- Volume 8, Issue 1, 1975

A single-step disk immunoimmobilistion method for the detection of motile salmonellae is described. It combines in a single step the use of the motility of these bacteria in soft agar, their selective growth on Salmonella-Shigella media, and their immobilisation by polyvalent H antiserum. The method is shown to be 10 to 10,000 times more sensitive than a standard method for detection of Salmonella under the experimental conditions used. The time required for detection is shortened to 24-48 h.

Immunodiffusion analysis of Mycobacterium lepraernurium indicated the presence of at least six antigens. Comparative analysis of the M. lepraemuriurn antigen-antibody system with similar systems established for other mycobacterial species, showed that M. lepraemurium shared up to two antigens with other species.

Although our observations are in accord with some of the studies on the antigenic mosaic of M. lepraemurium, they are in disagreement with the observations of Stanford (1973) concerning a close serological relationship of this organism to M. avium. This incompatibility cannot be explained satisfactorily at present.

We wish to thank Dr B. S. Tepper of the Johns Hopkins-Leonard Wood Memorial Leprosy Research Laboratory, Baltimore, Maryland, for supplying M. lepraemurium, Dr R. Merkal of the National Animal Diseases Laboratory, US Department of Agriculture, Ames, Iowa, for supplying the M. ohnei and Dr L. Levy of the United States Public Health Service Hospital, San Francisco, for the other mycobacterial strains used in this study. Our thanks are also due to the US-Japan Tuberculosis Panel’s Antigen Committee for supplying the M. tuberculosis (H37Rv) reference system.

This investigation was supported by the United States-Japan Cooperative Medical Sciences Program administered by the National Institutes of Allergy and Infectious Diseases of the National Institutes of Health, Education and Welfare (grant no. AI-0864-7), Bethesda, Maryland 20014, USA.

A solid, urea-containing medium buffered to pH 6.5 with a suitable mixture of KH2P04 and Na2HP04 produced enlarged T-mycoplasma colonies containing a white precipitate. This was absent from M. horninis colonies. The medium can be used for the isolation and identification of T-mycoplasmas.

We would like to thank Mr A. E. Pink for assistance with the analyses and Mr E. J. Kentish for the photography.

The in-vitro effect of EDTA-Tris-lysozyme solution on 16 pathogenic bacteria of medical or veterinary importance was determined. Marked decreases in bacterial count occurred with Pseudomonas aeruginosa, Escherichia coli, Moraxella osloensis and Campylobacter fetus, and smaller decreases with Salmonella typhimurium, Shigella boydii, Aeromonas hydrophila, Proteus mirabilis, Listeria monocytogenes and Erysipelothrix insidiosa. The test solution had no effect on Klebsiella ozaenae, Brucella canis, Corynebacterium pyogenes, Coryne. renale, Streptococcus equi and Staphylococcus aureus.

Aeromonas hydrophila was enteropathogenic in ligated ileal loops of rabbits, causing a fluid accumulation of 14-2.0 ml per cm of gut length. Gut reaction could be produced with an inoculum as low as 104 viable bacteria. There was no difference in the nature of the positive reactions given by strains isolated from diarrhoea1 and non-diarrhoea1 children and adults and from water. Plesiomonas shigelloides, on the other hand, did not cause a significant gut reaction. A. hydrophila multiplied in the ileal loop by about 105 whereas P. shigelloides did so at only 102-3. These experiments on an animal model thus indicated the enteropathogenic nature of A. hydrophila, but no definite conclusion could be drawn from this study on P. shigelloides.

We are grateful to Professor Hardas Singh, Head, Department of Microbiology, for his valuable suggestions and encouragement. We acknowledge with thanks the technical assistance of Mr Ram Achal Ram and Mr S. N. Pathak rendered during this study.

The pigment produced by three clinically isolated strains of Pseudomonas aeruginow has been shown to be pyomelanin. This pigment is also produced by three strains of the same species labelled as “var. erythrogenes” by the National Collection of Type Cultures, and by Aeromonas salmonicida. Melanin and pyorubrin may be distinguished by the differential effects of tyrosine and glutamate on their production in minimal salts medium; Furunculosis Agar is a suitable medium for differentiation of these pigments.

We are grateful to the Wellcome Trust for a grant enabling the publication of the colour plate.

A novel form of synergy has been observed to occur between cephalexin and certain other /3-lactam antibiotics. In the presence of cephalexin a reduction was found in the concentration of other /3-lactam agents needed to induce lysis of Escherichia coli; the effect was particularly marked when cephalexin was allowed to act for a short time before the addition of the second agent.

The basis of this type of synergy-which is not likely to be of therapeutic value-is discussed in terms of a theory previously put forward, which suggests that penicillins and cephalosporins have two distinct sites of action in Gram-negative bacilli.

We thank Glaxo Laboratories Ltd for financial assistance.

Erythrocyte-sensitising antigens (AE) were prepared from Vibrio cholerae serotypes, from El-Tor vibrio, Escherichia coli and Salmonella enteritidis by digesting the organisms with NaOH followed by precipitation with alcohol. When AE was used in indirect haemagglutination (IHA) tests, the results in a number of cases were somewhat more sensitive and more specific than those obtained in classical agglutination tests. No cross reactions occurred between V. cholerae serotypes and E. coli and S. enteritidis. Much of the reactive part of the AE was not sedimentable at 100,000 g for 1 h. The eluant from the V. cholerae AE on Sephadex G-200 yielded three fractions, one of which was the most active in IHA tests. Treatment of the AE with trypsin resulted in an appreciable increase in the heterotypic serum titres in IHA tests. The spectrophotometric absorption of the AE at 260 nm showed a hump that may have been indicative of the presence of nucleic acid. Treatment of the antigen with ribonuclease reduced its nucleic acid content but did not change to any significant extent the reactivity of the preparation. The AE antigen of V. cholerae was Molisch-positive and was capable of sensitising untanned erythrocytes in IHA tests. It is suggested that the reactive part of the AE antigen is a carbohydrate complex.

Bacterial activity and growth were monitored by following the changes of electrical impedance of cultures in liquid media. The signal is expressed automatically as a curve similar to growth curves produced by other methods. The technique offers a new, rapid and sensitive means of detecting active microorganisms and is potentially the basis of rapid automated systems in this field. The impedance changes indicate that the micro-organisms metabolise substrates of low conductivity into products of high conductivity and that the changes are due to the activity of the micro-organisms rather than increase in their numbers.

The activity of strains of Escherichia coli, Klebsiella aerogenes, Pseudornonas aeruginosa, Slaphylococcus aureus, and Streptococcus faecalis was detected within 2 h with inocula of 103-105 organisms per ml. Different reactions of bacteria in various media suggest that the method may be applied to the rapid identification of micro-organisms. The inhibitory effect of antibiotics on bacteria was demonstrated within 2 h, indicating that the method may be useful for the rapid determination of bacterial sensitivity to antibiotics and the rapid assay of antibiotics in serum. Correlation of response time to initial inoculum allows estimation of numbers of viable organisms. The sensitivity of the method allowed detection of activity due to Mycoplasma argininii within 3 h; this suggests that the method might be applicable to the rapid detection of other slowly growing organisms, such as mycobacteria.

Alpha protoxin of StaphyZococcus aureus “Wood 46” was activated by trypsin which had been coupled to carboxymethylcellulose, as indicated by the toxin’s ability to hydrolyse tosyl-arginine methylester (TAME). A Lineweaver-Burk plot of the degradation of TAME by toxin and trypsin showed that toxin had a greater affinity for the substrate than had trypsin. N-terminal aminoacid analyses of activated toxin suggested that leucine or isoleucine is the N-terminus, in contrast to protoxin, the N-terminus of which is histidine.

A strain of Staphybcoccus aureus has been isolated from a hospital environment over 3 months. Every isolate was lysed by phage 77, had high-level resistance to streptomycin, and was resistant to about 250 pg per ml of both tetracycline and sulphonamide; a combination of sulphamethoxazole and trimethoprim produced little bacteristatic synergy towards each isolate. All these organisms were thus considered to be “the same” the variation in other properties was probably due to rapid evolutionary change in vivo. The variation in sensitivity to methicillin and neomycin, and the absence of penicillinase production in some isolates, probably indicated loss of the relevant genes. Several isolates had probably acquired resistance to lincomycin by a one-step mutation in vivo. The usefulness of lincomycin and analogues in treating staphylococcal infections seems limited.

Cell-envelope polypeptides of eight phase-I and five phase-IV strains of Bordetella pertussis were compared by SDS-polyacrylamide gel electrophoresis. All phase4 strains gave a strikingly similar but complex pattern of protein bands, which did not appear to vary with known differences in heat-labile agglutinogens. Phase-IV strains gave the same pattern as phase-I strains, except that one band was missing and another was either much reduced or absent.

Envelopes from phase-I strains grown in Hornibrook medium rich in Mg2+ ions to produce “antigenically-modulated” C-mode cells gave a pattern of bands indistinguishable from phase-IV strains. A phase-IV strain grown in the high-Mg2+ medium gave the same pattern of bands as when grown in unmodified Hornibrook medium. We suggest that the two polypeptide bands that show changes may be responsible for one or more of the immunological or physiopathological activities that are lost during phase variation and antigenic modulation in B. pertussis.

Tests carried out on strictly anaerobic and facultatively anaerobic strains of “corrodens” bacteria, showed that although these organisms are relatively inactive biochemically, differentiation can be made on the basis of tests that demonstrate reduction of nitrite, hydrolysis of urea and 1-naphthyl acetate, decarboxylation of lysine and ornithine, and sensitivity to certain selective agents included in culture medium.

Plasma was found to be superior to serum in supporting the growth of all “corrodens” bacteria, and a combination of heated and unheated blood added to a nutrient base was shown to yield good growth.

Comparative studies are reported with various species of Bacteroides, Haemophilus, Bordetella and related genera.

Pseudomonas aeruginosa was found to produce a factor or factors that inhibited Cryptococcus neoformans and appeared to be extracellular because the anti-C. neofonnans activity was readily demonstrable in medium after the removal and killing of Pseudomonas organisms. Production of the inhibitor material was greatest in DST Agar after prolonged incubation and was reduced in the presence of glucose. A part of the inhibitory material was found to be chromatographically distinct from pyocyanin.

Antibiotic-resistance transfer between populations of donor and recipient strains of Escherichia coli was compIeteIy inhibited in broth by dense suspensions of Bacteroides fragilis. Comparable amounts of inert bacterial matter (formolised suspensions of E. coli or B. fragilis), or smaller numbers of viable B. fragilis, Streptococcus faecalis, Staphylococcus albus, Neisseria catarrhalis, or solutions of sodium taurocholate or glycocholate were only moderately inhibitory. Anaerobiosis had no effect upon plasmid transfer. Population densities of enteric organisms in these studies were similar to those found in faeces. The presence of dense cultures of B. fragilis provide a satisfactory explanation for the almost total inhbition of conjugation in the human gut. Other factors inhibiting conjugation to a lesser degree may reinforce the effect of B. fragilis in vivo.

As well as selecting for resistant organisms, antibiotics may also indirectly increase populations of R-factor-bearing organisms in the gut by interfering with the anaerobic flora and so permit an increase in the frequency of conjugation.

I am very grateful to Professor W. A. Gillespie for his interest and to Miss M. A. C. Jones for technical help. M. A. C. J. was supported by a Programme Grant to Professor M. H. Richmond from the Medical Research Council.

Agglutination tests were used to study the surface antigens of two recently described species of the genus Haemophilus, H. paraphrophilus and H. paraphrohaemolyticus, and their antigenic relationship to other members of the genus. The results obtained with a few strains of H. haemoglobinophilus and H. aphrophilus are also reported. The species H. paraphrophilus appears to be homogeneous; no major cross-reactions were observed. The species H. paraphrohaemolyticus contains at least three serotypes, of which two have been defined in terms of agglutination reactions. Cross-agglutinations occurred between one strain of H. paraphrohaemolyticus and strains of the other Vdependent species, H. parainjluenzae and H. parahaemolyticus. Of the Xdependent species, H. haemoglobinophilus seems to be homogeneous, and the species H. aphrophilus is not.

A non-specific antibody against horse blood in the medium occurred erratically and was present in only two antisera, those raised to H. aphrophilus strain Khairat and H. haemoglobinophilus strain no. NCTC8540.

Mutants lacking flagella or fimbriae (pili) or both were used for the preparation and absorption of rabbit antisera against the flagella of Pseudomonas aeruginosa. The results of agglutination, immobilisation and complementfixation tests indicate that the antisera obtained are specific for flagella.

The incorporation of nitrate into semi-solid agar for motility and immobilisation tests was found useful for the selection of actively motile cells and for the demonstration of specific anti body to flagella.

Strains of Escherichia coli causing urinary-tract infections were found to be more commonly haemolytic than were faecal strains. Strains from the introitus that subsequently gave rise to urinary-tract infections resembled urinary strains and those that did not give rise to infections resembled faecal strains.

α-Haemolysin production was closely associated, in strains from a variety of different sources, with the production of a haemolysin detectable in liquid medium and with cytotoxicity in tissue culture.

We are indebted to Professor F. W. O’Grady, who made available the introital and urinary strains of E. coli, and to Miss G. V. Martin for assistance with the tissue-culture work.

This investigation was supported by a grant from the St Mark’s Foundation.