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.

Family outbreak of dysentery caused by a rhamnose non-fermenting, ONPG-negative strain of Shigella sonnei phage type 6.

Three members of a Scottish family, with no history of foreign travel but who had recently visited Bristol, were infected by a strain of Shigella sonnei of phage type 6 (PT 6) that did not ferment rhamnose and was negative for o-nitrophenyl-beta-D-galactopyranoside (ONPG). The incident exposed limitations associated with commercial systems for the identification of strains of S. sonnei with atypical biochemical properties.

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.

Human infection in Tayside, Scotland due to Salmonella serotype Livingstone.

Livingstone was the third most common salmonella serotype isolated from cases of human salmonellosis in the Tayside region of Scotland in 1989-1991; latterly, it spread to Grampian region. The significant upsurge of Livingstone in these two Scottish regions was not matched by similar increases in its frequency of isolation from human cases of salmonellosis in other regions of Scotland or elsewhere in the UK. Although Salmonella Livingstone is usually associated in the UK with incidents of infection among poultry flocks, our detailed investigations found no clear evidence that poultry, eggs or poultry-related products were responsible for this outbreak. Most cases occurred in the summer months from July to September and many of the patients required hospital treatment. Other than one outbreak among geriatric patients in a long-stay hospital in north Tayside, most of the cases were sporadic. The extent of the outbreak, covering 3 years, was recognised mainly because Livingstone was previously an uncommon serotype in Tayside. There were few Livingstone isolations from non-human sources in Scotland in these same years. Possible sources of infection and predisposing factors among patients are discussed. Livingstone was not isolated in Scotland in 1992.

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.

In-vitro and in-vivo characteristics of TnphoA mutant strains of Salmonella serotype Gallinarum not invasive for tissue culture cells.

Insertion mutants of a strain of Salmonella serotype Gallinarum, the cause of fowl typhoid, were produced with transposon TnphoA. Eight mutants were identified as being less invasive in cell culture than the parent strain. Neither the parent strain nor the mutants showed mannose-sensitive haemagglutination of various red blood cell species. Although two mutants gave mannose-resistant (MR) haemagglutination of red cells of different animal species these MR activities could not be correlated with other characteristics in vitro or in vivo. The mutant strains were divisible into three classes by their patterns of invasiveness and adhesiveness in vitro and by changes in membrane proteins. Strains of classes 1 and 2 had single transposon insertions, detected by Southern hybridisation, of 8.9 and 2.4 kb EcoRV-digested chromosomal-fragments, respectively, and were slightly less invasive than the parent strain. They were no less virulent for chickens by the oral route than a mutant strain shown to be as invasive in vitro as the parent strain. The class-2 mutant was also less adhesive than the other strains. The single mutant strain of class 3 which contained insertions in several chromosomal fragments, was non-invasive in Vero cells (less than log10 1.0 cfu/ml recovered compared with log10 2.88 for the parent strain) and showed reduced virulence by both oral and intramuscular routes. The mutant strains of all classes were taken up equally rapidly from the blood by the spleen. Intramuscular immunisation of chickens with the class-3 mutant strain gave complete protection against oral challenge 3 weeks later with 1000 oral LD50 doses of the virulent parent strain.

A cost-effectiveness study of the management of intractable urinary incontinence by urinary catheterisation or incontinence pads.

STUDY OBJECTIVE: The aim was to compare the costs and effects of management of intractable urinary incontinence by urinary catheterisation or incontinence pads. DESIGN: This was a prospective, randomised study comparing catheterisation with pads, supplemented by additional data collected from patients with chronic indwelling catheters. Main outcome measures were costs of equipment, nursing time, patient preference, nursing preference, and clinical and bacteriological assessment of urinary infection. SUBJECTS: 78 intractably incontinent elderly female patients were randomly allocated to management by urinary catheter or pads and toileting. Supplementary data on equipment costs and nursing time were collected from 27 patients, of whom 22 were already catheterised at the time of the randomisation and five were catheterised by the nursing staff after the last date for entry into the randomisation. MAIN RESULTS: Of the 38 patients randomised to catheterisation, 14 refused consent so only 24 were catheterised on day 1 of the study. There was a rapid removal of catheters, especially in the first six weeks of the study and only four of the randomised catheter patients completed the full 26 weeks of the study. However, eight of the pads patients were catheterised between the 7th and 22nd week because of deteriorating general condition and all retained their catheters for the remainder of the study period. Of 35 patients who had experienced catheters and pads, 12 expressed a clear preference for catheters, 12 for pads, and 11 were undecided. Nurses were in favour of the use of pads, mainly because of concerns about urinary infection with catheters. Comparing costs for patients managed with catheters (532 patient weeks) or pads (903 patient weeks), catheter patients required less nursing time (15.4 v 29.0 h per patient per week) but equipment costs were higher (19.20-24.65 pounds v 8.79-11.35 pounds per patient per week), mainly because of the cost of catheter care (12.75 pounds per patient per week). Asymptomatic bacteriuria was prevalent in both groups but 73% of catheterised patients received treatment for clinical signs of infection compared with 40% of pads patients. Only 30% of patients who were treated had any generalised symptoms of infection. CONCLUSIONS: Use of catheters reduces nursing time but may increase weekly equipment costs depending on the cost of laundry. Despite the high dropout rate among patients randomised to catheters a minority of patients (12/35) expressed a clear preference for catheters and we believe that more patients with intractable incontinence should be given a trial of catheterisation to assess acceptability. Bacteriuria was prevalent in pads or catheter patients but no major episodes of invasive infection were noted in either group.

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.