- Volume 9, Issue 2, 1976

Continuous study, at intervals of 1 h, on the advancing edge of the swarm of Proteus vulgaris confirms that this is almost permanently composed of elongated swarmers, and that short, non-swarming forms arise in the interior of the culture where motion has already ceased. Previous errors have probably arisen from inaccurate sampling.

The occurrence of coliform bacilli carrying resistance-transfer factors (R factors) in children was studied. The frequency of R+ coliform bacilli as causes of urinary-tract infection acquired outside hospital was found to be similar to that in adults from the same geographical area and in the same years. The frequency of R+ coliform bacilli in the faeces in our children was also similar to that in the adult population, and oral chemotherapy produced similar changes in the faecal flora.

Meningococci of serogroups Z1 and 29E were examined serologically and shown to be identical. These meningococci should be designated either as group Z1. which has priority, or preferably by a new designation forming a logical sequence with the currently accepted serogroups.

Previous observers showed that many strains of Staphylococcus aureus stimulated the growth of Bordetellapertussis but we have found the reverse: the growth of all available strains of B. pertussis on charcoal-agar medium was inhibited by a standard strain of S. aureus; and 17 of 18 strains of S. aureus (as well as several other organisms) inhibited the growth of a standard strain of B. pertussis. All inhibiting colonies had an unusual brown colouration on the charcoal agar used in the investigation. Both the brown colouration and the inhibitory property were caused by acid production, probably from the starch in the medium. We therefore suggest that media containing starch and blood should not be used in studies of bacterial interference.

Clostridium difficile can be grown readily in Reinforced Clostridial Medium (RCM) containing 0·1-0·4% of o-, m- or p-cresol, or phenol. We recommend 0·2% of phenol or p-cresol in RCM for the isolation of this organism.

The characteristic “ cornfield ” growth in RCM in 25-ml Universal containers is described.

Glucose, fructose, galactose, mannose, raffinose, aesculin and mannitol are fermented with production of acid and gas; maltose, sucrose, glycogen, soluble starch and sorbitol are fermented with production of acid only. Lactose and rice starch are not fermented by any strain, and DL-methionine is not attacked. Nitrate is reduced to nitrite. Hydrogen sulphide and indole are not produced. Gelatin is attacked by all strains, but in some cases prolonged incubation is required. Hyaluronidase is produced, but not deoxyribonuclease. A lethal toxin appears to be produced.

Strains possess shared and strain-specific antigens.

A genetic analysis of resistance to antibiotics in methicillin-resistant Staphylococcus aureus was performed. Demonstration of plasmid-specific DNA either in transductants that had received antibiotic-resistance markers from multiply-resistant strains, or in segregants of methicillin-resistant strains that had lost unstable determinants except the one under study, indicated that markers of resistance to penicillin, chloramphenicol and neomycin are present on separate, mutually compatible plasmids. Absence of covalently closed circular DNA was demonstrated in transductants that were resistant to methicillin, tetracycline, erythromycin and streptomycin, as well as in segregants that had lost the penicillinase, chloramphenicol and neomycin plasmid, but were still resistant to methicillin, tetracycline, erythromycin, streptomycin and the sulphonamides. Analysis of plasmid DNA either in a 5-20% neutral sucrose gradient or by electron microscopy revealed the presence of three readily distinguishable plasmids. The molecular weights of these plasmids were estimated by comparing the sedimentation rate constants with those of known reference plasmids and by contour-length measurements. The molecular weight of the penicillinase plasmid was estimated to be 20 x 106 daltons, that of the chloramphenicol plasmid 3 x 106 daltons and that of the plasmid carrying the neomycin resistance marker 37 x 106 daltons.

To study the migration of Leptospira interrogans serotype pomona through the kidney, conventionally-reared mice aged 2 or 3 weeks were infected intraperitoneally with this organism. Within the first 4 days, the organisms migrated from the capillary lumina to the interstitial tissue and provoked an interstitial oedema. By the 10th day they were seen between the epithelial cells of the proximal convoluted tubules and by the 14th day many were located within tubular lumina. There was no evidence of viable leptospires within the cells of the proximal tubules, though occasionally structures resembling leptospiral fragments inside lysosomes were observed. At no stage during the study were glomerular lesions seen.

The interactions of a contractile, a filamentous and a small pyocine with a sensitive strain of Pseudomonas aeruginosa (no. P14) were examined in vivo. The purification procedure used yielded high-activity pyocine preparations that were not toxic to mice. The inhibitory activity of such preparations, when injected into mice by various routes, was retained for up to 24 h. However, high molecular-weight pyocines given intraperitoneally in the presence of a lethal dose of strain PI4 administered by the same route did not prevent the fatal outcome of infection unless they are given before or together with the bacteria. The small pyocine had no protective effect.

In burned mice infected with strain PI4, topical application of a filamentous pyocine failed to improve the chances of survival.

The results suggest that there is little future for pyocine therapy.

Catalase-negative vibrios can be isolated in large numbers from the affected intestinal mucosa of pigs suffering from a range of porcine enteropathies in which the mucosa has an adenomatous component. These vibrios cannot be distinguished from strains of Campylobacter sputorum subsp. mucosalis. The resemblance between these bacteria strengthens the case already made on morphological evidence that these enteropathies have a common origin.

Catalase-negative vibrios have also been isolated from the mouth and faeces of pigs. Some of these conform to the criteria established for the mucosalis subspecies but others can be differentiated from it.

An antigenic analysis shows that strains of the mucosalis subspecies are closely related antigenically, but that differences may allow separation of strains.

Synthesis of α-haemolysin by Escherichia coli was proportional to the amount of meat-broth factor present in the medium and not to bacterial growth.

The meat-broth component required for the synthesis of haemolysin was found to have several physical and chemical properties in common with α-haemolysin itself. Both molecules are trypsin-sensitive, acidic substances with similar elution volumes when subjected to gel-filtration chromatography with Sepharose 6B.

Isotopic labelling experiments in which the bacteria were grown on 14C-labelled meat broth showed that partially purified haemolysin preparations contained molecules of mammalian origin. Similar experiments in which 14C-glucose was used to label bacterial molecules showed that little or no material of bacterial origin was present in the haemolysin preparation.

These data suggest that α-haemolysin may be produced by bacterial modification of a molecule present in meat broth rather than by synthesis de novo within the bacterial cell.

Concentrated supernates of cultures of 98 strains of Staphylococcus aureus were screened for the production of epidermolytic toxin by (1) biological tests in 3-day-old mice, (2) double-diffusion precipitation tests against specific antiserum, and (3) the appearance of characteristic protein bands on thin-layer-gel isoelectric focusing. Positive results were obtained in all three of these tests with supernates from 11 of these cultures; the same 11 strains, and no others, produced epidermal splitting when newborn mice were challenged with viable organisms. Of the 14 phage-group-II strains included in the survey, eight (58 %) produced epidermolytic toxin. Three toxinogenic strains belonged to phage groups other than group II.

A radial-immunodiffusion test employing antiserum to purified epidermolytic toxin proved satisfactory for measuring amounts of epidermolytic toxin in excess of 200 εg per ml. The results of immunodiffusion tests indicated that six of the 11 positive strains produced two serologically distinct forms of epidermolytic toxin and that the remainder produced only one of these. A striking correlation was observed between the presence of toxin of serotypes i and ii and the occurrence of protein bands i and ii in thin-layer isoelectric-focusing gels.

These tests should facilitate the qualitative and quantitative assessment of the production of different serotypes of epidermolytic toxin by S. aureus in future surveys of strains isolated from toxic epidermal necrolysis of Ritter's type and impetigo.

The two forms of epidermolytic toxin previously designated ETA (pI = 7-0) and ETB (pI = 6η0) were detected by preparative isoelectric focusing of sero-type-i toxin. Evidence suggests that studies of the effect of heat should provide a means of investigating the relationship between the different molecular forms of epidermolytic toxin.

Isolations of reovirus-like agents (rotaviruses) were made from nine of 23 outbreaks of piglet diarrhoea on different farms and from both weaned and unweaned piglets. The viruses were shown to be morphologically and anti-genically similar to the rotaviruses of children and calves. Gnotobiotic piglets given intranasal inoculations of five different isolates developed acute gastroenteritis, and the virus was re-isolated from the faeces or intestinal contents. The piglet virus was not adapted to replicate in cell culture. We conclude that the pig rotavirus is commonly associated with outbreaks of gastroenteritis and is probably an important aetiological factor in this disease.

Tests with 140 strains representing Escherichia coli, Klebsiella aerogenes, K. ozaenae, Proteus mirabilis, P. vulgaris, P. rettgeri and P. morganii in a defined medium supplemented with 0·09m dimethylamine (DMA) and 0·1m potassium nitrate showed that at least 89% of the 136 strains able to reduce nitrates produced up to 9mM dimethylnitrosamine (DMN) in 70 h at 37°C. Four nitratase-negative strains produced DMN from DMA in the presence of sodium nitrate. Prolonged incubation was the most important factor in determining DMN production. Stasis and persistent infection in the urinary tract, by simulating prolonged incubation of a culture, may be of importance in determining whether the potential carcinogen, DMN, could be produced in vivo by bacterial action on DMA and nitrate in urine.