- Volume 2, Issue 4, 1969

Problems associated with the use of the lecithovitellin (LV) reaction for typing strains of Clostridium oedematiens have been defined and and investigated. The haemolysins of the various types of the organism were studied with particular reference to their application to serological neutralisation tests. A haemolysin (HL) neutralisation test has been developed with human red cells as the substrate; the test is recommended for use in parallel with the LV test system in typing procedures. The HL test is more sensitive than the LV test and is of particular value in identifying type-B strains, which frequently produce inadequate amounts of the β-antigen for detection in LV tests with unconcentrated culture products.

Thin-layer chromatographic procedures have been applied and extended to differentiate the various soluble products of Cl. oedematiens that contribute to LV activity in culture concentrates. It was confirmed that strains of types A, B and D consistently decompose lecithin; type-D strains are particularly active in this respect. Potent lipolytic activity was demonstrable in culture products of type-A strains. The lipolytic activity of these strains releases free fatty acids from triglyceride substrates in egg yolk and from the diglyceride products of lecithinase-C (phospholipase-C) activity. Diglycerides accumulate in reaction mixtures of type-B and type-D strains, which do not appear to produce a lipase.

The production, stability and partial purification of the lethal α-toxin were studied. A cytopathic factor (CPF I) in culture products of Cl. oedematiens of types A and B is almost certainly the α-antigen. In concentrated preparations of type-D cultures which contain high concentrations of β-antigen, a separate cytopathic effect was demonstrable. The cytopathogenicity test with CPF I provides a convenient in-vitro assay procedure for the α-antigen.

Results obtained with intradermal (Casoni) and complement-fixation tests (CFT) in cases of suspected hydatid disease in the United Kingdom studied over a period of 10 yr are reported, and a reassessment is made of the reliability of each test for diagnosis and prognosis. Because of a number of disadvantages, the Casoni test is not recommended for diagnosing current infection. It is useful, however, for indicating past or present infection in a community, and in these circumstances a potent antigen, capable of being used diluted, should be employed.

The CFT, properly standardised, is shown to be reliable for diagnosis and prognosis and for these purposes it is the test of choice. Sensitivity of the test is enhanced by chessboard titrations and by these means information is obtained relating to the antigenic complexity of hydatid fluids. Studies of CFT results have confirmed that cysts are found most commonly in the liver where they promote the highest titres. From the number of positive results, the greatest incidence of infection appears to be in the Greater London area, but the highest rate of infection acquired in Britain probably occurs in Wales.

Strains of Escherichia coli and Proteus mirabilis that appeared to have similar sensitivity to ampicillin when tested by conventional techniques, behaved differently when dense cultures were exposed to ampicillin and opacity readings were taken. These differences were accompanied by distinctive morphological changes revealed by the stereoscan electron microscope. The morphological changes and the very rapid fall in opacity of Escherichia on exposure to ampicillin suggest that lysis of enterobacteria by penicillins is not wholly due to destruction of osmotically sensitive spheroplasts liberated through defects in the cell wall.

The response of non-swarming strains of Proteus mirabilis to the drug differed from that of swarming strains. The pattern of opacity change was intermediate between that of swarming strains and Escherichia and the morphological response was distinctive.

Morphological evidence is presented of differences in the susceptibility of individual cells within a bacterial population. It is suggested that such intrapopulation differences in various lethal and non-lethal effects may account for the observed differences in response to ampicillin.

Some methicillin-resistant cultures of Staphylococcus aureus give equivocal results when tested in disk diffusion tests or tube dilution MIC tests incubated at 37°C.

Practically all resistant cultures have the following characters: (1) they give growth up to or to within 1 mm of a 10 μg methicillin disk in a disk diffusion test on nutrient agar or blood agar incubated at 30°C, and (2) they show a MIC of 25 μg methicillin per ml or greater after incubation for 18 hr at 30°C, or for 48 hr at 37°C, in a tube dilution test in nutrient broth.

Tests for growth at 37°C on methicillin salt agar, or on salt agar with a methicillin disk, are also generally reliable, but some batches of methicillin salt agar are relatively inhibitory for resistant staphylococci.

Routine sensitivity tests with cloxacillin give unsatisfactory results.

Sh. sonnei resistant to kanamycin in one household and, in another family, to nalidixic acid, is described. Resistance of this organism to these drugs is uncommon. Some individuals within each family excreted sensitive strains of the shigella. There was no history of previous exposure to either drug and the source of the resistant strains is unknown. As far as we know these strains neither arose from nor spread to the community. Kanamycin-resistance was determined by a transferable factor; no transfer of nalidixic acid-resistance could be demonstrated. The supplanting of the prevailing sensitive strains by new drug-resistant strains of Sh. sonnei is unexplained in both families.

Six patients with homograft aortic valves who developed endocarditis due to infection with Candida albicans or C. parapsilosis were found to have serum precipitins and high titres of agglutinins to several species of Candida. In 14 of 62 other patients undergoing open-heart surgery, a similar serological pattern developed in the 2nd wk after operation, suggesting the possibility that otherwise undetected candida infection was present. A further 13 of the 62 patients developed antibodies to a single species of Candida. Such patients, especially those with homograft valve replacements, should clearly be watched carefully for infective complications.

The technique of performing the Dienes test is described, as well as its application to the investigation of an outbreak of cross-infection with Proteus mirabilis. The test was found to possess a high degree of specificity. The frequency of reactions of identity among 48 proteus isolates obtained from unrelated sources and tested against themselves was 1 in 70, based on a total number of 1128 tests. These 48 isolates could be grouped into 35 “Dienes strains”

Compatible reactions were obtained between strains possessing major H-antigenic differences. This finding indicates that the relation between H-antigenic structure and inhibition in the Dienes test is probably not as close as has been previously suggested.

In a study of children attending hospital for up to 2 yr, strains of proteus repeatedly isolated from a child were almost always compatible with each other in the Dienes test. This indicates that each child retained his or her own strain and that the strains behaved consistently in relation to the Dienes test during the period of the study.

It is concluded that the specificity, reproducibility and simplicity of the Dienes test make it of great value for the detection of cross-infection with proteus, particularly in an ordinary hospital laboratory. The suggestion is made that the Dienes phenomenon could be due to colicine-like substances and that it could form the basis of a typing system.

Strains of Staphylococcus aureus isolated from toxic epidermal necrolysis and Ritter’s disease (named collectively “Ritter’s type” of TEN) and from extensive cases of impetigo contagiosa, in which the diagnosis of Ritter’s type of TEN was considered seriously, were assigned to group E (extensive lesions) and were compared with strains of S. aureus isolated from unequivocal impetigo contagiosa (group L—localised lesions); 24 of the 26 isolates examined belonged to phage-group II.

Strains from groups E and L were generally similar in their patterns of proteolytic activity, low incidence of positive egg-yolk reaction, possession of lipase and production of hyaluronidase. The difference lay in the many isolates in group E that produced a low-titre lysin active against rabbit red cells that was not α-haemolysin. The average titre of lysin active against rabbit erythrocytes in group E was 355 MHD per mi and in group L 1008 MHD per mi. Reasons are given for supposing that this low titre rabbit cell lysin is identical with staphylococcal δ-toxin. It is suggested that δ-toxin may play a part in the pathogenesis of Ritter’s type of TEN and extensive impetigo.

Receptor activity for the porcine enterovirus strains Talfan and V13 was found in homogenates of various tissues prepared from each of 20 piglets. Homogenates of the same tissues from 5 calves did not adsorb Talfan virus, but high adsorptive capacity for V13 virus was present in calf ileum, caecum and colon. Coxsackie B1 virus was adsorbed by homogenates of piglet and calf liver, cerebral cortex and cardiac muscle, and by piglet ileum, spleen and spinal cord. The adsorptive capacity of the homogenates was removed by ether treatment or centrifugation.

Small amounts of purified staphylococcus α-toxin caused an immediate increase in the rate of succinate oxidation by Krebs 2 mouse ascites tumour cells. This effect appeared to be specific for α-toxin and not to be due to contamination of the α-toxin preparation with staphylococcus δ-toxin. During interaction with Krebs 2 cells, the haemolytic activity of α-toxin decreased by more than 95 per cent. Streptolysin-S preparations also caused increased oxidation of succinate, but only when washed cells were pretreated with toxin. These effects can be explained by postulating an action of the toxins on the cell membrane that leads to increased permeability to succinate. The different actions of staphylococcus α-toxin and streptolysin S are discussed.

Colicines of group B produced by antigenically distinct strains of Escherichia coli may themselves be distinguished one from another antigenically. On the evidence from agglutination and precipitation experiments the antigenic behaviour of different B-colicines cannot be distinguished from that attributed to O-antigens of the bacteria producing them in culture. Attempts to separate individual factors for these antigenic and bactericidal properties were unsuccessful.

Most of a series of 16 conventional rats examined at various ages were found to have Streptobacillus moniliformis and Mycoplasma pulmonis in the nasopharynx and sometimes also in the lungs. Fifteen of the rats had histological evidence of chronic respiratory disease (CRD). Specific-pathogen-free (SPF) rats of the same strain, but kept in isolation, did not appear to carry these infections and had no histological evidence of CRD.

It was not possible to produce CRD in SPF rats by the intranasal inoculation of S. moniliformis, M. pulmonis or a mixture of the two. In contrast with this, lung homogenate from a naturally infected rat readily produced gross lesions of CRD in SPF rats, but the antibody titres to S. moniliformis and M. pulmonis remained low. The rats that had been given an inoculation of S. moniliformis showed little evidence of CRD, but had high antibody titres, and antibodies to the streptobacillus appeared in their progeny, which were in direct contact with their infected parents during a period of 7 mth. Inoculation of M. pulmonis failed to produce a significant antibody response.

Cortisone treatment of SPF rats given an inoculation of CRD agent without S. moniliformis and M. pulmonis promoted goblet-cell proliferation, but otherwise did not activate CRD. As cortisone activates CRD in naturally infected non-SPF rats, these findings suggest that the antibody response mechanism of SPF rats may be different from that of conventional rats.

Our findings suggest that neither S. moniliformis nor M. pulmonis plays an important part in the causation of CRD; the causal agent in the infective lung homogenate was not identified.

Cultures of Pseudomonas aeruginosa in milk were ingested by three normal people. The organisms were recovered from the faeces if 106 or more were ingested. They were present in the greatest number in the first specimen passed, provided this was 12 hr or more after administration of the organisms. The numbers excreted were less than those ingested and excretion did not continue longer than 6 days. Colonisation was achieved on one occasion when ampicillin was also administered.

T-mycoplasmas were isolated from the semen of 23 of 28 bulls at an A.I. Centre, and from the semen of each of 4 bulls on separate farms. Washings from the preputial cavities of 10 bulls each contained at least 105 organisms per ml. In all, T-mycoplasmas were isolated from either semen or the preputial cavities of 28 of 34 bulls examined. Although the organisms were isolated from semen, they were not isolated from testicular tissue, the vas deferens or the mucosal scrapings of the urethra of 2 slaughtered bulls. It seems that the mycoplasmas are confined to the preputial cavity and gain access to semen during ejaculation. It is suggested that the failure of the organisms to become established in other areas of the genital tract is due to the presence of a potent mycoplasma inhibitor that was detected in bull semen in this study. A similar inhibitor was found in bull serum. It is thermostable at 56°C and nondialysable. The ecology and pathological role of the T-mycoplasmas in cattle are discussed in relation to the findings in man.

Antigens that determine agglutination reactions, and are distinct from the O-, H- and Vi-antigens, were demonstrated in the type-1 fimbriae of bacteria in fimbriate-phase cultures of salmonellae. Most strains of salmonellae produced fimbriate cultures when grown for a sufficient period, e.g., 24–48 hr, at 37°C in aerobic static broth. The same strains produced non-fimbriate-phase cultures lacking fimbrial antigens when grown for only 6 hr in broth, for 12 hr in glucose broth or for 24 hr on an agar plate. The independence of the fimbrial antigens from the O-, H- and Vi-antigens was shown by the finding that an antiserum raised against a fimbriate-phase culture and freed from O-, H- and other non-fimbrial agglutinins by absorption with non-fimbriate-phase bacteria (“pure fimbrial antiserum”) strongly agglutinated fimbriate-phase bacteria, but did not agglutinate either non-fimbriate-phase bacteria or fimbriate bacteria that had been defimbriated by heating at 100°C.

Crude (unabsorbed) and pure (absorbed) fimbrial antisera to strains of several salmonella serotypes agglutinated fimbriate-phase bacteria of a wide variety of heterologous serotypes. The reactions indicated the presence of a common fimbrial antigen in all of 95 fimbriate strains in 79 serotypes of Salmonella and two strains each of Arizona and Citrobacter. Absorption findings suggested that other fimbrial antigens were present in addition to the common one in strains of certain serotypes, but that the same antigens were present in different strains of the same serotype. A common fimbrial antigen was demonstrated in strains of Shigella flexneri, Escherichia coli and Klebsiella aerogenes, but there was no sharing of antigens between this group of bacteria and the salmonella-arizona-citrobacter group.

Misleading cross-reactions may be obtained in diagnostic agglutination tests unless only non-fimbriate bacteria are used for the preparation of agglutinable suspensions and the production of diagnostic antisera. The development of cross-reacting fimbrial agglutinins in persons who have been infected with a salmonella or immunised with a TAB vaccine containing fimbrial antigens may lead to false-positive reactions being obtained in Widal tests.

A simple technique is described in which common laboratory reagents are used to determine the titre of anti-human-globulin-conjugates for use with a given lighting system.

V-dependent growth of Haemophilus paraphrophilus, which Zinnemann et al. (1968) found to take place only when cultures were incubated in air with 5–10 per cent. added CO2, or on a medium containing 0.6 per cent. NaCl, also takes place in the absence of either of these conditions if the cultures are incubated in a closed, moist atmosphere, as in a sealed plate or a sealed jar.

The findings here described do not provide arguments against recognition of the species Haemophilus paraphrophilus, since under conditions of normal humidity and air supply the unadapted, recently isolated strains grow sparsely, or not at all. Typical coccobacillary rods are formed only when cultures are grown in an atmosphere with added CO2 as well as an increased moisture content.

Fifteen per cent. of single serum samples from patients with chronic pulmonary tuberculosis with cavitation contained antibodies reacting with human and rabbit liver, lung and kidney homogenates. Some of the positive sera also gave false-positive reactions in Rose-Waaler tests and in tests for syphilis; a few contained weak anti-nuclear antibodies.