- Volume 7, Issue 1, 1974

Commercially available batches of bovine serum fraction (BSF) vary in their ability to promote the growth of Mycoplasma pneumoniae on glass. Poly-acrylamide gel electrophoresis shows the presence in BSF of three major components, which can also be separated by Sephadex chromatography. The slow moving component (SMC) in the gel appears to be the factor responsible for the adherence of M. pneumoniae to glass and its growth on that site; batches of BSF containing 6.15% of SMC possess high activity, those with less than 5% have low activity. Polyacrylamide gel electrophoresis is a simple and precise technique for determining growth-promoting activity of BSF.

A simple method is described for the assay of gentamicin and kanamycin in human serum. It is a modification of the monosaccharide hydrolysis method of Faine and Knight (1968) and will estimate low concentrations of antibiotic accurately within 3 hours. The method gives results similar to those obtained by a double-row tube-dilution growth inhibition technique, is unaffected by the presence of penicillins or cephalosporins, and requires only standard laboratory equipment. Because the method gives readily reproducible results and is easy to perform, it should commend itself to all laboratories dealing with patients receiving aminoglycoside therapy.

Of 358 carbohydrate tests carried out independently by two diagnostic laboratories on 28 Pasteurella pneumotropica isolates, the results of 273 were in agreement, 65 were judged uncertain by one laboratory, and 20 were contradictory. On the other hand, the following corroborated atypical reactions were found: negative catalase reaction (one strain), gas production in maltose (one strain), acid production in mannitol (one strain), and weak or absent indole production (14 strains).

A strain of P. pneumotropica from rats produced on subculture typical colonies together with a proportion of white, beta-haemolytic colonies of a variant that was indole negative, gave a stronger oxidase reaction, and acidified raffinose, trehalose and dulcitol, in addition to the carbohydrates acidified by the wild type.

The survival of “marked” strains of Escherichia coli bearing antibiotic-resistance-transfer (R) factors was studied in the faeces of healthy human volunteers for 7 days after ingestion. Two R factors, determining resistance to ampicillin/chloramphenicol/kanamycin/streptomycin/sulphonamides and to chloramphenicol/streptomycin/sulphonamides/tetracyclines were used in 14 experiments on 12 persons. Strains without R factors served as controls. Both R-factor-bearing and R-factor-free strains were identified by chromosomally determined resistance markers.

The R-factor-bearing organisms were isolated from the faeces in fewer numbers than the controls in 52 out of the 54 samples studied. In the two samples in which the reverse phenomenon was observed the effect was not marked. The fall in the number of R-factor-bearing organisms was generally progressive, and 4 days after ingestion the mean faecal population of R-factor-bearing organisms was only 0.5% of the control organisms. The relative decline in the R-factor population probably resulted from impaired vitality of organisms bearing these plasmids rather than from loss of R factors from the cell.

Seven strains of Mycoplasma hyorhinis obtained from different geographic areas were compared by growth-inhibition, metabolism-inhibition and latex-agglutination tests. Although serologically interrelated, the strains showed differences ranging from slight to profound, and therefore division into subtypes was suggested. Statistical analysis by the coefficient of concordance and by the rank correlation coefficient supported the finding that all three sero-logical methods are probably equally suitable for serological differentiation. Polyacrylamide gel electrophoresis revealed no appreciable differences among the strains.

A strain of Staphylococcus aureus (no. FAR4) has been isolated from a patient at frequent intervals over 18 months. This strain has previously been shown to lose resistance to several antibiotics (Lacey et al, 1973); during the last 6 months further changes have occurred in it in vivo.

1. Lincomycin resistance appeared after the patient had been treated with clindamycin. This was due to mutation to constitutive resistance to both lincomycin and erythromycin at a locus adjacent to that determining “indu-cible” resistance to erythromycin. Both loci are probably carried by an extrachromosomal element.

2. Three types of variation in the PF plasmid were observed, (a) Alteration in production of penicillinase from macroconstitutive to micro-constitutive, was not associated with any detectable change in plasmid size. This change gave the cell an advantage in vitro, because it was associated with a faster growth rate, and probably also in vivo, because the microconstitutive variants were isolated in increasing numbers after flucloxacillin therapy had ceased, (b) Complete loss of determinants for penicillinase production and for resistance to cadmium ions from the PF plasmid was associated with a decrease in plasmid size of about 4 × 106 daltons. This change was also associated with increased growth rate in vitro, (c) Loss of resistance to fusidic acid from the PF plasmid was associated with a decrease in plasmid size of about 1 × 106 daltons.

Clostridium perfringens type-A α-toxin (phospholipase C) was purified on a preparative scale by a simple, rapid, two-stage procedure involving precipitation of culture-supernatant fluids with ammonium sulphate at 35 to 50% saturation, followed by isoelectric focusing in a pH 4.6 gradient. Milligram yields of highly purified α-toxin with 10 to 15% recovery of activity were obtained in single fractions.

Two forms of α-toxin were identified by electrofocusing. The major peak of activity possessed a pI of 5.49±0.06(α A) and the minor, a pI of 5.25(α B). The former was free from detectable collagenase (pI 4.54), hyaluronidase (pI 4.73), θ-toxin (pI 6.56) and neuraminidase. It gave a single precipitin arc on immunoelectrophoresis, but analysis by SDS polyacrylamide disk-gel electrophoresis at high protein-loading revealed a major protein component of molecular weight 53,800 and two minor protein bands. Atomic emission spectro-scopy did not detect the presence of zinc in such preparations. The latter showed a single line of identity with α A in Ouchterlony gel diffusion tests and contained a protein component with the same molecular weight as that of α A.

Fractions α A and α B both possessed hot-cold haemolytic, phospholipase-C, and lethal activities. Both hydrolysed lecithin and sphingomyelin. Electro-focusing of α-toxin in the presence of 6m urea resulted in the detection of only one component, α urea, with a pI identical to α A. It also possessed all three biological activities of α-toxin. Removal of the urea and refocusing was accompanied by the reappearance of α B. The occurrence and formation of α B could not be interpreted in terms of artefactual causes of multiple forms of proteins identified by isoelectric focusing. These studies provided evidence favouring the suggestion that α A and α B could be related as conformers, although aggregation or polymerisation could not be entirely excluded as possible alternative explanations.

Seven strains of streptococci that grow on normal media only as satellite colonies to bacteria and fungi are described. Medium containing pyridoxine hydrochloride in concentration 0.2 µg per ml or greater supported normal growth of all these organisms. One strain grew also on medium containing 10 µg per ml of D-alanine instead of pyridoxine hydrochloride.

Tests for the identification of these strains and determinations of their antibiotic sensitivity were made on media supplemented with pyridoxine hydrochloride.

A concentration of pyridoxine hydrochloride of 1 µg per ml was without effect on the growth and colonial morphology of a series of potentially pathogenic organisms. Antibiotic-sensitivity tests on three selected organisms were likewise unaffected by the compound.

The frequency of occurrence and possible clinical significance of symbiotic streptococci is discussed. It is suggested that pyridoxine hydrochloride can usefully be incorporated in bacteriological media used for routine diagnosis.

One-hundred and one samples from a wide variety of sewers and water-drainage channels in Bristol, including some sewers that separately drained hospital and domestic premises, were examined for antibiotic-resistant coliform bacilli and for R factors. The antibiotic-sensitivity patterns of over 3000 coliform bacilli isolated were obtained and 1000 resistant strains were tested for R-factor transfer.

Hospital sewage contained more coliform bacilli with much higher proportions of resistant bacteria, more R factors, and a greater proportion of R factors carrying multiple resistance, than sewage from domestic and other sources. Despite these findings, it was calculated that less than 5% of the R factors in the sewage output of the City of Bristol originate in hospitals. The normal, healthy population appears to be by far the greatest reservoir of R factors in the community.

Aspergillus fumigatus shows a marked affinity for the bovine placenta in mycotic abortion. Aqueous extracts of foetal tissues and maternal uterus and caruncles from eight pregnant cows enhanced hyphal extension of A. fumigatus in vitro, and the foetal cotyledon extracts were the most active. Extracts of other maternal tissues had low activity with the exception of the spleen. The enhancement appeared to be due to accelerated germination, and the active material was of low molecular weight. It may play a part in the placental localisation in vivo.

A plasmid (PF) has been identified in 18 strains of Staphylococcus aureus that belong to phage-groups I, II, and III or are non-typable. This plasmid is characterised by: (a) carrying the genes for resistance to fusidic acid and cadmium ions, but not to arsenate or mercury ions, and for the production of D-serotype penicillinase, (b) A molecular weight of 16 million daltons, (c) the ability to exist with a “penicillinase plasmid” of compatibility-group I, and (d) a tendency to fragment. The PF plasmid has probably spread among the natural population of staphylococci.

Cell-free extracts of adenovirus-12-induced tumours were used to immunise CBA mice against transplantation of the homologous tumour cells. Greater immunity was obtained when heat-killed Mycobacterium butyricum was added to the tumour extract. Less conclusive results were obtained with Coryne-bacterium parvum. In contrast, tumour cell extract plus Bordetella pertussis, Escherichia coli or Staphylococcus aureus gave significantly less immunity than did the tumour extract alone. Listeria monocytogenes and Candida albicans had no effect on immunity to tumour challenge.

The enhancement or depression of immunity appeared to operate through cellular rather than humoral or antibody mechanisms.

Six hundred and seventy-seven strains of coagulase-negative Micrococcaceae isolated from human and animal sources were classified by Baird-Parker’s (1963) system. The human strains showed a predominance of staphylococci in the main clinical categories, although strains causing primary urinary-tract infections in otherwise healthy women were nearly all allocated to Micrococcus subgroup 3. Organisms isolated from the fur and skin of farm and zoo animals were predominantly micrococci, 170 out of 263 strains being allocated to Micrococcus subgroup 5. The generic separation of staphylococci from micrococci is briefly discussed.

A co-operative study of 28 cultures of Mycobacterium fortuitum by three laboratories, each using different methods, is described. Two agglutination types, four sensitin types, five immunodiffusion types, three lipids types and three biotypes were recognised amongst the strains. In most cases there was good agreement between different means of typing, and the strains could be divided into three groups. Twelve strains, including the type strains of Myco. ranae and Myco. fortuitum, formed a completely homogeneous group as judged by all methods used. A second and slightly less homogeneous group of seven strains included the type cultures of Myco. giae and Myco. peregrinum, and a third group, the least homogeneous, contained nine strains of unnamed organisms. It is suggested that the first two groups might be considered subspecies, Myco. fortuitum fortuitum and Myco. fortuitum giae (peregrinum) respectively.

Stools from 518 patients, suspected of having cholera, were examined. From 174 of these patients V. cholerae biotype eltor, serotype “Inaba” was isolated. All 174 were resistant to Mukerjee’s V. cholerae group-IV phage and to polymyxin B and colistin. They all agglutinated chicken RBC. Only 10 strains were positive by the tube haemolysis test with sheep RBC, but 172 were positive by the plate method. One hundred and twenty of the strains belonged to Mukerjee’s V. eltor phage-type IV and 53 to phage-type V; one strain was untypable. It is suggested that these strains may have originated from more than one source.