- Volume 6, Issue 3, 1973

The relationship between mucoid and non-mucoid strains of Pseudomonas aeruginosa isolated from the sputum of children with cystic fibrosis was investigated by typing the strains by pyocine production. The mucoid strains were usually untypable by the standard technique, but extraction and assay of the pyocines produced in liquid cultures showed that the strains belonged to recognised pyocine types. When mucoid and non-mucoid strains occurred simultaneously, they were of the same pyocine type; it is suggested that they are variants of the same strain.

Two patients suffered febrile reactions after being transfused with bank blood simultaneously contaminated with Enterobacter agglomerans and Pseudomonas fluorescens. The probable source of these organisms was the acid-citrate-dextrose solution used in the pilot tubes. Stock bottles of this were opened repeatedly and stored in the refrigerator in the meantime.

After being given tetracycline in drinking water (0.1 per cent.) for 5 days, rats were inoculated orally with Candida albicans on three occasions at 2-day intervals. Immediately after the first inoculation, the concentration of tetracycline in the water was reduced to 0.01 per cent. and this was maintained for the next 28 days. The rats’ mouths were regularly colonised by C. albicans, the organism being recoverable throughout the experiment. Histological examination showed the organism in the tissues in 75 per cent. of the animals. Infection of the dorsal surface of the tongue produced a striking change in the histological appearance of the lingual epithelium, reminiscent of that found in candidal leukoplakia in man.

Lactobacilli are generally not thought of as pathogenic, except in dental caries. Seven strains of lactobacilli obtained from various clinical conditions were fully investigated. They were identified as four strains of L. casei var. rhaninosus, two strains of L. piantarum and one strain of the subgenus Thermo-bacterium, which could not be further identified.

Some of these strains were believed to play a pathogenic role. In three cases the lactobacilli were repeatedly isolated from blood cultures taken during life; all these proved to be L. casei var. rhamnosus.

Groups of rabbits were given intratracheal injections of 5 x 107 or 108 yeast-forms of Candida albicans. Morphological transformation from yeasts to mycelia was seen in the tissues within 1 hr of injection. The lungs showed an intense cellular reaction with formation of bronchopneumonia, but this quickly and completely resolved and no progressive lesion developed. Rabbit lung seemed to be highly resistant to C. albicans infection.

Fungus entered the blood from the lungs and was disseminated to other viscera. Mycelial forms invaded polymorphonuclear leucocytes, large monocytes, blood vessels and renal tubular epithelium. Proliferation of large monocytes was responsible for fragmentation and destruction of the fungus in all the tissues except the spleen, where there was complete absence of reaction although fungal cultures were positive. The fungus was finally excreted by the kidney in which cortical abscesses developed. These healed slowly by scarring with occasional granuloma formation.

On the assumption that bactericidal action of drugs is probably required for the effective treatment of melioidosis, in-vitro experiments were performed with five chemotherapeutic agents and six Pseudomonas pseudomallei strains from clinical sources. Chloramphenicol, kanamycin, novobiocin and tetracycline, alone and in various combinations, were tested for bactericidal activity against reasonably heavy inocula by means of a tube-dilution technique with subculture for viable counts. Results are displayed by Loewe’s method of isobolograms in a simplified form. The novobiocin-tetracycline combination produced a kill of 99.4 to 99.8 per cent. with five of the six strains at concentrations at which neither drug had any effect when used alone (2.5 to 10 μg per ml of novobiocin and 2 to 5 μg per ml of tetracycline). The synergistic effect of this mixture was demonstrable with as little as 0.2 μg per ml of novobiocin, or 0.003 of the concentration needed for its comparable independent effect; in clinical use the latter could be achieved systemically only with unusually high dosage. No bactericidal effect was found with one strain.

Sulphamylon showed a strong bactericidal effect at concentrations that might be achieved locally by topical applications to wounds and burns.

The quantitative recovery of test strains of bacteria on blood agar media was compared in Baird and Tatlock anaerobic jars with and without additional CO2 in the gas mixture. Both the colony size and the colony count of Gramnegative anaerobes and some of the test strains of clostridia were increased in the presence of added CO2. An enhancing effect was also noted with two strains of haemolytic streptococci. The addition of 10 per cent. CO2 as a part of routine anaerobic procedure is recommended for the diagnostic service laboratory.

Amongst 778 yeasts isolated from clinical specimens and tested for sensitivity to 5-fluorocytosine, 4.1 per cent. had minimum inhibitory concentrations of 31 μg of 5FC per ml, or more, a lower percentage of primary resistance than in some reported series. Resistance was found in 4.5 per cent. of Candida albicans, 0.7 per cent. of Torulopsis glabrata and 5.6 per cent. of Cand. tropicalis. The minimum fungicidal concentrations for the resistant candida isolates greatly exceeded the minimum inhibitory concentrations, and some yeasts with low minimum inhibitory concentrations contained small numbers of highly resistant organisms.

The microcolony sizes of the aerobic and anaerobic cutaneous bacterial flora have been examined by a comparison of the number of viable aggregates and the total viable cells per cm2 of skin. The mean microcolony size was often of the order of 104 cells and counts in excess of 105 cells have been recorded. The importance of these microcolonies in relation to skin disinfection is discussed.

Resistotyping consists of testing bacterial strains against a set of arbitrarily chosen chemicals that show selective toxicity at a critical concentration. The pattern of resistances, which defines the resistotype, is believed to describe isolates for epidemiological purposes.

Such a system was devised for Shigella sonnei and 247 strains obtained from various outbreaks were tested. Thirty-four outbreaks in families, schools and localities were originally defined by their timing and geographical incidence, together with the colicine type and antibiotic resistance pattern of the strains isolated.

In general, strains from an outbreak belonged to the same resistotype. This was not altered in those strains that showed a change in antibiogram or colicine type. In some cases, strains from different sources but of the same colicine type could be differentiated into separate resistotypes, which corresponded to geographical outbreaks. In other instances, however, strains of the same resistotype were subdivided by their colicine type into different outbreaks.

In-vivo findings and in-vitro transfer of plasmids indicated that the resistotype markers were independent of the colicine type and antibiotic resistance markers. Thus, although the resistotype markers may also be episomally located, they are apparently not on the same episomes as the determinants of colicine type and antibiotic resistance.

All typing systems ultimately depend on the genetic stability of their markers, and it is theoretically impossible to devise a perfect system for epidemiology. This statement applies equally to phage and colicine type and to antibiogram and resistotype. In the case of S. sonnei, the method of resistotyping appears to be a useful adjunct for detailed epidemiology.

All of 670 apparently unrelated chloramphenicol-resistant strains of Escherichia coli of human and animal origin were multiply-resistant; over 66 per cent. of them were resistant to five or more antibiotics.

The resistance of 96 per cent, of 400 of the strains could be transmitted, often at a high rate, to E. coli strain K12F−. A lower and variable proportion of the strains transmitted their resistance to nine other enterobacterial strains, principally pathogens; the rate of transmission was much lower than that which occurred to E. coli strain K12F−.

Of 74 distinct strains of Staphylococcus aureus obtained from clinical sources, about 70 per cent. were resistant to high levels of streptomycin (MIC>1 mg per ml) and 30 per cent. resistant to lower levels (MIC about 100 μg per ml).

Streptomycin resistance in four strains exhibiting high-level resistance was stable on storage, whilst that in four strains with low-level resistance was unstable. Three of the latter were also resistant to other antibiotics; these resistances were also unstable, and in one of these strains (FAR4) there was evidence that streptomycin resistance was linked to erythromycin and neomycin resistance and to production of δ-haemolysin.

Strain 649 is resistant only to streptomycin (low-level resistance); plasmid DNA with a molecular weight of about 38 million daltons corresponding to this resistance was isolated from this strain. Attempts to correlate the CCC-DNA from the multi-resistant strain FAR4 with its resistances were unsuccessful.

Resistance to spectinomycin was not associated with resistance to streptomycin, but with erythromycin resistance or with neomycin resistance.

Subcutaneous immunisation of rabbits with crude cholera exotoxin induced a functional immunity in the gut to toxin challenge that was superior to that of non-immunised controls. Two injections given 3 wk apart gave markedly better protection than one injection. The functional immunity appeared to be of short duration, because the resistance 2 wk after a second injection was less than it was 4–5 days after immunisation. With prolonged storage of the crude toxin its immunogenicity increased markedly; this was related to aggregation of a considerable portion of the toxin. The correlation between the magnitude of immunity in the gut and the serum levels of neutralising antibodies was often poor suggesting that antibodies formed locally in the gut may be of significance for protection.

Although the simultaneous administration of staphylococci enhances the virulence for mice of certain Gram-negative bacilli, no such enhancement of virulence of gonococci could be demonstrated. On the contrary, the virulence of Staphylococcus aureus was enhanced by the simultaneous administration of gonococci, and fatal staphylococcal infections could be produced by normally non-lethal doses of staphylococci. However, mice were protected by previous immunisation with live staphylococci. The enhanced virulence of staphylococci is due to the action of gonococcal endotoxin on the cellular response of the mouse in the early stages of infection. The exact mechanism is not yet fully understood, but endotoxin administration produces leucopenia, and it is suggested that an important feature is the inhibiting effect of endotoxin on the migration of phagocytic cells to the site of infection.

The addition of sub-inhibitory concentrations of cephalothin, a competitive inhibitor of staphylococcal β-lactamase, to a culture of a penicillinase-producing Staphylococcus aureus containing a sub-inhibitory concentration of a hydrolysable penicillin (benzylpenicillin, phenoxymethylpenicillin, phenethicillin, propicillin, ampicillin or carbenicillin) was found to bring about an increase in the lag phase of the culture (Δt), during which there was a bactericidal effect. The extent of Δt depends upon certain enzymological parameters, of which a separate theoretical consideration has already been made (Hamilton-Miller, 1971a); the observed values of Δt were found to be in line with those predicted by means of the equation derived previously. Synergism between cephalothin and penicillins was also demonstrated by MIC determinations. The system used here to analyse synergism may have applications in other circumstances where antimicrobial agents are destroyed by resistant organisms.

Unstable resistance to methicillin has been demonstrated in an isolate of Staphylococcus epidermidis. Loss of resistance occurs rather slowly but consistently on nutrient agar kept at bench temperature. The resistant organism exhibits an “optimal zone” of inhibition by intermediate concentrations of the drug, and this is antagonised by cephalosporins and other penicillins. These effects are markedly modified by the medium, inoculum size and temperature. Neither of the phenomena has been observed with the sensitive variant. Against penicillin there is a paradoxical effect in that with light inocula the methicillin-resistant organism is much more resistant to penicillin than its sensitive variant, whereas the reverse holds for heavy inocula.

Phagocytosis of Neisseria gonorrhoeae in tissue cell-cultures may protect it from the lethal action of bactericidal agents. Type reversal from avirulent to virulent gonococci was not observed. We believe that maintenance of type I organisms in tissue cultures is too tedious and that snap-freezing in liquid nitrogen is more convenient.