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.

Grouping of Salmonella enterica serotype Montevideo strains by ribotyping and IS200 profiling.

One-hundred and twenty-one isolates of Salmonella enterica serotype Montevideo, representing different biotypes and incidents of infection detected in the UK between 1977 and 1995, were analysed by EcoRI ribotyping, PvuII ribotyping and IS200 fingerprinting. Among the isolates examined, 7 EcoRI ribotypes, 5 PvuII ribotypes and 55 IS200 profile types were recognized and 4 arbitrary groups defined. All 33 isolates of biotype 2d belonged to EcoRI/PvuII ribotype 1/1 and IS200 lineage A and comprised Group I. The other 88 isolates of biotype 10di and its variants were assigned to Groups II-IV. All 27 isolates in Group II were of EcoRI/PvuII ribotype 2/2 and IS200 lineage B. Among the 43 isolates in Group III, 42 of which were of EcoRI/PvuII ribotype 3/3, IS200 analysis identified 38 profiles in lineages C-I. Six EcoRI/PvuII ribotypes and 8 IS200 profiles, mostly in lineages C-E, were recognized among the 18 isolates in Group IV. The combined use of biotyping and ribotyping, and to some extent IS200 profiling, has enhanced our understanding of the clonal structure of serotype Montevideo and provides a basis for further study.

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.

Molecular fingerprinting of Salmonella serotype Glostrup.

Thirteen isolates of Salmonella serotype Glostrup (antigenic formula, 6.8:z10:e,n,z15) from various sources and countries were analysed by ribotyping and IS200 fingerprinting. Both methods provided a high index of strain discrimination by allowing detection of three ribotypes and eight IS200 fingerprints which, though generally related, were readily distinguishable. The findings of this analysis confirm the usefulness of ribotyping and IS200 fingerprinting for studying the epidemiology of rarely isolated salmonellae of serogroup C.

Assessment of strain relatedness among Salmonella serotypes Salinatis, Duisburg, and Sandiego by biotyping, ribotyping, IS200 fingerprinting, and pulsed-field gel electrophoresis.

Salinatis (antigenic formula, 4,12:d:eh:enz15) is a rare Salmonella serotype currently designated a triphasic variant of the diphasic serotype Duisburg (1,4,12,27:d:enz15) (underlining indicates that the O antigen is determined by phage lysogenization). Salinatis could also be related to serotype Sandiego (4,[5],12:eh:enz15), from which it might have been derived by loss of H-d flagellin genes. Nineteen Salmonella strains of serotypes Salinatis, Duisburg, and Sandiego were examined by biotyping, PvuII and SmaI ribotyping, IS200 fingerprinting, and pulsed-field gel electrophoretic profiling. Results from these methods, used alone or together, indicate that serotype Salinatis is more likely to be related to serotype Sandiego than to serotype Duisburg. For future lists of serotype names, it is recommended that Salinatis be considered a variant of Sandiego.

Molecular typing of Salmonella serotype Thompson strains isolated from human and animal sources.

One-hundred-and-thirteen isolates of Salmonella serotype Thompson from diverse sources in seven countries were characterized by PvuII ribotyping and IS200 fingerprinting. Ten PvuII ribotypes were observed. The predominant PvuII ribotype 1 represented a major clone of world-wide distribution but was not found in Australia; PvuII ribotypes 2 and 3 represented minor clones. HincII ribotyping discriminated subtypes within PvuII ribotype 1: HincII ribotype 1 was distributed widely but HincII ribotype 2 was found mainly in Scottish isolates. None of 101 isolates of PvuII ribotypes 1-3 contained copies of IS200. All 12 isolates of PvuII ribotypes 4-10 were from Australia and 7 of them contained copies of IS200 of 5 different profiles. These results suggest the existence of at least two lineages of Salmonella Thompson with a different geographical distribution. The finding that most isolates from man and poultry in Scotland belonged to the same ribotype (PvuII 1/HincII 2) and were IS200-negative suggests that poultry is an important source of human infection in Scotland.

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.

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.

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.

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.

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.

A miniaturized biotyping system for strain discrimination in Escherichia coli.

A two-tier miniaturized scheme of eight tests was devised for biotyping strains of Escherichia coli in microwell plates. Primary biotypes were defined by positive and negative reactions in tests for fermentation of raffinose, sorbose, dulcitol and 2-deoxy-D-ribose and for decarboxylation of ornithine when read after specified periods of incubation; subtypes were identified within primary biotypes according to results in secondary tests for rhamnose fermentation, lysine decarboxylation and motility. The method gave reproducible results on different occasions of testing. Among 100 E. coli strains from various sources, 26 of the 32 possible primary biotypes and 56 full biotypes, as defined by results in both primary and secondary tests, were identified, thus demonstrating a high index of strain discrimination (D = 0.98). The scheme is recommended as a simple, reliable, inexpensive and efficient method of differentiating strains of E. coli.

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.

Numerical index of the discriminatory ability of biotyping and resistotyping for strains of Escherichia coli.

A discrimination index was applied to assess the value of biotyping and resistotyping, used alone or together, for the subspecific discrimination of Escherichia coli. The index was high when 599 strains from a wide variety of sources were examined by full biotyping with 10 tests. Although the discrimination achieved was lower when a sub-collection of 333 strains was examined, that finding probably indicated that many of the latter strains were of urinary origin and represented a limited number of uropathogenic clones. Combined biotyping and resistotyping provided a higher level of strain discrimination than either method used on its own. Results suggest that these typing methods may be used with confidence for the discrimination of strains of E. coli.

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.

Genetic population structure, clonal phylogeny, and pathogenicity of Salmonella paratyphi B.

Genetic diversity and relationships among 123 strains of Salmonella paratyphi B (serotype 1,4,[5],12:b:[1,2]) were estimated from an assessment of electrophoretically demonstrable allelic variation at 24 chromosomal enzyme gene loci. Fourteen electrophoretic types, marking clones, were distinguished, the phylogeny of the clonal lineages was reconstructed, and biotype and other phenotypic characters were mapped onto this structure. Most d-tartrate-negative strains are members of an abundant, globally distributed clone (Pb 1) that is polymorphic for many biotype characters (including d-tartrate utilization), bacteriophage type, rRNA pattern, and colicin M and phage ES18 sensitivity. This clone is largely responsible for S. paratyphi B enteric fever in humans. In contrast, d-tartrate-positive strains (formerly known as S. java) occurred in all seven of the clonal lineages identified by population genetic analysis, although most d-tartrate-positive isolates belong to only two clones (Pb 3 and Pb 4), which vary in frequency geographically. Monophasic strains represent four closely related clones forming a distinctive phylogenetic lineage. The Kauffmann hypothesis of convergence in serotype among distantly related cell lineages through recombination (via phage transduction or other means) may account for the considerable genotypic diversity among clones of S. paratyphi B. Pb 4, Pb 6, and Pb 7 are more closely allied with clones of S. typhimurium and S. saintpaul than with other clones of S. paratyphi B. Sensitivity or resistance to colicin M and phage ES18 and the electrophoretic pattern of the rRNA, which were incorporated into a recently proposed scheme for the identification of types of S. paratyphi B, individually or in combination fail to mark clones or other meaningful phylogenetic subdivisions.

Immunological and genetical relatedness of type-1 and type-2 fimbriae in salmonellas of serotypes Gallinarum, Pullorum and Typhimurium.

The fimbriae of 50 strains of serotype Gallinarum and 35 strains of serotype Pullorum of the genus Salmonella were compared with the type-1 fimbriae of serotype Typhimurium strains by immune electron microscopy and dot blot hybridization tests with gene probes for type-1 fimbriation in Typhimurium. The fimbriae of Gallinarum and Pullorum strains were coated with Typhimurium type-1 fimbrial antiserum and probes hybridized strongly with DNA of Gallinarum and Pullorum strains under stringent conditions. Furthermore, when Typhimurium type-1 fimbrial antiserum, that had been absorbed with fimbriate Gallinarum or Pullorum bacteria, was used in immune gold labelling experiments, it was shown that residual antibody recognized sites of possible adhesin incorporation at intervals along the length of Typhimurium type-1 fimbriae. These findings suggest that the type-2 fimbriae produced by all Gallinarum and Pullorum strains are non-adhesive forms of adhesive, type-1 fimbriae. This observation is of interest because type-1 fimbriae have never been reported in naturally occurring strains of these two avian-adapted serotypes.

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.

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.

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.