- Volume 3, Issue 2, 1970

Osmotic protection by the addition of sodium chloride to the level of 750 m.osmoles per kg was required to support an adequate growth of penicillininduced L-forms of Streptococcus faecalis on a serum agar medium. This requirement was not reduced by the addition of spermine or by the acidification of the medium.

L-form preparations that gave typical L-form colonies on serum agar containing 0.5M-NaCl without added penicillin, but no colonies on isotonic serum agar, were tested for the ability to survive in the serum and tissues of mice. The L-forms were rapidly killed when incubated in vitro in mouse serum unprotected by the addition of NaCl, and disappeared rapidly after being injected into the bloodstream or peritoneal cavity. Osmotic lysis was probably the cause of their death and disappearance, since the mouse serum did not contain antibodies capable of killing the L-forms, and treatment with cortisone, which inhibits phagocytosis, did not affect the rate of their clearance from the peritoneum. Although the L-forms slowly underwent lysis in mouse urine, ones that were injected into the urinary bladder were probably also removed to a large extent by the voiding of the urine.

The findings show that L-forms were not virulent in mice.

Despite repeated attempts, and the use of a variety of cultural methods, no osmotically fragile persisting organisms, or L-forms, could be detected in the kidneys of mice with enterococcal pyelonephritis after they had been treated with ampicillin. L-forms added to the kidneys were readily recovered after processing of the tissue in a Teflon grinder, and destruction during processing could not explain the failure to detect variants in urine plated directly on to L-form agar. No L-forms were detected in the kidneys after suppression of renal defence systems with cortisone. It is suggested that the failure of penicillin-damaged enterococci to grow as L-forms was due to inimical physiological conditions in the mouse kidney.

An identification method based on the failure of methicillin (5 μg) and oxacillin (1 μg) disks to inhibit enterococci on Mueller-Hinton Agar was evaluated. All strains tested of Streptococcus faecalis and its varieties and of S. faecium grew up to the edge of the disks; inhibition of S. faecium var. durans and of S. bovis was variable. In contrast, S. equinus and almost all streptococci not belonging to group-D streptococci showed inhibition zones of 10 mm or more.

The methicillin test seems suitable for use in the clinical laboratory in view of the scarcity of S. faecium var. durans, S. bovis and S. equinus in pathological material from man and of the margin of error of the traditional tests for enterococci.

One hundred and seventy strains of meningococci isolated in the UK from 1966 to 1968 were tested for sensitivity to sodium sulphadiazine. Eighty-eight strains were isolated from CSF or blood and 82 strains from postnasal swabs and other sources. Seven strains were resistant to sulphonamides: one was highly resistant (MIC 100 μg per ml), two were moderately resistant (MIC 50 μg per ml) and four were slightly resistant (MIC 6.4 or 10 μg per ml). One hundred and sixteen strains, including the seven sulphonamide-resistant strains, were tested for sensitivity to six antibiotics: benzyl-penicillin, ampicillin, erythromycin, tetracycline, chloramphenicol and cephaloridine. None of the strains showed any increase in resistance to these antibiotics.

One hundred and sixty strains were typed serologically: 15 (9 per cent.) were group A, 51 (32 per cent.) were group B and 94 (59 per cent.) were untypable.

The use of 1-hydroxy-2-naphthoic acid and N-phenyl-p-phenylenediamine allowed oxidase activity to be detected cytologically in bacteria. The demonstration of this activity seemed to be correlated in the main with known oxidase activity of these bacteria as determined by more conventional means. The method was applied to the presumptive identification of oxidase-positive bacteria in direct smears of clinical material, and this procedure sometimes assisted in the presumptive diagnosis of infectious disease due to these oxidase-positive bacteria.

Of 125 clinical specimens with only oxidase-negative bacteria on the direct smear, there were ten (8 per cent.) from which oxidase-positive bacteria Pseudomonas aeruginosa) were cultured. In 39 clinical specimens that showed oxidase-positive bacteria on direct microscopy, there were 32 in which a presumptive diagnosis of pseudomonas or alkaligenes infection was made; Ps. aeruginosa or Alkaligenes faecalis was cultured from 28 (85 per cent.) of these 32 specimens.

A vervet monkey kidney cell line designated GL-V3 has been established. After 75 passages it still has the morphology of epithelial cells and its chromosome complement is aneuploid. Cultures of GL-V3 are susceptible to a wide range of viruses. No latent viruses were detected and the cells are not neoplastic when implanted in the cheek pouches of hamsters.

Three sublines of HeLa cells were infected with Mycoplasma orale type 1, M. hominis and M. hyorhinis and compared with mycoplasma-free cells for their ability to support the growth of adenovirus types 2 and 31, herpes simplex virus, Sabin’s poliovirus type 1, parainfluenza virus type 2, respiratory syncytial virus and vaccinia virus. Titres of herpesvirus and adenovirus tended to be reduced in the cells infected with mycoplasmas, but titres of the other viruses were generally raised by such infection. The largest differences in titre in either direction were approximately ten-fold and were obtained with adenovirus type 2 (reduction) and vaccinia virus (increase). Much smaller effects were noted with the other viruses.

With herpesviruses and adenoviruses, which have a known requirement for arginine, the antagonistic effect of the two arginine-metabolising mycoplasmas, M. orale type 1 and M. hominis, was more than that of M. hyorhinis, which does not metabolise arginine. With the other viruses, the higher titres obtained in the mycoplasma-infected cells were independent of the species of mycoplasma. The cell lines infected with M. hyorhinis were difficult to maintain for long periods on account of the cytopathic effects of this species, and were, therefore, less suitable for titration of the more slowly growing herpes- and adeno-viruses.

It is concluded that mycoplasma contamination of a HeLa cell line may interfere with the titration of some viruses, but the effects are relatively small and probably do not constitute a serious hazard in routine diagnostic virology.

Typing of strains of Herpesvirus hominis according to the method of Pauls and Dowdle can be performed in BHK cells instead of in primary rabbit kidney cells. The modified method has the advantage of a reduction in incubation time and the convenience of working with a semi-continuous cell line. The concentration of virus antigen inoculated into rabbits to produce typing antisera has an effect on the cross-reactivity of the sera obtained. Higher immunising doses of virus produce antisera with higher degrees of cross-reactivity than is found in antisera produced in response to low immunising doses of virus.

A simple technique was developed for the intramammary inoculation of mice. Experimental mastitis was produced in lactating mice with strains of Staphylococcus aureus, Streptococcus agalactiae, Corynebacterium pyogenes, Pseudomonas aeruginosa and Escherichia coli that had originally been isolated from cases of bovine mastitis. There was evidence that the spectrum of pathogenicity of the different bacterial strains in the bovine species was to some extent paralleled by differences in pathogenicity in the mouse. Mastitis could be produced in BSVS mice by the inoculation of comparatively small numbers of staphylococci. The appearance and histology of the affected mammary glands resembled mastitis produced in the bovine species by the bacterial species concerned. The size of the mouse mammary gland was very convenient for histological and other studies requiring examination of the whole gland. Experimental mastitis in the mouse provides a useful model for certain fundamental studies and for experiments, such as those of a screening nature, prior to studies on cattle.

The carbohydrate fermentation reactions of Clostridium oedematiens were critically assessed with particular reference to problems that are encountered in the test procedure. Frequent failures of growth occurred in currently accepted basal media, and a test procedure was developed with a medium that supports consistent growth of this demanding anaerobe. The medium consists of nutrient broth and freshly prepared meat particles; acid production was measured with a pH meter. The results of carbohydrate fermentation tests with 26 strains of CI. oedematiens representing at least one strain of each type suggest that the fermentation patterns may be of value when strains are encountered that do not fit readily into the present serological scheme of classification. The advantages of the fermentation test procedure developed in the present study are discussed in relation to the disadvantages of other fermentation test systems for anaerobic organisms.

Probably because CI. tetani is a well-defined species whose recognition is rarely difficult, no recent attempts have been made to clarify the present uncertainties about its gelatinolytic and proteolytic activity. In the present study of 71 strains of the organism, all of them were shown to produce gelatinase, and none was proteolytic.

A proportion of the strains (49 of 71) produced a fibrinolytic enzyme, which behaved like a kinase.

A proportion of the strains (40 of 71) produced a deoxyribonuclease.

None of the strains produced phosphatase.

All 71 strains produced an intense diffuse opacity on milk agar, and complete precipitation of casein in milk broth due to the production of a rennin-like enzyme. This enzyme was present in culture-supernatants, was active under both aerobic and anaerobic conditions, and was specifically inhibited by tetanus antitoxic sera. With the exception of CI. oedematiens types A and B, the reactions of all other clostridia tested on milk agar and in milk broth were dissimilar to those of CI. tetani.

Commercial horse tetanus antitoxic serum inhibits the swarming growth of CI. tetani in plate cultures. Advantage may be taken of this in the separation of other anaerobic organisms from mixtures with CI. tetani.

An experimental model of S. choleraesuis infection was set up in mice to study the effects of therapy. Furazolidone was superior to chloramphenicol, ampicillin, tetracycline, streptomycin or tylosin, as assessed by both mortalities and the carriage of salmonellae in the spleen. Furazolidone at 100 mg per kg daily for 7 days completely protected infected mice and eliminated the organism from the spleen. Apparent toxicity of furazolidone was observed in infected, but not in normal, mice with doses of 200 mg per kg daily for 7 days. The effects of dose rate, duration of treatment, and the time at which treatment was initiated were examined. A carrier state occurred in all groups treated with the antibiotics. When the spleen was examined at 24 hr after the last dose, falsenegative results were obtained compared with an examination 3 wk later. The possible reasons for this are discussed.

The complement-fixing antibody formed by infants in response to a primary RS virus infection has a different fixation pattern, with a higher antigen requirement, than antibody present in adult serum and maternal antibody in the serum of infants. Differentiation between actively and passively acquired antibodies in paired sera from single infants enabled serodiagnoses to be made in cases where the presence of maternal antibody would otherwise have obscured the development of the infant’s own antibodies. Possible reasons for the differences in fixation pattern are discussed.

A case of gas gangrene arising as a complication of exploratory ureterotomy in a patient with chronic bilateral renal infection is described. The bacteriological diagnosis was established on the second day after onset; a toxigenic strain of Clostridium welchii type A was isolated from the wound. Proteus mirabilis was isolated from the wound and from the patient’s bloodstream.

Combined therapy included prompt surgical treatment and repeated local instillations of H2O2 and a saline solution containing 1000 IU penicillin per ml. Parenteral antibiotic therapy included 20 M.units of penicillin daily, and pyrrolidinomethy 1 tetracycline and spiramycin. A dose of 10,000 units of Cl. welchii antitoxin was given intravenously in a polyvalent clostridial antitoxic serum, followed by 30,000 units as monovalent serum when the diagnosis was confirmed.

The patient’s condition improved dramatically within a few hours of the start of the combined therapy and recovery was complete within 8 days.

The pathogenesis, diagnosis and current methods of treatment of gas gangrene are discussed, with special reference to post-operative gas gangrene.

The situation of enteropathogenic strains of Escherichia coli in the porcine intestine of naturally occurring and experimentally reproduced cases of E. coli diarrhoea was studied mainly by fluorescent staining techniques. Three different enteropathogenic strains were found on villi of the small intestine, but only small numbers of these bacteria were found on the colonic mucosa except when they were contiguous with the mass of bacteria in the lumen. A non-entero-pathogenic strain of E. coli was found on villi of the small intestine in small numbers. Occasional Gram-positive bacilli were observed on a few villi in the intestines of two healthy piglets.

Coliform attachment to villi was found to be most marked when diarrhoea was clinically well established, but such attachment was also sometimes observed during the early clinical stages of diarrhoea. It is therefore considered that adhesion of E. coli to small-intestinal villi is a significant lesion of porcine diarrhoea associated with enteropathogenic strains of E. coli.

The characteristics of a Gram-positive bacterium isolated from a chronic sore throat are described. It possessed a streptobacillary morphology and was capable of growing only upon a solid surface. Its physiology and chemical composition, and those of Gram-positive variants derived from it, suggest a relationship with both Streptococcus and Corynebacterium, especially the latter.

The isoquinoline drugs UK2054 and UK2371 were tested for activity against rhino-viruses. UK2054, but not UK2371, decreased the yield of rhinovirus types 2, 4, 9 and 43 from HeLa cells maintained in medium containing the drug. This activity was not demonstrable in a semi-continuous line of human embryo lung fibroblasts, HEL-218. In a double-blind, placebo-controlled trial, human volunteers were experimentally infected with rhinovirus type 9 and given prophylactic and therapeutic UK2054 by mouth. No significant antiviral activity was found in this trial.

The bacterial flora of axillary hairs from persons with and without trichomycosis axillaris has been examined. Affected hairs bore a larger number of diphtheroid species than control hairs and a higher proportion of isolates from trichomycosis were found to belong to Evans’ group D.