Characterisation of urinary isolates of Escherichia coli by multiple typing: a retrospective analysis.

Cultures of Escherichia coli of urinary origin were examined after storage for 12 to 15 years by a combination of the techniques of biotyping, resistotyping, haemagglutinin typing, and colicin typing (multiple typing) which allowed strain identification to be made even when variation in one or more typing characters had occurred in vivo. Biotyping and resistotyping used in conjunction were sufficient to allow identification of as many as 106 of 110 pairs of E. coli examined. In the four pairs of isolates for which it was necessary to extend strain identification profiles to include data from other typing methods, probable strain identification was achieved for all but one pair. The multiple typing approach seems useful for retrospective analysis of stored cultures of E. coli.

Methods for the detection of haemagglutinins in Aeromonas.

When grown in specified conditions and tested by a rocked-tile method, 40 of 41 isolates of two species of Aeromonas formed simultaneously at least two haemagglutinins among which were: (i) a mannose-sensitive haemagglutinins with strongest activity for guinea-pig or fowl red cells, formed by all of 31 isolates of A. hydrophila and 9 of 10 isolates of A. punctata ss. caviae; (ii) a haemagglutinin, sensitive to L-fucose or D-mannose, that reacted with human red cells and which was formed by all 41 isolates; and (iii) a mannose-resistant 'tanned red cell' haemagglutinin formed by 29 isolates of A. hydrophila and one isolate of A. punctata ss. caviae. Results emphasise that for the fullest possible identification of haemagglutinins produced by Aeromonas spp., strains should be cultured in a variety of conditions and tested with a wide range of red-cell species.

Salmonellae of serotypes gallinarum and pullorum grouped by biotyping and fimbrial-gene probing.

When salmonellae of serotypes Gallinarum (50 isolates) and Pullorum (36 isolates), that produce non-adhesive (type-2) fimbriae, were tested for their reactions in biochemical tests, 81 (94%) were found to belong to three distinct biochemical groups, I-III. Interaction of HinfI-digested DNA of both Gallinarum and Pullorum with a probe of accessory genes of type-1 fimbriation in serotype Typhimurium gave one type of Southern hybridisation pattern that was readily distinguished from that of Typhimurium strains. With a probe of the Typhimurium fimbrial subunit gene, Pullorum isolates were separated into strongly and weakly probe-reactive groups which showed restriction fragment-length polymorphism; these latter groups corresponded to biochemical groups II and III, respectively.

Biotyping of Escherichia coli.

We examined the results of tests with 22 substrates for their ability to discriminate a series of 917 strains of Escherichia coli collected from different sources. The tests with three of the substrates were discarded because of difficulties in performance or interpretation, and another nine substrates because they provided little discrimination. The tests used to obtain biotype profiles for strains were those for the fermentation of dulcitol, D-raffinose or sucrose or both, L-rhamnose and L-sorbose, the decarboxylation of L-lysine and L-ornithine, the hydrolysis of aesculin, motility, and prototrophy. Observations on several series of cultures from different sources showed that biotype characters were stable in vivo and after storage on non-selective medium. The biotype profiles obtained were as reliable as partial O serotyping for the routine subtyping of strains of E. coli isolated from the urine of patients with long-term urinary-tract infections and those from other sources in different patients. Biotyping and O serotyping used in conjuction offered a very fine degree of strain discrimination.

A biotyping scheme for the subspecific discrimination of Escherichia coli.

A two-tier system for biotyping Escherichia coli gave a fine and reliable differentiation of strains and is capable of future extension by the addition of new types and new tests. Strains were allocated to a primary biotype (1-16) by their reactions in four primary tests in culture media containing raffinose, sorbose, ornithine or dulcitol. Subtypes were distinguished within the primary biotypes by reactions in six secondary tests for rhamnose fermentation, lysine decarboxylation, aesculin hydrolysis, motility, type-1 fimbriation and prototrophy. Full biotypes were designated by letters indicating subtype reactions appended to primary type numbers. A series of 599 strains (1242 cultures) of E. coli from diverse sources was classified into 16 primary biotypes and into 213 full biotypes. The biotype characters of a strain were generally stable during its spread in the natural environment and in non-selective media used for storage.

P fimbriation among biochemically inactive strains of Escherichia coli of the group formerly called Alkalescens-Dispar.

Biochemically inactive, non-motile strains of Escherichia coli of the group formerly known as Alkalescens-Dispar (AD) and of known AD serogroups were analysed for biotype, resistotype, type-1 fimbriation, P fimbriation and haemolysin production. All strains of AD serogroups O1 and O2 examined were P-fimbriate and of closely related bio-resistotypes, suggesting that they may be representatives of two E. coli P-fimbriate clones, members of which have not infrequently been isolated from infected urine.

Demonstration by immuno-electronmicroscopy of antigenic heterogeneity among P fimbriae of strains of Escherichia coli.

The identification of P fimbriae on urinary strains of Escherichia coli isolated from urine was made by observation of the patterns of mannose-resistant and eluting haemagglutination of erythrocytes of seven animal species (and including those of human p phenotype), and by haemagglutination-inhibition tests with hydatid-cyst fluid known to contain an analogue of P-fimbrial receptor. In tests with five different, pure P-fimbrial antisera prepared in rabbits, agglutinin titres of 37 P-fimbriate strains revealed differences in their reactivity; immuno-electronmicroscopy studies with the same five antisera showed that P-fimbriate strains were markedly different in the extent to which their P fimbriae were coated with antibody. The antigenic heterogeneity observed among P fimbriae is discussed with regard to the development of P-fimbrial vaccines.

Phenotypic and genotypic discrimination of strains of Salmonella serotype Eimsbuettel from human and animal sources.

One hundred isolates of Salmonella serotype Eimsbuettel from various human, animal and environmental sources in six countries were typed and shown to belong to five ribotypes, five biotypes and eight different ribotype/biotype groups, one of which, ribotype 3/biotype 5, was represented among isolates from all six countries. Most of the Eimsbuettel isolates from Scotland belonged to ribotype 1/biotype 3, which was the epidemic strain involved in a large outbreak centred in a Glasgow maternity hospital in 1986. That strain was also responsible for almost all the human infections that occurred in the west of Scotland in the years of this study. However, isolates from human cases in the east of Scotland belonged to either ribotype 2/biotype 1 or ribotype 3/biotype 5, groups not found in the west of Scotland. Representatives of all three ribotype/biotype groups causing human infection in Scotland were also found among isolates from poultry or poultry-associated materials. Plasmids were carried by only 14% of isolates and so provided little additional strain discrimination. However, plasmid analysis suggested that Salmonella Eimsbuettel of ribotype 2/biotype 1 had the potential to enter the human food chain in the UK via meat or bone meal, animal feed and poultry.

Distribution of insertion sequence IS200 among different clonal lines of the related Salmonella serotypes Livingstone and Eimsbuettel.

The copy number and genetic location of IS200 have provided evidence of strain relatedness in many serotypes of Salmonella. In this study, 100 isolates of the related serotypes Livingstone (6,7:d:l,w) and Eimsbuettel (6,7,14:d:l,w), representing 10 ribotype/biotype (RT/BT) groups isolated from human and non-human sources in seven countries over a 26-year period, were examined for their IS200 profiles. The distribution of IS200 in strains of these serotypes was limited, being present in all 53 isolates of ribotype 1 (RT1) and its variant type RT6, in one of five isolates of RT5 but in none of 42 isolates of RTs 2, 3 or 4. Although the seven IS200 profiles identified in RT1 isolates were of little value for further discrimination within different biotype groups, they were extremely valuable for confirming serotype: isolates of RT1/BT8/IS200 profile A (or its variants) and those of RT1/BT3/IS200 profile B (or its variants) were almost invariably associated with serotypes Livingstone and Eimsbuettel, respectively.

An attempt to identify the evolutionary origin of a novel serotype of Salmonella enterica isolated from harbour porpoises.

The isolation since 1991 of a new serotype of Salmonella enterica (antigenic formula 4,12:a:-) from harbour porpoises (Phocoena phocoena) at post-mortem examination raised the question of its evolutionary origin. Representative strains of S. enterica serotype 4,12:a:- and strains of eight other serotypes of serogroup 04 with phase-1 flagellar antigen H 'a' were examined by EcoRI ribotyping, IS200 fingerprinting and PCR-based profiling. Statistical analysis of results of multiple typing showed that strains of Salmonella serotype 4,12:a:- were genetically distant from those of antigenically similar salmonella serotypes, none of which seemed likely to be the progenitor of the 'porpoise' serotype.

Characterisation of strains of Salmonella serotype Livingstone by multiple typing.

Isolates of Salmonella serotype Livingstone (6,7:d:1,w) from man, water and various animals and animal products in Canada, England, France, Israel and Scotland were examined for ribotype, biotype and plasmid profile. Analysis by these methods indicated that an epidemic strain of Livingstone of ribotype 1/biotype 8/plasmid-type 6 was responsible for the major upsurge of Livingstone infection that occurred in man in Tayside (Scotland) between 1989 and 1991; that type was also isolated from spring water, animal feed and poultry. Livingstone isolates of ribotype 1/biotype 8 with plasmid profiles other than type 6 were also present in Scotland, England and France at that same time. Among representative Livingstone isolates from England, a strain of ribotype 2/biotype 1 was predominant in man and poultry products between 1988 and 1992, although strains of other ribotypes (1, 3 and 4) were also present. Strains of ribotype 3 of different biotypes were obtained from poultry and animal feed sources in Canada. A strain of ribotype 5/biotype 3 caused human infections in Israel between 1968 and 1992. Ribotyping, biotyping and plasmid profile analysis used together have helped to trace the sources and extent of spread of human infections caused by Salmonella Livingstone.

Mannose-resistant and eluting haemagglutinins and fimbriae in Escherichia coli.

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.

Discrimination of urinary strains of Escherichia coli by five typing methods.

A series of 156 cultures of Escherichia coli isolated from sequential urines from 20 patients with urinary-tract infection was examined by biotyping, resistotyping, haemagglutinin typing, O serotyping and antibiogram typing. From 10 of the patients, we repeatedly isolated cultures of a single strain; from each of the other 10 patients, different strains were discriminated. All cultures were typable by biotyping and resistotyping, and those techniques should prove practicable for laboratories not able to perform complete serological analysis. Only a minority (43.6%) of the cultures was typed with a limited range of 24 commercial O sera. Maximum discrimination of strains was achieved by the combined use of several of these techniques.

Ribosomal-RNA patterns of Escherichia coli, Salmonella typhimurium and related Enterobacteriaceae.

rRNA sequences are usually highly conserved among species. In Enterobacteriaceae we have shown that Salmonella typhimurium does not have an equivalent to the 23S rRNA of Escherichia coli but its 23S rRNA is cleaved in vivo into two smaller species. This cleavage appears to be a result of a difference between the S. typhimurium and E. coli rRNA sequences rather than to differences in ribonuclease activity. We have surveyed a wide range of Enterobacteriaceae for the presence or absence of 23S rRNA and found this rRNA species to be present in all strains of E. coli, Shigella and Citrobacter and all salmonellae examined except S. typhimurium. All S. typhimurium cultures, isolated at different times and from several different countries, lack an intact 23S rRNA. Thus, the presence or absence of this rRNA species is an excellent diagnostic characteristic for S. typhimurium.

Types of Salmonella paratyphi B and their phylogenetic significance.

The substrates inositol, rhamnose, d-tartrate and m-tartrate used in fermentation tests with 338 cultures of Salmonella paratyphi B differentiated strains in some phage types to give information that could be used in epidemiological investigations. Xylose in Bitter's medium, the fifth substrate by which 13 of a potential 32 biotypes were identified, differentiated few cultures with the negative character. The possession of a specific type of outer-membrane protein receptor for colicin M or bacteriophage ES18 and the particular type of ribosomal ribonucleic acid present, defined three groups among the phage-typed and biotyped cultures. The possibility that the serotype S. paratyphi B contains clones of different phylogenetic origin and the consequent implications for nomenclature are discussed.

Evolutionary origin and radiation of the avian-adapted non-motile salmonellae.

Multilocus enzyme electrophoresis was employed to estimate chromosomal genotypic diversity and relationships among 131 isolates of the non-motile Salmonella biotypes Gallinarum and Pullorum (serotype 1, 9, 12:-:-) that cause fowl typhoid and pullorum disease, respectively. Thirteen electrophoretic types (ETs), marking clones, were distinguished, and construction of a neighbour-joining phylogenetic tree revealed three lineages: one consisted of five ETs of Gallinarum, a second included seven ETs of Pullorum, and a third was represented by a single ET (Ga/Pu 1) that is intermediate between those of the other two lineages in both multilocus enzyme genotype and biochemical properties. Enzyme genotype analysis and comparative nucleotide sequencing of the phase 1 flagellin gene (fliC), the hook-associated protein 1 gene (flgK), and the 6-phosphogluconate dehydrogenase gene (gnd) identified serotype Enteritidis (1, 9, 12:g, m:-) as a close relative of the non-motile salmonellae. In most strains of biotype Gallinarum, the fliC gene is complete, intact and identical in sequence to that of Enteritidis, but isolates of three ETs had a stop codon at position 495. The fliC sequences of the ETs of Pullorum differed from that of Enteritidis in having non-synonymous changes in either two or three codons and a synonymous change in one codon. The sharing of distinctive alleles at three metabolic enzyme loci and a stop codon in flgK indicates that the non-motile salmonellae are monophyletic and that their most recent common ancestor was non-motile. Since diverging from that ancestor, the Pullorum lineage has evolved more rapidly than the Gallinarum and Ga/Pu 1 lineages.

An interesting problem of strain identification with urinary isolates of Escherichia coli.

Twenty-one cultures of Escherichia coli, recovered over a 5-year period from 14 urine specimens from a patient, were examined by multiple typing techniques selected for their reproducibility, their proven strain differentiating ability and for their suitability for use in small laboratories. Six isolates were readily identified as belonging to three distinct strains. Despite the finding that the remaining 15 isolates showed minor variations in resistotype and serotype characters, all 15 isolates belonged to the same full biotype. They were probably representatives of a fourth strain distinct from the other three strains identified, but even complete serological analysis could not resolve the problem of their strain identity.

Subspecies discrimination of staphylococci from revision arthroplasties by ribotyping.

Sixty-six cultures of Staphylococcus spp. were obtained from bone and tissue samples collected from 37 patients during revision arthroplasties and were speciated and ribotyped to assess strain diversity in each species. There were 10 ribotypes among 51 isolates of S. epidermidis, three among three isolates of S. capitis, two among four isolates of S. aureus and two among two isolates of S. simulans. One ribotype was found among each of: two isolates of S. warneri; two isolates of S. haemolyticus and single isolates of S. cohnii and S. saprophyticus. Low molecular weight bands of ribotype patterns characterized one or two related species whereas high molecular weight bands were useful for distinguishing types within species. Specimens from 17 patients yielded more than one isolate of Staphylococcus spp. In 13 of these patients the isolates were representatives of a single species but in only eight did ribotyping show the isolates to be identical. The findings of multiple species and ribotypes from samples taken from the same patient may have implications for understanding the nature of infection in revision arthroplasty and for antibiotic therapy.

Strain discrimination of a novel serotype of Salmonella from harbour porpoises (Phocoena phocoena) by molecular techniques.

The relatedness of 41 isolates of Salmonella of a novel serotype (antigenic formula 4,12:a:-) of serogroup B, obtained from harbour porpoises (Phocoena phocoena) stranded at various sites around the coastline of Scotland, was assessed by two molecular typing methods. Ribotyping showed that these isolates belonged to seven EcoRI (E) ribotypes and 11 PstI (P) ribotypes that were, in each case, distinct but closely related. Combined ribotyping data identified 15 different E/P ribotypes, the most common of which, E1/P1, was represented by 15 isolates from 14 animals stranded on both east and west coastlines. Strain discrimination achieved by E/P ribotyping was high (D=0.84). IS200 profiling revealed only three different fingerprints and strain discrimination by this method alone was poor (D=0.39). When E/P ribotyping and IS200 profiling were used together, they revealed the existence of 17 different types among the 41 isolates which formed two distinct, but related, groups of Salmonella serotype 4,12:a:-. This information should prove helpful in future studies examining the mode of transmission of this novel salmonella serotype and its association with disease in harbour porpoises.

Biotyping of Escherichia coli in microwell plates.

A simple, inexpensive scheme of eight tests for biotyping strains of Escherichia coli in microwell plates is described. The tests comprise primary tests for the fermentation of raffinose, sorbose, ornithine, dulcitol and 2-deoxy-D-ribose, and secondary tests for rhamnose fermentation, lysine decarboxylation and motility. Among a collection of 75 clinical isolates of Esch. coli from 12 patients, 18 full biotypes designated according to their positive and negative reactions in the eight tests were distinguished. These biotypes gave an indication of the natural history of patients' infections. Because it provides excellent and reliable type discrimination, biotyping can be used in a combination with other typing techniques to resolve local epidemiological problems involving Esch. coli.