Biomedical subjects
Publications and source records attributed to J P Duguid.
Evidence on the extent of the part played by infected hens' eggs in causing salmonella food-poisoning is inconclusive. The role of freshly cooked shell eggs is currently much exaggerated. Prevention should be sought through improved catering practices and kitchen hygiene, and attempts to eradicate salmonellas from laying flocks are likely to be ineffective.
The nature of the adhesive capacity of three hemagglutinating Escherichia coli strains that had earlier been described as nonfimbriated was studied. The strains that were isolated from human disease adhered to human buccal and urinary tract epithelial cells, an adhesion that was not inhibited by D-mannose. By crossed immunoelectrophoresis it was shown that the three strains produced a common antigen, Z1, developed after growth at 37 degrees C but not 18 degrees C. One of the strains produced an additional antigen, Z2, of almost the same electrophoretic mobility in crossed immunoelectrophoresis. A mutant of this strain deficient of its polysaccharide K antigen had maintained the adhesive capacity, indicating that the K antigen was not responsible for adhesion. A further mutant of the acapsular mutant produced a strongly reduced amount of the Z antigens and had lost the ability to adhere. The Z1 (and Z2?) antigens were therefore deemed to be responsible for adhesion. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of cells of the three strains, a heavy Coomassie-blue stained line was seen, indicating the presence of a protein subunit of molecular weight slightly above 14,400. By immunoblotting with absorbed antiserum, it was shown that this protein was the same as that detected by crossed immunoelectrophoresis. Protease from Streptomyces griseus, but not trypsin, digested the protein. Heating to 100 degrees C did not affect it. By immunoelectron microscopy of embedded and sectioned bacteria that had first been treated with specific antisera and ferritin-labeled antirabbit immunoglobulin, the protein adhesin-antibody complex was found to surround the bacteria as a heavy capsule. After negative staining with uranylacetate (pH approximately 4), the capsule appeared as a mesh of very fine filaments. The possible role of this capsule in the pathogenesis of disease is discussed.
When fimbriate (Fim+) strains of Salmonella typhimurium were grown in static broth, many bacteria were in the fimbriate phase and bore fimbrial mannose-sensitive haemagglutinin (MSHA) that enabled them to adhere to guinea-pig and other erythrocytes and to agglutinate them in rocked tile and static settling tests. When either Fim+ or Fim- strains were grown on phosphate-buffered nutrient agar, the bacteria formed a diffusible, mannose-resistant haemagglutinin (MRHA) that gave dispersed sediments with sheep and pig erythrocytes in static settling tests, but without evidence of bacterial adhesion to the erythrocytes. On exposure, from above or from below, to cultured HEp2 and HeLa cells for 30 or 90 min at 37 degrees C, motile MSHA-rich, MRHA-negative broth-grown bacteria adhered to the cells in large numbers (e.g., 20-100/cell), but motile MSHA-negative, MRHA-negative broth-grown bacteria and non-motile MSHA-negative, MRHA-rich agar-grown bacteria adhered in only small numbers (usually less than 1/cell). Thus, strong adhesiveness of bacteria for cultured cells in vitro appears to depend upon the presence of MSHA, not MRHA, and as Fim- (MSHA-negative) strains of S. typhimurium are known to be highly infective in animals, a strong reaction in the in-vitro model does not reflect a property of the bacteria essential for infectivity in vivo.
Twenty-three strains of Escherichia coli with mannose-resistant and eluting (MRE) haemagglutinins with eight different patterns of substrate specificity were examined by a variety of electronmicroscope methods. In 12 strains, the presence of MRE haemagglutinins with broad-spectrum patterns 1, 3 and 4 was correlated with that of fimbriae. In 11 strains with MRE haemagglutinins with the less common patterns 6, 7 var., 8, 9 and 10, fimbriae were not found on bacteria in MRE+ cultures, indicating that the latter haemagglutinins are non-fimbrial.
The production of fimbriate (Fim+) recombinants was observed in transductional crosses between different pairs of wild-type strains of different biotypes of Salmonella typhimurium. Fim+ recombinants were readily produced in transductions from Fim+ donor strains to Fim- recipient strains and, less frequently, between some pairs of Fim- strains, for example, between almost any strain of the firn biogroup (Fim- Inl- Rha- Bxyl-) and many strains of the non-FIRN Fim- biogroup. None of numerous crosses between different pairs of FIRN strains gave Fim+ recombinants, suggesting that the fim mutation was present at the same intragenic site in all FIRN strains. FIRN strains are thought to have descended from a single ancestral FIRN bacterium which originated by a series of mutations from a strain of the common biotype 1a (Fim+ Inl+ Rha+ Bxyl+). Two FIRN-like (Fim- Inl+ Rha- Bxyl-) strains that did not yield Fim+ recombinants in crosses with FIRN strains were probably wild-type Inl+ mutants from FIRN strains.
Both cultures of Escherichia coli were examined for mannose-sensitive (MS) haemagglutinin in rocked-tile tests with guinea-pig red cells at ambient temperature, and agar plate cultures were examined for mannose-resistant eluting (MRE) haemagglutinins against 14 species of red cells in tests mixed at 3--5 degrees C in the presence of .05% (w/v) D-mannose. Ox, sheep, human, pig, horse, guinea-pig, and fowl red cells were required to detect the various patterns of MRE haemagglutination with the different species of cells. Of 387 strains in 155 O serogroups, 95 formed both MS and MRE haemagglutinins (MS+/MRE+), 198 formed only MS (MS+/MRE-), 21 only MRE (MS+/MRE+), and 73 neither (MS-/MRE-). Strains of more than one of these types, and MRE+ strains with different cell specificities were found in many of the serogroups. Some strains in 144 O serogroups had MS haemagglutinin and some in 50 an MRE haemagglutinin. The presence of MS haemagglutinin in a culture was invariably associated with the presence of type-1 fimbriae on the bacteria. All MS+ strains shared a common antigen in their type-1 fimbriae and three groups of these strains possessed also a group-specific fimbrial antigen. The presence of certain kinds of MRE haemagglutinin in over half the MRE+ strains was associated with that of type-MRE fimbriae, but fimbriae were not detected in the other MRE+ strains. The antigens of the MRE haemagglutinins in different strains were heterogeneous and differed from those of the type-1 fimbriae of MS+ strains. Three series of strains from normal faeces, and from patients with infantile diarrhoea and urinary-tract infections each included a minority possessing neither type of haemagglutinin, but this observation did not preclude a role of the haemagglutinins in colonization or pathogenicity.
A series of 2092 cultures of Salmonella typhimurium isolated from human, animal and other sources in 57 countries were differentiated into 204 phage types and 19 primary and 147 full biotypes. Different biotypes belonged to the same phage type and different phage types to the same biotype, so the combination of typing methods differentiated strains more finely than either method alone: 574 different ;phage type/biotypes' were distinguished in 1937 cultures belonging to the 204 recognized phage types.The combination of biotyping with phage-typing was valuable in studying the phylogeny and spread of epidemic strains by distinguishing clones of different biotype within the same phage type and by confirming the relationship between cultures isolated from widely dispersed clones and that between cultures isolated before and after a clone had undergone variation in phage type, biotype, colicin type or antibiotic-sensitivity pattern.A widespread outbreak of infection with S. typhimurium phage type 141 in Scotland comprised independent dissemination of three clones of different biotypes, 1f, 9f and 31bd. During its epidemic spread in cattle in Britain between 1962 and 1969, another strain underwent variations in phage type (type 44 to type 29), biotype (type 26a to types 26d, 26bd, 26dgi, 26dz and 26i) and antibiotic sensitivity. A group of 275 non-fimbriate, non-inositol-fermenting and non-rhamnose fermenting (FIRN) strains, particularly associated with avian infections and thought to be clonal in origin, contained 27 phage types and 22 full biotypes in the primary biotypes 29-32.
A genotypically non-fimbriate (Fim-) strain of Salmonella typhimurium and a genotypically fimbriate (Fim+) strain derived from it by spontaneous mutation were compared for pathogenicity in mice. The two strains caused similar numbers of infections and deaths in groups of mice challenged by intraperitoneal inoculation, and nearly similar numbers in groups challenged by inoculation on to the conjunctiva, but the Fim+ strain caused many more infections (plus 26%) and deaths (plus 40%) than the Fim- strain when the inoculation was by mouth. Faecal cultures were made at intervals up to 120 days in the mice surviving after oral or conjunctival challenge and S. typhimurium was isolated more commonly from the animals challenged with the Fim+ strain (906 isolations from 384 animals infected out of 877 challenged) than from those challenged with the Fim- strain (614 isolations from 341 animals infected out of 877 challenged). The greater opportunity for faecal dissemination enjoyed by Fim+ bacteria may account for the preponderance of Fim+ over Fim- strains of S. typhimurium found in mammalian sources.
A new, two-tier system for biotyping Salmonella typhimurium gives a finer and more reliable differentiation of strains than the Kristensen scheme and is capable of future extension by the addition of new types and new tests. Strains are allocated to a primary type (1-32) by their reactions in five primary tests with Bitter's xylose medium, meso-inositol, L-rhamnose, d-tartrate and m-tartrate. Subtypes are distinguished within the primary types by reactions in ten secondary tests, which include observations for flagella and type-1 (haemagglutinating) fimbriae. Full biotypes are designated by letters indicating the subtype reactions appended to the primary-type numbers. A series of 2030 strains of S. typhimurium collected from many different sources and countries during 53 years was classified into 19 of the 32 potential primary biotypes and into 144 full biotypes. Of the series, 14% (275) were non-fimbriate inositol-nonfermenting rhamnose-nonfermenting (FIRN) strain in primary biotypes 29-32. Most other strains were fimbriate and rhamnose fermenting. Observations on several series of cultures isolated from different human or animal sources in the same epidemic showed that the biotype characters of a strain were generally stable during its growth in the natural environment and in the unselective media used for isolation and storage. Most non-fermenting strains gave rise to fermenting mutants on prolonged incubation in the substrate-containing--and therefore selective--test medium, and false-positive results from this cause were avoided by making the definitive readings of tests after a short, carefully chosen period of incubation. A genealogical tree has been drawn to show how eighteen observed primary biotypes may have evolved from a presumed archetypal ancestor of biotype 1.
The colour-change and lead acetate tests for fermentation of d-, l- and m-tartaric acids and citric acid used in the Kristensen scheme for biotyping Salmonella typhimurium were found to be unreliable because, whatever the conditions of culture, they gave different results in replicate tests of the same strains. Many genotypically non-fermenting strains gave inconsistent reactions due to the emergence of fermenting mutant bacilli in some of their test cultures. No reliable test was found for the fermentation of citric acid.A ;turbidity' test was found to give consistent and reliable results with the three tartaric acid isomers. It demonstrated fermentation by the significantly greater amount of growth obtained in a 24 hr. culture in Oxoid peptone water with added isomer than in a control culture without isomer. Lewis & Stocker's (1971) plateinhibition test for fermentation of m-tartrate, which identifies m-tartrate-negative strains because m-tartrate inhibits their growth on citrate- or glycerol-containing minimal medium, was found to be as reliable as, and easier to read than, the turbidity test.Use of the turbidity test for d- and l-tartrates and the plate-inhibition test for m-tartrate in biotyping 1435 strains of S. typhimurium showed that many strains had previously been mistyped by the lead acetate test and distinguished 16 new biotypes in addition to the 22 biotypes already recognized.
The ability to form type 1 fimbriae (Fim(+)) was readily transduced to 159 out of 161 wild-type motile Fim(-) FIRN strains of Salmonella typhimurium with phage P22 propagated on a Fim(+) donor strain. Fim(+) clones were isolated from about 35% of tests after the fimbriate bacteria in the transduction mixture had been enriched by culture in aerobic static broth for 48 to 96 hr. A Fim(+) transductant was isolated from only 1 out of 280 tests made with 10 nonmotile recipient FIRN strains that were nonflagellate (Fla(-))- or possessed "paralyzed" flagella (Fla(+) Mot(-)), though motile variants from these strains were fully competent in yielding Fim(+) transductants. The property of motility was thought to facilitate the selective outgrowth of Fim(+) transductant bacteria by enabling them to migrate aerotactically to the surface of the broth where their fimbriae permitted them to float and grow in a pellicle stimulated by the free supply of atmospheric oxygen.
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Competitive mixed cultures were grown from inocula of a large number of bacteria of a genotypically nonfimbriate (fim(-)) strain of Salmonella typhimurium and a small number of a genotypically fimbriate (fim(+)) variant strain that formed type 1 fimbriae and had been derived from the fim(-) strain by phage transduction. The fim(+) strain differed from the fim(-) strain in fermenting l-rhamnose (rha(+)), and the viable fim(+) and fim(-) bacteria present in the cultures after different periods at 37 C were counted differentially in platings on rhamnose media. When the cultures were grown under aerobic static conditions in tubes of nutrient broth, the fim(+) bacteria rapidly outgrew the fim(-) bacteria, so that, although starting as a small minority (e.g., 1 in 10(7)), they approached or surpassed the number of the fim(-) in 48 hr. A pellicle consisting of fimbriate bacteria was formed on the surface of the broth between 6 and 24 hr, and it is thought that the advantage of access to atmospheric oxygen enjoyed by these bacteria in the pellicle enabled them to outgrow the fim(-) bacteria confined in the oxygen-depleted broth. The fim(+) bacteria did not show selective outgrowth in mixed cultures grown in broth aerated by continuous shaking, in static broth incubated anaerobically in hydrogen, and on aerobic agar plates, i.e., under conditions not allowing an advantage from pellicle formation. The outgrowth of fim(+) bacteria in aerobic static broth was prevented by the addition of alpha-methylmannoside, a substance that inhibits the adhesive and early pellicle-forming properties of bacteria with type 1 fimbriae. A motile flagellate (fla(+)) variant of a fim(-)fla(-) strain of S. typhimurium outgrew its parent strain in mixed cultures in aerobic static broth, but the selective advantage conferred by motility was weaker than that conferred by fimbriation.
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