Staphylococcal binding with immobilized proteins in the bacteriosorption reaction]

The affinity binding of staphylococci with fibronectin (FN), fibrinogen (FG) and IgG was studied by means of the bacteriosorption test developed by the authors. For this purpose, the suspensions of staphylococci of the strains under study in physiological saline containing 0.1% of Tween-20 was applied in drops onto the areas of polystyrene Petri dishes sensitized with consecutive dilutions of FN, FG, gamma globulin (GL) and ovalbumin (control). The results of the test, manifested as spots formed by the adsorption of staphylococci, were evaluated by visual examination. The test showed that all 62 strains of coagulase-positive staphylococci (S. aureus) were adsorbed on immobilized FG and FN, and 49 of them (79%) were also adsorbed on immobilized GL. The specificity of adsorption was confirmed by negative controls and by the inhibition of the reaction with the solutions of 4 above-mentioned proteins. Out of 32 strains of coagulase-negative staphylococci (S. saprophyticus and S. epidermidis), 20 strains (62.5%) were not adsorbed on any of the proteins, and the remaining 12 strains were nonspecifically adsorbed on all 4 proteins. This new test is proposed for use as an auxiliary method for the identification and typing of micro-organisms.     

Influence of Food Microorganisms on Staphylococcal Growth and Enterotoxin Production in Meat

Forty-four microorganisms were studied for their influence on staphylococcal growth and enterotoxin production. Inhibition was found to be more common than stimulation. Two types of inhibition were observed: inhibition of staphylococcal growth, and inhibition of enterotoxin formation with no apparent effect on growth. By use of a plate test, 12 of the 44 food microorganisms were found to inhibit staphylococcal growth at 35 C. Of the 12, 3 also inhibited growth at 25 C. No significant differences in inhibition were observed with the 15 strains of enterotoxigenic staphylococci. In meat slurries, inhibition of staphylococcal growth was found to be greater at 25 C than at 35 C. Results on inhibition obtained from the plate test could not be correlated with the effect of the organisms in slurries. Environmental conditions were found to affect markedly the influence of food microorganisms on staphylococci. Of the 44 food microorganisms studied, only Bacillus cereus was observed to stimulate significantly staphylococcal growth and enterotoxin formation. Stimulation was more pronounced with Staphylococcus aureus 196E than with other strains of enterotoxigenic staphylococci. Bacillus megaterium and Brevibacterium linens were inhibited by staphylococci. These organisms were completely inhibited when inoculated in mixed cultures with staphylococci. In pure cultures, good staphylococcal growth was found to be accompanied by enterotoxin production; however, in the presence of food microorganisms, good staphylococcal growth occurred without the formation of detectable levels of enterotoxin A.     

Use of Shake Cultures in a Semisolid Thioglycolate Medium for Differentiating Staphylococci from Micrococci

The standard diagnostic test for differentiating staphylococci from micrococci is based on the ability of the former to produce acid anaerobically in a glucose-containing growth medium. This test has been modified to provide greater convenience, easier interpretation of results, and better correlation with deoxyribonucleic acid (DNA) base composition. In the modified test, shake cultures in Brewer's fluid thioglycolate medium with 0.3% agar added are observed for growth in the anaerobic zone of the tubes. This test was applied to 125 strains of staphylococci and micrococci, and all except two strains gave results that were consistent with other criteria. Of particular interest were eight strains of Micrococcus saprophyticus and three strains of M. lactis that have a DNA composition of 30 to 37% guanine plus cytosine (GC). All 11 of these cultures produced anaerobic growth and thus would be classified as staphylococci. Strains of M. lactis that have a high GC content in their DNA grew only aerobically. Some cultures of staphylococci produced characteristic band patterns of anaerobic growth and other cultures produced only a few anaerobic colonies from an inoculum of 10(6) to 10(7) cells. These observations suggest some interesting genetic and metabolic capabilities in such cultures.     

Rapid detection of methicillin resistance in teicoplanin-resistant coagulase-negative staphylococci by a penicillin-binding protein 2' latex agglutination method

Thirty-five clinical isolates of coagulase-negative staphylococci with decreased glycopeptide sensitivity were examined by a penicillin-binding protein (PBP2′) latex agglutination (LA) test and were compared to the detection of the mecA gene by PCR, and oxacillin susceptibility determined minimum inhibitory concentrations. The latex test demonstrated high sensitivity and specificity for detecting methicillin resistance in coagulase-negative staphylococci after PBP2′ induction with oxacillin.     

Rapid test for the serological separation of staphylococci from micrococci.

A simple test for the serological separation of staphylococci from micrococci is described, which is based on the quite different cell wall peptidoglycan structures of these two genera. Antisera to (pentaglycyl-epsilon-amino-n-hexanoic acid)20-albumin agglutinated without exception all staphylococci and gave no positive reaction with micrococci or other bacterial cells. To obtain a good reaction, it was necessary to extract the cells with hot trichloroacetic acid for 30 min. Antisera to (tri-L-alanyl-epsilon-amino-n-hexanoic acid)22-albumin reacted strongly with micrococci containing oligo-L-alanine bridges in their peptidoglycan, but did not agglutinate staphylococci or other bacteria lacking alanine interpeptide bridges.     

Rapid test for the serological separation of staphylococci from micrococci

A simple test for the serological separation of staphylococci from micrococci is described, which is based on the quite different cell wall peptidoglycan structures of these two genera. Antisera to (pentaglycyl-epsilon-amino-n-hexanoic acid)20-albumin agglutinated without exception all staphylococci and gave no positive reaction with micrococci or other bacterial cells. To obtain a good reaction, it was necessary to extract the cells with hot trichloroacetic acid for 30 min. Antisera to (tri-L-alanyl-epsilon-amino-n-hexanoic acid)22-albumin reacted strongly with micrococci containing oligo-L-alanine bridges in their peptidoglycan, but did not agglutinate staphylococci or other bacteria lacking alanine interpeptide bridges.     

Cytidine deamination assay to differentiate Staphylococcus aureus from other staphylococci

AIMS: Adoption of the property of cytidine (cytosine-beta-d-riboside) deamination in staphylococci to distinguish Staphylococcus aureus from other staphylococci. METHODS AND RESULTS: A total of 560 staphylococcal strains were examined. The test demonstrated a sensitivity of 97.1% and a specificity of 98.8%. Of the 249 S. aureus strains (115 oxacillin-resistant) 58 strains were coagulase-negative S. aureus and another 16 strains were clumping factor-negative S. aureus. The 74 deficient S. aureus strains were identified by 16S rRNA gene sequencing and further investigated by spa typing and 13 spa types were found. CONCLUSIONS: The cytidine deaminase test (CDT) is useful especially for distinguishing coagulase- and clumping factor-negative S. aureus from other staphylococci and the results correlated well with 16S rRNA sequencing and the polymerase chain reaction (PCR) amplification of the nuc gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Cytidine deamination assay differentiates S. aureus from other staphylococci. This method is fast (6 h) and reliable in distinguishing between non-S. aureus and the defective (coagulase-negative, clumping factor-negative) S. aureus isolates which could have major consequences for therapy.     

Stimulation of the respiratory burst of the neutrophils from healthy subjects by different Staphylococcus aureus concentrations

Preopsonized live and heat-killed S. aureus stimulated, without the washing of serum, the luminol-dependent chemiluminescence of human neutrophils obtained from healthy donors. The intensity of chemiluminescence was evaluated by the index of stimulation with staphylococci, with due consideration for their concentration. With the microbe/phagocyte ratio equal to 10:1, these indices had the maximum values when both live and killed staphylococci were used. At high concentrations of staphylococci, especially live ones, all indices were low (those for live staphylococci had negative values) and uniform. As the concentration of the antigen decreased, individual features in the reaction of each donor became apparent. With the microbe/phagocyte ratio equal to 100:1, stimulation with live and killed staphylococci induced the identical fluorescence of neutrophils. The capacity of nonopsonized staphylococci for inducing chemiluminescence was poorly pronounced. For this reason, the test system using S. aureus at low concentrations was proposed for the prognostication of this infection, while the ratio 100:1 can be used for the evaluation of the opsonin-phagocytic system in case of a developed purulent process.     

Limitations of Fibrinogen-Polymyxin Medium in Detecting Coagulase-Positive Staphylococci in Raw Milk

A fibrinogen-polymyxin medium and Staphylococcus Medium 110 were used in the isolation of coagulase-positive staphylococci in raw milk. Results indicated that both media allow the growth of some rods and of many coagulase-negative cocci. A significantly greater number of coagulase-positive staphylococci were identified by the tube test than were revealed by halo formation on fibrinogen-polymyxin medium.     

Slide Agglutination Method for the Serological Identification of Neisseria gonorrhoeae with Anti-Gonococcal Antibodies Adsorbed to Protein A-Containing Staphylococci

A rapid slide agglutination test has been developed for the identification of Neisseria gonorrhoeae that are primarily detected as oxidase-positive colonies in gonococcal cultures. The technique is based on the specific nonimmune reactivity between the Fc portion of immunoglobulin (Ig)G and staphylococcal protein A. IgG molecules adsorbed to stabilized staphylococci will thereby become oriented with their antigen-reactive sites that are directed outwards. Protein A-containing staphylococci with unabsorbed anti-gonococcal antibodies gave positive co-agglutination reactions with gonococci but also with meningococci, some Moraxella, Haemophilus, and Pseudomonas strains. These crossreactions were eliminated by absorption of the anti-gonococcal antiserum with meningococcal and Moraxella organisms prior to the coating of reagent staphylococci. In the routine culture diagnosis of N. gonorrhoeae the use of specific gonococcal reagent staphylococci gave concordant results with fermentation procedures and immunofluorescent techniques.     

THE OXIDASE ACTIVITY OF STAPHYLOCOCCI

SUMMARY: By the use of Kovacs'(1956) test, oxidase activity was demonstrated in 23 of 66 strains of coagulase negative staphylococci (or micrococci) but in none of 82 strains of coagulase positive staphylococci. Less sensitive methods showed fewer reactions or failed to demonstrate them at all. Oxidase activity could not be correlated with other biochemical features.     

Die Verteilung des Membranpotentials in der Zellkultur

Zusammenfassung Das Membranpotential kultivierter Zellen in derDeckglaskultur läßt sich unter entsprechenden Voraussetzungen mit guter Reproduzierbarkeit messen und unterliegt einer leicht asymmetrischen Normalverteilung. Die Deckglaskultur erweist sich somit als günstig für Untersuchungen über die Beziehungen zwischen dem elektrischen Polarisationszustand der Membran und anderen Zelleigenschaften und Funktionen.

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Summary The membrane potential of the cultured cells in the cover-glass culture can be measured by the corresponding suppositions with a good repeatability and it is subject to a standard distributing being easy asymmetrical.The cover-glass culture is with it favourable for the inquiries between the electrical condition of the polarization of the membran and other cell properties and functions.

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Herrn Dr. med. habil. L.Döhner vom Institut für Medizinische Mikrobiologie sei auch an dieser Stelle für die ständige Lieferung der Kulturen und für wertvolle Hinweise und Anregungen gedankt. Frl. D.Lüders danke ich für gewissenhafte technische Assistenz.     

Quicolor: A novel system for rapid antibacterial susceptibility testing

Early determination of antibacterial susceptibility increases the success of therapy, decreases unnecessary use of antibacterials and side effects and lowers the overall healthcare costs. We have evaluated a rapid antibacterial susceptibility test, Quicolor (Salubris Inc., Massachusetts, USA), which is based on a rapid culture medium that indicates growth early by changing its colour. Quicolor proved to be a reliable rapid test for determining antibacterial susceptibility, having an overall agreement of 97.6% with the conventional CLSI sisk diffusion susceptibility test results. Between two methods overall agreement was 96.7% for Enterobacteriaceae, 96.8% for staphylococci and 94.2% for non-fermentative bacteria. There was only 0.6% major discrepancy in Enterobacteriaceae, 1.7% in staphylococci and 0.9% in non-fermentative bacteria. Since the test provides results in 3.5–6 h, it can provide the means to choose the right treatment regimen the same day the infectious agent is isolated.     

Participation of Oryza sativa leaf wax in appressorium formation by Pyricularia oryzae

Appressorium formation of Pyricularia oryzae P2 on cover-glass coated with each of the components of rice leaf wax was examined. Wax esters, aldehydes and alcohols, having polar groups and low contact angles, promoted appressorium formation, but alkanes, non-polar molecules having high contact angles, had no effects. Germination of conidia, however, was not aftected with those constituents.     

孢粉相对密度及其在石油地质中的应用——以东营凹陷古近纪为例

为了充分利用已有的非定量分析孢粉资料,开展孢粉相及孢粉与烃源岩关系研究,引入孢粉相对密度概念,定义为孢粉样品鉴定中每个盖片数的孢粉个数。根据牛28井相同井段对比分析,孢粉相对密度的变化趋势与地层中孢粉实际含量的变化趋势一致。以东营凹陷古近纪为例,探讨了孢粉相对密度在石油地质中的应用。可以利用孢粉相对密度进行层序划分对比,孢粉相对密度在区域上的变化具有同步性。通过对孢粉相对密度在横向上分布规律研究,能为研究孢粉量的区域分布特征提供依据。     

Sensitivity to polyvalent therapeutic staphylococcal bacteriophage as a supplementary criterion of staphylococcal pathogenicity]

It is recommended to use the capacity of pathogenic staphylococci to be lysed by polyvalent therapeutic staphylococcal bacteriophage in the capacity of an additional simple and accessible criterion of staphylococcus pathogenicity. Of 147 strains of the pathogenic plasmacoagulating staphylococci 101 were lysed by the phage and of 166 nonplasmocoagulating nonpathogenic strains--only 6. This test correlated with the other signs of staphylococcus (lecithinase and hemolytic activity). The simplicity and sufficient specificity of this test permits to use it in any practical laboratory. Polyvalent diagnostic phage can be used on the basis of therapeutic bacteriophage by its additional adaptation to the pathogenic strains of staphylococcus.     

Occurrence of Coagulase-Positive Staphylococci in Cheddar Cheese

Samples (13) from several lots of cheddar cheese incriminated in staphylococcal food poisoning and 343 samples of cheddar cheese purchased over a 3-year period in retail markets were examined quantitatively for coagulase-positive staphylococci with the smear plate technique. Of the food-poisoning samples, 11 contained coagulase-positive staphylococci in numbers that ranged from 50 to several million per g. Of the 343 market cheese samples, 20% contained coagulase-positive staphylococci in concentrations ranging from less than 50 to more than 200,000 per g. The phage patterns of 64 of 89 cultures isolated from the food-poisoning samples placed them in the miscellaneous phage group (44A) or in phage group IV and the miscellaneous group (42D/44A); 14 had phage patterns that involved group III, the group with which food poisoning has usually been associated. In contrast, over 50% of 104 cultures from the market cheese, which were typed at 100 times the critical test dilution, had phage patterns that involved group III. Of nine selected cultures isolated from the food-poisoning cheese, three (all in phage group III) were positive for enterotoxin by intravenous injection test of cats.     

Rapid Method of Determining Coagulase Activity During Staphylococcal Bacteriophage Typing

The plate test, a modification of the slide test described by Cadness-Graves was developed for the rapid identification of coagulase-positive staphylococci in conjunction with bacteriophage typing. An evaluation of 1,145 cultures by three coagulase-determination methods, the slide, tube, and plate tests, indicates that the plate test is as accurate as the slide tests, and the plate test agrees 97.7% with the tube test.     

AN INVESTIGATION INTO THE CAUSE OF THE SPONTANEOUS AGGREGATION OF FLAGELLATES AND INTO THE REACTIONS OF FLAGELLATES TO DISSOLVED OXYGEN : PART II.

Spontaneous aggregations of flagellates are formed under the cover-glass because the organisms are attracted to and remain in regions where the concentration of dissolved oxygen is less than the saturation concentration under atmospheric partial pressure. These regions of lessened oxygen content arise towards the center of the liquid beneath the cover-glass, owing to the oxygen consumed by the flagellates in respiration not being replaced here by the solution of atmospheric oxygen, as it is along the edges of the liquid. The flagellates, however, are insensitive to the attraction of regions of lessened oxygen concentration when the oxygen concentration throughout the liquid is above a certain value. Therefore, for the aggregations to form, either the initial concentration of dissolved oxygen must be below this limiting value, or an interval of time must first elapse after the making of the preparation until the respiration of the organisms has reduced the oxygen concentration throughout the liquid down to this limiting value. The aggregations will then form because the flagellates have become positively chemotropic to the lower concentration of oxygen at the center of the liquid. Once established, such an aggregation of flagellates does not remain long in the same form. An area free from flagellates appears at the center of the aggregation so that the organisms lie in a circular band surrounding the clear area. The latter increases in size and its bordering band of flagellates in diameter, the band gradually becoming less circular and more square in shape, if the cover-glass is a square one. The clear central area is a region where the oxygen consumption of the flagellates has reduced the oxygen content to such a low value that the organisms are forced to leave the region. They collect in a band where the concentration of dissolved oxygen is an optimum for them. It is the equilibrium position between the oxygen consumed at the center and that diffusing in from the edges of the liquid. As the consumption at the center is more rapid than the replacement from the edge, the flagellate band moves outwards until it becomes stationary at a position where the rates of consumption and replacement of oxygen are equal. Although the flagellates collect in this manner in regions of optimum oxygen concentration, yet greater concentrations of dissolved oxygen have no injurious effect on them. Concentrations of dissolved oxygen lower than the optimum have the effect of inhibiting the movement of the flagellates. They recover their activity, however, immediately they are given access to dissolved oxygen again. Work done in the past on chemotropism of flagellates will have to be revised in the light of the above facts, since the oxygen content of solutions used has never been taken into account.     

In-vitro bacteriocin-mediated antagonism by oral streptococci against human carrier strains of staphylococci

All strains of oral streptococci tested and specially those of Streptococcus mutans, Strep. sanguis and Strep. mitior produced more than one distinct bacteriocin-like substance with variable inhibitory activity on 20 indicator staphylococci. Inhibitory activity was comparatively higher on nasal strains of Staph. aureus and Staph. epidermidis than on strains of both species isolated from the mouth. Nineteen of 20 staphylococcal indicators were inhibited by 1-12 of the 12 effector streptococci. Sensitivity of nasal staphylococci to bacteriocins (frequency of positive inhibitory tests and total inhibition zone diameters) was significantly higher (P less than 0.001, chi 2 test and P less than 0.05, t test respectively) than that of oral ones. The sensitivity of nasal over oral Staph. aureus (P less than 0.001 and P less than 0.01) and of oral Staph. epidermidis over oral Staph. aureus (P less than 0.01 and P less than 0.05) was also significantly higher. The evaluation of variability of inhibitory patterns of bacteriocins produced by streptococci (p-typing), of sensitivity patterns of staphylococci to bacteriocins (s-typing) and of the significantly higher sensitivity of nasal over oral staphylococci to bacteriocins from the epidemiological and ecological viewpoints are discussed.