Lysostaphin lysis procedure for detection of Staphylococcus aureus by the firefly bioluminescent ATP method.

The objective of this study was to examine the use of lysostaphin as an ATP-extracting agent for the estimation of Staphylococcus aureus cell number by a rapid bioluminescent ATP method. The results of the study showed that lysostaphin (22 U/ml) was able to lyse most of the S. aureus cells (greater than 99.9%) at room temperature in 1 min; ATP of S. aureus cells extracted by the lysostaphin lysis procedure was stable for 24 h in the presence of EDTA; there was a linear relationship between the ATP content and the number of S. aureus cells (ranging from 10(4) to 10(6) CFU/ml); and the lysis of S. aureus cells by lysostaphin allowed estimation of the number of S. aureus cells in mixed cultures and in meat samples.     

Rapid detection of Staphylococcus aureus using bioluminescent enzyme immunoassay

A bioluminescent enzyme immunoassay (BLEIA) method for detecting protein A-bearing Staphylococcus aureus was developed using biotinylated firefly luciferase. The BLEIA was able to detect protein A at one pg ml-1 and 103 cfu ml-1 level of Staph. aureus. The BLEIA showed significant signals with overnight cultures of all 24 Staph. aureus strains, and the BLEIA did not show any significant signals with overnight cultures of all 44 strains of coagulase-negative staphylococci and the other genus bacteria. After 5 h cultivation beginning at approximately 50 cfu ml-1, the BLEIA was able to detect all 35 Staph. aureus strains isolated from healthy humans.     

Prevention of Staphylococcus aureus lysis

Staphylococcus aureus S-6 cells grown in chemically defined media often lysed after exponential growth. Lysis could be prevented by the addition of alanine or proline before the culture reached stationary phase.     

Salt-induced cell lysis of Staphylococcus aureus

Cell lysis was efficiently induced in Staphylococcus aureus by the addition of 0.3 m NaCl to exponentially growing cultures at 30°C. When cells harvested at the exponential phase were incubated in buffer with NaCl, autolysis occurred. Treatment with chloramphenicol failed to induce cell lysis by NaCl. The effects of NaCl on growing cells and harvested cells were inhibited by the addition of sodium polyanethole sulfonate, subtilisin, cardiolipin, and lipoteichoic acid. These agents diminished the activity of a cell wall-lytic enzyme liberated from the cells in the presence of NaCl. Lysis induced by salt appears to be catalyzed by a similar lytic enzyme in growing and harvested cells.     

Prevention of Staphylococcus aureus lysis.

Staphylococcus aureus S-6 cells grown in chemically defined media often lysed after exponential growth. Lysis could be prevented by the addition of alanine or proline before the culture reached stationary phase.     

Comparative Inhibition of Methicillin-resistant Strains of Staphylococcus aureus by Lysostaphin and Otner Antibiotics

Sixteen methicillin-resistant strains of Staphylococcus aureus obtained from Europe were found to be sensitive to the lytic activity of lysotaphin. With only minor exceptions, the strains were found to be sensitive to novobiocin, erythromycin, fusidic acid, and lincomycin, and slightly less sensitive to vancomycin and chloramphenicol. All strains were resistant to tetracycline, penicillinase-sensitive penicillins (benzylpenicillin, ampicillin, and propicillin), penicillinase-resistant penicillins (methicillin, nafcillin, ancillin, oxacillin, cloxacillin, and dicloxacillin), and two cephalosporin antibiotics (cephalothin and cephaloridine).     

Efficient Adsorption of Lysostaphin on Bacterial Cells of Lysostaphin-Resistant Staphylococcus aureus Mutant

A simple and efficient method for the purification of staphylolytic endopeptidase (lysostaphin) contained in culture supernatant of Staphylococcus simulans biovar staphylolyticus strain by adsorption of the enzyme on bacterial cells of lysostaphin-resistant S. aureus mutant was successfully devised. Lysostaphin was sufficiently adsorbed on the heat-killed mutant cells derived from S. aureus Cowan I and efficiently eluted by 3 M KSCN. Enzyme preparation obtained by a single procedure of the affinity purification was pure enough for practical use. The yield of the enzyme was 25 mg from 1 liter culture and recovery rate was 64%.     

Continuous-flow bioluminescent dtermination of ATP in platelets using firefly luciferase immobilized on epoxy methacrylate

Firefly luciferase was immobilized on epoxy methacrylate beads and used for a continuous-flow assay of ATP extracted from platelets. The immobilized luciferase had a half-life of 3 days at 25°C; there was a 25% recovery of luciferase activity upon immobilization, and ca 50 reactors were made from 1 mg of commercial enzyme. The sensitivity of the assay was 0.3 pmol of ATP, and the response was linear between 1 and 500 pmol of ATP. The content of platelets obtained with the present method correlated well with those obtained using soluble luciferase.     

Electron microscopy of Staphylococcus aureus cell wall lysis

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Mu?oz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018-2024. 1966.-A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents.     

The use of the firefly bioluminescent reaction for the rapid detection and counting of mycobacterium BCG

A rapid test for the counting of Mycobacterium BCG, based on firefly luciferase assay of bacterial ATP has been evaluated. Three different methods for the extraction of ATP from mycobacterial cells were examined. Extraction with n-butanol proved to be the best method. The amount of ATP extracted correlated well with the number of colony forming units. It was found that the ATP content per colony forming particle of BCG varied only slightly after various periods of cultivation.     

Nuclease Production and Lysostaphin Susceptibility of Staphylococcus aureus and Other Catalase-Positive Cocci

Some strains of Staphylococcus epidermidis and Micrococcus sp. produce nucleases. However, thermal stability was shown to be unique to the nucleases of S. aureus. In addition, two micromethods for susceptibility testing to lysostaphin were more precise and convenient than anaerobic glucose fermentation in distinguishing between the genera Staphylococcus and Micrococcus.     

A nonuniform sequential procedure for diagnosing Staphylococcus aureus

The possibility of the sharp differentiation of S. aureus in the nonuniform successive statistical identification procedure with the use of signs ranked by their differential information content. The procedure starting from the signs with greater information content permits the identification of S. aureus in 100% of cases.     

Tandem coagulase/thermonuclease agar method for the detection of Staphylococcus aureus.

In optimizing previously reported coagulase agar media to obtain a rapid, reliable, and inexpensive coagulase test agar, variations in plasmas, pH, buffer system, fibrinogen, and fibrinolytic inhibitor were investigated. The agar with the following composition was determined best for the demonstration of coagulase production by Staphylococcus aureus: 25 ml of 15% bovine fibrinogen (fraction I, type I, citrated, Sigma Chemical Co.), 25 ml of rehydrated rabbit plasma (coagulase plasma ethylenediaminetetraacetic acid, Difco), 10.0 mg of soybean trypsin inhibitor (Schwarz/Mann), and 450 ml of brain heart infusion agar (Difco). In additional studies involving 7 different temperatures and 11 heating times, the thermal destruction of microbial nucleases on plate count agar and coagulase test agar was investigated. Heating the plates for 2.5 h at 65 degrees C destroyed all heatlabile nucleases, but not thermonucleases of S. aureus. A tandem agar plate method for the identification of S. aureus was developed. Coagulase and thermonuclease activity of 50 colonies can be detected on a single agar plate. Suspect S. aureus colonies isolated on various selective media are transferred to coagulase test agar, the plates are incubated at 37 degrees C for 18 h, and the coagulase reaction is recorded. The plates are then heated at 65 degrees C for 2.5 h, overlaid with toluidine blue-metachromatic diffusion agar, and reincubated at 37 degrees C for 3 h, and the thermonuclease reaction is recorded. Studies based on 88 enterotoxigenic S. aureus strains and 133 and 48 suspect S. aureus strains isolated from fresh salami mixtures on mannitol salt and tellurite-polymyxin-egg yolk agars, respectively, demonstrated 100% agreement between the tandem agar plate method and standard coagulase and thermonuclease tests. Overall, the tandem agar plate method is a rapid and convenient approach contributing to the identification of S. aureus from foods.     

基于重组溶葡球菌酶和ATP生物发光技术特异定量检测金黄色葡萄球菌

基于重组溶葡球菌酶和ATP生物发光法建立特异定量检测金黄色葡萄球菌的方法。优化设计合成溶葡球菌酶序列,构建重组表达载体pQE30-Lys,转化至大肠杆菌M15并诱导表达,镍柱纯化得到目的蛋白。利用重组溶葡球菌酶和ATP生物发光法特异定量检测金黄色葡萄球菌并与平板计数对比。成功表达了重组溶葡球菌酶,并建立了特异定量检测金黄色葡萄球菌的方法,与平板计数具有显著线性关系。本研究建立的将重组溶葡球菌酶和ATP生物发光法相结合的检测方法操作快捷简单,具有良好的应用前景。     

Suppression of penicillin-induced lysis of Staphylococcus aureus by cibacron blue 3G-A

The effect of cibacron blue 3G-A (CB) on bacteriolysis induced by penicillin G was investigated using Staphylococcus aureus FDA 209P. Penicillin-induced lysis was completely inhibited by 30 microM CB. However, the bactericidal effect of penicillin G was not influenced by CB. These results indicate that a bacteriolytic process is not essential for penicillin to kill S. aureus.     

Gentle lysis of Staphylococcus aureus at low temperature.

A low-temperature lysis procedure for Staphylococcus aureus is described. It is simple and requires no special equipment.     

Efficacy and Safety of Topical Lysostaphin Treatment of Persistent Nasal Carriage of Staphylococcus aureus

The efficacy of lysostaphin nasal spray and Neosporin ointment (Burroughs Wellcome & Co.) in altering nasal carriage of Staphylococcus aureus was studied with persistent carriers in an institution for mentally retarded children and adults. Treatment for 5 days with either agent significantly reduced carriage rates. This effect persisted through the 5th day after therapy with lysostaphin but not with Neosporin. By the 11th day after therapy, carriage rates in the treatment and control groups were not significantly different. Except for a single immediate wheal and flair skin test reaction, no other evidence of adverse reactions to topical lysostaphin was detected. No consistent changes in hemagglutination-inhibition titers to lysostaphin were observed after therapy. Lysostaphin appears to be slightly more effective than conventional topical antimicrobial therapy in reducing nasal carriage of staphylococci in this rigorously defined population of persistent carriers.     

Improved method for electroporation of Staphylococcus aureus

We have developed a significantly improved method for the electroporation of plasmid DNA into Staphylococcus aureus. The highest transformation efficiency achieved with this procedure was 4.0 x 10(8) transformants per microgram of plasmid pSK265 DNA. This represents a 530-fold improvement over the previously reported optimum efficiency of 7.5 x 10(5) transformants per microgram of plasmid DNA after electroporation of S. aureus cells [9]. Identical results were obtained when electrocompetent cells, which had been stored frozen at -80 degrees C, were used. The improved efficiency is due primarily to the use of a modified medium (designated as B2 medium) and secondarily to the use of 0.1-cm cuvettes. Several other plasmids (pI258, pMH109, and pSK270) were also electrotransformed into competent cells using our procedure, and for each plasmid, the transformation efficiency was significantly reduced compared to that observed when pSK265 DNA was used. With respect to plasmid pI258, the transformation efficiency was 3500-fold higher than that reported previously for transformation of this plasmid into S. aureus RN4220 [9]. The optimized electroporation procedure was less successful in transforming other staphylococci. Electrocompetent cells of S. aureus ATCC 29213 and S. epidermidis ATCC 12228 produced 5.5 x 10(5) and 5 x 10(3) transformants per microgram of pSK265 DNA, respectively.     

Accelerated procedure for the enumeration and identification of food-borne Staphylococcus aureus.

A procedure was developed for accelerating to 29 h the enumeration and identification of both healthy and stressed cells of Staphylococcus aureus in foods. Baird-Parker agar medium was incubated for 24 h; S. aureus was identified within 5 additional h by using a simplified thermonuclease test.