A chemically defined medium for the production of staphylococcal beta hemolysin.

It was recently found that alcohols can confer hemolytic properties on certain species of yeast. Here, it is reported that alcohol can promote hemolysis by various species of staphylococci, including strains of Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus hominis. In order to study this novel phenomenon in S. aureus and S. epidermidis, strains that exhibit this phenomenon (e.g. S. aureus 8325-4, COL, SH1000, S. epidermidis), as compared with strains that exhibited little alcohol-enhanced hemolysis (e.g. S. aureus 8325-4 DeltaTRAP, RN6911) were examined. Both ethanol and n-butanol caused upregulation of the virulence regulator-RNAIII, with a concomitant increase in the production of alpha, beta and gamma-hemolysins in strain 8325-4. In S. aureus COL and SH1000, there was an increase in RNAIII but no change in transcription levels of alpha, beta and gamma hemolysins. Staphylococcus epidermidis stain sofi exhibited increased RNAIII and beta hemolysin production. Staphylococcus aureus mutant strains (8325-4 DeltaTRAP and RN6911) showed no change in the transcription level of the RNAIII regulator and the above hemolysins. Increased hemolysis in S. aureus COL, SH1000 and mutant strains may be caused by other hemolysins (not regulated by RNAIII) or through other mechanisms such as hyperoxidation or cytotoxic lipids.

Journal Article A Study of Enterotoxin and Alpha and Beta Hemolysin Production by Certain Staphylococcus Cultures Get access Michael J. Surgalla, Michael J. Surgalla From the Department of Bacteriology and Parasitology, The University of Chicago Search for other works by this author on: Oxford Academic PubMed Google Scholar K. Eileen Hite K. Eileen Hite From the Department of Bacteriology and Parasitology, The University of Chicago Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 76, Issue 1, January 1945, Pages 78–82, https://doi.org/10.1093/infdis/76.1.78 Published: 01 January 1945 Article history Received: 25 November 1944 Published: 01 January 1945

Escherichia coli isolated from cases of bacteremia and from a variety of urinary tract infections were characterized according to serotype (O:H antigenicity), K type (possession of K1, K2, K3, K12, or K13), hemagglutination (HA) type, and production of beta-hemolysin. Results obtained with the bacteremia and urinary tract infection isolates were similar except for more hemolytic isolated from urine than from blood (42 versus 29%) and more K1+ isolates from blood than from urine (50 versus 29%). A close correlation was found between Ha type VI (production of fimbriae which mediate mannose-resistant HA of human and African green monkey erythrocytes) and the production of hemolysin or K1 capsular antigen or both. Most (95 of 98, or 95%) of the HA type VI+ blood isolates and most (146 of 164, or 89%) of the HA type VI+ urine isolates produced hemolysin or K1 or both, in contrast to 22 and 26%, respectively, of those belonging to HA types other than HA type VI. Also, 76% of all hemolytic and 70% of all K1+ isolates belonged to HA type VI. Remarkably few of the HA type VI+ isolates (13%) and even fewer of the HA type VI- isolates (3%) produced both K1 and hemolysin; these belonged mainly to serotypes O16:H6, O18:H7 and O2:H4. Other major serogroups were usually K1+/hemolysin- (O1, O7) or K1-/hemolysin+ (O2, O4, O6). At least 74% (262 of 351) and possibly as many as 83% (293 of 351) of those isolates which produced mannose-resistant HA of human erythrocytes were classified as HA type VI+; 31 isolates produced mannose-resistant HA with all erythrocytes tested. Taking serogroup and serotype into consideration, we conclude that the E. coli fimbrial hemagglutinin(s) responsible for the HA type VI phenotype will prove to be the same as the virulence-associated mannose-resistant adhesins of uropathogenic E. coli which other investigators have characterized as unique fimbrial antigens detectable by mannose-resistant HA of human erythrocytes.

Strains of Aeromonas hydrophila isolated from skin infections of common freshwater fish in Bangladesh were tested for enterotoxin production, hemolysin production, and any correlation between these two activities. We also tested the resistance patterns of A. hydrophila to different drugs, especially in relation to ampicillin. The A. hydrophila strains produced an enterotoxin that was related to their beta-hemolytic activities. Production of beta-hemolysin may thus be an indicator of enterotoxicity. As 50% of the strains of A. hydrophila were found to be susceptible to 12.5 micrograms of ampicillin per ml, media containing this antibiotic may not be suitable for their isolation.

Staphylococcal beta hemolysin from the 681 strain of Staphylococcus aureus grown in a Heart Infusion dialysate semisolid medium under 10% carbon dioxide was obtained in an immunoelectrophoretically pure form by a combination of procedures of precipitation with 2 volumes of acetone followed by chromatography on diethylaminoethyl cellulose at pH 6.0. The acetone precipitation procedure did not show any deleterious effect on the hemolytic activity of the beta hemolysin unless the precipitate was left in contact with the acetone for at least 4 hr. The crude preparations contained two types of beta hemolysin. One of these represented the major portion of the total activity of beta hemolysin and behaved as a cation. The other represented a minor (1/5,000) portion of the total beta hemolysin activity and behaved as an anion. These active principles were designated as cationic and anionic beta hemolysins, respectively. An unexpected increase in the total beta hemolysin activity of the crude preparations was noted when these were concentrated by dialysis against polyethylene glycol (20 m). This effect was probably due to polyethylene glycol. A further unexpected increase in the titer of the acetone-precipitated preparations occurred when these were lyophilized. The reason for this incremental increase is not known. It may be due to fragmentation of the beta hemolysin.

Introduction and Aim: Presence of virulence factors may cause increased persistence of Enterococci in the healthcare environment, increase ability to colonise inpatients and thereby result in the transmission of infection. The present study was performed to detect the presence of virulence traits among the clinical strains of Enterococci and to determine its association between virulence factors and susceptibility to various antibiotics. Materials and Methods: Clinical isolates of Enterococci were identified to species level by conventional method and Vitek 2 automated method and subjected to antibiotic susceptibility testing using disk diffusion and Minimum Inhibitory Concentration (MIC) method. Presence of hemolysin, gelatinase and biofilm was detected by phenotypic method. Results: Out of 708 isolates from urine 39 (5.51%) Enterococcus faecalis, and 3 isolates each of Enterococcus faecium (0.42 %) and Enterococcus durans (0.42 %) were biofilm producers. Beta hemolysin production was detected in 342 (48.30%) E. faecalis obtained from urine and 9/48 (18.75 %) from pus. Out of the isolates studied, 9/774 (1.16 %) isolates were found to be positive for beta-hemolysin production, gelatinase and biofilm production. All the 9 (100.00 %) isolates were resistant to penicillin, high level gentamicin, ciprofloxacin and levofloxacin. Conclusion: Virulence factors described in Enterococci enable these organisms to colonise patient tissue, increase resistance to antimicrobial agents and aggravate infection outcome.

166 Staphylococcus aureus septicemia strains were phage grouped and tested for lipolytic activity, protein A content, alpha, beta and delta hemolysin activity and toxic shock syndrome toxin (TSST-1) production. These strain characteristics were correlated to the clinical features of the infections. Patients infected with phage group II strains showed the lowest mortality but were more prone to develop internal abscesses. Lipolytic activity and protein A positivity was found in most strains and no correlation to phage group or clinical signs could be shown. Alpha hemolysin was the most common of the investigated hemolysins though it was only produced by 58% of 88 investigated strains. Beta and delta hemolysin production was found in 25% and 24% of the strains, respectively. The lowest frequency of alpha hemolysin production (25%) was found among phage group I strains, especially those producing TSST-1, where only 1 of 12 strains was positive. The overall frequency of TSST-1 production was 18% in 88 tested strains and most positive strains were non-hemolytic. These results indicate that hemolysin production does not seem to be required for a strain to be invasive.

Haque, Riaz-ul (Ohio State University, Columbus), and Jack N. Baldwin. Purification and properties of staphylococcal beta-hemolysin. I. Production of beta-hemolysin. J. Bacteriol. 88:1304-1309. 1964.-Highest activity of beta-hemolysin was observed when buffered saline (pH 7.0) containing 0.001 m magnesium sulfate was used as a diluent, and the tubes were incubated at 37 C for 80 min and then refrigerated for 30 min. Either Heart Infusion semisolid agar or a dialysate of Heart Infusion containing 0.3% agar was suitable for the production of large quantities of beta-hemolysin. The concentration of beta-hemolysin in semisolid and broth cultures was greatest after incubation for 24 hr. Continued incubation resulted in a loss of active hemolysin in broth cultures but not in semisolid agar cultures. Incubation in atmospheres containing 20% carbon dioxide greatly enhanced the production of beta-hemolysin. The presence of fermentable sugars inhibited the production of beta-hemolysin. Highest yields of beta-hemolysin were obtained when the initial pH of the medium was 5.5 to 5.8.

Three variations of a synthetic growth medium were used to study the folic acid and p-aminobenzoic acid (PABA) requirements of Propionibacterium. P. shermanii, P. freudenreichii, P. thoenii, and P. arabinosum synthesize folic acid and do not require PABA or folic acid. P. pentosaceum, P. jensenii, and P. rubrum are stimulated by folic acid or PABA, but do not show an absolute requirement. P. peterssonii shows a requirement for either PABA or folic acid. The addition of 300 mug of sulfadiazine per ml did not inhibit growth of propionibacteria in the synthetic medium, synthetic medium plus PABA, or synthetic medium plus folic acid. P. freudenreichii was not inhibited even when 500 mug of sulfadiazine per ml was added to the synthetic medium, nor did it degrade sulfadiazine significantly. Trimethoprim totally inhibited the growth of Propionibacterium. Radioactive sulfadiazine was transported by sulfadiazine-sensitive Escherichia coli but not by P. freudenreichii, indicating that the sulfadiazine resistance of propionibacteria could be mainly due to their inability to transport sulfonamides.

The mode of action of highly punned beta hemolysin derived from the R-1 strain of Staphylococcus aureus has been investigated. Sphingomyelin was absent from lipid extracts of sheep erythrocytes treated with beta hemolysin when compared to normal cells. A correlation was also established between the sphingomyelin content of other erythrocyte species and their sensitivity to beta hemolysin. Further investigations revealed that sphingomyelin is hydrolyzed to yield phosphorylcholine and N-acyl sphingosine. Thus, the mode of action of the beta hemolysin is like that of phospholipase C. Various phosphate compounds other than sphingomyelin, including RNA, glycerophosphate, phenyl-phosphate, phosphatidylethanolamine, and phosphatidylcholine were tested as substrates, but virtually no hydrolysis was observed. In contrast with the results of other workers, R-1 beta hemolysin did not release detectable amounts of carbohydrate from staphylococcal cell walls.

fish with an incidence of ( 20% and 42% ) respectively. All the isolates were subjected to different tests to detect some phenotypic characteristics related to virulence: coagulase, DNase, TNase, slime and hemolysin production. In the present study all examined isolates were coagulase positive and the production of DNase, TNase, slime and beta hemolysin was shown by (90 and 96.83), (83.33% -90.48%), (73.33%- 65.8%) and (80% - 92.06%) of chicken and fish isolates respectively. Coagulase gene was detected by PCR and the amplified products of the examined

Twenty-one strains of Verocytotoxin-producing Escherichia coli (VTEC) that hybridized with DNA probe CVD419 were examined for the ability to produce haemolysin. With solid media, all strains produced most haemolysin when grown in blood agar tubes and least when grown on blood agar plates incubated in air. Haemolysin production was increased considerably by incubating blood agar plates in an atmosphere comprising 8% carbon dioxide, 40% hydrogen and 52% nitrogen at 37 degrees C for 16 h, followed by 6 h at 21 degrees C in air. Haemolysin production was also increased when strains were grown on L-agar containing the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid) prior to subculture on blood agar. Intracellular haemolysin was detected in five out of the 21 strains of E. coli grown on L-agar in the atmosphere described above, but haemolysin was not detected in L-broth culture supernatants. The haemolysins lysed guinea pig, mouse and ferret erythrocytes, but not human, rabbit, rat, turkey or chicken erythrocytes. Also, the addition of calcium ions to culture media was not required for haemolytic activity. It was concluded that haemolysins produced by VTEC appear to be quite distinct from E. coli alpha-haemolysin and resemble a form of beta-haemolysin.

When 168 fresh clinical isolates of Staphylococcus aureus were examined for their reactions on a medium containing 1 part in 100,000 crystal violet 50.6% of strains produced a purple appearance, 39.3% produced a white appearance and 10.1% produced a yellow appearance. Purple-reacting isolates were significantly associated with both invasive infections (P less than 0.01) and hospital origin (P less than 0.001). There were no significant associations between the crystal violet reactions and either animal contact or other properties previously reported to be characteristic of white and yellow-reacting strains (beta haemolysin and bovine coagulase production). The results of phage typing showed associations between susceptibility to group III phages and purple-reacting strains and between phage group II susceptibility and white and yellow-reacting strains. There was also a highly significant association between white reactions on crystal violet agar and susceptibility to lysis by a combination of all three groups (that is, I + II + III) and white-reacting strains were significantly more susceptible to lysis by phages 94 and/or 96, whether as a restricted pattern or as part of a broader pattern. The purple reaction on crystal violet medium may be a reliable marker of the 'hospital staphylococcus'.

Three simple, rapid, and accurate methods, i.e., the derivative ratio spectra-zero-crossing method (method I), double divisor-ratio spectra derivative method (method II), and column reversed-phase high-performance liquid chromatographic (RP-HPLC) method (method III) were developed for the simultaneous determination of doxylamine succinate (DOX), pyridoxine hydrochloride (PYR), and folic acid (FA) in their ternary mixtures and in tablets. In methods I and II, the calibration graphs were linear in the range of 2.5-80, 1.0-40, and 1.0-30 microg/mL for DOX, PYR, and FA, respectively. In the HPLC method, the separation of these compounds was performed using mobile phase consisting of 0.05 M phosphate buffer (pH 6.3)-methanol-acetonitrile (50 + 20 + 30, v/v/v), and UV detection was performed at 263 nm. Linearity was observed between the concentrations of the analytes and peak areas [correlation coefficient (r) > or =0.9998] in the concentration range of 1.0-200, 4.0-600, and 4.0-600 microg/mL for DOX, PYR, and FA, respectively. The standard deviation of retention time in method III was 0.011, 0.015, and 0.016 for DOX, PYR, and FA, respectively. The precision studies for all of the methods gave relative standard deviation values of <2%. The results obtained from the methods were statistically compared by means of Student's t-test and the variance ratio F-test. It was concluded that all of the developed methods were equally accurate, sensitive, and precise. These methods could be applied to determine DOX, PYR, and FA in their combined dosage forms.

Methods are described for the aseptic culture of Drosophila. All the factors necessary for normal development can be extracted from yeast in water-soluble form. A chemically defined medium sufficient for almost normal growth is described and includes : casein (and gelatin) as a source of amino-acids, dextrose, cholesterol, ergosterol, yeast nucleic acid, inositol, biotin, aneurin hydrochloride, riboflavine, nicotinic acid, pyridoxin hydrochloride, Ca-pantothenate, choline chloride, thymine and folic acid. For completely normal development an alkali-soluble fraction must be added. This fraction shows nucleoprotein-like reaction, but it is neither arginine nor nucleic acid. Folic acid is essential for pupation. Attention is drawn to the need for caution in gene-action studies with synthetic media which do not give completely normal development even in the wild type.