- Volume 12, Issue 1, 1979

A collection of 39 staphylococcal strains of known peptidoglycan structure was cultivated on media supplemented with different peptones or with glycine or inosine. The susceptibility to lysostaphin 50 mg/litre was then checked by a plate-dilution method. The results of the test were dependent on the conditions of growth.

Another collection of 403 staphylococcal strains from international collections, clinical material and healthy human skin was also tested for susceptibility to lysostaphin. No uniform pattern of the lysostaphin sensitivity in individual staphylococcal species was found.

The lysostaphin-susceptibility test thus seems to be of little value in the classification of staphylococci for the purposes of medical microbiology.

The enhancement by exogenous ferric iron, both systemic and local, of the infectivity of 120 strains of bacteria, representing 17 genera, was measured in the skin of guinea-pigs. Systemic iron enhanced only 23% of 115 strains, and local iron 49% of 71 strains. Systemic iron, by an apparently anti-inflammatory action, depressed the size of lesions produced by 27 of the non-enhanced strains from nine of the genera tested.

For most strains, the degree of enhancement was small, ranging from 2- to 8 fold, and often evident only with the more effective local iron; among these were some near-saprophytes like Mycobacterium phlei, M. smegmatis, Bacillus cereus and Clostridium bifermentans. Substantial enhancement, from 14- to 50-fold, was observed with the more pathogenic among the strains tested: namely BCG, Corynebacterium ovis, C. murium, Listeria monocytogenes, Erysipelothrix rhusiopathiae, Cl. perfringens, Cl. septicum, Cl. oedematiens, and some strains of Klebsiella spp., Proteus spp. and Aeromonas hydrophila.

The enhancement of BCG by a single dose of iron given locally with the inoculum was only feebly manifest after 7 days, but substantial after 14-19 days, indicating the decisive effect of interference with an early humoral defence on the establishment of chronic infection some time later.

Insofar as guinea-pigs whose antibacterial defences are lowered by substantial amounts of exogenous iron in the circulation represent human subjects at risk of infection because of clinical states characterised by excess of available iron, the results of the survey suggest that only a minority among the environmental bacteria can take advantage of the decreased resistance associated with such states; but that this minority is likely to include the more virulent strains in the environment.

By means of spore-agglutination and fluorescent-antibody techniques, three serological types were identified among 84 strains of Clostridium sporogenes. Four spore antigens were identified, designated A, B, C and D. A, B and C were specific for the respective types whilst D was a group antigen shared by strains of the three types. The spore antigens had corresponding somatic antigens; the type-specific somatic antigens were designated I, 11, 111, and the shared somatic antigen IV. The flagellar antigens were found to be type specific and were designated 1,2 and 3; no common flagellar antigen was detected.

The results of precipitation tests with spore extracts depended on the method of testing. By a capillary-tube ring method there were cross reactions among the three types of C. sporogenes, whilst by immunodiffusion in agar layers the reactions were generally type specific. In a disintegrated spore extract, two non-protein antigenic components, possibly polysaccharide, were detected by means of immunoelectrophoresis. This extract showed cross reaction with antisera to strains of all types of C. sporogenes by the ring test and by immunodiffusion

The intranasal infection of infant rats with Haemophilus influenzae type b can be considerably enhanced by prior infection of the rats with influenza virus. When influenza virus A/England/939/69 was used to infect the animals a minimum of 104.0 EID5O was required to enhance H. influenzae infection; infection with 4×106 H. influenzae bacteria was needed to reveal this enhancement and infant rats two days old at the time of virus inoculation had to be used. By this method, nine strains of influenza virus were assessed for their ability to enhance H. influenzae infection, and the results were compared with their known virulence for man. The results showed a close correlation in this respect for all of the viruses, except strain A/PR/8/34.

The replication of these viruses in infant-rat turbinates and lungs was also studied; virus concentrations in turbinate tissues 48 h after infection showed a close correlation with virulence for man. Thus, three influenza virus strains known to be virulent for man reached concentrations in infant-rat turbinates ranging from 104.8 to 105.7 EBID50/0.05 ml at 48 h; the concentrations of six viruses known to be attenuated or non-infectious for man grew less well in infant rat turbinates, and reached concentrations at 48 h of 101.0 to 103.5 EBID50/0.05 ml.

The results are discussed in relation to the use of the infant-rat model for assessment of the attenuation of candidate live influenza virus vaccine strains.

Variants that had lost some of their antibiotic-resistance determinants were selected from a multiple-antibiotic-resistant strain of Staphylococcus aureus.

When tested by subcutaneous injection into guinea-pigs, and measured as the number of cocci needed to produce a skin lesion of an arbitrarily chosen diameter, the virulence of strains fell progressively with loss of resistance determinants. When the staphylococci were injected intracutaneously into mice, however, the results were less easy to interpret, but loss of resistance appeared to be associated with a reduction of the slope of the dose-response line.

There was no association between the antibiogram of the strains and their production of certain enzymes and haemolysins.

A slide-agglutination test (PH test) for the identification of all 0 serotypes of Vibrio cholerae (particularly NAG vibrios) has been developed. The reaction involves the flagellar H-antigen complex which is a species characteristic in vibrios. The novel feature of the test is cell sensitisation by 1.5% phenol which is incorporated in the saline used to make suspensions. Phenol treatment increases H but reduces 0 sensitivity without affecting gross cell morphology. It is suggested that this low concentration of phenol causes damage to the outer envelope membrane which normally covers the flagellum and acts as a barrier to H antibody in the living vibrio.

The species-specificity of the PH test with H antiserum has been established with 96 cultures of V. cholerae and allied species. Of the 53 cholera and NAG vibrios, 52 reacted positively, the sole exception being an auto-agglutinable strain. None of the 43 control cultures reacted. The success rate was therefore greater than for conventional bacteriological tests. The test was equally effective with stock cultures or unknown freshly isolated strains from several sources. Unabsorbed, whole-cell (OH) antiserum was also used successfully, and this simplification is recommended for routine use. The advantages of the PH test in clinical work are the ease and rapidity with which it enables NAG serotypes to be identified

Two hundred and forty-one strains of resident skin bacteria comprising 93 isolates of Propionobacterium acnes and 148 of Micrococcaceae derived from 36 acne patients and 8 control subjects were screened for their ability to inhibit 32 indicator strains, including 20 strains of P. acnes and 12 strains of Staphyzococcus epidermidis derived from patients with all grades of acne and from normal skin. Fifty-three strains (22%) showed some activity against at least one indicator strain. Both broad- and narrow-spectrum inhibition was detected. Inhibitory isolates of P. acnes outnumbered inhibitory Micrococcaceae by four to one. There was a low frequency of inhibition of S. epidermidis by Micrococcaceae (2.7%) and by P. acnes (1.1%) and a higher frequency of inhibition of P. acnes by Micrococcaceae (9.5%) and by P. acnes (40.8%). Furthermore, 81.8% of the subjects sampled possessed strains inhibitory to P. acnes. The significance of this finding is, as yet, unknown. No difference in the prevalence of active strains in normal (20%) and acne (22.5%) skin was detected. These findings suggest that the possession of inhibitory strains and conversely the possession of sensitive strains does not predispose to acne.

In Candida albicans the continued increase in dry weight, in cell volume and in hyphal length during 5FC treatment is mainly due to increased amount of carbohydrate despite the decreased amounts of nucleic acids. Incorporation studies with 32PO4 (for RNA) in C. albicans and with 3H-thymidine-monophosphate (for DNA) in a thymidine-utilising strain of Saccharomyces cerevisiae have shown that the decreased amounts of nucleic acids were due to an inhibition of synthesis of RNA and DNA by 5FC. Nuclear-staining techniques on the hyphal phase of C. albicans showed that 5FC inhibits nuclear division. The changes in amounts of protein during 5FC treatment do not wholly explain the changes in cell size although 14C-histidine incorporation experiments showed that protein synthesis continued in the presence of 5FC. 14C-glucose incorporation in the presence of 5FC showed an initial accelerated synthesis of carbohydrate with a maintained level of synthesis after 16 h. This abnormal pattern of synthesis correlates with the increase in amount of carbohydrate and in cell size and hyphal elongation. 5FC inhibits DNA synthesis, and all manifestations of unbalanced growth that culminate in the cell volume changes appear to be a consequence of that inhibition.

The virulence of six strains of staphylococci in experimental subcutaneous infection in mice of three age groups (3, 10 and 21 days) was studied. The results showed an age-related susceptibility to infection, in that the newborn mice were more susceptible than older mice to death and lesion formation. Resistance, i.e., ability to survive challenge of 107 c.f.u., developed at about 5 days. The strains used varied in toxin and enzyme pattern and there were marked differences in response to challenge as measured by mortality and lesion development. The Staphylococcus epidermidis strain was least virulent, while some strains of S. aureus produced lesions at low doses (104 c.f.u.). Two distinct types of lesion were observed, abscesses and necrotic lesions. Development of necrotic lesions appeared to be correlated with the ability to produce toxin in vitro.

The nasopharyngeal acquisition of meningococci was followed in healthy military recruits during their primary training. The production of antibodies to meningococcal serotype protein antigens and serotype lipopolysaccharide antigens accompanied the carrier state. Bactericidal antibody to protein serotype 2 was generated in response to the carriage of meningococci of low virulence that carried this antigen.

Cytomegalovirus (CMV) infection of CD1 mice was investigated by immunofluorescence and virus titration. The salivary glands were obviously prime targets for the virus, infection involving initially perivascular interstitial cells and then spreading to acinar cells. The submaxillary gland was the most affected and, in mice infected at 3 wk of age, virus was recoverable from saliva for up to 58 days. The ovaries became infected, especially after intravenous injection of virus into pregnant mice, but it was corpora lutea and not ova that were involved. In the adrenals, the medulla was most heavily infected in suckling mice, the cortex in older mice. Virus was not recoverable from mammary glands or milk. Infection of respiratory epithelial cells did not occur, and in the skin only the dermis became infected, leading to the appearance of bald patches.

Among mice surviving neonatal infection, about half continued to carry infectious virus in the kidneys for 12-26 mth, and a smaller number carried virus in the salivary glands. This was seen after infection with a virulent, salivary-gland-passaged strain of virus and also with an attenuated tissueculture-passaged strain. Nearly all these surviving mice had chronic glomerulonephritis and hyaline acellular deposits of unknown nature in the adrenals and salivary glands.

The presence of capsular polysaccharides (K antigens) and their relation to phagocytosis and sensitivity to the lytic action of serum of 26 strains of E. coli isolated from stools of healthy volunteers and from blood cultures were studied. Four of 12 strains isolated from stool cultures and 12 (86%) of the 14 strains isolated from blood cultures possessed K antigen. Three of the 12 strains isolated from stool cultures and seven of the 14 isolated from blood cultures were resistant to uptake by polymorphonuclear leucocytes; these resistant strains contained large amounts of K antigen. By contrast 10 strains, three with low amounts of K antigen and seven without detectable amounts of K antigen, were readily phagocytosed. Thus it appears that K antigen renders E. coli resistant to phagocytosis.

Only four (15%) of the 26 strains were sensitive to serum lysis and there was no correlation between the presence of K antigen and the resistance to serumlysis.

Twelve hundred and thirty three sera were tested in parallel by the single radial haemolysis (SRH) and by the simplified haemagglutination inhibition (HAI) technique in routine use in this laboratory. In 1203 (97.6%) of these sera the results were in agreement. The remaining 30 (2.4%) sera were re-tested by both methods because on initial testing the results obtained by the two methods did not agree; only three specimens remained discrepant.

The SRH is a simple test to perform and discriminates clearly between positive and negative sera. It is particularly suitable for screening large batches of sera.

The effects of rapid and slow rates of freezing in liquid nitrogen, storage in liquid nitrogen for 12 months, and the rate of subsequent thawing on the viability and growth of M. Zeprae in the mouse footpad were studied. Some loss of viability of M. Zeprae was detected, and this was found to be associated with the freezing process, rather than with storage or thawing. Slow freezing was less deleterious than quick freezing, with a loss of viability of 90% compared with 98%. The growth pattern of M. Zeprae was unaffected except for a delay in the appearance of growth caused by the loss of viability, though there was some evidence of an increased lag phase of one strain, possibly due to the repair of sublethally damaged organisms.

Mepartricin, a polyene antibiotic with candidacidal and trichomonicidal activity, was found to be without toxic effects for human polymorphonuclear leucocytes; the drug seems to be unable to enter the human cells. Some synergism between the antifungal activities of mepartricin and of human leucocytes is seen if Candida cells are pre-incubated with sub-lethal concentrations of the drug.