Salmonella in Black-headed gulls ( Larus ridibundus); prevalence, genotypes and influence on Salmonella epidemiology.

During a period of 3 years, 1998-2000, 1047 faecal swabs from Black-headed gulls were sampled at one location in Southern Sweden. Salmonella spp. was found in 28 individuals (2.7%) and the dominating serotype found was S. Typhimurium (83%). Twenty-five per cent of the Salmonella-infected gulls were later recaptured and re-sampled. We found that Salmonella infection in Black-headed gulls was of short duration, and that infection in this bird species was predominantly expressed as carriage without disease manifestations. All S. Typhimurium isolates were subjected to antibiotic resistance profiling and molecular characterization by pulsed-field gel electrophoresis and IS200 polymerase chain reaction. The S. Typhimurium gull isolates were compared to human and domestic animal isolates of the same serotype and phage type. We found genetic relatedness of S. Typhimurium DT195 isolates from gulls, domestic animals and humans, indicating that Black-headed gulls might play a role in the spread of S. Typhimurium in Sweden.

Travel-associated non-typhoidal salmonellosis: geographical and seasonal differences and serotype distribution.

The Swedish database on notifiable communicable diseases was used to identify 24 803 cases of travel-associated non-typhoidal salmonellosis from the period 1997-2003. Serotype data were available for 24 358 (98.2%) of these cases, which were compared with a data set from the same period of 16 255 randomly selected Swedish residents with a history of recent overnight travel outside Sweden. The highest risk of disease was seen in travellers returning from East Africa (471/100 000 travellers; 95% CI 294-755), or the Indian subcontinent (474/100 000; 95% CI 330-681). Children aged 0-6 years were at higher risk than travellers of other ages (OR 2.4; 95% CI 2.1-2.8). Some distinct seasonal patterns could be distinguished, with highest (adjusted) risk in December in East Asia, and in August in Europe. Marked geographical differences in serotype distribution were noted. Salmonella Enteritidis was especially dominant in Europe, but was much less common in Africa, Asia and America, where the variety of circulating serotypes was greater. Overall, the two data sets provided important information on travel risks which are also likely to apply to travellers from other western countries.

[Anthrax--the Swedish perspective]. / Antrax--det svenska perspektivet.

The recent occurrence in the USA of deliberate release of virulent Bacillus anthracis in letters sent to three media corporations and to the American senate has led to a great anxiety in Sweden and elsewhere in Europe. Numerous letters have been suspected to contain B. anthracis spores and several have contained powder of different types. In none of the tested letters collected by the Swedish police have we been able to detect anthrax bacilli. Powder containing letters have been tested with either bacterial isolation and/or B. anthracis specific PCR. Anthrax is a disease found naturally in herbivores and is occasionally spread to humans. It is caused by the gram-positive rod B. anthracis that was discovered by Robert Koch in 1876. Beginning in the 1930s many states have developed B. anthracis for use as a weapon. A few releases of the bacteria have been reported before October 2001. B. anthracis causes three forms of disease, cutaneous, pulmonary and gastro-intestinal. The pulmonary form is the most dangerous and may lead to death merely one to two days after onset of severe symptoms. This is due to the rapid growth and release of several potent toxins that engage the immune system and promote tissue destruction. B. anthracis infection can be treated with several antibiotics, among which quinolones and tetracyclins have been recommended. Diagnosis can readily be achieved by microscopy, bacterial isolation and PCR at the Swedish Institute for Infectious Disease Control and the Swedish Defence Research Agency. Antibiotics relevant for treatment of B. anthracis infections are already stockpilled in our country. Further actions to strengthen our capability to deal with bioterrorism are ongoing.

Salmonella in sub-Antarctica: low heterogeneity in Salmonella serotypes in South Georgian seals and birds.

The number of human visitors to Antarctica is increasing rapidly, and with it a risk of introducing infectious organisms to native animals. To study the occurrence of salmonella serotypes in sub-Antarctic wildlife, faecal samples were collected from gentoo penguins, macaroni penguins, gray-headed albatrosses, black-browed albatrosses and Antarctic fur seals on Bird Island in the South Georgian archipelago during the austral summer of 1996 and 1998. In 1996, S. havana, S. typhimurium and S. enteritidis were isolated from 7% of gentoo penguins and 4% of fur seals. In 1998, however, 22% of fur seals were found to be infected with S. havana, S. enteritidis and S. newport. All isolates, except one, showed identical pulsed-field gel electrophoresis-patterns within each serotype, irrespective of sampling year and animal reservoir. No significant antibiotic resistance was found. The very low heterogeneity in the salmonella isolates found could either indicate a high genetic adaptation of the bacteria to the environment or a recent introduction of salmonella into the area.

Comparative analysis of PCR versus culture for diagnosis of ulceroglandular tularemia.

PCR and culture were comparatively evaluated for their abilities to demonstrate Francisella tularensis in wound specimens from tularemia patients during an outbreak in Sweden in 1998. For transport of the specimens used for PCR, a buffer solution containing a nuclease inhibitor was used, and for transport of the specimens used for culture, a commercial transport system was selected after experimental comparison of various systems. Of 40 patients with culture- and/or serology-verified ulceroglandular tularemia, PCR detected F. tularensis DNA in 30 (75%) patients, whereas culture detected bacterial growth in 25 (62%) patients. Compared to data from a previous study, the present inclusion of a nuclease inhibitor in the transport medium did not improve the sensitivity of the PCR, whereas the sensitivity of the culture procedure was significantly increased by selection of the system used for transport. Among eight patients with clinically suspected tularemia but with negative serology and culture, specimens from four patients showed detectable DNA. In three of these patients the diagnosis was verified by the demonstration of an F. tularensis-specific T-cell response in vitro. In conclusion, PCR was more sensitive than culture for demonstration of F. tularensis in wound specimens. Besides, we showed that tularemia may proceed without development of serum antibodies, and in these patients, PCR may be of special importance for verification of the diagnosis.

Structural studies of the O-antigenic polysaccharide from Escherichia coli O167.

The structure of the O-antigenic polysaccharide from Escherichia coli O167:H5 has been investigated. Sugar and methylation analyses, fast-atom-bombardment mass spectrometry and 1H- and 13C-NMR spectroscopy were the main methods used. The structure of the repeating unit of the polysaccharide was found to be: [formula in text]. Oligosaccharide derivatives of the polysaccharide were obtained by HF solvolysis and by a Smith degradation. Furthermore, base treatment of the polysaccharide led to a degraded polymeric material. For the methylated polysaccharide the amide linkage between alanine and the galacturonic acid residue was reductively cleaved with LiBD4 in ethanol, to give, among other things, a 3-O-methyl galactose derivative.

Structure of the O-polysaccharide from the LPS of a Hafnia alvei strain isolated from a patient with suspect yersinosis.

The carbohydrate backbone of the Hafnia alvei strain Y166/91 lipopolysaccharide (LPS) was isolated by mild acid hydrolysis followed by gel permeation chromatography and studied by NMR spectroscopy and methylation analysis. Treatment with periodate and hypoiodite gave a modified polysaccharide which was also characterised. It was concluded that the polysaccharide has the following structure with two distinct regions, which are connected. The chain length parameters m and n were not determined but the ratio m/n is approximately 2. [-->3)-beta-D-Galp-1(1-->3)-alpha-D-Galp-(1-->]m[-->3)-alpha-D-Galp-(1-- >3)- beta-D-Galf-(1-->]n From the present data it is not possible to determine whether it is the Galp-Galp chain or the Galp-Galf chain that is connected to the core. The structure found here is identical to that suggested for the O-specific polysaccharide chain of Klebsiella pneumoniae O1K2 (NCTC 5055) LPS [O. Kol, J.-M. Wieruszeski, G. Strecker, J. Montreuil, and B. Fournet, Carbohydr. Res., 217 (1991) 117-125; O. Kol, J.-M. Wieruszeski, G. Strecker, B. Fournet, R. Zalisz, and P. Smets, Carbohydr. Res., 236 (1992) 339-344].

Biochemical phenotypes of Salmonella Livingstone isolated from humans, animals and feedstuffs in Sweden.

Salmonella Livingstone is occasionally isolated from humans, animals and feedstuffs in Sweden. To follow the spread of infection and trace the source of isolates, adequate typing methods are needed. We have developed an automated typing system based on biochemical fingerprinting of bacteria (the PhP system) for typing of different Salmonella serotypes. The system measures the kinetics of various biochemical reactions of bacteria grown in liquid medium in microtiter plates and uses numerical techniques to identify biochemical phenotypes (BPTs) among the tested strains. In the present study we used a set of 16 highly discriminatory tests to differentiate strains of Salmonella of serotype Livingstone and evaluated the system for its discriminatory ability using a collection of 34 unrelated human isolates of S. Livingstone. We also used the system to investigate BPTs of 45 Livingstone strains isolated from animals and feedstuffs in Sweden between 1987 and 1991. Altogether 19 different BPTs were found among human isolate giving a diversity index (Di) of 0.930. In contrast, most strains isolated from animals and feedstuffs in Sweden belonged to 2 dominating BPTs (Di = 0.704). One of these contained 17 strains mainly isolated during 1992 whereas the other contained 18 strains isolated between 1987 and 1991. None of the Swedish human isolates were identical to those of animals and feedstuffs. These findings suggest that 2 different BPTs of Salmonella Livingstone strains are particularly common among animals and feedstuffs in Sweden and that they are not related to human cases of enteritis in this country. We also conclude that biochemical fingerprinting with the PhP system is a reliable and highly discriminatory method for detecting epidemic strains of Salmonella Livingstone.

Variations in biochemical phenotypes and phage types of Salmonella enteritidis in Germany 1980-92.

The Phene Plate system for typing Salmonella serotypes (PhP-S) is a simple automated typing method based on biochemical fingerprinting. It gives a quantitative value of the metabolism of various substrates by measuring the speed and intensity of each reaction. The 'biochemical fingerprint' of each isolate is used to calculate similarities among the tested strains with a personal computer program. We used this system to examine a collection of 86 strains of Salmonella enteritidis isolated from human sporadic cases in Germany between 1980 and 1992. Twenty-three biochemical phenotypes (BPTs) consisting of 9 common (C) and 14 single (S) BPTs were identified. BPTs C2 and C4 containing 20 and 36 strains respectively accounted for 65% of the isolates. Strains of BPT C2 were found over a wide period of time whereas strains of BPT C4 were isolated during the period between 1988 and 1992. With phage typing, 11 discrete phage types (PTs) and 18 strains designated as non-specific type (NST) were identified. PTs 4 and 8 with 39 and 17 strains respectively were the dominant PTs. Strains of PT 8 were isolated over a wide period of time whereas all (except one) strains of PT 4 were isolated between 1988 and 1992. Combination of biochemical fingerprinting and phage typing divided the strains into 25 phenotypes (BPT:PTs). Whilst phenotype C2:8 was found over a number of different years, phenotype C4:4 was isolated only between 1988 and 1992. These findings indicate the presence of one persistent and one recently emerged phenotype among S. enteritidis strains in Germany.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of the PhP system for biochemical-fingerprint typing of strains of Salmonella of serotype Typhimurium.

The Phene Plate (PhP) system of biochemical fingerprinting of bacteria is a computerised typing system, based on quantitative measurements of the kinetics of several biochemical reactions of bacteria grown in liquid medium in microtitration plates. For each isolate tested, it yields a biochemical fingerprint comprising several kinds of quantitative data which are useful for establishing similarities among strains with a personal-computer program. In this study, a set of 16 specific substrates was chosen to differentiate strains of Salmonella of serotype Typhimurium. The system was evaluated for its typability, reproducibility and discriminatory power in tests with a collection of 100 epidemiologically unrelated Typhimurium strains and results were compared with those obtained by phage typing. At an identity level of 0.980, strains were assigned by this method to 51 biochemical phenotypes (BPTs), giving a diversity index of 0.963 and a resolution index of 0.210. In contrast, 24 phage types (PTs) were identified among these isolates (a diversity index of 0.901). The combined use of biochemical fingerprinting by the PhP system and phage typing discriminated 82 phenotypes (a diversity index of 0.994). Stability of markers in each of the methods was also evaluated after subculture of 20 strains for 21 consecutive days. Only nine biochemical reactions were found that were subject to small, but measurable, changes for at least one isolate. These changes slightly decreased the mean similarity coefficients among strains but the overall BPTs of the strains showed changes in four strains (20%). In contrast, eight strains (40%) showed changes in their PTs after this treatment. It is concluded that the PhP system is a highly discriminatory and reproducible method for typing Typhimurium strains. It is easy to perform, and may be used alone or in combination with phage typing in epidemiological studies of Typhimurium strains.

The use of biochemical fingerprinting, phage typing and antimicrobial-susceptibility testing in the detection of epidemic strains of Salmonella of serotype Typhimurium in Iran.

A collection of 86 strains of Salmonella of serotype Typhimurium isolated from children with gastroenteritis in Tehran, Iran was examined for biochemical phenotype, phage type and antibiotic-resistance pattern. Twenty-seven biochemical phenotypes (BPTs), 14 discrete phage types (PTs) and 18 resistotypes (RTs) were identified. Fifty-three strains (62%) belonged to two major and probably related BPTs, whereas the other 33 isolates belonged to less common BPTs. The two predominant BPTs contained 26 strains of the same PT and 23 strains of the same RT. Different PTs and RTs of strains with similar BPT were sometimes observed, possibly reflecting antibiotic pressures in Iran. These results suggest that two major "clones" of Typhimurium strains are particularly common in Iran and, although each method alone adequately detected these and other less common "clones", biochemical fingerprinting provided additional information about relationships among strains.

Identification of Salmonellae with the 4-methylumbelliferyl caprilate fluorescence test.

We have tested 750 Salmonella strains and 130 strains of other species of the family Enterbacteriaceae with the 4-methylumbelliferyl caprilate reagent (MUCAP) test. The MUCAP test is a fluorescence test for rapid identification of Salmonella strains. The non-Salmonella strains were strains sent for identification as suspected Salmonella strains and thus have phenotypes similar to those of Salmonella strains. All 748 tested Salmonella strains of subgroups I, II, III, and IV were positive in the MUCAP test. Of the two tested rare Salmonella subgroup V strains, one was positive and the other was negative. In the selected material containing strains with phenotypes similar to those of Salmonella strains, only one Hafnia alvei strain of 130 Enterbacteriaceae bacteria tested was positive. The fluorescence of the H. alvei strain, the six tested Salmonella dublin strains, and the positive Salmonella subgroup V strain was weaker than that of the other salmonellae. The MUCAP assay is simple and is performed within 5 min. With an almost 100% sensitivity for Salmonella strains, apart from a single Salmonella subgroup V strain, we found the MUCAP test to be a convenient complement to traditional biochemical identification methods, especially for atypical and unusual Salmonella strains.

Studies of the binding activity of phage G13 to synthetic trisaccharides analogous to binding structures in Salmonella typhimurium and Escherichia coli C core saccharide. Correlation between conformation and binding activity.

Phage G13 binds to the carbohydrate part of lipopolysaccharides from rough mutants of Salmonella and Escherichia coli as the first event of infection. Equilibrium dialysis inhibition studies with native and synthetic trisaccharides as inhibitors suggested that phage G13 recognizes branched oligosaccharides having 6-O-alpha- or 7-O-alpha-glycosyl groups with alpha-Man(1----3) [alpha-Man(1----6)]Man (Man[Man]Man) and alpha-Glc(1----3)-[alpha-Hep(1----7)] alpha-Hep(1----3) alpha-Hep(1----5)Kdo as the smallest saccharides with inhibitory activity (Wollin et al., 1989). Of four synthetic analogues to Man[Man]Man only Man(1----3)[alpha-Gal(1----6)]alpha-Man-OMe (Man[Gal]-Man) and alpha-Glc(1----3)[alpha-Hep(1----7)]alpha-Hep-OMe (Glc[Hep]Hep) inhibited the binding of labelled E. coli C core nonasaccharide ligand to G13 with activities which were 10- and 15-fold lower than Man[Man]Man. The trisaccharides alpha-Man(1----3)[alpha-Glc(1----6)[alpha-Man-OMe (Man[Glc]Mann) and alpha-Man(1---3)[alpha-Tal(1----6)]alpha-Man-OMe (Man[Tal]Man) showed no inhibition at concentrations 75-fold higher than Man[Man]Man. Minimum energy conformation calculations of the saccharides using the GESA method showed that the 6-O-alpha-Man group in Man[Man]Man and the 7-O-alpha-Hep group SL805 pentasaccharide expose their OH-2 and OH-3 groups in a similar way and these are postulated to be key structural features for binding activity. The importance of hydroxy groups at certain positions is implied from the fact that both manno- and galacto-isomers are active. We also conclude that the O6-C6-C5-O5-C1 region of the 3-O-alpha-glycosyl group in the Man[Man]Man trisaccharide, or part of it, is important for the G13 binding activity.

Plasmid-identified Staphylococcus saprophyticus isolated from the rectum of patients with urinary tract infections.

Among 15 strains of Staphylococcus saprophyticus of various origin, 13 presented different plasmid patterns, making plasmid identification a useful epidemiological marker. In a consecutive study of 14 young female patients with urinary tract infection caused by S. saprophyticus, 6 patients were simultaneously positive for the same bacterium in the stools. Three paired samples contained the identical plasmid-identified clone of S. saprophyticus indicating that the rectum may be a reservoir of this urinary pathogen.

Interaction between phage G13 and its oligosaccharide receptor studied by equilibrium dialysis.

The reversible binding of phage G13, a phi X174-like single-strand DNA phage, to a 3H-labelled nonasaccharide from the lipopolysaccharide of its natural host Escherichia coli C was studied with equilibrium dialysis. The binding constant (Ka) was determined to 1.3 x 10(7) M-1 in Scatchard and Lineweaver-Burk plots. Approximately one saccharide bound per G13 phage particle which suggests that only one of the 12 spikes in each G13 virion was engaged in the phage/receptor saccharide interaction. Equilibrium dialysis inhibition experiments with saccharides from lipopolysaccharides of an isogenic series of Salmonella typhimurium mutants showed that hepta- and pentasaccharides from two G13-sensitive bacteria, i.e., with efficiencies of plating of 0.1-1.0 compared to E. coli C, were efficient inhibitors with Ka-values greater than or equal to 1.2 x 10(7) M-1. The octa- and hexasaccharides from two G13 resistant strains, with efficiency of plating less than or equal to x 10(-4), were either greater than 1000-fold or greater than 15-fold less efficient as inhibitors with Ka-values less than or equal to 8.8 x 10(5) M-1. The results show that phage G13 binds in a specific and reversible way to penta-, hepta-, and nonasaccharides from G13 sensitive bacteria with the specificity residing in the hexose and heptose region of the core lipopolysaccharide.

The conformation of core oligosaccharides from Escherichia coli and Salmonella typhimurium lipopolysaccharides as predicted by semi-empirical calculations.

The preferred conformation of the hexose and heptose regions of core saccharides from Enterobacteriaceae lipopolysaccharides was calculated. The Hard Sphere Exo Anomeric (HSEA) approach was used and the minimum energy conformation of the Salmonella typhimurium and Escherichia coli R1, R2, R3, R4 and K12 cores calculated. The results indicate that most of the cores are sterically crowded, with small degrees of freedom, and that the hexose and heptose parts form two separate regions. The core structures exhibit a 'front'-side and a 'back'-side, the former being similar for all the structures and the latter being characteristic for each core type.

Definition of the phage G13 receptor as structural domains of trisaccharides in Salmonella and Escherichia coli core oligosaccharides.

The interaction between phage G13 and different bacterial and synthetic oligosaccharides has been studied using equilibrium dialysis inhibition. The results, and conformational analysis of the oligosaccharides, make us conclude that the phage G13 carbohydrate receptor is a conformational domain involving three sugar residues. The following trisaccharide elements contain the domain: alpha-D-Galp-(1----3)-[alpha-D-Galp-(1----6)]-alpha-D-Glcp, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)-alpha-D-Manp , and alpha-D-Glcp-(1----3)-[L-gly-alpha-D-man-Hepp-(1----7)]-L-gly-alph a-D- man-Hepp. Thus two structures, either a hexose substituted with alpha-D-glycopyranosyl groups in the 3- and 6-positions, or a heptose substituted with such groups in the 3- and 7-positions are functional G13 binding sites. Such domains are present in several cores of lipopolysaccharides from Salmonella and Escherichia coli species. Some cores, e.g. those from S. typhimurium chemotypes Ra, Rb1 and Rb2, contain two such domains. The identification of two G13 receptor domains within different core saccharides could explain the broad host range of this phage.

Lysogenic conversion of Salmonella typhimurium bacteriophages A3 and A4 consists of O-acetylation of rhamnose of the repeating unit of the O-antigenic polysaccharide chain.

Lysogenization of Salmonella typhimurium with either of the bacteriophages A3 and A4 results in O-acetylation of the L-rhamnose residues of the O-polysaccharide chain of the lipopolysaccharide of the bacterial cell envelope. The O-acetyl group is found on both O-2 and O-3 of the L-rhamnosyl residues. This lysogenic conversion prevents the adsorption of the A3 and A4 phages and also greatly reduces the rate of adsorption of phage P22 to the O-polysaccharide chain as measured by binding studies with whole bacteria. Isolated lipopolysaccharide from A3- and A4-lysogenized bacteria was also inefficient in inactivating these phages: the concentration required for 50% inactivation was 10,000-fold higher than that for lipopolysaccharide from S. typhimurium not lysogenized by any A phage. Binding of phages A3 and A4 is accompanied by hydrolysis of the alpha-1,3 linkage between rhamnose and galactose in the tetrasaccharide repeating unit of the O-polysaccharide. Phage hydrolysis generates saccharides of various lengths, the majority being dodecasaccharides, i.e., equivalent to three repeating units. It is surmised that O-acetylation of the rhamnosyl residue interferes with phage A3, A4, and P22 infection by preventing binding to and hydrolysis of the O-polysaccharide chain, the initial step in the phage infection cycle. The new O-acetyl-rhamnose entities did not elicit specific antibodies in rabbits in accordance with earlier experiences. The O-acetylation of O-2 and O-3 of rhamnose is a new, hitherto unknown, modification of the O-polysaccharide chain of S. typhimurium.

Salmonella typhimurium mutants defective in UDP-D-galactose:lipopolysaccharide alpha 1,6-D-galactosyltransferase. Structural, immunochemical, and enzymologic studies of rfaB mutants.

The biochemical defect in a class of Salmonella typhimurium mutants (rfaB) defective in biosynthesis of the lipopolysaccharide core is described. Structural, immunochemical and enzymologic studies showed that: (i) the core polysaccharide completely lacked the branch alpha 1,6-D-galactosyl residue of the normal lipopolysaccharide as shown by methylation analysis and 1H nmr spectroscopy; (ii) the mutant lipopolysaccharides acted as acceptors for transfer of D-galactose from UDP-D-galactose into alpha 1,6 linkage to the proximal D-glucosyl residue of the core in a reaction catalyzed by an enzyme activity present in extracts from rfaB+ cells; (iii) the UDP-D-galactose:(glucosyl)lipopolysaccharide alpha 1,6-D-galactosyltransferase activity was absent from extracts of rfaB cells.