Henning Brandis

Production, Purification and Chemical Properties of an Antistaphylococcal Agent Produced by Staphylococcus epidermidis

An antibacterial agent produced by Staphylococcus epidermidis strain 5 was isolated from culture supernatants; its spectrum of activity is restricted to staphylococci and micrococci. The bacteriocin-like substance was purified to homogeneity by column chromatography on Servachrome XAD-2, CM-Sephadex C-25 and Sephadex G-50. Chemical analysis showed it to be a peptide with a molecular weight of about 6000 and an isoelectric point of about 10.5. Purified material was rapidly inactivated by trypsin, chymotrypsin and pronase and it was relatively heat-stable. After acidic hydrolysis 10 amino acid residues and two unidentified ninhydrin-positive substances were detected. Production of the bacteriocin-like substance was not inducible with mitomycin C.

An avidin-biotin ELISA for the detection of staphylococcal enterotoxins A and B

The avidin-biotin system was incorporated into the enzyme-linked immunosorbent assay (ELISA) to establish a new detection method for staphylococcal enterotoxins A and B in culture supernatants. Staphylococcal protein A (SPA) does not interfere significantly with the procedure. The test shows good sensitivity for the toxins and the ease and rapidity with which the assay can be performed make it a valuable tool for the routine detection of enterotoxins.

Plasmid Involvement in Production of and Immunity to the Staphylococcin-like Peptide Pep 5

The staphylococcin-like peptide Pep 5 is produced by the penicillin resistant strain Staphylococcus epidermidis 5. This strain is immune to the peptide. Plasmid analysis of S. epidermidis 5 by agarose gel electrophoresis and electron microscopy demonstrated five plasmids with molecular weights ranging from 5.8 X 10(6) to 29 X 10(6). Variants of S. epidermidis 5 not producing Pep 5 or which had become penicillin sensitive were induced by various curing treatments. Strains lacking the 13.9 X 10(6) mol. wt plasmid (pED502) had lost penicillin resistance, and those lacking the 12.3 X 10(6) mol. wt plasmid (pED503) failed to produce Pep 5. pED503 is also responsible for the immunity of the producer cell to Pep 5. Plasmid pED502 could be transformed into S. aureus RN 981 which then became resistant to penicillin. pED503 could not be transformed into S. aureus RN 981, but could be transformed into S. epidermidis 5 variants previously cured of this plasmid; the transformants then regained the properties of Pep 5 production and immunity.

Mode of Action of the Staphylococcin-like Peptide Pep 5 and Culture Conditions Effecting its Activity

Pep 5, a staphylococcin-like peptide produced by Staphylococcus epidermidis 5 was bactericidal for staphylococci and micrococci (Fig. 1). A stable L-phase variant of Staphylococcus aureus, but not of Streptococcus faecalis, was susceptible, too, indicating that the selectivity of Pep 5 is not due to the presence of a cell wall receptor. Addition of Pep 5 to cultures of the indicator strain caused immediate inhibition of DNA, RNA, protein, and polysaccharide synthesis (Fig. 5). The action of Pep 5 could be inhibited by increasing the cation content of the medium (e.g., to 100 mM Mg2+ or 600 mM K+) (Fig. 3), indicating a primary binding to anionic sites on the cell wall. Sublethally injured cells could be rescued by the addition of trypsin or 1 M KCl (Fig. 4).

Interaction of the Staphylococcin-like Peptide Pep 5 with Cell Walls and Isolated Cell Wall Components of Gram-Positive Bacteria

Unlike bacteriocins of Gram-negative bacteria, the strongly basic staphylococcin-like peptide Pep 5 lacked specific receptor mediated binding to sensitive Gram-positive bacteria. Studies with whole cells, purified cell walls, teichoic acids, and lipoteichoic acids strongly suggested that it binds reversibly via electrostatic interaction to negatively charged groups. Thus, Pep 5 binding could be reversed by sufficiently high concentrations of monovalent (K+, 150-250 mM) and divalent (Ca2+, 15-30 mM) cations (Fig. 1, 2) and by low pH (pH 2), where Pep 5 binding groups are protonated. Cells of Staphylococcus cohnii 22 with a reduced teichoic acid content showed a reduced Pep 5 binding capacity (Fig. 3). The results indicate that teichoic, teichuronic, and lipoteichoic acids are the unspecific cell wall binding sites for Pep 5.

Falcivibrio grandis gen. nov. sp. nov., and Falcivibrio vaginalis gen. nov. sp. nov., a New Genus and Species to Accommodate Anaerobic Motile Curved Rods Formerly Described as “Vibrio mulieris” (Prévot 1940) Breed et at. 1948

Summary Strains of anaerobic, motile, curved rods, formerly designated “ Vibrio mulieris ”, wereisolated from human vaginal secretions. They were investigated for their biochemical, morphological and cytological behaviour and compared to similar strains from other authors. The strains studied were divided into two species on the basis of their physiologicalactivities and morphology. The first species comprises saccharolytic strains with a crescent cell shape and a GC base ratio of 50-53, the second species asaccharolytic strains with a less crescent, but often coryneform cell shape and a GC base ratio of 53-54. The Gram reaction of the organisms is variable. Electron microscopy of the cell wallcould not elucidate the Gram type clearly. The saccharolytic strains are described as Falcivibrio grandis gen. nov. sp. nov., theasaccharolytic strains as Falcivibrio vaginalis gen. nov. sp. nov. The new genus cannot be assigned to any of the recognized families.

Species distribution of coagulase-negative staphylococci isolated from clinical sources

The distribution of coagulase-negative staphylococcal species from clinical material was investigated in order to detect possible tropisms. 398 strains of staphylococci (336 coagulase-negative and 62 coagulase-positive strains) and 3 micrococcus strains were isolated. Differentiation between the two genera was achieved by testing sensitivity to lysostaphin and anaerobic growth in thioglycolate. Four S. hominis strains reacted like micrococci in these tests. Staphylococci were classified according to the Kloos and Schleifer biotyping scheme in a modified way on microtitre plates. Additionally coagulation and reduction of methylene blue milk were determined and the test proved to be useful. Identified coagulase-negative species included S. epidermidis (205 strains), S. hominis (64), S. haemolyticus (35), S. warneri (15), S. capitis (5), S. simulans (6), S. saprophyticus (5), and S. species (1). A high predominance of S. epidermidis was found among isolates from nasopharyngeal (91%) and tracheobronchial (80%) tracts and most other localizations. A different distribution of species was observed in urine and vaginal swabs where non-S. epidermidis species accounted for over 50% of identified strains. The majority of S. hominis and S. haemolyticus strains was isolated from these sources. S. simulans, like S. saprophyticus was found in urine specimens only and occurred predominantly in women of the sexually active age.

Isolation and Mode of Action of a Staphylococcin-like Substance Active Against Gram-positive and Gram-negative Bacteria

Screening of non-phage group II Staphylococcus aureus strains for antagonistic substances revealed one particular strain, S. aureus D91, to excrete a substance with a wide spectrum of activity; both Gram-positive and Gram-negative bacteria were susceptible. The staphylococcin-like substance D91 produced by this strain was partially purified by column chromatography on Sephadex G-50, DEAE-cellulose, Phenyl Sepharose CL-4B and Sephadex G-200. A molecular weight of 76000 was estimated by gel filtration. The activity was heat sensitive but was not affected by hydrolytic enzymes except for pronase. The protein character of substance D91 was confirmed by gel electrophoresis and subsequent staining with Coomassie blue. The action exerted on sensitive bacteria was bacteriostatic rather than bactericidal. Biosynthesis of DNA, RNA, protein and polysaccharides were inhibited simultaneously in both Escherichia coli and Staphylococcus aureus. Active transport of glutamic acid was stopped in both S. cohnii and E. coli, whereas glucose uptake was inhibited in E. coli only. The substance induced a slow efflux of 86Rb+ from proloaded cells of S. cohnii and E. coli. The antagonistic activity of S. aureus D91 was eliminated by ethidium bromide at a rate of 47.6% suggesting that plasmids may be involved in its production.

Purification and Properties of LIQ 4, an Antibacterial Substance Produced by Streptococcus faecalis var. liquefaciens K 4

Summary: An antibacterial substance (LIQ 4) produced by Streptococcus faecalis var. liquefaciens K 4 was isolated in extracellular and cell associated form. It markedly inhibited the growth of Gram-positive bacteria, whereas only a few Gram-negative bacteria were susceptible. LIQ 4 was purified by hydrophobic chromatography (Servachrome XAD-2; octyl-Sepharose CL-4B; Sephadex LH 60) and Sephacryl S-200. TLC yielded a fluorescent spot as the active component and several inactive ninhydrin-positive substances. These contaminating peptides strongly adsorbed to LIQ 4 and could only be removed by repeated reversed phase HPLC. Furthermore, HPLC separated LIQ 4 into seven closely related substances. All showed strong fluorescence under UV light, stained yellow with ninhydrin, and contained aspartate and lysine after acid hydrolysis. The molecular weight was estimated by Amicon ultrafiltration to be less than 2000. LIQ 4 was stable at 80°C (30 min) and pH 2, but considerably inactivated above pH 8. It was apparently affected by proteolytic enzymes, but the activity could be fully restored upon heating.

Correlation between enterotoxigenicity, tested by different ELISA-techniques, antibiotic resistance patterns and phage groups of Staphylococcus aureus strains.

A group of 596 Staphylococcus aureus strains isolated from various clinical sources or implicated in food poisoning was investigated for enterotoxins A and B (SEA and SEB) production. The conventional ELISA techniques (competitive and sandwich ELISA) were compared with a newly developed avidin-biotin ELISA in their ability to detect the enterotoxins. The avidin-biotin system was not remarkably influenced by SPA up to 10 micrograms/ml. A semi-quantitative competitive ELISA for the detection of staphylococcal protein A (SPA) in culture supernatants was carried out in parallel. The strains isolated in cases of food poisoning showed different antibiotic resistance patterns, whereas the strains from clinical sources were selected for either methicillin or penicillin resistance only. The strains isolated in food poisoning outbreaks (FP strains) were enterotoxin A positive in 22%, enterotoxin B positive in 11%, and SEA + SEB positive in 9% of cases. The strains with resistance to penicillin only (PER strains) produced SEB in 26%, SEA in 14%, and both toxins in 7% of the cases. The methicillin-resistant strains (MCR strains) produced SEA in 59% of cases, whereas SEB was produced in 6% only (SEA + SEB: 20%). 37% of the SEA producers belonged to phage group III (SEB: 30%; SEA + SEB: 25%) and 12% (SEB: 11%; SEA + SEB: 9%) to phage group I. 26% of the SEA-producing and 37% of the SEB-producing strains (SEA + SEB: 23%) were non-typable.