- Volume 37, Issue 3, 1992

In the evaluation of treatment failure in Streptococcus pyogenes pharyngitis it is necessary to distinguish between persistence of the original streptococcus and acquisition of a new strain. We used the analysis of restriction fragment length polymorphism (RFLP) of total DNA and of ribosomal DNA (rDNA) regions (ribotypes) as epidemiological tools to compare 43 pre- and post-treatment S. pyogenes strains obtained from 20 patients. In 16 cases pre- and post-treatment strains gave indistinguishable RFLP patterns of total DNA, strongly suggesting relapse with the same strain. However, in four cases different patterns were obtained for the pre- and post-treatment isolates, indicating recurrence due to the acquisition of a new strain. Ribotyping did not improve discrimination among strains. Thus, analysis of DNA RFLP is a promising method for distinguishing recurrence from relapse in failures of pharyngitis treatment.

An outer-membrane protein (OMP) was isolated from a clinical strain of Bacteroides distasonis. Changes in growth media did not appreciably affect the appearance of this protein in crude outer-membrane preparations examined by SDS-PAGE. However, the proportion of the protein relative to other OMPs was greater in 24-h cultures than in 48-h cultures. The protein could not be readily solubilised by various conventional detergent extraction techniques but treatment of the insoluble material at 100°C with SDS released the protein, as did overnight extraction at 37°C with SDS. This OMP was heat-modifiable, and thus was similar to the OmpA protein of Escherichia coli, with a faster mobility on SDS-PAGE when solubilised at 25°C than at 100°C. The critical temperature for conversion was between 80°C and 90°C. Because of the characteristic heat-modifiability, the protein was called B. distasonis HMP-1 (heat modifiable protein-1). Overnight exposure to EDTA or NaCl at 37°C favoured conversion of the 25°C form to the 100°C form. In intact cells, the protein was labelled by a cell-surface radio-iodination procedure, and thus is at least partially exposed at the cell surface. No reactions between the B. distasonis HMP-1 and antibodies to either E. coli OmpA or E. coli porin were found by Western blot analysis. A B. distasonis OM preparation containing predominantly HMP-1 had pore-forming ability in a liposome assay. This study is the first report of the isolation and characterisation of a heat-modifiable OMP in Bacteroides, and it is the first description of pore-forming activity in a Bacteroides OM fraction.

Single specimens of diarrhoeal stool from 676 patients, mostly aboriginals aged less than 5 years, admitted to Alice Springs Hospital, central Australia, for diarrhoea between Sept. 1988 and Feb. 1989, were examined for Campylobacter spp. by culture on a blood-free medium with selective supplement (BFM; Oxoid) and blood agar overlaid with a membrane filter (FM). Campylobacter spp. were isolated on either BFM or FM or both from 225 patients. Campylobacter spp. were isolated on BFM alone from 75 patients and on FM alone from 213 patients (p < 0.001; χ2 test). Most campylobacters isolated on BFM were C. jejuni. All C. jejuni subsp. doylei, all “C. upsaliensis” except one, all C. laridis, C. fetus subsp. fetus and several uncharacterised Campylobacter isolates were isolated on FM only. C. jejuni was isolated on BFM but not FM from several patients, and vice versa. Serotyping of C. jejuni and C. coli isolated from both media showed the serotypes recovered from the two media to be different in some patients. In some patients concurrent infection with several species or serotypes (up to five) of Campylobacter, or both, was shown for the first time by the use of FM. We conclude that the use in combination of a selective medium and a non-selective medium with a filtration technique are better than either medium alone for the isolation of Campylobacter spp.

The ability of Staphylococcus aureus conjugative plasmids to mobilise non-conjugative resistance plasmids from clinical isolates of S. aureus and S. epidermidis was studied. Plasmids which could not be transferred by transduction or mixed-culture transfer were transferred from phage-typable and non-typable S. aureus and from S. epidermidis. Plasmids encoding single resistance determinants were transferred by mobilisation whereas multiple-resistance plasmids were transferred as co-integrates between the conjugative and non-conjugative plasmids. This study demonstrates that mobilisation is a useful tool for the transfer and study of staphylococcal plasmids and illustrates how antibiotic resistance could be transferred between staphylococci in vivo.

Twenty-six clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) collected from six Australian hospitals by a National Staphylococcal Study Group were examined by analysis of restriction fragment length polymorphisms (RFLPs) of chromosomal DNA with pulsed field gel electrophoresis. Digestion with the restriction endonuclease SmaI produced 13–17 bands of 7–700 kb. The digestion patterns were easily distinguished and isolates could be classified into 17 groups based on their RFLPs. Isolates giving a pattern associated with one group were from four hospitals in four different states. In another group, the isolates responsible were from three hospitals in two states and in a further group, the isolates were derived from two hospitals in different states. The remaining groups comprised only one member each. The method has promise for typing and studying the epidemiology of MRSA.

Staphylococcus aureus mutants lacking pigment, or expressing only low levels of pigment, were more sensitive to oleic acid than were the parent strain and mutants making more pigment than the parent. One class of mutants (colour index 5), although possessing significant levels of pigment, were nevertheless quite sensitive to oleic acid. This suggested that only certain carotenoids in the biosynthetic pathway were capable of imparting resistance to fatty acids. The phenotypic expression of pigment also affected the sensitivity of a strain to oleic acid. The parent S. aureus strain 18Z, when grown to express its maximal pigment potential, was more resistant to oleic acid than when it was grown to express minimal levels of pigment.

A new typing method for Staphylococcus epidermidis was developed. Four biotinylated lectins—wheat germ agglutinin (WGA), soy bean agglutinin (SBA), lentil agglutinin (LCA) and Concanavalin A (ConA)—were added to immobilised whole cells of coagulase-negative staphylococci (CNS) in microtitration plates. The amount of bound lectin was measured by peroxidase-conjugated avidin followed by a peroxidase reaction. The method was compared to antibiotic-resistance analysis, phage typing, plasmid DNA profiles and slime production. A total of 113 isolates of CNS from 21 patients was investigated and 71 strains of CNS, including 64 strains of S. epidermidis, were detected if all typing methods were taken into consideration. If only one typing method was used the highest discriminatory power among the S. epidermidis isolates was obtained with the lectin-binding assay which allowed 49 different strains to be detected. If the lectin-binding assay was combined with plasmid-profile analysis, all 64 different strains could be identified. The typability of lectin-binding assay was 96.9% among the S. epidermidis isolates and 25 different lectin-binding patterns were established among the 64 strains. The highest number of strains belonging to one lectin-binding pattern was 13 (20.3%). The assay was reproducible, easy to perform, relatively inexpensive and therefore applicable to large scale typing of S. epidermidis.

An elastase of Staphylococcus epidermidis was purified by ion exchange chromatography on CM-Sepharose and characterised. Its Mr is c. 21 kDa, its optimal temperature for activity is 42°C and the pH optimum is 6.8. The enzyme is activated by cysteine and other SH-donators and inhibited by l-trans-epoxy-succinylleucylamido-(4-guanidino)butane (E64), an inhibitor of cysteine proteases, but not by 3,4-dichloroisocoumarin (3,4-DCI), an inhibitor of serine proteases. This finding suggests that the elastase of S. epidermidis is a cysteine protease. Because S. epidermidis elastase degrades human sIgA, IgM, serum albumin, fibrinogen, and fibronectin, this enzyme may be regarded as a virulence factor.

Correlation between cytotoxin production and sporulation was demonstrated when a Clostridium difficile culture was inoculated into fresh broth to give an initial count of < 10 vegetative cells/ml with no spores. Under these conditions, cytotoxin was produced and released during sporulation. Addition of a sporulation inhibitor (acridine orange, 30 mg/L), resulted in a marked decrease in both sporulation and cytotoxin production, despite there being no change in the number of vegetative cells in the culture. These results indicate that sporulation might be closely related to cytotoxin production.

The distribution of oral Streptococcus milleri carbohydrate type antigens in other viridans streptococcus species was examined. Rantz-Randall extracts of cells of the test strains grown in broth containing glucose were allowed to react with typing or grouping antisera for S. milleri serotypes a–k, or Lancefield groups A–G and K. Of 93 strains comprising more than 12 streptococcal species that included S. mutans and S. sanguis complexes, only 15 S. salivarius strains and one S. mitis strain were immunologically related to S. milleri serotype f. Unlike S. milleri strains, S. salivarius type f strains belonged to Lancefield group K, whereas the S. mitis strain was closely related to S. milleri serotype f but did not react with any of the Lancefield grouping antisera tested. Results suggest that oral S. milleri strains can be distinguished serologically from other oral viridans streptococci and that the typing antisera used in our researches might differentiate S. milleri isolates from the mouth from those associated with systemic purulent infections.

Monoclonal antibodies (MAbs) were produced to an outer-envelope preparation from Serpulina (Treponema) hyodysenteriae, the aetiological agent of swine dysentery. Three MAbs (isotype IgG1) were obtained. All three recognised a 16-kDa antigen that was common to most strains of S. hyodysenteriae of different serotypes but was absent from non-pathogenic, porcine intestinal spirochaetes. Immunofluorescence and immunogold labelling studies showed that the 16-kDa antigen was exposed on the surface of intact spirochaetes. The MAbs agglutinated freshly grown cultures of spirochaetes and inhibited growth of S. hyodysenteriae strains in vitro.

The susceptibility of a strain of Giardia lamblia to benzimidazole carbamates, 5-nitroimidazoles, nitrofurans and other drugs was studied in vitro. Albendazole was the most active compound, with a 50% inhibitory concentration (IC50) of 0.01 mg/L and a minimal lethal concentration (MLC) of < 0.04 mg/L; the IC50 of mebendazole was 0.06 mg/L and the MLC < 0.5 mg/L. Among the 5-nitroimidazoles tested, ornidazole was the most effective (IC50 0.12 mg/L); tinidazole, metronidazole, secnidazole and hemezole were less active. Nifuroxazide, etofamide and nalidixic acid exhibited modest anti-giardial activity; quinfamide did not inhibit the growth of the parasite at a concentration of 200 mg/L. Albendazole and mebendazole are promising candidates for clinical use and should be further evaluated.