- Volume 3, Issue 1, 1970

The course of infection after the intracerebral inoculation of high-virulence and low-virulence strains of Bordetella pertussis into mice was followed by making serial viable counts of the number of bacteria in the brain.

With all strains, some 90 per cent. of the inoculum was lost within 1 hr, but by 24 hr the number of bacteria in the brain reached or exceeded the number inoculated.

When small doses (c. 5 X 103) of high-virulence strains were given, the bacterial count increased continuously until death. With one strain (no. G353) there was a pause in multiplication when the viable count reached 106, but this was not seen with 2 other high-virulence strains (no. 18/323 and 2-Atox).

Challenge doses of low-virulence strains smaller than 105 organisms gave rise to a transient infection, the maximum count was reached on the 1st or 2nd day, and most brains examined after 4 days contained less than 50 Bord. pertussis bacteria.

Resistance of the mouse brain to low-virulence strains was not due to the action of mouse complement or to the occurrence of modulation. The intracerebral injection of a small dose of a low-virulence strain made the mouse brain resistant to reinfection 4 1/2 days later by a low-virulence but not by a high-virulence strain.

When the interval between injections was reduced to 2 days, however, mice infected with a low-virulence strain were somewhat resistant to reinfection with a high-virulence strain.

Intracerebral infection of mice with low-virulence strains of Bordetella pertussis was greatly enhanced by the prior administration of cortisone acetate subcutaneously or of vitamin-A alcohol or streptolysin O intracerebrally.

The infections did not appear to stimulate the release of lysosomal enzymes in the brain.

When normal mice were X-irradiated to the head, with the body shielded, subsequent intracerebral infection with low-virulence strains was very much enhanced. Irradiation of the head alone did not affect the survival of actively immunised mice after the intracerebral injection of either high-virulence or low-virulence strains, but irradiation of the body, with the head shielded, significantly reduced the number of survivors after infection with a high-virulence strain.

It appears that a cerebral glial response controls intracerebral infections with low-virulence strains in normal mice, but that circulating leucocytes play a major role in controlling infections with high-virulence strains in immunised mice.

The main types of phospholipids present in erythrocytes, namely lysolecithin, lecithin, phosphatidyl ethanolamine, phosphatidyl serine and sphingomyelin, were found to be degraded by enzymes produced by parasitic and saprophytic leptospirae. No basic difference was found between the degradation of the phospholipids bound in serum lipoproteins and that of the phospholipids bound in the membranes of erythrocytes. The parasitic leptospirae were differentiated into two groups according to their ability to degrade the choline-containing phospholipids: (1) strains degrading lecithin and sphingomyelin, and (2) strains degrading neither lecithin nor sphingomyelin. The saprophytic leptospirae degraded lecithin but not sphingomyelin.

The activity of the enzymes of the different strains of leptospirae in degrading phospholipids was correlated with the haemolytic activity of the strains. Calcium ions were found to activate both the degradation of lecithin, phosphatidyl ethanolamine and phosphatidyl serine by saprophytic leptospirae and the haemolysis caused by these leptospirae. Calcium ions did not activate the degradation of erythrocyte phospholipids and the haemolysis caused by the parasitic leptospirae.

These findings suggest (1) that the haemolytic action of leptospiral haemolysin is due to an enzymatic degradation of phospholipids in the erythrocyte membrane, (2) that the “haemolysin” of leptospirae consists of more than one enzyme (phospholipase), (3) that the enzymes constituting the “haemolysin” of the parasitic leptospirae differ from those constituting that of the saprophytic leptospirae, and (4) that two main factors determine the susceptibility of erythrocytes from different animal species to the haemolytic action of leptospirae, namely, the enzyme composition of the haemolysin and the phospholipid composition of the erythrocyte membrane.

Over a period of 20 mth all the patients with tracheostomy in an intensive care unit for major injuries were examined frequently, often daily, for the presence of Pseudomonas aeruginosa in the tracheal mucus. During the same period the hands of staff and the inanimate environment in the Unit were examined weekly for the presence of Ps. aeruginosa. Numerous isolates of Ps. aeruginosa from these sources were typed by phage and serological methods.

Eight outbreaks occurred in which patients had infection with Ps. aeruginosa in the trachea, and there were seven periods between outbreaks when Ps. aeruginosa was not found in samples from tracheostomies. Strains of Ps. aeruginosa isolated in successive outbreaks were of different types. Strains isolated from the environment were usually of types already present in tracheostomies. More rarely strains of types already present in the environment were subsequently found in tracheostomies. There was no endemic infecting strain in patients, though a single type was isolated from sinks, as well as strains of other types that were transient, throughout almost the whole period of the study. This strain from sinks caused infection in patients during four of the eight outbreaks, but was not the first strain to cause infection except in the first outbreak.

Of the numerous sources from which Ps. aeruginosa was isolated, and from which patients might become infected, the hands of nurses, physiotherapists and other members of staff seemed to present a special hazard of transferring infection. Other potentially important sources were plastic washing bowls, food and food mixers, and suction apparatus. Many sites commonly associated with Ps. aeruginosa contamination, e.g., nailbrushes, dishcloths, floors and floor mops, sinks and sink mops, were found to be contaminated. Samples of air occasionally yielded a few Ps. aeruginosa. Respiratory ventilators were used on some of the patients who became infected with Ps. aeruginosa, but the evidence suggested that these were not an important source of infection. Blower-humidifiers sometimes became contaminated, but experimental study of this equipment suggested that it was not a likely source of infection. Self-infection may have occurred in one patient.

A number of recommendations were made, including the use of disposable plastic gloves for a wide range of nursing procedures, the supply of individual plastic washing bowls disinfected daily, and improvements in the arrangement for suction. Sometimes infection seemed to be due to lapses in technique caused by ignorance or by pressure of work. Prevention of infection of tracheostomies remains a challenge to vigilance and discipline.

An investigation of the incidence of erythrasma in a healthy young adult population is described. The axillae, groins and toe-webs were examined and clinical erythrasma, showing characteristic coral-red fluorescence under Wood’s light, was found in 19 per cent. of the 754 students, with lesions occurring mainly in the toe-webs. A diphtheroid organism, fluorescing when grown on tissue culture medium no. 199, could be cultured from 42 per cent. of the fluorescent skin areas and from 13 per cent. of apparently healthy, non-fluorescing sites. These organisms were divided into 8 groups on the basis of their reactions in 8 biochemical tests. There did not appear to be one particular group involved with clinical lesions, nor was any one strain predominant among the students and staff of the different colleges visited.

Chickens given an intravenous injection of human serum albumin (HSA) produced a rapid rise of antibody to a peak at 8–9 days, followed by a rapid decline. Plasma cells containing anti-HSA appeared in the spleen at 24 hr after injection. Evidence is presented that early produced antibody causes the localisation of antigen (HSA), presumably as complexes, to cells in the white pulp of the spleen at the periphery of ellipsoids (Schweigger-Seidel sheaths) by 24–32 hr. Inspection of a series of tissue sections shows that antigen-bearing cells appear to migrate through the white pulp and subsequently to appear (88 hr–6 days) as dendritic cells within germinal centres, which originated in the angle between the diverging penicillary arterioles at their point of origin from the central arteriole of the white pulp. A hypothesis is advanced that accounts for the formation of germinal centres by a process of progressive capture and aggregation of lymphocytes at the surface of antigen-bearing dendritic cells. This process is discussed in relation to the progressive increase of avidity in antibody synthesised later in the immunological response or after a secondary stimulus.

Between 1953 and 1968, 42 of 3384 women attending a subfertility clinic were found to have genital tract tuberculosis.

The examination of endometrial biopsies by guinea-pig inoculation was the most reliable method of detecting tuberculous endometritis, and was more sensitive than histological and cultural methods. Sixteen of 31 patients would not have been identified without the use of animal inoculation.

The tuberculin test was a useful preliminary for those patients who required a diagnostic endometrial examination.

Patients with a history of primary tuberculosis in early adult life or of pneumonia complicated by pleurisy, effusion or empyema were significantly more likely to have genital tract tuberculosis than were women without such a history. The past and family histories, however, gave no clue to the diagnosis in nearly half of the patients with pelvic tuberculosis.

The isolation of Cardiobacterium hominis from two patients with bacterial endocarditis is reported, and the clinical features are described.

These strains of C. hominis produced small, shiny, convex colonies visible within a week on first isolation. After further subcultures, colonies were visible within 2 days. Isolation was achieved on blood or chocolate media with added CO2. When examined later the strains did not require CO2, but growth was improved by moisture.

They gave a positive oxidase reaction and produced indole, but did not produce catalase or reduce nitrates; this pattern of reactions should serve to distinguish them from strains of Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans and Streptobacillus moniliformis.

Fluid contaminated with fungi was inadvertently administered intravenously to two patients. In both instances the bottle was cracked. However, apparently intact bottles can also show visible signs of growth. Both patients were treated with amphotericin B in a dose of 0.25 mg per kg body weight for 24 hr without toxic effects. There was complete recovery. It is suggested that all bottles used for intravenous therapy should be carefully inspected before use.

Many chemical compounds exert selective toxicity within a species of microorganism. This fact can be used to define a profile of a strain based on its resistance to selected compounds: the profile of the strain is referred to as its “resistogram”. The compounds are so chosen that the organisms do not meet them under natural circumstances. Therefore, unlike the antibiotics, the compounds do not lend selective advantage to resistant elements within the population and for epidemiological purposes the resistogram type would be expected to remain stable.

The potential value of the new approach is illustrated by its application to the species Escherichia coli. A simple and rapid method of resistogram typing based on eight substances was evolved. This was applied to the study of 50 strains from urinary infections. In these strains somatic serotype and source were known, giving an indication of the extent of their diversity. Good agreement was found between serotyping and resistogram typing. In a few instances different strains fell into a single resistogram type, but in other cases strains of the same serotype from different patients could be distinguished by means of the resistogram.

This paper describes the isolation and characterisation of a new mycoplasma species for which the name Mycoplasma dispar is proposed. Many strains of this glucose-fermenting organism were isolated in an enriched glucose calf-serum medium from pneumonic lungs of calves, but not from healthy lungs of calves or cows. It could be isolated in medium free of known bacterial inhibitors, and when subcultured in this medium showed no evidence of reversion to bacterial forms.

The general characteristics of these strains were those of mycoplasmas; these included cultural and metabolic characters, morphological appearance in stained films and electron micrographs, growth inhibition by specific antiserum and a DNA base composition of 28.5 per cent. GC. However, the group showed some unusual features, including failure to pass readily through membrane filters of 450 nm pore diameter, poor growth in conventional mycoplasma media and production of atypical colonies on GS agar medium.

The serological identity of strains classified as M. dispar was demonstrated by growth and metabolic inhibition tests. They were serologically distinct from other recognised bovine mycoplasmas and from a wide range of mycoplasmas from other sources.

Ninety-six penicillinase-producing strains of Staphylococcus aureus, all members of the phage-groups I and III, were examined for sensitivity to mercury, cadmium and arsenate.

Strains resistant to mercury, arsenate and cadmium were nearly always multiple-antibiotic resistant and produced large amounts of A-type penicillinase, much of it extracellular. They included representatives of a few widespread types of “hospital” staphylococci.

Other strains, that were sensitive to mercury but resistant to arsenate and cadmium appeared, though less frequently, to occur endemically in hospitals; they were generally sensitive to antibiotics other than penicillin and although they produced variable amounts of penicillinase, much of it was extracellular. Their penicillinase was either of the A-type or the C-type; in strains with A-type penicillinase the determinants for resistance to arsenate and cadmium appeared to be located on the penicillinase plasmid, but in strains with C-type penicillinase they appeared to be chromosomal. Penicillin-negative variants appeared relatively frequently in all but one of the cultures, suggesting that the genetic determinant for penicillinase production was on a plasmid; in one culture, however, there was evidence that it was chromosomal. Resistance to other antibiotics was not genetically linked with penicillinase production.

Fourteen serologically distinct mycoplasmas from various avian and mammalian sources were stored frozen in liquid medium. At -70°C, the number of viable organisms of only three of these mycoplasmas decreased by 1 log10 after 3 yr. Four of the same mycoplasmas of human origin were stored at -50°C and the number of viable organisms remained undiminished after 2 yr. On the other hand, at -30°C the viability titres of 8 of 11 mycoplasmas decreased by at least 1 log10, but by no more than 2 log10 after a similar period of time. Lyophilisation caused an immediate decrease in the number of viable organisms of most but not all mycoplasmas so treated. The addition of several “stabilising” fluids, used in the lyophilisation of viruses and bacteria, was not helpful. These had no immediate protective effect and some of them rapidly killed the organisms during subsequent storage. Cultures of ten mycoplasmas in liquid medium containing 2 per cent. bovine plasma albumin were lyophilised and samples of each lyophilised culture were stored in parallel at 37°C and at 4°C. They all contained viable organisms after storage at 37°C for 15–18 mth; of 7 cultures tested after 27–34 mth, 4 of them still contained viable organisms. All the lyophilised cultures stored at 4°C contained viable organisms, seven of them in undiminished numbers, after 27–34 mth.

A coagulase-positive strain of Staphylococcus aureus grown in vitro (strain O) was passaged in rabbits to increase virulence. The passaged organisms grown in vitro (strain P) were used to infect rabbits from which organisms grown in vivo (strain V) were isolated.

By LD50 measurement in rabbits, strain V was 25 times more virulent than strain P and 6000 times more virulent than strain O. On incubation in rabbit serum, strain V multiplied whilst strains P and O decreased in viable number (strain O the more rapidly) during the first 3 hr, recovering subsequently. In lysates of polymorphonuclear leucocytes, strain V multiplied or was slightly reduced in number; strains P and O decreased in number (strain O the more rapidly).

The findings suggest that resistance to serum and polymorph bactericidins is an important attribute of the virulence of staphylococci and that the substances responsible are qualitatively different or quantitatively increased on growth in vivo.

The resistance to phagocytic uptake and intracellular killing of staphylococci grown in vivo and in vitro has been compared. Strains O (original) and P (rabbit passaged) grown in vitro and strain V (isolated directly from rabbits infected with strain P) did not differ in their susceptibility to ingestion by polymorphs. Intracellular killing was most marked in the 1st hr after phagocytosis; survival of strain V was substantially greater than that of strains O and P. This is in agreement with earlier observations on the resistance of these strains to the bactericidins of polymorph lysates. In contrast to behaviour in lysates, strain P was not significantly different from strain O in resistance to phagocytic killing. It is suggested that the virulence difference between strains P and O depends on the properties of the phenotypic form that each assumes in vivo.

In mixed culture experiments, great variation was found between different strains of Escherichia coli and salmonellae in their ability to transmit or to receive antibiotic resistance (R factors).

Resistance transmission was demonstrated to occur in the alimentary tract of chicken given strains by mouth, but only between strains that were known to be good donors and recipients in vitro and able to colonise the alimentary tract reasonably well. Sometimes, the R factors appeared to infect the recipient organisms in the alimentary tract in epidemic form. Some strains of S. typhimurium, including one of phage-type 29, were outstandingly good recipients in vivo; organisms of these strains that had received R factors were not uncommonly found in the liver as well as in the faeces of experimental chicken. It was not necessary to give each chick both the prospective donor and the prospective recipient strain. For example, transfer occurred in chicken that were mixed together after some had been given a prospective donor strain of E. coli and others a prospective recipient strain of S. typhimurium.

During transfer studies in chicken, feeding with a diet containing an antibiotic that featured in the transmissible resistance pattern of the prospective donor strain occasionally had a suppressive effect on the numbers of prospective recipient organisms in the faeces. Usually, however, it led to a great increase in the proportion of R-factor-containing recipient organisms, both in the faeces and in the liver. Chloramphenicol did not appear to influence the situation. This was because the drug is absorbed in the anterior part of the alimentary tract and well before the caeca, the organs in which resistance transfer was found to occur.

Resistance transfer from E. coli to S. typhimurium was also demonstrated to occur in the alimentary tract of calves but not of pigs. Poor colonisation of the alimentary tract by S. typhimurium was the probable reason for the failure in pigs; transfer from E. coli to E. coli, however, was demonstrated in these animals.

The results are discussed with particular reference to the high incidence of antibiotic-resistant strains of S. typhimurium in intensively reared calves.

Of 167 strains of salmonellae isolated from poultry in England and Wales in 1968, 7 (4.2 per cent.) were resistant to antibiotics; 3 strains were resistant to tetracyclines, streptomycin and sulphonamides, 2 to streptomycin and sulphonamides, and 2 to tetracyclines; they were all sensitive to ampicillin, neomycin, chloramphenicol, furazolidone and nalidixic acid. The incidence of resistance amongst the Salmonella typhimurium strains examined was 10.9 per cent. and 0.9 per cent. amongst the other strains. All the resistance was of the transmissible type.

A small minority of Staphylococcus aureus strains produce a substance referred to as “aureocin”. It appears to inhibit all coagulase-positive staphylococci, unless they themselves produce this substance.

I should like to thank Dr W. Shepherd for his support and encouragement and Mrs H. D. Landau for phage typing the staphylococci involved.