Yolk Reaction Research Articles

Enterotoxigenic and Antimicrobic Susceptibility Profile of Staphylococcus aureus Isolates from Fresh Cheese in Croatia.

Certain Staphylococcus aureus strains harbour staphylococcal enterotoxin genes and hence can produce enterotoxin during their growth in food. Therefore, food can be a source of staphylococcal food poisoning, one of the most common food-borne diseases worldwide. Epidemiological data show that S. aureus is often present in raw milk cheeses, and consequently, cheeses are often the source of staphylococcal food poisoning outbreaks. The aim of this study was to determine the phenotypic characteristics of S. aureus isolates from fresh cheese, including antibiotic susceptibility; the presence of classical sea-see enterotoxin genes through molecular methods; and the isolate's ability to produce SEA-SEE enterotoxins in vitro through reversed passive latex agglutination. A total of 180 coagulase-positive staphylococci were isolated from 18 out of 30 cheese samples, and 175 were confirmed as S. aureus through latex agglutination and API STAPH tests. All isolates possessed phenotypic characteristics typical for S. aureus, with certain variations in the egg yolk reaction (18.3% of the isolates showed a weak reaction and 28% no reaction at all) and haemolysis pattern (36.6% of the isolates produced double-haemolysis and 4.6% were non-haemolytic). Antibiotic resistance was observed in 1.1% of the isolates and to mupirocin only. Real-time PCR detected the sec gene in 34 (19.4%) isolates, but most isolates (80.6%) were not enterotoxigenic. For all 34 (19.4%) strains that carried the sec gene, the RPLA method detected the production of the SEC enterotoxin in vitro. For those enterotoxigenic strains, the possibility of enterotoxin production in fresh cheese could not be ruled out.

Incidence of enterotoxigenic Staphylococcus aureus in milk and Egyptian artisanal dairy products

Staphylococcus aureus (S. aureus) is one of the most concerning foodborne pathogens due to its ability to produce wide varieties of heat-stable enterotoxins. Hence, this study was designed to investigate the prevalence of the enterotoxigenic S. aureus strains and their enterotoxins in milk and in some representative artisanal Egyptian dairy products. Additionally, the prevalence of Methicillin-Resistant S. aureus (MRSA; antibiotic resistant strain) was considered throughout the study. Three-hundred and fifty samples (n = 350) were randomly collected from Assuit city in upper Egypt, including market cow's milk, yoghurt, Domiati cheese, Kareish cheese, Ras cheese, cooking butter and small-scale ice cream. The identification of the S. aureus strains was done using conventional biochemical method (catalase test, egg yolk reaction test, anaerobic utilization of mannitol and coagulase test) and PCR. Furthermore, Staphylococcal enterotoxins genes (SEA and SED) were detected by PCR while the classical enterotoxins were examined in 30 samples of Ras cheese and small-scale ice cream using ELISA technique. S. aureus was isolated from 142 out of 350 (41%) [95% CI: 35.4–45.7%] examined samples at levels of up to 5.79 log10 cfu/ml or g. The prevalence of S. aureus was significantly (P < 0.05) higher in small scale ice cream (74%) than in other food categories with a mean count of 3.74 ± 1.03 log10 cfu/g. MRSA strains were recovered from 54 (15%) [95% CI: 11.6–19.2%] samples with a log mean value of 3.54 ± 0.523 log10 cfu/ml or g. In addition, seventeen (30%) of the S. aureus isolates tested by PCR were found to be positive for enterotoxin genes. Out of these, fifteen strains carried one gene and only two strains harbored both A and D genes. Additionally, 4 (7.4%) of MRSA isolates from the strains recovered from Ras cheese were enterotoxigenic. Finally, the enterotoxin E (SEE) was determined in 8 out of 30 (26.67%) examined samples that were contaminated with high level of enterotoxigenic S. aureus. It is concluded that additional preventive measures must be applied in the artisanal dairy processing chain in Egypt to avoid milk contamination and consumers' exposure to enterotoxigenic strains of S. aureus.

Phenotypic and molecular characterization ofStaphylococcus aureusstrains of veterinary, dairy and human origin

Austrian veterinary (n=91), dairy (n=86), and human strains (n=48) of Staphylococcus aureus were tested for various phenotypic properties including clumping factor, egg-yolk reaction, production of thermonuclease and susceptibility to 14 antibiotics. In addition the expression of enterotoxins (A-E), and the presence of enterotoxin genes sea to sej and tst was determined. Significant differences in antimicrobial susceptibility were found with 84.6% of veterinary, 57.0% of dairy, and 20.8% of human strains susceptible to all antibiotics tested (P<0.0005). More human strains produced enterotoxins (41.7%) than veterinary (9.9%) and dairy strains (12.6%) while 40.7% and 38.5% of veterinary, 47.7% and 52.3% of dairy, and 77.1% and 87.5% of human strains were se- and tst-positive, respectively. AFLP analysis revealed nine clusters with over- or under-representation of strains with specific characteristics. Strains clustered according to origin (veterinary, dairy, and human) and/or presence of toxin genes and antimicrobial resistance.

Characterization of enterotoxigenic Staphylococcus aureus strains isolated from bovine mastitis in north-east Switzerland

Thirty-four strains of enterotoxin-producing Staphylococcus aureus obtained from milk samples of 34 dairy cows suffering from mastitis from 34 different farms in north-east Switzerland were identified and further characterized by pheno- and genotypic methods. This included the identification of staphylococcal enterotoxin (SE) types, an antibiotic resistance testing, the appraisal of hemolysis, the egg yolk reaction, the detection of the clumping factor and protein A by means of a latex agglutination, the PCR amplification of a S. aureus specific part of the gene encoding the 16S–23S rRNA “intergenic spacer” region and a species specific part of the 23S rRNA-gene, the PCR amplification of the clumping factor ( clfA) gene, the X region and the IgG-binding region of the protein A ( spa) gene, the coagulase ( coa) gene and additionally a macrorestriction analysis of the chromosomal DNA. Within the 26 cultures which formed a single SE, there were 23 SEC- and three SED-formers. Eight cultures were SEAD formers. It was remarkable that 22 SEC formers were also positive for TSST-1. Eighteen of the 23 SEC-formers could be classified as being of the same phenotype. Most of the cultures of one enterotoxin type also showed a great uniformity in the size and number of repeats of the X region as well as in the size of the IgG-binding region of protein A gene and in the size of the coagulase gene. Macrorestriction analysis revealed 11 PFGE patterns. These were in part only different from each other in a few fragments and thus displayed close clonal relations. The results of the present investigation show that a broad distribution of identical or closely related enterotoxin-producing S. aureus clones seem to contribute to the bovine mastitis problem in north-east Switzerland.

Response surface studies on cholesterol reduction in egg yolk using #ps01β-cyclodextrin

The effects of β-cyclodextrin (BCD) and reaction time on the recovery of cholesterol from egg yolk were studied at 25°C based on response surface methodology. The developed response model is of the form: Y = 5.585 + 429.77 X c + 0.534 X t 2, where Y is the predicted cholesterol recovery (%), X c is the BCD/egg yolk ratio (kg/kg) and X t is the reaction time (h). Cholesterol recovery increased with the both BCD/egg yolk ratio and reaction time and there was no optimum value. The model predicted a cholesterol recovery of 94.5% at a BCD/egg yolk ratio of 0.163 over a reaction time of 6 h whereas experiments showed a cholesterol recovery of 87.7%.

Simultaneous isolation of MRSA and Pseudomonas aeruginosa using a novel selective and differential PMAC agar

PMAC agar, a novel, selective and differential medium has been developed and was subjected for evaluation of its selective and differential capability of methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa from other bacteria such as Bacillus, Micrococcus, Gram-negative bacteria and drug resistant ones. Growth of MRSA and P. aeruginosa on PMAC agar was facilitated and their colonies were easily differentiated. Colonies of MRSA after 24 approximately 48 h incubation at 35 degrees C were small (2 to 4 mm in diameter), smooth and egg-yolk reaction positive. On the other hand, P. aeruginosa with pigment production (pyocianin, fluorescin or pyomelanin) formed large (2.5 to 7.0 mm in diameter), brownish black or brown colonies with a creamy edge. PMAC agar did not allow to grow unwanted bacteria tested except certain species formerly classified to Pseudomonas such as Burkholderia and Stenotrophomonas. However multi-drug resistant strains such as Enterobacter cloacae, Serratia marcescens and Acinetobacter calcoaceticus formed extremely small colonies. PMAC agar is recommended as a novel, useful medium for isolation, differentiation and presumptive identification of MRSA and P. aeruginosa from clinical and environmental sources.

Lecithinase reaction of Staphylococcus aureus strains of different origin on Baird-Parker medium

Staphylococcus aureus produces one or more enzymes with lipolytic activity, but differences between strains have been reported (Owens and John 1975; O'Toole 1987; Rollof et al. 1987). The biological and biochemical properties of these enzymes have been investigated and results were recently reviewed (Kötting et al. 1984). Baird-Parker medium (Baird-Parker 1962) is a selective medium commonly used for the isolation of Staph. aureus. The presence of egg yolk in this medium permits the detection of two reactions due to lipolytic activity of staphylococci: (1) Lecithinase reaction, a zone of precipitate in the medium surrounding the colonies; and (2) Lipase reaction or 'pearly layer', an iridescent film in and immediately surrounding colonies, visible by reflected light (iridescent sheen or 'oil in water'). In this study, human and bovine strains, previously biotyped according to the scheme of Devriese et al. (1984), were compared for production of a zone of precipitation, lecithinase reaction, on Baird-Parker medium. Bovine and human strains of Staph. aureus were compared for production of the egg yolk reaction (lecithinase reaction) on Baird-Parker medium and the results were related to their biotypes and site of origin of the sample. Human strains and strains biotyped as human biotypes had higher percentage of positive results than bovine isolates and/or biotypes. However, all strains isolated from body sites of heifers produced a positive reaction regardless of the biotype.

Newly developed MRSA medium

We have developed a new selective medium, tentatively named MR(SA)2, for rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) from clinical specimens. MR(SA)2 medium contained modified Müller-Hinton agar supplemented with 75 g of NaCl, 10 g of mannit, 20 mg of bromcresol purple, 20 g of egg-yolk, 4 mg of oxacillin, and 12.5 mg of ceftisoxime per 1,000 ml. There were no differences between the growth of MRSA strains on this medium at 30 C and that of 37 C. This medium can detect the egg-yolk reaction instead of the coagulase reaction. By single streaking of a test material on the surfaces of MR(SA)2 agar, MRSAs can easily be distinguished from methicillin-resistant coagulase-negative staphylococci (MR-CNS), other bacteria and fungi from their colony morphologies in a quantitative manner. A few MRSA strains would not form colonies on this medium because of their susceptibilities to ceftisoxime, but this may not inpede its use, since most MRSA strains isolated from clinical materials showed resistance to ceftisoxime. From the above results, the MR(SA)2 medium may be suitable for rapid detection of MRSA and MR-CNS.

Positive reaction in mouse ligated intestinal loop assay with nonenterotoxigenic and nonhemolytic strains of Staphylococcus aureus.

Nonenterotoxigenic and nonhemolytic strains of Staphylococcus aureus exhibited a positive reaction in the mouse intestinal loop assay except for a strain negative for the egg yolk reaction. Edema and swelling in all positive loops, increased bacterial cell numbers within intestinal loops, and extremely close adhesion to or inclusion of numerous bacterial cells in HeLa cells after inoculation of the strains were observed. These results suggest a possible invasive ability of the strains.

Differences in the egg yolk reaction on Baird-Parker medium between bovine and human strains of coagulase-positive staphylococci

Strains of coagulase-positive staphylococci isolated from cases of bovine mastitis did not produce halos on Baird-Parker medium whereas strains from cases of human infections did.

An improved selective medium, EPGS agar, for isolating Staphylococcus aureus

Egg yolk-Polymix B-Glycine-Salt (EPGS) agar was devised for the rapid isolation of Staphylococcus aureus from feces of patients with food poisoning. The medium consisted of 10g of mannitol, 3g of glycine, 5g of lithium chloride, 10mg of polymyxin B sulfate, 25mg of phenol red, 18g of agar, and 900 ml of distilled water. It was autoclaved at 121°C for 15 minutes. 100ml of a 30% egg yolk emulsion were added to the medium after it had cooled to 50°C. It was found that the presence of 0.3% glycine, 0.5% litium chloride and 0.001% polymyxin B sulfate in the medium showed a considerable inhibitory effect on Bacillus subtilis strains, while the medium had enhanced the growth of all S. aureus strains used in the experiment.When the EPGS agar was applied to feces of healthy people and patients with food posisoning, S. aureus was detected more effectively than by the Mannit Salt Egg Yolk (MSEY) agar. On the EPGS agar, colonies with egg yolk reaction could be counted as specifically S. aureus after an incubation time of 24 hrs, whereas the MSEY agar required 48 hrs to grow S. aureus from feces of patients with food poisoning.The EPGS agar may be useful for a routine laboratory test for detection of S. aureus in feces of patients with food poisoing.

Tween-esterase activity in Enterobacter sakazakii

Seventy-one of 73 E. sakazakii strains isolated in Czechoslovakia produced Tween 80 esterase; its production was slower and less intensive than in Serratia, vibrios and aeromonads. The E. sakazakii strains did not produce lecithinase (yolk reaction was negative for 8 days). The following set of tests was recommended for verification of E. sakazakii identification: pigment (was produced by 100% of strains), Tween 80 esterase (was positive in 97.3%), mucate (negative in 100%) and sorbitol (negative in 100%).

Detection of Staphylococcus aureus in bovine mastitis and some characteristics with special reference to enterotoxin producibility and coagulase types of isolates.

During the period from november, 1979 to August, 1980, 332 samples of mastitis udders of cattle killed at the Tokyo Shibaura Slaughter House were investigated for the presence of Staphylococcus aureus. The isolates were tested for biochemical characteristics, enterotoxin production and coagulase typing. Eighty-seven S. aureus were isolated from the 85(25.6%) samples of 332 mastitis udders examined. Twenty-nine(33.3%) of the 87 strains produced enterotoxins A, B or C, and 24(82.8%) of them produced enterotoxin C. All except one strain, fell into either of types I through VIII in coagulase typing. Types VI(34.5%) and V(20.7%) were dominant among the eight coagulase types. Of the 87 strains 49(56.3%) coagulated bovine plasma irrespective of 100% in rabbit plasma coagulation. Twenty-nine(33.3%) isolates produced fibrinolysin, 82(94.3%) fermented mannitol, 40(46.0%) were positive in egg-yolk reaction, and all the strains grew on blood agar containing 1:200, 000 crystal violet showing the A type colonial character. Some of the staphylococal strains of bovine origin were negative in bovine plasma coagulation test, but positive in fibrinolysin. It is suggested from these results that S. aureus of bovine origin might be one of the causative agents for food poisoning.

Sensitivity to Lytic Agents and DNA Base Composition of Several Aerobic Spore-Bearing Bacilli

The authors studied the possible relationship between a genetic characteristic, like DNA base composition, and certain phenotypic characteristics, i.e., sensitivity to lytic agents, morphology of colonies, and biochemical reactions in 34 strains of spore-bearing bacilli. From the results obtained two groups of bacilli have been identified. The first group includes the species B. subtilis, B. pumilus, B. licheniformis, and B. firmus and one strain of B. megaterium. The mean value of the GC% of the DNA is 44.22 +/- 1.76. All the strains examined are highly sensitive to lysozyme and resistant to sodium lauryl sulphate (S.L.S.); the surface colonies have a "rhizoid" appearance and the microcolonies on slide microculture are star-shaped. The second group includes the species B. cereus, B. cereus var. mycoides, B. anthracis, and B. thuringiensis. The mean value of the GC% of the DNA is 33.65 +/- 0.59. All the strains belonging to this group are resistant to both lysozyme and S.L.S., and the surface macro-colonies and the microcolonies have a "medusae head" appearance. The two groups also have certain different biochemical reactions; e.g., anaerobic growth and the egg yolk reaction, with few exception, are negative for the first group and positive for the second; furthermore, the strains in the first group (with rare exceptions) cause fermentation in the three carbohydrates, glucose, arabinose, and xylose, while glucose only is fermented by all strains with one exception in the second group. The position of B. megaterium is not yet clear, although one strain may certainly be included in the first group. Lysis by lipase is extremely variable and does not correlate with any of the other characteristics studied. The other species studied in relation to the characteristics, considered in our research (B. coagulans, B. macerans, B. polymyxa, B. laterosporus, B. alvei, B. circulans, B. stearothermophilus, and B. brevis), are not susceptible to grouping, either in the first, or in the second or even in a separate group.

コアグラーゼ陽性ブドウ球菌の卵黄寒天培地における卵黄反応についての一考察

コアグラーゼ陽性ブドウ球菌の卵黄寒天培地における卵黄反応についての一考察

Characterization of“Pasteurella” piscicidaIsolated from White Perch and Cultivated Yellowtail

The morphological, physiological, and biochemical characteristics of twelve "Pasteurella" piscicida strains isolated from white perch and yellowtail are described and the present uncertain taxonomic status of the organisms is discussed. The organisms isolated were gram-negative rods showing bipolar staining and pleomorphism. No spores or flagella were observed. They were non motile and viscid colonies were formed. Growth was observed in a temperature range of 20 to 30 C and the salinity range of growth was between 0.5% and 4.0%. They were aerobic and facultative anaerobic. Oxidase and catalase were produced. Nitrates were not reduced to nitrites. Lysine and ornithine decarboxylases were not produced but arginine dihydrolase was produced. The egg yolk reaction was positive. Tween 80 and tributyrin were decomposed. Phosphatase was produced. Beta hemolysis was revealed on a medium containing defibrinated blood from chickens or carp but not from mammals. Methyl red and Voges-Proskauer tests were positive, and acetoin was produced from 2,3-butanediol. Glucose was fermented without gas production. Acid was produced from fructose, mannose, galactose, sucrose, maltose, dextrin and glycerol. These organisms differed from all other members of the genus Pasteurella with respect to nitrate reduction, arginine dihydrolase, Voges-Proskauer and methyl red tests. The formation of viscid colonies and inability to grow in a medium without sodium chloride or at 37 C were additional characteristics of these organisms.

Characterization of three Aeromonas and nine pseudomonas species by extracellular enzymes and haemolysins.

Strains from type culture collections and clinical isolates belonging to the Aeromonas and Pseudomonas genera were identified with conventional tests. Production of extra-cellular enzymes and haemolysins were detected by simple plate agar methods. The following enzymes were found to be of special value for a rapid and simple classification of certain species in both genera: potease (casein and gelatin agar), lecithinase (lecithin agar), and deoxyribonuclease (DNA agar). Elastase, staphylolytic enzyme, lipase, ribonuclease, amylase, and egg yolk reaction were other enzymes studied. However, these tests were not positive for more than 90% of any species. A. hydrophila, A. salmonicida, and P. aeruginosa were haemolytic on agar containing rabbit erythrocytes.

The Egg Yolk Reaction Produced by Several Species of Bacteria

Summary. A study of the egg yolk reaction produced by various species of bacteria has shown that it is caused by the hydrolysis of phosphatidylcholine. Staphylococcus aureus and Serratia marcescens degrade phosphatidylcholine by a series of hydrolytic reactions which are initiated by phosphatidylcholine: cholesterol o‐acyltransferase. The degradation of phosphatidylcholine results in the formation of a sub‐surface, opaque homogeneous zone around colonies growing on egg yolk agar and a collar of lipid material floating on the surface of egg yolk broth. This reaction may not be shown by all species growing in egg yolk media but it can be demonstrated if enzyme extracts are used.

Production of enzymes and toxins by hospital strains of Pseudomonas aeruginosa in relation to serotype and phage-typing pattern.

SUMMARY We studied 148 strains of Pseudomonas aeruginosa isolated from nonbacteriaemic infections and healthy carriers (group I) and 50 strains isolated from patients with bacteriaemia (group II). More than 90 per cent. of the strains in both groups produced lipase, esterase, protease, and a haemolysin most active on ox and rabbit erythrocytes. Between 60 and 90 per cent. gave a positive egg-yolk reaction, produced nuclease (DNAase and RNAase), lecithinase, elastase, staphylolytic enzyme, and another haemolysin most active on sheep and horse erythrocytes. No significant difference between the two groups in the formation of any of these factors was found. The strains of the two groups showed more than 100 different phage-typing patterns when tested with a set of 22 phages; only a few strains were untypable and no phage-typing pattern predominated. About one-third of all the strains belonged to Habs′ serotype 6 and the rest of the strains were rather evenly distributed between the other serotypes without any difference between the two groups.

Characterization of Staphylococcus aureus in a Pediatric Burn Unit

A one-year study on an endemic strain of Staphylococcus aureus phage type 84/85 in a children's burn unit is described. The endemic strain rapidly colonized the burns and nares of acute patients after admission but was not isolated from a patient on admission. Nonendemic strains of S. aureus found on some new patients were mostly non-phage typable and did not prevail in burns. The endemic strain was rarely isolated from the nares and skin of reconstructive patients or from the nares of hospital personnel. The endemic strain did colonize the oral cavity, normal skin, and intestinal tract of some acute patients. Endemic and nonendemic strains of S. aureus from the burned children were compared in their biochemical activities and antibiotic sensitivities to two groups of S. aureus from one other local and one Danish burns unit. The latter groups of strains represented different combinations of staphylococcal phage group III strains. Each of the four groups of strains differed in production of hemolysins, Tween 80 hydrolysis, egg yolk reaction, and proteolysis of casein and gelatin. All of the strains were uniformly sensitive to gentamicin, oxacillin, and cephalothin. Only 4 of 162 strains tested were methicillin resistant. The endemic S. aureus strains of phage type 84/85 were uniformly resistant to eight other antibiotics including lincomycin and clindamycin. The endemic strain was not the known cause of a clinically documented infection in a group of 82 acute patients studied. The possible role of S. aureus strains of phage group III in burn grafting problems is discussed.