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Repair of thermal injury of Staphylococcus aureus.

Iandolo, John J. (University of Illinois, Urbana), and Z. John Ordal. Repair of thermal injury of Staphylococcus aureus. J. Bacteriol. 91:134-142. 1966.-Exposure of Staphylococcus aureus MF 31 to sublethal temperatures produced a temporary change in the salt tolerance and growth of the organism. After sublethal heat treatment at 55 C for 15 min, more than 99% of the viable population was unable to reproduce on media containing 7.5% NaCl. The data presented demonstrate that thermal injury, in part, occurred owing to changes in the cell membrane, which allowed soluble cellular components to leak into the heating menstruum. When the cells were placed in a limiting medium, complete recovery did not occur, regardless of the incubation time. The temperature and the pH which produced the optimal rate of recovery were similar to those described previously for the multiplication of uninjured cells. However, the rate of recovery as well as the unchanging total count during recovery indicated that cell multiplication was not a factor during the recovery process. The nutrient requirements for the complete recovery of injured cells consisted of a solution containing an energy source, such as glucose, a mixture of amino acids, and phosphate. The use of the metabolic inhibitors, penicillin, cycloserine, 2,4-dinitrophenol, and chloramphenicol, did not inhibit recovery. Actinomycin D, however, completely suppressed recovery. This result implied that ribonucleic acid synthesis was particularly involved; this inference was substantiated by radio tracer experiments. The rate at which label was incorporated in the nucleic acid fraction paralleled that of recovery and the return of salt tolerance.

Amino Acids↗

Color of cultures of Staphylococcus epidermidis determined by spectral reflectance colorimetry.

Brown, Richard W. (National Animal Disease Laboratory, Ames, Iowa). Color of cultures of Staphylococcus epidermidis determined by spectral reflectance colorimetry. J. Bacteriol. 91:911-918. 1966.-A colorimeter with a reflectance attachment was used to study pigment production by Staphylococcus epidermidis strains grown on a medium containing Trypticase Soy Agar (BBL) and cream. The color of each culture was first characterized by reflectance colorimetry for dominant wavelength, purity, and luminous reflectance (Y) and was then classified visually into 1 of 10 color grades. There was not complete agreement in grading colors by the two methods, inasmuch as cultures that were considered more pigmented in relation to other cultures by the reflectance method were sometimes graded visually as less pigmented, and vice versa. Nevertheless, when the cultures were visually graded as being more pigmented, there was a concomitant increase in the average values of dominant wavelength and purity with a decrease in Y for the cultures in each higher grade. Thus, the nonpigmented cultures had the lowest dominant wavelength and purity values but the highest Y (brightness) values, whereas the most pigmented cultures had the highest dominant wavelength and purity values, but the lowest Y values. These results indicated that the cultures did not produce pigments of different hues (greenish-yellow, yellow, yellowish-orange) each with high, medium, and low degrees of purity and brightness. The value (1 - z), where the chromaticity coordinate z = Z/(X + Y + Z), was found to be proportional to the purity value. An inverse relationship between the tristimulus Z and purity values was also demonstrated. All cultures tested by the reflectance method were also classified according to the type of spectral absorption curve obtained with pigments extracted from the cultures with methanol. A comparison of these methods indicated that determining the type of spectral absorption curve would be better for differentiating strains of S. epidermidis, whereas the use of the reflectance method would be better for determining differences of pigment production within strains.

Color↗

Electron microscopy of Staphylococcus aureus cell wall lysis.

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Muñoz, 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.

Bacteriolysis↗

Inhibition of protein synthesis by spermine in growing cells of Staphylococcus aureus.

Friedman, Mischa E. (Hebrew University, Jerusalem, Israel), and Uriel Bachrach. Inhibition of protein synthesis by spermine in growing cells of Staphylococcus aureus. J. Bacteriol. 92:49-55. 1966.-Staphylococcus aureus SFL 9725 incorporated valine-C(14) into cellular protein. This incorporation was inhibited by spermine when the pH of the culture was adjusted to 7.8, and the inhibition was antagonized partially by Mg(++). The incorporation of C(14)-labeled leucine, phenylalanine, lysine, arginine, and possibly glutamic acid was inhibited to a much greater extent than that of alanine, glycine, or threonine. The uptake of spermine-C(14) by S. aureus cells was rapid. More than 50% of the radioactivity resided in the soluble extract. The protein fraction of the soluble extract contained 99% of the recovered label, whereas the ribonucleic acid and deoxyribonucleic acid fractions contained little or no spermine-C(14).

Amino Acids↗

Biological properties of alpha-toxin mutants of Staphylococcus aureus.

McClatchy, J. K. (The University of Texas Southwestern Medical School, Dallas), and E. D. Rosenblum. Biological properties of alpha-toxin mutants of Staphylococcus aureus. J. Bacteriol. 92:575-579. 1966.-Twenty nonhemolytic mutants of Staphylococcus aureus were isolated after treatment of a hemolytic strain with ultraviolet light or nitrous acid. Thirteen strains isolated were completely lacking in the synthesis of alpha toxin or immunologically related proteins, presumably the result of a single mutational event. Although the strains were nonhemolytic on rabbit blood-agar plates, six of them retained the dermonecrotic and lethal activities usually associated with staphylococcal alpha toxin, as well as slight hemolytic activity for rabbit erythrocyte suspensions. The active mutants and one inactive mutant produced a protein that reacted immunologically with antibody to alpha toxin. Mutations which alter the alpha toxin molecule can effect the lethal, dermonecrotic, and hemolytic activities separately or in varying ratios.

Antibodies↗

Genetic recombination between alpha-toxin mutants of Staphylococcus aureus.

McClatchy, J. K. (The University of Texas Southwestern Medical School, Dallas), and E. D. Rosenblum. Genetic recombination between alpha-toxin mutants of Staphylococcus aureus. J. Bacteriol. 92:580-583. 1966.-A demonstration of genetic recombination between Staphylococcus aureus nonhemolytic mutants was attempted by means of transduction. The results of two-point reciprocal transductions placed the mutants into two genetic groups. Recombination within each group was not detectable within the limits of the method, but hemolytic recombinants were obtained in transductional crosses when donor and recipient were from different groups. At least two genetic loci are therefore involved in alpha-toxin production. The 11 mutants of group II were fibrinolysin-negative. The recombinants were always found to be restored to fibrinolysin production as well as to alpha-toxin production. These data suggest the existence of a pleiotropic gene simultaneously affecting the synthesis of both alpha toxin and fibrinolysin. The nine mutants of group I were fibrinolysin-positive. Group I members are postulated to be alpha-toxin structural mutants. Three mutants were also negative for bound coagulase, but no linkage was observed between the locus controlling bound coagulase and the loci for either fibrinolysin or alpha-toxin production.

Coagulase↗

Significance of chromogenic variants in studies of virulence factors of Staphylococcus aureus.

Parisi, Joseph T. (Duquesne University, Pittsburgh, Pa.). Significance of chromogenic variants in studies of virulence factors of Staphylococcus aureus. J. Bacteriol. 92:589-591. 1966.-Large numbers of chromogenic variants were isolated from cultures of a parent strain of Staphylococcus aureus growing in Brain Heart Infusion (Difco). The parent strain and four selected chromogenic variants were tested for either quantitative or qualitative differences in the production of extracellular substances associated with virulence. Quantitative differences were found in the ability of these strains to produce coagulase and hyaluronidase, whereas qualitative differences were found in the production of plate hemolysins, bound coagulase, opacity in an egg yolk medium, and a proteinase. In view of the rate and extent of the occurrence of these chromogenic variants, their presence in an inoculum could lead to inaccurate results in in vitro studies of staphylococcal virulence.

Coagulase↗

Clumping of Staphylococcus aureus in the peritoneal cavity of mice.

Kapral, Frank A. (Philadelphia General Hospital, Philadelphia, Pa.). Clumping of Staphylococcus aureus in the peritoneal cavity of mice. J. Bacteriol. 92:1188-1195. 1966.-Nonencapsulated strains of Staphylococcus aureus inoculated into the peritoneal cavity of mice are promptly clumped by the interaction of fibrinogen with the bound coagulase present on the bacterial surface. Some of the pre-existing leukocytes adhere to the staphylococcal clumps during the 1st hr, but phagocytosis is minimal. During the 2nd hr, there is an influx of neutrophils into the region, and these form a thick layer around the staphylococcal clumps and, apparently, prevent further egress of toxin. Leukocytes in proximity to the organisms undergo degeneration, but cells located externally maintain an effective barrier and, thus, confine the organisms. The encapsulated Smith strain of S. aureus is not clumped under these circumstances, presumably because the capsule prevents the bound coagulase-fibrinogen interaction.

Animals↗

Agar medium for the selective enumeration of coagulase-positive Staphylococcus from the rat alimentary tract.

A selective agar medium (medium J(1)) is proposed for the quantitative enumeration of egg yolk-positive (EYP) and egg yolk-negative (EYN) Staphylococcus pyogenes from the digestive tract and feces of the rat. This medium, buffered at pH 5.0, is composed of acid casein hydrolysate and yeast extract with 7.5% sodium chloride, 1.6% sodium pyruvate, 0.0008% 2,3,5-triphenyltetrazolium chloride (TTC), and 6% egg yolk emulsion. Inoculation is by the pour-plate method and incubation is at 38 C in a water-jacketed incubator for 36 hr. Colonies of S. pyogenes reduce TTC; EYP strains are surrounded by a halo of opacity; and EYN strains may be surrounded by a red halo, but no opacity. Small, white colonies of S. epidermidis may develop, but Micrococcus, and all other groups of Staphylococcus recognized in the rat intestinal flora, are inhibited. Other bacterial genera, notably Bacillus, Corynebacterium, Proteus, and Streptococcus, are also inhibited.

Agar↗

Comparative study using various methods for identification of Staphylococcus species in clinical specimens.

Coagulase-negative staphylococci (CNS) play a predominant role in nosocomial infections. Rapid, reliable identification of these organisms is essential for accurate diagnosis and prompt effective treatment of these infections. Quite recently, the VITEK 2 g-positive (gram-positive [GP]) identification card (bioMérieux) has been redesigned for greater accuracy in the identification of gram-positive cocci. We compared the BD Phoenix (Becton Dickinson) and VITEK 2 (bioMérieux) automated microbiology systems, using their respective update version cards, and the API ID32 STAPH test. The glyceraldehyde-3-phosphate dehydrogenase (gap) gene-based T-RFLP (terminal restriction fragment length polymorphism) method was used for verifying the results. In total, 86 clinical isolates of CNS and 27 reference strains were analyzed. The results show that for identification of CNS, the automated identification methods using the newest VITEK 2 and BD Phoenix identification cards are comparable. However, API ID32 STAPH revealed more correct results compared to both automated microbiology systems. Despite the increased performance of the phenotypic automated identification systems compared to the former versions, molecular methods, e.g., the gap-based T-RFLP method, still show superior accuracy in identifying Staphylococcus species other than Staphylococcus aureus.

Bacterial Typing Techniques↗

Identification of the capsular polysaccharides in Staphylococcus aureus clinical isolates by PCR and agglutination tests.

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. The predominance of two capsular polysaccharides, types 5 and 8, on the surface of clinical isolates led to the development of a conjugate vaccine (StaphVAX) based on capsular polysaccharides types 5 and 8 conjugated to a carrier protein. We have studied the capsular phenotypes and genotypes of 195 isolates representative of all clinical syndromes that encompassed both hospital and community-acquired infections. These isolates were mainly detected in France between January 2001 and December 2004. In this population, most of clinical isolates (87%) expressed either capsular polysaccharide type 5 (42%) or 8 (45%), whereas 13% were nontypeable by the serotyping method with antibodies specific to capsular polysaccharide type 5 or 8. These 26 nontypeable strains were further serotyped and were demonstrated to express the cell wall surface antigen 336, a polyribitol phosphate N-acetylglucosamine, which resembles cell wall teichoic acid. Among methicillin-resistant Staphylococcus aureus (MRSA) strains, we found a predominance of serotype 5 for 64% of strains, whereas MSSA isolates were predominantly capsular serotype 8 (60%). All S. aureus clinical isolates included in the present study have been investigated by PCR method, demonstrating that all isolates carried either the cap5 or the cap8 locus.

Agglutination Tests↗

First report of septicemia caused by an obligately anaerobic Staphylococcus aureus infection in a human.

In this case report, we describe the first instance of septicemia caused by an obligately anaerobic Staphylococcus aureus in a human. A 45-year-old man presented with septicemia, septic arthritis, and multiple pulmonary abscesses, which were caused by an obligately anaerobic S. aureus. The clinical and microbiological features that led to the diagnosis are discussed. Genotyping cannot at present reliably separate S. aureus subsp. aureus from S. aureus subsp. anaerobius, but phenotypic characteristics suggest that the present isolate is a previously undescribed strain of anaerobic Staphylococcus aureus.

Anaerobiosis↗

Heterogeneity of methicillin-susceptible Staphylococcus aureus strains at a German University Hospital implicates the circulating-strain pool as a potential source of emerging methicillin-resistant S. aureus clones.

Recently, we demonstrated rapid dissemination of different methicillin-resistant Staphylococcus aureus (MRSA) clones at the Institute for Microbiology at the University of Magdeburg (B. Ghebremedhin, W. König, and B. König, Eur. J. Clin. Microbiol. Infect. Dis. 24:388-398, 2005). The majority of them harbored the readily transmissible mec cassette type IV. Thus, theoretically, methicillin-susceptible Staphylococcus aureus (MSSA) might capture the mecA gene from circulating MRSA, or MRSA strains might catch mobile toxin genes from MSSA. Therefore, we characterized MSSA strains circulating at the University Hospital in Magdeburg. Among a total of 84 MSSA strains under study, about 40% possessed the tst (toxic shock syndrome toxin) gene and up to four additional enterotoxin genes. tst-positive MSSA strains belonged to all known agr groups (I to IV) and to 14 different spa types (t008, t012, t015, t019, t024, t056, t065, t127, t133, t162, t271, t287, t399, and t400), and they were classified by multilocus sequence typing (MLST) as ST1, ST8, ST30, ST39, ST45, ST101, ST121, ST395, and ST426. In contrast, simultaneously circulating MRSA strains (n = 24) harbored in general two or three genes of the enterotoxin gene cluster, and the tst-positive MRSA isolates belonged to the well-known epidemic types ST22, ST45, and ST228 and were classified as spa types t001, t028, and t032. From our results, one may conclude that the pool of circulating MSSA strains is an important parameter with regard to the epidemiology of hospital- and community-acquired MRSA clones and their potential virulence.

Anti-Bacterial Agents↗

First case of osteomyelitis caused by "Staphylococcus pettenkoferi".

"Staphylococcus pettenkoferi" (proposed name) was identified as an unusual agent of osteomyelitis in a diabetic foot infection. The phenotypical tests used failed to give a good identification. Molecular 16S rRNA gene and rpoB sequencing allowed us to correctly identify this new species of coagulase-negative staphylococcus responsible for this chronic infection.

Bacterial Proteins↗

Direct identification of Staphylococcus aureus in blood culture fluid with a commercial latex agglutination test.

A commercial latex agglutination slide test (SeroSTAT Staph, Scott Laboratories, Inc., Fiskeville, R.I.) accurately identified Staphylococcus aureus when applied directly to blood culture fluid containing staphylococci. This latex agglutination test exhibited 100% accuracy when 30 seeded aerobic and anaerobic radiometric blood cultures (15 strains of S. aureus, 15 strains of other staphylococcal species) were tested blindly. In 36 actual clinical specimens yielding 16 isolates of S. aureus and 20 isolates of Staphylococcus epidermidis, 94.4% accuracy was achieved. The latex agglutination test provided positive test results before objective criteria of blood culture positivity such as radiometric growth indices and Gram stains became positive.

Aerobiosis↗

Rapid identification of Staphylococcus aureus and Streptococcus pneumoniae from blood cultures.

Simultaneous application of the lysostaphin sensitivity test for identification of Staphylococcus aureus and the deoxycholate test for the identification of Streptococcus pneumoniae was evaluated for reliability in rapid identification (1 h) of these organisms from blood cultures by using BACTEC 6B and 7C bottles. The procedure was applied to 127 cultures, 74 lysostaphin tests and 53 deoxycholate tests. Lysostaphin-tested organisms included 23 S. aureus, 40 Staphylococcus epidermidis, 1 Listeria sp., 1 Peptococcus sp., 2 Micrococcus sp., 2 diphtheroids, and 5 mixed cultures. Deoxycholate-tested organisms included 14 S. pneumoniae, 32 Streptococcus spp. (not S. pneumoniae), 1 Listeria sp., 1 Peptococcus sp., 2 diphtheroids, and 3 mixed cultures. Analysis of the data revealed positive and negative predictive values of 95.8 and 94%, respectively, for the lysostaphin test and 100 and 97.4%, respectively, for the deoxycholate test. The combined tests provide rapid reliable identification or elimination of S. aureus and S. pneumoniae in blood cultures.

Blood↗

Misidentification of mucoid variants of Staphylococcus aureus by standard laboratory techniques.

Ten atypical strains of Staphylococcus aureus were examined and compared with previously characterized S. aureus strains. Five of the strains were sent to two hospital microbiology laboratories for identification. All of the mucoid strains were misidentified when tested by standardized coagulase, DNase, and mannitol fermentation criteria, as well as by an automated identification method, but were accurately identified by the API system (Analytab Products, Plainview, N.Y.). These results illustrate the possibility of misidentification when reliance is placed on a few key characteristics rather than a thorough characterization of unusual Staphylococcus isolates.

Bacteriological Techniques↗

Slime production by bovine milk Staphylococcus aureus and identification of coagulase-negative staphylococcal isolates.

Staphylococcus aureus isolates from bovine milk were assessed for capsule or slime production. When pure S. aureus cultures in milk were inoculated directly into serum-soft agar constituted with a modified staphylococcus 110 medium, 100% of the isolates grew with diffuse colony morphology. Diffuse colony morphology was rapidly lost on subculture and was more rapidly lost in brain heart infusion-serum-soft agar. No evidence was seen for encapsulation in India ink preparations or by the clumping factor test. It was concluded that freshly isolated S. aureus strains produce slime, not true capsules. During examination of the 84 milk samples that grew staphylococci in addition to S. aureus (27.4%), a significant number of coagulase-negative staphylococcal species were encountered and identified by conventional tests as S. simulans (41.7%), S. xylosus (11.9%), S. epidermidis (3.6%), S. saprophyticus (3.6%), S. hyicus (2.9%), S. cohnii (1.2%), S. haemolyticus (1.2%), and S. warneri (1.2%). Five isolates (6.0%) were not identified. Attempts were also made to identify the isolates by the API Staph-Ident system, which gave an overall accuracy of 45.2%. The susceptibilities of the isolates to a variety of antibiotics were determined, and they appeared to be less resistant than human clinical isolates.

Animals↗