Molecular epidemiology of Salmonella enterica serovar Agona: characterization of a diffuse outbreak caused by aniseed-fennel-caraway infusion.

During 2002-2003 increased numbers of notified salmonellosis due to S. enterica serovar Agona were observed in Germany. In order to understand the recent spread of this serovar and to trace the route of infection to its source, a new phage-typing scheme and pulsed field gel electrophoresis (PFGE) were used to analyse these isolates. By using 14 bacteriophages, 52 phage types were distinguished among the S. Agona strains. PFGE also differentiated 52 different patterns. A combination of both methods generated 94 clonal types among 165 S. Agona strains originating from Germany and other countries including the United States, United Arab Emirates, Turkey, India, Austria and Finland, indicating a great biological diversity within this serovar. However, 36 recent S. Agona isolates from infantile gastroenteritis in Germany, from an untreated batch of aniseed imported from Turkey and from fennel-aniseed-caraway infusion (packed in tea bags) revealed clonal identity indicating their epidemiological relatedness as a new source of infection. It is suggested that strains of S. Agona will continue to be of public health concern, and that phage typing together with PFGE typing should be applied as reliable and rapid tools for epidemiological subtyping and future monitoring.

A foodborne outbreak of Salmonella enterica subsp. enterica serovar Madelia at a silver anniversary reception.

Reported here is an outbreak of Salmonella enterica subsp. enterica serovar Madelia infection that occurred among 44 persons attending a silver anniversary reception in Hesse, Germany. Isolates of Salmonella Madelia are extremely unusual, and no outbreaks associated with this serotype have been reported previously. Forty-two attendees were interviewed and information was obtained from each of them regarding demographic and clinical characteristics and food consumed at the reception. Twenty-four attendees submitted stool samples for microbiological testing, and 10 of these were culture-positive for S. Madelia. Twenty-three attendees met the case definition of infection, while 18 met the clinical case definition (i.e. vomiting or diarrhoea within 3 days of consuming food at the reception) and five had asymptomatic infection with S. Madelia. The most likely vehicles of infection were tortellini and a red pesto sauce.

[Intestinal yersiniosis. Clinical importance, epidemiology, diagnosis, and prevention]. / Enterale Yersiniosen. Klinische Bedeutung, Epidemiologie, Diagnostik und Prävention.

Yersinia enterocolitica and Y. pseudotuberculosis are causative agents of yersiniosis in humans and animals that have to be separated from Y. pestis, the causative agent of plague, representing a separate clinical and epidemiological entity. Intestinal yersiniosis may manifest in humans as (1) enteritis, (2) terminal ileitis, mesenteric lymphadenitis, or pseudoappendicitis, and (3) septicemia leading to focal abscesses in spleen and liver. The intestinal infection may be followed by reactive arthritis in a number of cases. Y. enterocolitica and Y. pseudotuberculosis are distributed worldwide but occur mainly in moderate or subtropical climates. The most important reservoirs are rodents, lagomorphs, and birds for Y. pseudotuberculosis and domestic animals, especially pigs, for Y. enterocolitica. All Y. pseudotuberculosis isolates may be considered as pathogenic whereas Y. enterocolitica strains can be subdivided into pathotypes of different virulence. The differentiation of pathotypes by determination of the biovar and demonstration of the 75-kb virulence plasmid is therefore of diagnostic importance. Preventive measures include avoidance of direct infection by contact with infected reservoir animals and practice of good hygiene during slaughtering as well as in food production and preparation of meals.

[Dehydroepiandrosterone metabolism by Epidermophyton floccosum]. / Metabolismus von Dehydroepiandrosteron durch Epidermophyton floccosum.

Steroid hormones may be relevant for the fungus-host relation in dermatophytoses. In contrast to most other hosts of dermatophytes, humans are characterized by a high cutaneous concentration of the adrenal androgen dehydroepiandrosterone (DHEA) and its sulphate (DHEAS). To investigate whether the strictly anthropophilic dermatophyte Epidermophyton floccosum can metabolize this steroid hormone, cultures of E. floccosum were supplemented with DHEA. After 5 days of incubation the steroids in the culture supernatants were extracted and differentiated by gaschromatography and massspectrometry (GC-MS). The results show that a nearly complete metabolization of DHEA by E. floccosum leads to the formation of multiple new steroids/metabolites some of which have not been reported before. Therefore, this fungus could possibly mediate the hormone regulated cutaneous defense mechanisms of the host by an intraepidermal metabolization of DHEA.

[Not Available]. / Infektionen des Menschen durch enterohämorrhagische Escherichia coli (EHEC) in der Bundesrepublik Deutschland von 1998 bis 2001 Prävalenz und Typenspektrum : Prävalenz und Typenspektrum.

Intestinal infections in Germany due to enterohemorrhagic E. coli bacteria (EHEC) between 1998 and 2001 reveal a large scale of biological diversity of their pathogens. However, no dramatic increase of their clinical importance and public health implications has been observed. As strains of serovar O157:H7 have continuously declined as causative agents, other serovars such as O26:H11 and O103:H2 have replaced them. The great diversity of the EHEC pathogens might point to a great number of various infection routes and sources. Since recently new pathogenic factors of EHEC bacteria have been detected (especially by the sequencing of the genome of EHEC), it is currently not possible to define a clear-cut difference between human pathogens and nonhuman pathogens. The enhanced surveillance of EHEC pathogens with respect to their biological diversity and dynamics, their epidemic spread, and their infection routes and sources remain an essential task of the public health authorities.

Elucidation of the role of functional amino acid residues of the small sialidase from Clostridium perfringens by site-directed mutagenesis.

Bacterial sialidases represent important colonization or virulence factors. The development of a rational basis for the design of antimicrobials targeted to sialidases requires the knowledge of the exact roles of their conserved amino acids. A recombinant enzyme of the 'small' (43 kDa) sialidase of Clostridium perfringens was used as a model in our study. Several conserved amino acids, identified by alignment of known sialidase sequences, were altered by site-directed mutagenesis. All recombinant enzymes were affinity-purified and the enzymatic characteristics were determined. Among the mutated enzymes with modifications in the environment of the 4-hydroxyl group of bound sialic acids, D54N and D54E exhibited minor changes in substrate binding. However, a reduced activity and changes in their pH curves indicate the importance of a charged group at this area. R56K, which is supposed to bind directly to sialic acids as in the homologous Salmonella typhimurium sialidase, showed a 2500-fold reduced activity. The amino acids Asp-62 and Asp-100 are probably involved in catalysis, indicated by reduced activities and altered temperature and pH curves of mutant enzymes. Exchanging Glu-230 with threonine or aspartic acid led to dramatic decreases in activity. This residue and Y347 are supposed to be crucial for providing a suitable environment for catalysis. However, unaltered pH curves of mutant sialidases exclude their direct involvement in protonation or deprotonation events. These results indicate that the interactions with the substrates vary in different sialidases and that they might be more complex than suggested by mere static X-ray structures.

Lack of sialidase activity in Candida albicans and Candida glabrata.

Because several micro-organisms having close contact to animal hosts and man produce sialidase (EC 3.2.1.18) as a tool for adhesion and invasion, we investigated two Candida species for the presence of this enzyme. Two sensitive assays, a fluorometric test with 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid and a radiometric test with 3H-labelled sialyllactitol as sialidase substrates, were applied to detect sialidase activity. None of 40 Candida albicans and 10 C. glabrata strains grown in three different media exhibited sialidase activity, but the positive control Ophiostoma stenoceras produced sialidase under comparable conditions. Our surprising negative findings are divergent from an earlier positive report, which may be due to strain selection or bacterial contamination. These results indicate that sialidase is probably of no relevance in cutaneous or mucosal candidoses.

Enzymatic and molecular properties of the Clostridium tertium sialidase.

Clostridium tertium metabolizes sialoglycoconjugates via a secreted sialidase [EC 3.2.1.18] and an intracellular acylneuraminate pyruvate lyase [EC 4.1.3.3]. The sialidase was enriched 1,900-fold from the culture medium with a specific activity of 0.7 U per mg protein. It exhibits a temperature optimum of 50 degreesC and tolerates mercury ions at relatively high concentrations (50% inhibition at 5.2 mM Hg2+). The sialidase gene was detected on two restriction fragments (HincII, HindIII) of chromosomal DNA and their correct recombination resulted in an active enzyme expressed by Escherichia coli. The structural gene is represented by 2,319 bp encoding a protein of 773 amino acids with a molecular mass of 85.4 kDa. The first 28 amino acids possess the character of a signal peptide. The protein reveals a FRIP-region and four Asp-boxes common in all bacterial sialidases. It has 42.6% identical amino acids when compared with the sialidase of Clostridium septicum and 64.8% with a sialidase gene amplified from Macrobdella decora. A further open reading frame was detected 30 bp downstream from the sialidase gene, which exhibits significant homology with acylneuraminate pyruvate lyases. For the first time, both genes were found in close proximity on a bacterial chromosome, probably being part of one operon.

Effect of cysteine modifications on the activity of the 'small' Clostridium perfringens sialidase.

The 'small' (43 kDa) sialidase of Clostridium perfringens is inhibited by low concentrations of mercury ions. For the investigation of possible functional roles of the enzyme's four cysteine residues at the amino acid positions 2, 282, 333 and 349, they were separately altered to serine by site-directed mutagenesis. The four mutant sialidases expressed in E. coli and purified by metal chelate chromatography were markedly reduced in specific activity when compared to the wild-type enzyme but with the exception of C282S exhibited similar K(M)-values indicating an unchanged mode of substrate binding. The substrate specificity was also conserved for C2S, C282S, and C333S. Only the C349S sialidase exhibited a higher relative activity with colominic acid and the alpha2,6-linked sialic acid of sialyllactose compared to the alpha2,3-linked isomer than the other mutants. Chemical modifications with the thiol-blocking reagents N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB) and HgCl2 had little effect on the C282S sialidase, e.g., 6% inhibition by 5 mM NEM compared to reductions in activity between 65 and 90% for the wild-type and other mutant enzymes, supporting the idea that among the enzyme's cysteines, Cys-282 has the highest structural or functional significance. The results also explain the higher mercury tolerance of Salmonella typhimurium and Clostridium tertium sialidases, which have the positions equivalent to Cys-282 altered to Val and Thr, respectively, indicating that the thiol group of Cys-282, despite being situated near the active site, is not involved in catalysis.

[Dermatophytes do not produce sialidase in vitro]. / Dermatophyten bilden in vitro keine Sialidase.

Sialidase (EC 3.2.1.18) is a pathogenicity factor of many microorganisms, and may also play a role in adhesion of dermatophytes to the epithelia of their hosts by the hydrolytical cleavage of terminal, negatively charged sialic acids of glycoconjugates on the cell surfaces, thus allowing fungal lectins to bind to the subterminal sugars. Therefore, 116 strains of seven species of dermatophytes were investigated for sialidase production. Two highly sensitive, quantitative sialidase assays were applied to cell homogenates and culture supernatants from seven different media of the fungi, but were always negative for sialidase activity. However, sialidase activity was always detected in Ophiostoma stenoceras used as a positive control cultivated in parallel; the enzyme was inducible by sialylated mucins. A sialidase-dependent pathomechanism for dermatophytes appears unlikely based on the results presented.

Cloning, sequencing and expression of the acylneuraminate lyase gene from Clostridium perfringens A99.

The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a noncoding region with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected. The 3'-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the 'large' sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species.

Expression and purification of a recombinant "small" sialidase from Clostridium perfringens A99.

A 1.4-kb gene encoding the "small" sialidase isoenzyme of Clostridium perfringens A99, including its own promoter, was previously cloned in and expressed by Escherichia coli JM 101. Since all attempts to purify this enzyme to homogeneity were unsuccessful, a new strategy was developed. The structural gene was amplified by means of a PCR technique and inserted into the plasmid vector pQE-10, transferring a six-histidine affinity tag (His6) to the N-terminus of the protein. In order to minimize proteolytic degradation of the sialidase protein, the gene was subcloned into the Escherichia coli strain BL21(DE3)pLys S with reduced protease activity. The sialidase production was increased about 2.5-fold when compared with that of the original clone. The enzyme, released by lysozyme treatment of the bacterial cells, was purified by metal chelate chromatography on Ni-nitrilo-triacetic acid agarose to apparent homogeneity in SDS-PAGE. The 42-kDa protein was enriched 62-fold with a yield of 82% and a specific activity of 280 U mg-1. A total amount of 1 mg sialidase was obtained from 1 liter of bacterial culture. For future studies, including crystallization experiments, the histidine affinity tag was removed from the sialidase enzyme by aminopeptidase K. The sialidase was then separated from aminopeptidase K by ion-exchange chromatography, resulting in an overall yield of 83% and a specific activity of 305 U mg-1 using 4-methylumbelliferyl- alpha-D-N-acetylneuraminic acid under standard conditions. The two forms (with or without the histidine tag) of sialidase exhibited similar kinetic properties when compared to the wild-type enzyme.

Diversity in the properties of two sialidase isoenzymes produced by Clostridium perfringens spp.

Clostridium perfringens produces two sialidases, one of which has a molecular mass of 71 kDa and is secreted, while the 'small', 43 kDa isoenzyme remains in the cells. The secreted, higher molecular mass sialidases of two different clostridial strains, DSM756T and A99, exhibit maximum activity at pH 5.5 and at 51 or 55 degrees C, respectively. The molecular mass of both enzymes is 71 kDa in SDS-PAGE and 63 kDa as determined by gel-filtration, which indicates the absence of subunits. Natural sialidase substrates are hydrolyzed at comparably high rates, e.g. the glycoproteins fetuin and bovine submandibular gland mucin, the homopolymer colominic acid, and the ganglioside mixture from bovine brain. The partially purified 'small' isoenzyme from C. perfringens A99 cells had similar properties to the corresponding recombinant sialidase isolated from the Escherichia coli host. It is located inside the clostridial and E. coli cells and exhibits maximum activity at pH 6.1 and 37 degrees C. A relative molecular mass of 32,000 was found with FPLC gel-filtration chromatography, while primary structure analysis yielded a value of 43,000. It differs a significantly from the 'large' isoenzyme by substrate specificity. Preferred substrates are oligosaccharides, while other, more complex sialoglycoconjugates are hydrolyzed only at very low rates. alpha 2,3-linkages are hydrolyzed much faster than alpha 2,6-bonds.

Gene structure of the 'large' sialidase isoenzyme from Clostridium perfringens A99 and its relationship with other clostridial nanH proteins.

Clostridium perfringens possesses two sialidase isoenzymes of different molecular weight. Almost 90% of the gene encoding the 'large' form was found on a 3.1 kb chromosomal fragment (Sau3AI) of strain A99 by hybridization with probes developed from the N-terminal protein sequence and from commonly conserved sialidase motifs ('Asp-boxes'), whereas the remaining 3'-terminal part was detected on a 2.1 kb fragment (Hind III) of chromosomal DNA. After combination of both fragments, the resulting E. coli clones expressed sialidase activity, the properties of the recombinant sialidase corresponding with those of the wild type enzyme. The entire chromosomal fragment of 3665 bp encompasses the complete sialidase gene of 2082 bp corresponding to 694 amino acids, from which a molecular weight of 72,956 for the mature protein can be deduced. The first 41 amino acids are mostly hydrophobic and probably represent a signal peptide. The sialidase structural gene follows a non-coding region with an inverted repeat and a ribosome-binding site. Upstream from the regulatory region, another open reading frame (ORF) was detected. The 3'-terminus of the sialidase structural gene is directly followed by a further ORF of unknown function, which possibly encodes a putative permease or the acylneuraminate pyruvate-lyase involved in sialic acid catabolism. The primary structure of the 'large' isoenzyme is very similar to the sialidase of Clostridium septicum (55% identical amino acids), whereas the homology with the 'small' form of the same species is comparatively low (26%).

The sialidase superfamily and its spread by horizontal gene transfer.

Sialidases (neuraminidases, EC 3.2.1.18) belong to a class of glycosyl hydrolases that release terminal N-acylneuraminate (sialic acid) residues from glycoproteins, glycolipids, and polysaccharides. These enzymes are common in animals of the deuterostomate lineage (Echinodermata through Mammalia) and also in diverse microorganisms that mostly exist as animal commensals or pathogens. Sialidases, and their sialyl substrates, appear to be absent from plants and most other metazoans. Even among bacteria, sialidase is found irregularly so that related species or even strains of one species differ in this property. This unusual phylogenetic distribution makes sialidases interesting for evolutionary studies. The biochemical diversity among bacterial sialidases does not indicate close relationships. However, at the molecular level, homologies are detectable, supporting the hypothesis of a common sialidase origin and thus of a sialidase superfamily. Some findings indicate that sialidase genes were recently transferred via phages among bacteria. The proposal of a sialidase origin in higher animals is suggested by the presence of apparently homologous enzymes in this kingdom, supporting the idea that some microbes may have acquired the genetic information during association with their animal hosts.

Isolation and properties of the natural and the recombinant sialidase from Clostridium septicum NC 0054714.

The natural sialidase of Clostridium septicum was purified and characterized in parallel with the recombinant enzyme expressed by Escherichia coli. The two enzymes exhibit almost identical properties. The maximum hydrolytic activity was measured at 37 degrees C in 60 mM sodium acetate buffer, pH 5.3. Glycoproteins like fetuin and saponified bovine submandibular gland mucin, most of them having alpha(2-6) linked sialic acids, are preferred substrates, while sialic acids from gangliosides, sialyllactoses, or the alpha(2-8) linked sialic acid polymer (colominic acid) are hydrolysed at lower rates. alpha(2-3) Linkages are more rapidly hydrolysed than alpha(2-6) bonds of sialyllactoses. The cleavage rate is markedly reduced by O-acetylation of the sialic acid moiety. These properties are similar to those of other secreted clostridial sialidases. The enzyme exists in mono-, di- and trimeric forms, the monomer exhibiting a molecular mass of 125 kDa, which is close to the protein mass of 111 kDa deduced from the nucleotide sequence of the cloned gene.

An immunoassay for the rapid and specific detection of three sialidase-producing clostridia causing gas gangrene.

A rapid and methodologically unusual diagnostic test was developed for the specific detection of Clostridium perfringens, C. septicum and C. sordellii, which cause clostridial myonecrosis. Sialidases (EC 3.2.1.18) secreted by these bacterial species were bound to polyclonal antibodies raised against the respective enzyme and immobilized onto microtiter plates. The activity of bound sialidase was determined with the fluorogenic substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid. The assay permits the detection of a minimum sialidase activity of about 0.1-1 mU/ml of sample solution within 2 h. The sensitivity of the test was reduced by about three-fold for sialidase activities in samples containing 50% serum. Only a few, low cross-reactivities, which never exceeded 10% of the homologous reaction, were observed with 12 sialidases from other bacterial sources. Clinical isolates of the three clostridial species were analysed by this assay and gave positive signals in the homologous test. The assay for the detection of C. perfringens was applied to nine samples from patients suspected to be suffering from clostridial myonecrosis. There was a high correlation between the results of the immunoassay and the bacteriological analysis of infection.

Effects of site-specific mutations on the enzymatic properties of a sialidase from Clostridium perfringens.

Three site-specific mutations were performed in two regions of a sialidase gene from Clostridium perfringens which are known to be conserved in bacterial sialidases. The mutant enzymes were expressed in Escherichia coli and, when measured with MU-Neu5Ac as substrate, exhibited variations in enzymatic properties compared with the wild-type enzyme. The conservative substitution of Arg 37 by Lys, located in a short conserved region upstream from the four repeated sequences common in bacterial sialidase genes, was of special interest, as KM and Vmax, as well as K(i) measured with Neu5Ac2en, were dramatically changed. These data suggest that this residue may be involved in substrate binding. In addition to its low activity, this mutant enzyme has a lower temperature optimum and is active over a more limited pH range. This mutation also prevents the binding of an antibody able to inhibit the wild-type sialidase. The other mutations, located in one of the consensus sequences, were of lower influence on enzyme activity and recognition by antibodies.

Cloning, sequencing and expression of the sialidase gene from Actinomyces viscosus DSM 43798.

Chromosomal DNA from Actinomyces viscosus was digested with restriction endonucleases and the fragments ligated with pUC-vectors were used to transform Escherichia coli cells. Clones bearing the required sialidase gene were detected by spraying the colonies with the fluorogenic sialidase substrate MU-Neu5Ac. The identity of the cloned sialidase was confirmed after the 5700-fold enrichment and comparison with the purified enzyme of A. viscosus. Both sialidases were identical with regard to molecular mass, substrate specificity tested with sialyllactoses, and the inhibition of their activity by heterologous antisialidase antibodies. The sequenced insert (EMBL accession number X62276) revealed a mol% G + C of 68.2, typical for A. viscosus. An open reading frame of 2739 bp follows a sequence with dyad symmetry and an AG-rich region, and codes for 913 amino acids representing a molecular mass of 113 kDa. The conserved amino acid sequence [Ser-X-Asp-X-Gly-X-Thr-Trp] typical for bacterial sialidases was found at five positions in the predicted amino acid sequence. The gene of this enzyme is expressed by E. coli, despite the low relatedness of both species.

Purification and properties of cloned Salmonella typhimurium LT2 sialidase with virus-typical kinetic preference for sialyl alpha 2----3 linkages.

Subclones containing the Salmonella typhimurium LT2 sialidase gene, nanH, were expressed in Escherichia coli from multicopy derivatives of pBR329. The cloned sialidase structural gene directed overproduction of sialidase polypeptide which was detected as the major soluble protein species in cell-free extracts. Overproduced enzyme was purified to near electrophoretic homogeneity after 65-fold enrichment using conventional preparative techniques. Unlike all previously investigated sialidases, S. typhimurium sialidase was positively charged (pI greater than or equal to 9.0). Km, Vmax, and turnover number of the purified sialidase, measured using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUNeu5Ac), were 0.25 mM, 5,200 nmol min-1, and 2,700 s-1, respectively. These values are the highest yet reported for a sialidase. Sialidase was inhibited by 2-deoxy-2,3-didehydro-N-acetyl-neuraminic acid at unusually high concentrations (Ki = 0.38 mM), but not by 20 mM N-acetylneuraminic acid. Divalent cations were not required for activity. The pH optimum for hydrolysis of MUNeu5Ac was between 5.5 and 7.0 and depended on the assay buffer system. Substrate specificity measurements using natural sialoglycoconjugates showed a 260-fold kinetic preference for sialyl alpha 2----3 linkages when compared with alpha 2----6 bound sialic acids. The enzyme also efficiently cleaved residues from glycoproteins and gangliosides, but not from mucin or sialohomopolysaccharides. S. typhimurium sialidase is thus the first bacterial enzyme to be described with influenza A virus sialidase-like kinetic preference for sialyl alpha 2----3 linkages and to have a basic pI.