In vitro studies of haemolysis by some staphylococci grown in chemically defined media

Staphylococcus aureus (five strains) and Staph. epidermidis (one strain) have been evaluated for comparative growth and haemolysin titre in both brain heart infusion (BHI) and in developed, nutritionally adequate, chemically defined media (CDMs) varying only in amino acid composition. The ability to show a particular haemolytic profile was strain-dependent and the haemolytic titre (HU50/ml) was both strain- and medium-dependent. Highest titres of both alpha and beta type haemolysins were obtained in BHI. Maximum titres were in general detected in the late exponential phase in both CDMs and BHI. Titres declined during the stationary phase in CDMs. Staphylococcus epidermidis produced a delta-type haemolysis profile on BHI-based blood agars, but only rabbit blood was sensitive in agars based on a developed, chemically defined medium (CDM/A; 13 amino acids) in which all six staphylococci grew. The addition of yeast extract to CDM/A increased alpha haemolysin titre, but suppressed beta haemolysin formation; beta haemolysin was, however, detected in yeast extract/phosphate-buffered saline. Strain Wood 46 degraded haemoglobin, but only in (initially) whole blood; red blood cell-free haemoglobin-rich plates (BHI) were unaffected during growth. A novel haemolytic profile is described for Staph. aureus NCTC 8532 growing on blood agars based on CDM/A and may relate to the production of methaemoglobin during haemolysis.     

Polar lipids of Staphylococcus strains analysed by fast atom bombardment mass spectrometry

This study used fast atom bombardment mass spectrometry (FAB MS) to obtain detailed information on polar lipids of Staphylococcus by examining 23 isolates. Eighteen major anions were found in the range m/z 199–297, consistent with the presence of carboxylate anions. A further 21 major anions were found in the higher mass regions of m/z 609–805, consistent with the presence of phospholipid anions. In Staph. aureus, Staph. epidermidis, Staph. haemolyticus and Staph. hominis , the most intense peaks putatively assigned as carboxylate ions were consistent with presence of C_15:0, followed by C_17:0 except in the case of Staph. epidermidis. The major phospholipid anions were consistent with the presence of PG(30 : 0), PG(32 : 0) and PG(33 : 0). It is concluded that Staphylococcus has a characteristic polar lipid profile and that qualitative and quantitative differences may be seen between Staph. aureus and Staph. epidermidis.     

Detection and recovery of sublethally-injured enterotoxigenic Staphylococcus aureus

AIMS: To determine whether sublethally-injured (acid- or heat-shocked) Staphylococcus aureus cells are recoverable using selective agar overlays. METHODS AND RESULTS: Brain Heart Infusion (BHI) Agar overlaid with either Baird-Parker Agar (BPA) or Gram-Positive Agar (GPA) was compared in the ability to resuscitate heat- and acid-shocked enterotoxigenic Staph. aureus. BHI/BPA overlays allowed for greater recovery of both heat- and acid-shocked cells than BHI/GPA, although the former was not selective and allowed growth of bacteria other than Staph. aureus. No significant difference existed in percent recovery of heat- and acid-shocked cells between the two overlay approaches. Significant differences were noted in counts on BHI/GPA plates and straight selective GPA/GPA plates, however. Viability of heat- and acid-shocked Staph. aureus was also examined using fluorescence microscopy, the relative counts of which correlated well to the calculated percent recovery on selective agar overlays. CONCLUSIONS: This work has shown that an improved agar overlay technique increases the sensitivity of the standard plate count while enumerating sublethally-injured enterotoxigenic Staph. aureus compared with direct plating onto selective media. SIGNIFICANCE AND IMPACT OF THE STUDY: These data emphasize the need to develop practical and cost-effective methods that reliably detect and enumerate sublethally-injured pathogens such as Staph. aureus.     

Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications

Aim: To evaluate an inter‐generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Methods and Results: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap‐extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r‐αCS specifically detected 42‐kDa and 33‐kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild‐type toxins in vitro. Conclusions: The r‐αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild‐type toxins in vitro. Significance of the Study: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co‐infections like gangrenous ischaemia, gangrenous mastitis, etc.     

The adherence of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli on cotton, polyester and their blends

The adherent behaviour of the Gram-positive Staphylococcus aureus and Staphylococcus epidermidis and the Gram-negative Escherichia coli on cotton, polyester and their blends through contact in aqueous suspensions was studied. Staphylococcus epidermidis was found to adhere to fabrics much more so than Staph. aureus. The adherence of both Staph. epidermidis and Staph. aureus to fabrics increased as the content of polyester fibres in the fabrics increased. The attachment of E. coli to all fabrics was very low and was not affected by the fibre contents. Total numbers of adherent bacteria on cotton and polyester fabrics were related directly to the concentrations of the bacterial suspensions. The extents of adherence, expressed by the percentage of adherent bacteria from the suspension, however, were independent of the concentration. The length of contact with bacteria was also found to affect the adherence of bacteria on fabrics studied.     

The adherence of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli on cotton, polyester and their blends

The adherent behaviour of the Gram-positive Staphylococcus aureus and Staphylococcus epidermidis and the Gram-negative Escherichia coli on cotton, polyester and their blends through contact in aqueous suspensions was studied. Staphylococcus epidermidis was found to adhere to fabrics much more so than Staph. aureus. The adherence of both Staph. epidermidis and Staph. aureus to fabrics increased as the content of polyester fibres in the fabrics increased. The attachment of E. coli to all fabrics was very low and was not affected by the fibre contents. Total numbers of adherent bacteria on cotton and polyester fabrics were related directly to the concentrations of the bacterial suspensions. The extents of adherence, expressed by the percentage of adherent bacteria from the suspension, however, were independent of the concentration. The length of contact with bacteria was also found to affect the adherence of bacteria on fabrics studied.     

Action of egg lysozyme on representatives of the family Micrococcaceae. The action on staphylococci

The results of the study on the effect of various concentrations of lysozyme on staphylococci of three species, i.e. Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus saprophyticus are presented. The study was performed with the use of the turbidimetric method. It was found that all the staphylococci had a common tendency for changing the optic density (OD) of the suspensions under the effect of lysozyme which was different from that in Micrococcus luteus. However, the dynamics of decreasing the OD in Staph. saprophyticus was characterized by some properties which provided a supposition that the structures of the cell walls in Staph. aureus and Staph. epidermidis were close, while the cell walls in Staph. saprophyticus had some unknown characteristics.     

Novel haemolysins of Salmonella enterica spp. enterica serovar Gallinarum

Haemolysins of Salmonella are important due to their probable role in pathogenesis of systemic salmonellosis and use in sub-serovar level typing. The present study was undertaken to determine haemolytic potential of Salmonella Gallinarum strains through phenotypic and genotypic methods. Amplification of haemolysin gene (clyA) and cytolysin gene (slyA) was attempted in order to determine their role in haemolysin production. Study on 94 strains of S. Gallinarum revealed the production of two types of haemolysis viz., beneath the colony haemolysis (BCH) or contact haemolysis and clear zone haemolysis (CZH). Haemolysis was observed on blood agar prepared with blood of cattle, buffalo, sheep, goat, horse, rabbit, guinea pig, fowl, and human blood group A, B, AB and O. Although, haemolysis was also observed on blood agar prepared with whole blood, clarity of zone was more evident on blood agar made from washed erythrocytes. Clear zone haemolysis was best observed on blood agar prepared with washed erythrocytes of goat and a total of 12% (11 of 94) S. Gallinarum strains under study produced CZH on it. The clyA gene could not be detected in any of the 94 strains under study, while slyA gene could be amplified uniformly irrespective of haemolytic potential (CZH) and haemolytic pattern (BCH) of the strains. The study suggested that the two types of haemolysis (CZH and BCH) observed among S. Gallinarum strains may not be due to either slyA or clyA gene products and thus there may be some other gene responsible for haemolytic trait in Gallinarum serovar. Different haemolytic patterns of strains under study indicated multiplicity of haemolysins in S. Gallinarum.     

Comparative studies of selective media for recovery of Staphylococcus aureus from natural waters

Several selective media currently used for the enumeration of Staphylococcus aureus from different sources were evaluated in order to establish their quantitative recovery, specificity and degree of selectivity, using different types of water samples. The highest selectivity and reliability in the enumeration of Staph. aureus from the samples was obtained on Borrego-Florido-Romero-0 (BFR-0) and KRANEP agars. The method that produced the highest recovery of Staph. aureus was BFR-0 agar with membrane filter and incubation at 36°C for 48–72 h.     

Comparative studies of selective media for recovery of Staphylococcus aureus from natural waters

Several selective media currently used for the enumeration of Staphylococcus aureus from different sources were evaluated in order to establish their quantitative recovery, specificity and degree of selectivity, using different types of water samples. The highest selectivity and reliability in the enumeration of Staph. aureus from the samples was obtained on Borrego-Florido-Romero-0 (BFR-0) and KRANEP agars. The method that produced the highest recovery of Staph. aureus was BFR-0 agar with membrane filter and incubation at 36 degrees C for 48-72 h.     

Isolation and characterization of staphylococci from goats milk produced in Northern Ireland

Goats milk was examined for total viable bacteria and staphylococci (Baird-Parker medium, Schleifer & Kramer's (SK) medium, SK medium with a reduced sodium azide content (SKR) and SK and SKR with 5% added sheep blood). Staphylococcus aureus, Staph. epidermidis and Staph. simulans were the predominant species isolated overall. SK medium proved inhibitory with respect to the isolation of Staph. caprae and Staph. chromogenes. This was reduced by plating on the modified SK media. Representative strains of each species isolated were examined for production of enterotoxins A-E. Enterotoxin C alone was produced by 35% of the Staph. aureus strains tested. None of the other staphylococcal species examined produced any of the known enterotoxins.     

Mechanism of haemolysis by Vibrio vulnificus haemolysin

The haemolytic action of Vibrio vulnificus haemolysin (VVH) was compared to that of streptolysin O (SLO). Both were cholesterol-binding haemolysins, but differed in the release of haemoglobin (Hb). In the first step of haemolysis, the haemolysins were temperature-independently bound to the cholesterol site on the target erythrocyte membrane. This was followed by the rapid release of K+, which is an intra-erythrocyte marker. Hb was then released, in different ways. In the case of VVH, Hb was released slowly after a relatively long lag, whereas with SLO, Hb was released as rapidly as K+. Haemolysis by VVH was inhibited by the addition of 30 mM-dextran 4 (mean Mr 4000), which is considered to be an effective colloid-osmotic protectant. The results therefore indicated that haemolysis by VVH (like that by Escherichia coli alpha-haemolysin and Staphylococcus aureus alpha-toxin) was caused by a colloid-osmotic mechanism. Both K+ and Hb release caused by VVH proceeded temperature-dependently, and the membrane fluidity of liposomes prepared with lipids extracted from sheep red blood cell membranes increased above 20 degrees C. These results suggest that the temperature-dependence of the haemolysis by VVH is due to the requirement for an increase in the membrane fluidity during the formation of a transmembrane pore.     

Staphylococci associated with the rumen of young and wild ruminants

Staphylococcus warneri, Staph, xylosus, Staph. saprophyticus, Staph. epidermidis, Staph. sciuri subsp. lentus, Staph. sciuri subsp. sciuri and Staph. cohnii subsp. urealyticum were the most frequently occurring staphylococci in the rumen content and wall of young and wild ruminants. Staphylococcus warneri formed a high percentage mainly among 2–9-week-old ruminants. Staphylococcus hominis was found only in mouflons. Staphylococcus gallinarum was detected only in calves. Staphylococcus aureus was the predominant representative of coagulase-positive staphylococci in the rumen of wild ruminants. Most of the strains examined could not be identified as known species.     

A Staphylococcus aureus-specific oligonucleotide probe derived from 16S rRNA gene sequences

Genes encoding 16S rRNA were sequenced from 16 species of Staphylococcus. Sequence analysis highlighted a potential Staph. aureus -specific region and a complementary oligonucleotide probe was synthesized and its specificity tested. Northern blotting indicated molecular specificity, and dot blots to RNA from Staph. aureus, Staph. capitis, Staph. caprae, Staph. carnosus, Staph. caseolyticus, Staph. cohnii, Staph. epidermidis, Staph. gallinarum, Staph. haemolyticus, Staph. hominis, Staph. hyicus, Staph. saprophyticus, Staph. sciuri, Staph. simulans, Staph. warneri and Staph. xylosus indicated species-specificity.     

Inhibition of Staphylococcus aureus by the commensal bacteria of human milk

AIMS: To study the bacterial diversity in expressed human milk with a focus on detecting bacteria with an antimicrobial activity against Staphylococcus aureus, known as a causative agent of maternal breast infections and neonatal infections. METHODS AND RESULTS: Random isolates (n = 509) were collected from breast milk samples (n = 40) of healthy lactating women, genotypically identified, and tested for antimicrobial activity against Staph. aureus. Commensal staphylococci (64%) and oral streptococci (30%), with Staph. epidermidis, Strep. salivarius, and Strep. mitis as the most frequent isolates, were the predominant bacterial species in breast milk. One-fifth of Staph. epidermidis and half of Strep. salivarius isolates suppressed growth of Staph. aureus. Enterococci (Ent. faecalis), isolated from 7.5% of samples, and lactic acid bacteria (LAB) (Lactobacillus rhamnosus, Lact. crispatus, Lactococcus lactis, Leuconoctoc mesenteroides), isolated from 12.5% of samples, were also effective against Staph. aureus. One L. lactis isolate was shown to produce nisin, a bacteriocin used in food industry to prevent bacterial pathogens and spoilage. CONCLUSIONS: Expressed breast milk contains commensal bacteria, which inhibit Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains inhibitory against the pathogen Staph. aureus have potential use as bacteriotherapeutic agents in preventing neonatal and maternal breast infections caused by this bacterium.     

Variation in the behaviour of enterotoxigenic Staphylococcus aureus after heat stress in milk

The survival of several strains of Staphylococcus aureus after heat stress in different menstrua was not logarithmic and F-values were determined to express their resistance to heat. Of the strains tested, Staph, aureus 234 (enterotoxin B) was the most heat resistant and Staph. aureus 790 (enterotoxin E) was the most heat sensitive. Buffalo milk gave the best protection to all the strains of Staph. aureus against heat, followed by cow's milk; phosphate-buffered saline gave the least protection. Soyabean casein digest agar gave maximum recovery of survivors followed by brain heart infusion and Baird-Parker medium. At 50°C there was no marked variation in coagulase production by the surviving strains but at 55 and 62–5dE C there was complete loss of coagulase activity. There was a decreased deoxyribonuclease (DNase) production by all the strains of Staph. aureus after heat stress. Heat-treatment at 55 and 62mD5dE C resulted in loss of enterotoxin production by all the survivors except S₆ and 234, the surviving cells of which still prodused enterotoxin B after heat treatment at 55dE C. Most of the survivors regained lost characteristics such as coagulase, DNase and enterotoxin production after four to five passages through BHI which suggests that subculture of Staph. aureus recovered from heat-processed milk is necessary to avoid false results.     

Relation of human serum antibody against Staphylococcus epidermidis cell surface polysaccharide detected by enzyme-linked immunosorbent assay to passive protection in the mouse

A specific and rapid enzyme-linked immunosorbent assay (ELISA) has been applied for the detection of immunoglobulins to Staphylococcus epidermidis cell surface polysaccharides in human serum. Positive IgG, IgM and IgA titres of more than 1: 6400, 1: 1600 and 1: 400 were observed with this assay against passive protective human serum. However, IgG, IgM and IgA titres of less than 1: 400, 1: 100 and 1: 50 were shown in non-protective serum. When the cross-reactivity of passive protective human serum to homologous and heterologous cell surface polysaccharides was examined by inhibition test with ELISA, remarkable inhibition was shown with homologous cell surface polysaccharide, whereas no inhibition was observed with heterologous substances. According to these results, the quantitation of human serum antibody by the ELISA method against Staph. epidermidis cell surface polysaccharide was found to be significant for the demonstration of passive protective activities against Staph. epidermidis.     

Characterization of staphylococcal gamma-lysin.

gamma-Lysin was purified from Staphylococcus aureus strains Smith 5R and PG23 (a toxic shock syndrome isolate) by a combination of heparin-agarose and hydroxylapatite chromatography. Both strains produced two haemolytic components, designated gamma 1 and gamma 2. Though each component was weakly haemolytic they acted synergistically to potentiate haemolysis on rabbit, sheep and human blood. Rabbit and sheep erythrocytes were more sensitive to lysis by gamma-lysin than human erythrocytes. The molecular mass of gamma 1 was 32 kDa and its pI value was 9.4. gamma 2 had a molecular mass of 36 kDa and a pI value of 9.3. While both trypsin and papain acted synergistically with gamma 2 to induce increased haemolysis, no such synergism was seen with gamma 1. Also, protease inhibitors acted to inhibit synergism between gamma 1 and gamma 2. These findings suggest that gamma 1 could be a protease.     

Study of haemolytic activity of some Campylobacter spp. on blood agar plates

A total of 152 strains of Campylobacter jejuni, C. coli, C. laridis and C. fetus subsp. fetus were tested for haemolysis on blood agar plates. Distinct haemolysis was detected in 92.3% (96/104) of strains of C. jejuni and 21.7% (5/23) of strains of C. coli on sheep blood heart infusion agar after incubation for 4 d microaerobically at 42 degrees C. Haemolysis was also detected on horse blood heart infusion agar. Haemolysis was not detected at 37 degrees C except with one of 50 strains of C. jejuni tested at this temperature, which was weakly positive. Campylobacter laridis was not haemolytic; C. fetus subsp. fetus, which does not grow at 42 degrees C, showed no haemolysis at 37 degrees C. Blood agar (Oxoid, BA Base No. 2) was not suitable for testing for haemolysis by these organisms. A microaerobic gas mixture containing hydrogen is better than that containing nitrogen because the medium has a brighter colour, making haemolysis easier to detect. There was no synergistic haemolysis with Staphylococcus aureus or Streptococcus agalactiae. The plate haemolysis test as described here may aid differentiation within the thermophilic campylobacters.     

A simplified system for biotyping Staphylococcus aureus strains isolated from different animal species

D evriese , L.A. 1984. A simplified system for biotyping Staphylococcus aureus strains isolated from different animal species. Journal of Applied Bacteriology 56 , 215–220.
A biotyping system for Staphylococcus aureus strains is proposed which is a simplified version of biotyping procedures described in the literature. It differentiates Staph. aureus strains from man and animals into host-specific ecovars and biotypes which are not host-specific. With the help of tests for βhaemolysin, staphylokinase, coagulation of bovine plasma and the crystal-violet reaction, the origin of many but not all Staph. aureus strains can be determined: 604 of 809 strains from man. poultry, cattle, pigs, goats, rabbits and foods could be alloted to four ecovars which are typically associated with man, poultry, sheep and goats and cattle. The other strains belonged to five non-host specific biotypes.