we used bacteriophage λ Red-mediated recombination, or “recombineering,” to uncover new L4 and L22 alleles that confer macrolide resistance in Escherichia coli. We randomized residues at the tips of the L4 and L22 loops using recombineered oligonucleotide libraries and selected the mutagenized cells for erythromycin-resistant mutants. These experiments led to the identification of 341 resistance mutations encoding 278 unique L4 and L22 proteins—the overwhelming majority of which are novel. Many resistance mutations were complex, involving multiple missense mutations, in-frame deletions, and insertions. Transfer of L4 and L22 mutations into wild-type cells by phage P1-mediated transduction demonstrated that each allele was sufficient to confer macrolide resistance. Although L4 and L22 mutants are typically resistant to most macrolides, selections carried out on different antibiotics revealed macrolide-specific resistance mutations. L22 Lys90Trp is one such allele that confers resistance to erythromycin but not to tylosin and spiramycin. Purified L22 Lys90Trp ribosomes show reduced erythromycin binding but have the same affinity for tylosin as wild-type ribosomes. Moreover, dimethyl sulfate methylation protection assays demonstrated that L22 Lys90Trp ribosomes bind tylosin more readily than erythromycin in vivo. This work underscores the exceptional functional plasticity of the L4 and L22 proteins and highlights the utility of Red-mediated recombination in targeted genetic selections.