Synergistic hemolytic reactions between staphylococci and Micrococcus lylae

The primary culture of a clinical specimen obtained from a dog with an acute squamous eczema revealed three different bacterial species which demonstrated synergistic hemolytic activities on sheep blood agar plates. The three cultures were identified as beta-hemolytic Staphylococcus intermedius, as a coagulase-negative staphylococcal species, producing a delta-like hemolysin and as non-hemolytic Micrococcus lylae. The coagulase-negative staphylococcal species as well as M. lylae produced synergistically with beta-hemolytic S. intermedius zones of complete hemolysis. The occurrence of three different synergistically active bacterial species from one clinical specimen might be of clinical significance.     

CAMP-reaction among skin isolates obtained from a dog with an acute squamous eczema

The primary culture of a clinical specimen obtained from a dog with an acute squamous eczema revealed 3 different bacterial cultures. Two of these cultures, a beta-hemolytic Staphylococcus aureus and a group B streptococcal culture, demonstrated synergistic hemolytic activities on this primary culture plate. The group B streptococcus had the serotype surface antigens Ib/c, protein antigen c in its c beta component.     

In vitro effect of combinations of antimicrobial agents and EDTA-tromethamine on certain gram-positive bacteria

Combinations of EDTA-tromethamine and 7 antimicrobial agents (chloramphenicol, nalidixic acid, oxytetracycline, penicillin, polymyxin-B, streptomycin, and a triple sulfonamide preparation) were tested for synergistic activities against Staphylococcus aureus, Corynebacterium renal, Listeria monocytogenes, Erysipelothrix rhusiopathiae, and a beta-hemolytic streptococci. Two in vitro tests were used--minimal inhibitory concentrations of the drugs and a 2-dimensional Microtiter checkerboard technique. A slight synergistic action was seen when L monocytogenes was exposed to combinations of penicillin-EDTA or streptomycin-EDTA.     

Purification and properties of cholesterol oxidase and choline phosphohydrolase from Rhodococcus equi.

Cholesterol oxidase (CO) and choline phosphohydrolase (CPH) exoenzymes were isolated from culture supernatants of Rhodococcus equi ATCC 33701 and their hemolytic and cytotoxic activities examined. The purifications involved differential ammonium sulphate precipitation, ion exchange and gel filtration chromatography. A purification of 32.8-fold and a yield of 0.3% of CO were determined by synergistic hemolysis of sheep red blood cells (SRBC) presensitized with Staphylococcus aureus beta toxin. The enzymatic activity of CO was also demonstrated by oxidation of aqueous cholesterol suspensions. The activity of CO was reversibly inhibited by concentration. A purification of 412.4-fold and a yield of 1.7% of CPH were determined by hydrolysis of p-nitrophenyphosphorylcholine. Purity of both exoenzymes was confirmed by immunoblotting. On sodium dodecyl sulphate polyacrylamide gel electrophoresis, the CO had a molecular mass (Mr) of 60 kd and the CPH a Mr of 65 kd. Choline phosphohydrolase did not hydrolyse sphingomyelin. Sphingomyelinase C (SMC) activity was however demonstrated in concentrated culture supernatants. This dissociation of SMC from CPH activity indicates that R. equi produces two distinct phospholipase C exoenzymes, a CPH and a SMC. Both CO and CPH combined, or individually, did not lyse native SRBC even with subsequent chilling of the cells at 4 degrees C ("hot-cold" treatment). Purified CO lysed beta toxin-sensitized SRBC. The CPH showed only minor hemolytic activity against such sensitized SRBC even at high concentrations. Combination of CO and CPH in lysis of beta toxin sensitized SRBC showed only minor additive rather than synergistic effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

蒲公英提取物联合多种抗生素对奶牛乳房炎金黄色葡萄球菌的体外协同抑菌实验

为研究蒲公英提取物联合多种药物对奶牛乳房炎金黄色葡萄球菌的体外协同抑菌效果,以期应用于临床,减少抗菌药的使用量,降低细菌耐药性和兽药残留。试验选取3种来源(河北,东北,北京)的蒲公英提取物,通过高效液相色谱仪对3种不同来源的蒲公英提取物的主成分及含量进行测定并分析,选取后期试验所需的蒲公英提取物来源。并应用微量肉汤稀释法测定多种抗生素对金黄色葡萄球菌的MIC值(最小抑菌浓度),随后测试蒲公英提取物与抗菌药物联合用药,是否对金黄色葡萄球菌具有协同效果。试验结果表明,不同来源的蒲公英提取物主成分含量差异较大,含有单咖啡酰酒石酸、咖啡酸、菊苣酸、绿原酸,但含量比例不同。随之测得不同来源蒲公英提取物对金黄色葡萄球菌的敏感程度也有差异,临床用药前有必要进行药敏试验科学精准的选择更合适的药物。蒲公英提取物与金霉素、恩诺沙星、阿莫西林、氨苄西林、头孢噻呋、头孢喹肟等联合用药,对金葡菌的体外抑菌实验有良好的协同作用,体外能显著降低抗菌药物的用量。     

亚抑菌浓度穿心莲提取物对减缓金黄色葡萄球菌α-溶血素介导的A549细胞损伤作用的影响

金黄色葡萄球菌是一种广泛存在的重要致病菌,可引起人类和动物许多严重的感染。另外,α-溶血素是金黄色葡萄球菌最重要的毒力因子之一。本研究通过乙醇回流提取方法,获得中草药穿心莲提取物,然后通过最低抑制浓度(MIC)值测定、溶血活性测定、Western blotting、RT-PCR、LDH及Live/dead等方法研究分析穿心莲提取物对金黄色葡萄球菌α-溶血素分泌的影响及对A549细胞的保护作用。试验结果表明,穿心莲提取物对金黄色葡萄球菌的MIC值为2048 μg/mL,且呈剂量依赖性地抑制金黄色葡萄球菌α-溶血素分泌,并能有效减缓金黄色葡萄球菌菌液上清对A549细胞的损伤。提示,穿心莲提取物是一种潜在的抗金黄色葡萄球菌感染的药物。     

Comparison of the antibacterial activity of honey from different provenance against bacteria usually isolated from skin wounds

The antibacterial activity of honey samples provided by apiarists and honey packers was tested against microorganisms usually isolated from skin wounds. The antibacterial activity was tested using the well-agar diffusion assay. The honey samples were tested without dilution, and at 75, 50, 30, and 10% (w/v) dilution. Most of the undiluted honey samples inhibited the growth of Staphylococcus aureus and Staphylococcus epidermidis. Some honey samples provided by apiarists also inhibited the growth of S. aureus even at 50% dilution. Undiluted honey samples also inhibited the growth of Staphylococcus uberis, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae, although to a lesser extent. No inhibition of Micrococcus luteus and Enterococcus faecalis growth was detected. The diameters of the inhibition zones generated by honey samples provided by apiarists were larger than those generated by honey samples provided by honey packers. This observation may be explained by considering the provenance of the honey samples.     

构树叶提取物对金黄色葡萄球菌的抑制作用及机理研究

为了研究构树叶提取物的抑菌作用及其机理,以金黄色葡萄球菌为供试菌,采用牛津杯法测定构树叶提取物的抑菌效果,通过测定供试菌生长曲线、细胞膜渗透性和蛋白质合成来研究构树叶提取物的抑菌机理。结果表明,构树叶水提取物和75%乙醇提取物抑菌圈直径分别为17.5和18.0 mm,最小抑菌浓度均为6.8 mg/mL,最小杀菌浓度均为12.50 mg/mL。构树提取物能显著抑制金黄色葡萄球菌蛋白质合成,从而抑制其生长,但对其细胞膜的通透性影响不大。因此,构树叶提取物通过抑制金黄色葡萄球菌蛋白质合成发挥其抑菌效果。     

Comparison of three commercial rapid agglutination test kits for identification of coagulase positive staphylococci from foods and animals.

Three rapid agglutination assays for the identification of Staphylococcus aureus Monostaph (Bionor A/S, Skien, Norway), Staphyslide-Test (BioMerieux, Lyon, France) and Staph-Rapid-Test (Roche, Basel, Switzerland), were compared. A total of 104 Gram-positive, catalase positive cocci were tested: Nineteen Staphylococcus reference strains comprising 15 spp. (4 strains were coagulase positive), and 7 Micrococcus reference strains comprising 4 spp.; 22 food isolates comprising 13 S. aureus, 8 coagulase positive Staphylococcus spp., and 1 Micrococcus sp.; 56 animal isolates comprising 11 S. aureus, 9 S. hyicus subsp. hyicus, 2 S. intermedius, 15 coagulase positive and 19 coagulase negative Staphylococcus spp. Totally 54 strains were coagulase positive. Considering agglutination of a coagulase positive strain as a correct identification, Monostaph, Staph-Rapid-Test, and Staphyslide-Test correctly identified 52 (96.3%), 47 (87.0%) and 48 (89.0%) of the coagulase positive staphylococci, respectively. Monostaph, Staph-Rapid-Test and Staphyslide-Test showed 1 (2.0%), 4 (8.0%) and 4 (8.0%) false positive reactions respectively. Monostaph, Staph-Rapid-Test and Staphyslide-Test gave 0 (0.0%), 6 (5.8%) and 7 (6.7%) non-interpretable reactions, respectively. Monostaph may be a good alternative to the tube-coagulase test for rapid and reliable identification of coagulase positive staphylococci from both food and veterinary sources. However, false negative reactions may occur with coagulase positive strains of S. hyicus subsp. hyicus and S. intermedius.     

Inhibition of Dermatophilus congolensis by substances produced by bacteria found on the skin

Bacteria, isolated from the skins of clinically normal sheep, were tested for inhibitory activity against Dermatophilus congolensis grown in vitro. Out of 85 bacterial isolates, 19, mainly Bacillus spp., showed zones of inhibition when grown together with D. congolensis. The inhibitory activity was shown to be due to the metabolites released by the bacteria.     

Effect of chosen lactic acid bacteria strains on Staphylococcus aureus in vitro as well as in meat and raw sausages

The present study was aimed at determining the influence of 15 strains of lactic acid bacteria on the growth of 2 Staphylococcus aureus strains in vitro as well as in meat and raw sausages. The investigations were performed within the framework of three alternate stages which differed in respect to the products studied, the number of Lactobacillus sp. strains and, partly, methodological approach. The study also considered water activity (a(w)) and pH of the products investigated. The results obtained are demonstrated in 5 diagrams. It was found that among 15 strains of Lactobacillus aureus investigated only one strain, Lactobacillus helveticus T 78, showed antagonistic effect on studied strains of Staphylococcus aureus both in vitro as well as in meat and raw sausages. Five other strains of Lactobacillus spp. displayed the antagonistic effect in vitro only. The temperature and incubation time of sausages, but also the type of sausage stuffing were found to have a distinct or slight influence, respectively, on the antagonistic interaction between the bacteria. However, this phenomenon was affected by neither a(w) nor pH.     

Effects of Tryptophan Analogs, Mitomycin c and Acridine Orange on the Production of Filtrable Hemolysin by Escherichia coli

Bacteriostatic doses of 5-methyltryptophan and of 7-azatryptophan exert a complete inhibition on the in vitro production of filtrable hemolysin by Escherichia coli. This inhibition is readily overcome by L-tryptophan, and does not seem to be specific but secondary to an interference with de novo protein synthesis and cell multiplication as is observed with chloramphenicol in sensitive strains. Although the influence of the two tryptophan analogs on hemolysin production and cell multiplication is similar, their mechanism of action at the molecular level appears to be different.

The addition of 50 µg/ml 7-azatryptophan which causes an unbalanced growth characterized by an arrest of the cellular division and an increase of cell size, blocks the production of an active hemolysin. Exposure to 4 µg/ml 5-methyltryptophan also prevents cell multiplication and hemolysin production but no sign of unbalanced growth is evident. Mitomycin C in concentrations sufficient to prevent increase in the number of viable units provokes an extreme elongation of E. coli cells and, apparently, does not stop the synthesis of hemolysin. In blocking the production of hemolysin the three inhibitors of protein synthesis used in this study were more effective than mitomycin C an agent known to affect deoxyribonucleic acid synthesis and to induce extrachromosomic genetic factors. Results of conjugation experiments also described here support the finding of other workers that the genetic factor that controls the production of the filtrable hemolysin in E. coli can be transmitted by conjugation. Acridine orange eliminated the hemolytic property from a large proportion of the population of a hemolytic strain which did not carry the R factor, but was little effective in the strains which had received both the R factor and the hemolytic character by conjugation.

     

A comparison of extracted proteins of isolates of Dermatophilus congolensis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting.

Antigenic diversity within a collection of 18 isolates of Dermatophilus congolensis from different Continents was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blotting with sera from cattle with clinical dermatophilosis using whole cell extracts obtained by three methods and one extract of extracellular products of D. congolensis. One of the methods involving the release of a lysostaphin-solubilized protein (LSP) of whole cells of D. congolensis revealed a number of discrete and easily-identifiable bands in SDS-PAGE which were found suitable for characterizing protein patterns and was, therefore, subsequently used for a comparative analysis of the proteins of all the D. congolensis isolates. Six electropherotypes (ET) of D. congolensis were identified among the 18 isolates using the protein profiles based on the presence of four protein bands at Molecular weights (MW) 62, 28, 17.4 and 16.4 kDa. The ETs were found among isolates from different animal species and from different sources with ET1 consisting of three bovine and two equine isolates; ET2, two bovine and three ovine isolates; ET3, two bovine isolates; ET4, two bovine isolates; ET5, one bovine and one ovine isolates and ET6, two bovine isolates. Immunoblotting of the extracts of D. congolensis isolates with sera from cattle with clinical dermatophilosis infection demonstrated protein bands of MW ranging from 9 kDa to 188 kDa. Sera from chronic dermatophilosis infection demonstrated a 28 kDa protein which was immunodominant in the LSP extracts of all the 18 isolates of D. congolensis tested while sera from mild infections demonstrated mainly the 62 kDa protein in the same extracts. However, many protein bands were demonstrated in surface membrane (TSMP) and extracellular protein extracts with sera from only mildly infected animals. The protein patterns observed in all isolates of D. congolensis revealed global antigenic similarities and distinct differences among isolates which could not be associated with either geographic, climatic or host factors. Also sera from infected animals from endemic regions of dermatophilosis could not differentiate isolates of D. congolensis. This suggests the possibility that such sera must have come from animals that had been infected by a multitude of D. congolensis strains present in the herd environment and strains an animal could have come across during the 'ritual' annual cross-country migration of the cattle herds.     

Toxigenic characteristics of Clostridium perfringens type C in enterotoxemia of domestic animals.

Eleven Clostridium perfringens type C strains isolated from fatal cases of hemorrhagic enterotoxemia of Canadian calves, a piglet, and a foal were studied for the production of soluble antigens. All the isolates from calves and a foal failed to produce delta toxin, but were capable of producing large amounts of lethal beta toxin. A strain isolated from a piglet produced delta, but very little beta toxin. Other differences were relatively minor. The results indicated that young domestic animals may be susceptible to all subtypes of C. perfringens type C. A simple method of using blood agar plates coated with type A antiserum for demonstration of hemolytic patterns was found advantageous in differentiation of C. perfringens strains.     

重组鹅β-防御素7蛋白的原核表达及其生物学特性分析

为探讨鹅β-防御素7(Av BD7)的生物学特性,将鹅Av BD7基因亚克隆到大肠杆菌原核表达载体p Pro EX HTa的EcoRⅠ和XhoⅠ双酶切位点上,构建重组表达质粒p Pro EX-Av BD7,将重组质粒转化到大肠杆菌Rosetta感受态细胞中,用异丙基-β-D-硫代半乳糖苷(IPTG)对其菌液进行诱导表达。经N-三(羟甲基)甲基甘氨酸十二烷基硫酸钠聚丙烯酰胺凝胶电泳(Tricine-SDS-PAGE)分析,该重组蛋白大小为10~15 ku,与预期大小结果一致。重组鹅Av BD7蛋白纯化后,通过菌落计数的方法测定其体外抗大肠杆菌、鸡白痢沙门氏菌、金黄色葡萄球菌、四联球菌、枯草芽孢杆菌等抑菌活性,盐离子浓度对其抗菌活性的影响及其对鸡红细胞的溶血活性。结果显示,重组鹅Av BD7蛋白对所测定的5种细菌均有显著抗菌活性(P0.05),且其抑菌活性随蛋白浓度的增加而增强。高浓度盐离子(150 mmol/L)显著抑制重组鹅Av BD7蛋白的抗菌活性(P0.05)。重组鹅Av BD7蛋白对鸡红细胞没有溶血活性(P0.05)。由此可见,重组鹅Av BD7蛋白具有广谱抗菌活性,高浓度盐离子显著降低其抗菌活性,且该重组蛋白不具有溶血活性。     

Comparative Studies on Corynebacter1Um Pyogenes Toxin Formation in Monocultures and Mixed Cultures: The Demonstration of a Stimulating Effect of Peptococcus Indolicus and Stuart-Schwan Cocci

The growth and the toxin (i.e. hemolysin) producing capacity of Corynebacterium pyogenes were studied in monocultures and in co-cultures with 1 or more of the organisms frequently accompanying it in summer mastitis in cattle (Peptococcus indolicus, Stuart-Schwan cocci, Bacteroides melaninogenicus subsp. levii, Fusobacterium necrophorum and Streptococcus dysgalactiae) or with organisms seldom associated with summer mastitis (Streptococcus uberis, Streptococcus agalactiae, non-toxic staphylococci and Escherichia coli).Pc. indolicus, and to some extent also Stuart-Schwan cocci, stimulated the growth as well as the hemolysin producing capacity of Gb. pyogenes (Table 1) while Str. dysgalactiae, Str. uberis, Str. agalactiae, E. coli and the majority of the staphylococci reduced these activities. Most F. necrophorum strains stimulated the growth, but not the hemolytic activity. With B. melaninogenicus the results were inconclusive.The effect of Pc. indolicus appeared to be associated with the production of a filterable factor (Tables 2 and 3).Mouse toxicity and hemolytic activity of culture filtrates were closely correlated (Table 4).     

Cellular immune responses of the rat to experimental infection with Dermatophilus congolensis

The host cell-mediated immune response was examined following experimentally-induced infection of rats with Dermatophilus congolensis, the causal agent of the skin disease dermatophilosis. Mononuclear cells (MC) isolated from Wistar rats 10 days following the induction of a third infection underwent a strong and specific proliferative response, as assessed by a [3H]thymidine incorporation assay, when cultured with various concentrations of inactivated D. congolensis cocci. Using specific monoclonal antibodies in an indirect fluorescent antibody test, this in vitro response was found to be characterised by a large expansion of the W3/25 (T-helper phenotype) population to form 56% of the total. Finally, the primed and stimulated MC were assessed for their ability to produce factors capable of inhibiting macrophage migration. The culture supernatants of D. congolensis-stimulated MC from infected rats caused significant migration inhibition of normal rat peritoneal exudate cells, whilst the supernatants of similarly-stimulated MC from naive rats failed to cause significant inhibition. The results show that a MC subpopulation becomes primed following experimentally-induced infection with D. congolensis and becomes activated after subsequent, in vitro, exposure.     

Protection activity of a novel probiotic strain of Bacillus subtilis against Salmonella Enteritidis infection

The activity of 240 bacterial isolates screened from the gastrointestinal tracts of native chickens were evaluated for use as a potential probiotic in food animal production in order to protect against animal diseases and reduce pathogenic contamination of human food products. In observing the antagonistic activity of 117 bacilli isolates, 10 of these isolates exhibited higher growth inhibition of seven foodborne pathogens, including Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Vibrio cholerae. Beneficial probiotic criteria from these isolates - which included non-pathogenicity, acid and bile salt tolerance, hydrophobicity, and adhesion to intestinal epithelial cells - exhibited that one isolate of NC11 had the most potential as a probiotic. 16S rRNA gene sequencing showed that this NC11 isolate was Bacillus subtilis. This B. subtilis NC11 was sensitive to all antibiotics and was not cytotoxic to intestinal epithelial cells. Reduction of S. Enteritidis attachment to the surfaces of intestinal epithelial cells via action of a cultured medium from B. subtilis NC11 was observed by scanning electron microscopy. B. subtilis NC11 cells, as well as the bacterial cultured medium or the cultured medium adjusted to pH 7, significantly inhibited S. Enteritidis invasion (P<0.01) of intestinal epithelial cells. This study indicates that B. subtilis NC11 has characteristics of a potential probiotic, and exhibits strong inhibition activity against S. Enteritidis infection to intestinal epithelial cells.     

Dual infection of a white-tailed deer by Dermatophilus congolensis and Alternaria alternata.

Infection by both Dermatophilus congolensis and Alternaria alternata was found in a 5 1/2-year-old, female white-tailed deer (Odocoileus virginianus). Encrusted lesions characteristic of dermatophilosis were observed on the hocks, flanks, and back. Giemsa-staining of smears of material from beneath the crusts revealed branching filaments, transversely and longitudinally divided into packets of coccoid cells typical of D congolensis. Hyphae morphologically consistent with those of A alternata were found in methenamine-silver- and hematoxylin-and-eosin-stained sections of tissue from the ears, flanks, and back. Nutrient agar cultures inoculated with tissue from an ear and hindlimb of the deer yielded, respectively, A alternata and D congolensis.     

Bacterial flora of the conjunctiva and nasal cavity in normal and diseased captive bustards.

A survey was carried out to describe the normal aerobic bacterial flora of the conjunctiva and nasal cavity of captive houbara bustards (Chlamydotis undulata), kori bustards (Ardeotis kori), and white-bellied bustards (Eupodotis senegalensis) maintained at the National Avian Research Center, Abu Dhabi, United Arab Emirates. A total of 58 samples were examined from the nasal cavity and 55 samples from the conjunctiva of healthy bustards. There was no bacterial growth in 45% of conjunctival samples. Bacteria isolated from the conjunctiva of healthy birds included Micrococcus spp., Staphylococcus auricularis, Staphylococcus xylosus, Staphylococcus capitis, Staphylococcus warneri, Bacillus spp., and Enterobacter amigenus. Bacteria isolated from the nasal cavity of healthy birds included Bacillus spp., Micrococcus spp., S. auricularis, S. xylosus, Staphylococcus simulans, Staphylococcus saprophyticus, Staphylococcus hyicus, Staphylococcus cohnii, Staphylococcus sciuri, Aerococcus spp., and Providencia rettgeri. These findings were compared with bacterial isolates from bustards with clinical signs of ocular or upper respiratory tract diseases. Mycoplasma spp., Pseudomonas aeruginosa, Pseudomonas stutzeri, Proteus mirabilis, Escherichia coli, Klebsiella spp., Aeromonas hydrophila, and Staphylococcus aureus were the pathogenic bacteria isolated from the conjunctiva of 34.3% bustards with ocular discharges. Mycoplasma spp., P. aeruginosa, Pseudomonas spp., P. mirabilis, E. coli, Klebsiella pneumoniae, and S. aureus were the pathogenic bacteria isolated from the nasal cavity of 74% bustards with upper respiratory tract diseases.