First report of macrolide resistance gene erm(T) harbored by a novel small plasmid from Erysipelothrix rhusiopathiae.

The macrolide resistance gene erm(T) was identified for the first time in a porcine Erysipelothrix rhusiopathiae isolate from swine in China. The novel 3,749-bp small plasmid pER29, which carries erm(T), had a G+C content of 31% and four distinct open reading frames. The presence of pER29 increased by at least 128-fold the MICs of clindamycin and erythromycin for E. rhusiopathiae. The fitness cost of pER29 could be responsible for the low frequency of erm(T) in E. rhusiopathiae.

Erysipelothrix rhusiopathiae neuraminidase and its role in pathogenicity.

The role of the enzyme neuraminidase in pathogenicity of the bacillus Erysipelothrix rhusiopathiae was studied. Different substances with low and high molecular weight were tested as inducers of E. rhusiopathiae neuraminidase biosynthesis. It was found that macromolecular complexes induce the secretion of the enzyme. K(M) values for different substrates showed that the affinity of the E. rhusiopathiae neuraminidase increases in parallel with the enlargement of the molecular weight of glycoproteins. Results from the rabbits skin test confirmed the role of E. rhusiopathiae neuraminidase as a factor of pathogenicity with spreading functions.

Unusual substrate specificity of the peptidoglycan MurE ligase from Erysipelothrix rhusiopathiae.

Erysipelothrix rhusiopathiae is a Gram-positive bacterium pathogenic to many species of birds and mammals, including humans. The main feature of its peptidoglycan is the presence of l-alanine at position 3 of the peptide stem. In the present work, we cloned the murE gene from E. rhusiopathiae and purified the corresponding protein as His6-tagged form. Enzymatic assays showed that E. rhusiopathiae MurE was indeed an l-alanine-adding enzyme. Surprisingly, it was also able, although to a lesser extent, to add meso-diaminopimelic acid, the amino acid found at position 3 in many Gram-negative bacteria, Bacilli and Mycobacteria. Sequence alignment of MurE enzymes from E. rhusiopathiae and Escherichia coli revealed that the DNPR motif that is characteristic of meso-diaminopimelate-adding enzymes was replaced by HDNR. The role of the latter motif in the interaction with l-alanine and meso-diaminopimelic acid was demonstrated by site-directed mutagenesis experiments and the construction of a homology model. The overexpression of the E. rhusiopathiae murE gene in E. coli resulted in the incorporation of l-alanine at position 3 of the peptide part of peptidoglycan.

Neuraminidase production by Erysipelothrix rhusiopathiae.

In order to characterise neuraminidase activity by Erysipelothrix, 85 isolates of Erysipelothrix spp. from a variety of sources including human clinical, marine and terrestrial animals, and the environment were investigated for neuraminidase production. Neuraminidase activity was detected by a peanut lectin haemagglutination method. The effects of media, incubation conditions and pH on the production and activity of neuraminidase were also investigated. Enzyme activity was detected only in the supernatants of the isolates of Erysipelothrix rhusiopathiae which had been incubated in cooked meat broth and Todd Hewitt broth supplemented with horse serum after 16 and 36 h incubation at 37 degrees C. The maximum titres were reached at 40 h in cooked meat broth and 56 h in Todd Hewitt broth supplemented with horse serum. All 58 isolates and the type strain (ATCC 19414) of E. rhusiopathiae produced detectable neuraminidase activity with titres between 10 and 320. The optimal pH for the enzyme activity varied among the isolates with a pH between 6.0 and 7.0 covering the highest enzyme activity of the most. There was no statistically significant difference in the level of neuraminidase activity between isolates from different sources (p > 0.05). Neuraminidase activity was not detected in the non-pathogenic Erysipelothrix spp. such as E. tonsillarum. Neuraminidase was detected only in E. rhusiopathiae suggesting its possible role as a virulence factor. Enzyme production and activity were medium and pH dependent. The peanut lectin haemagglutination assay is a simple, rapid and sensitive method and is particularly useful for the analysis of multiple samples.

Possible role of neuraminidase in the pathogenesis of arteritis and thrombocytopenia induced in rats by Erysipelothrix rhusiopathiae.

The role of the neuraminidase produced by Erysipelothrix rhusiopathiae (E. rhusiopathiae) in the pathogenesis of arteritis and induced thrombocytopenia was examined using young and adult rats. There was a close correlation between bacterial invasion, desialation and cell infiltration in the common iliac artery. E. rhusiopathiae induced arteritis from the second and third day after inoculation with 3 X 10(8) viable bacteria in the young and adult rats, respectively. This delay with age was closely related to the increase of free sialic acid in the plasma. The sites invaded by E. rhusiopathiae coincided with the desialated lesions, and the bacteria invaded the periarterial region which was always accompanied by desialation when examined with FITC-conjugated peanut lectin. The free sialic acid in the plasma was, at least partly, considered to originate from the desialation of the arterial wall caused by E. rhusiopathiae. The platelet number decreased significantly after inoculation. The sialic acid content of the platelets prepared from circulating blood at 12 and 18 hours after inoculation showed a slight decrease and decreased further when the platelets were incubated with the bacteria. Platelets obtained from circulating blood within 24 hours after inoculation or incubated with the bacteria had demonstrated desialated sites as detected by immunofluorescent staining with FITC-conjugated peanut lectin. In conclusion, free sialic acid in the plasma was considered to be a good marker of the desialation of the arteries caused by E. rhusiopathiae, and the neuraminidase produced by the bacteria would be a key to solve the pathogenesis of the arteritis and thrombocytopenia.

Adhesion of Erysipelothrix rhusiopathiae to cultured rat aortic endothelial cells. Role of bacterial neuraminidase in the induction of arteritis.

The adhesion of Erysipelothrix rhusiopathiae (E. rhusiopathiae) to the cultured confluent monolayer of rat aortic endothelial cells (EC) and the role of neuraminidase in the interaction between EC and E. rhusiopathiae were examined. One EC line was obtained by collagenase treatment of rat aorta. The EC showed a typical cobblestone appearance and possessed the factor VIII related antigen. When cultured more than two weeks after reaching confluence, the EC formed a vascular plexus-like appearance. E. rhusiopathiae began to adhere to EC within 2 minutes after the beginning of culture and adhered at a constant rate for 20 minutes. The adhesion of bacteria to EC was closely related to the release of sialic acid from the EC. Significantly more bacteria adhered to neuraminidase treated EC, and bacterial adhesion was inhibited dose-dependently by N-acetylneuraminic-lactose, which is the substrate of bacterial neuraminidase. It is concluded that bacterial neuraminidase plays an essential role in initiating the interaction between EC and E. rhusiopathiae, which would contribute to the genesis of arteritis.

Enzymatic profiles of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillae.

The enzymatic activities of 39 strains of Erysipelothrix rhusiopathiae and 34 of E tonsillae were determined with the API ZYM system. The profiles of these two species were very similar, differing solely in N-acetyl-beta-glucosaminidase activity. Whereas 90 per cent of strains of E rhusiopathiae exhibited strong activity with N-acetyl-beta-glucosaminidase, positive reactions were observed for this enzyme in only 24 per cent of strains of E tonsillae. These results support previous DNA-DNA hybridisation studies and suggest that E tonsillae is a new species of the genus Erysipelothrix.

Hyaluronidase is not essential for the lethality of Erysipelothrix rhusiopathiae infection in mice.

To investigate the role of hyaluronidase in the pathogenicity of Erysipelothrix rhusiopathiae, transposon Tn916 was transferred from Enterococcus faecalis CG110 to a virulent strain of E. rhusiopathiae, and hyaluronidase-deficient mutants were isolated. A virulence assay in the mice showed that of the seven hyaluronidase-deficient mutants tested, six mutants were avirulent, but that one mutant, designated AST121, was as virulent as its parental strain. Western immunoblotting with a monoclonal antibody specific to the capsule, a major virulence factor of the organism, revealed that all of the avirulent mutants had lost the capsular antigen, whereas the mutant AST121 did not. These results suggest that the lack of virulence of the six hyaluronidase-negative mutants could be due to a loss of the capsule and that hyaluronidase does not contribute to the lethality of E. rhusiopathiae infection in mice.

Characterization of ß-galactoside phosphorylases with diverging acceptor specificities.

Glycoside phosphorylases are a special group of carbohydrate-active enzymes, with characteristics in between those of glycoside hydrolases and glycosyl transferases. The phosphorylases from family GH-112 are exceptional because they employ galactose-1-phosphate instead of glucose-1-phosphate as glycosyl donor. Different acceptor specificities have been observed in this family, ranging from l-rhamnose to GlcNAc, GalNAc and a combination of the latter. Three new phosphorylases from previously unexplored branches of the phylogenetic tree of family GH-112 have now been characterized to shed more light on this divergence in acceptor specificity. The enzymes from Erysipelothrix rhusiopathiae and Streptobacillus moniliformis were found to prefer GalNAc as acceptor, while that from Anaerococcus prevotii displays similar activities on GalNAc and GlcNAc. These results confirm the correlation between the amino acid residue at position 162 and the enzyme's specificity, i.e. a threonine in the former group and a valine in the latter. However, mutagenesis of residue 162 did not allow the rational transformation of the substrate preference, as the substitution of valine by threonine in the enzyme from Bifidobacterium longum did not tighten its specificity towards GalNAc. Unexpectedly, introducing an isoleucine at position 162 increased the preference for GlcNAc as acceptor, which illustrates that the structure-function relationships in ß-galactoside phosphorylases are not yet completely understood. Several other positions have also been examined by mutational analysis but true determinants of the acceptor specificity in family GH-112 could not be identified.

A taxonomic study on erysipelothrix by DNA-DNA hybridization experiments with numerous strains isolated from extensive origins.

The purpose of this study was to clarify the taxonomic relationship between all the serovars and species of the genus Erysipelothrix by performing DNA-DNA hybridization experiments, the customary criterion for separation of bacterial genospecies. A total of 93 strains were isolated from a wide variety of sources, including pigs affected with acute or chronic erysipelas, other diseased animals, healthy animals, fish, retail meats, and environmental materials from throughout the world during the period 1958 to 1996. The present data on phenotypic characterization and DNA relatedness values demonstrate that 24 strains (96%) of E. tonsillarum are avirulent for swine, whereas 39 strains (66%) of genomic E. rhusiopathiae induced generalized or local urticarial lesion in swine after intradermal inoculation. This observation suggests that genomic E. tonsillarum has little etiological significance. Three minor groups contained several strains which exhibited minimal association with each type strain of E. rhusiopathiae and E. tonsillarum. In conclusion, it was confirmed that members of the E. rhusiopathiae and E. tonsillarum groups resemble each other in regard to many phenotypic characteristics, but differ in their ability to produce acid from saccharose and in their pathogenicity for swine. The genus Erysipelothrix certainly contains two main species: E. rhusiopathiae and E. tonsillarum.

[The virulence of erysipelothrix rhusiopathiae strains and their neuraminidase production (author's transl)]. / Die Virulenz von Erysipelothrix rhusiopathiae-Stämmen und ihre Neuraminidase-Produktion

We studied the virulence of 20 Erysipelothrix rhusiopathiae strains in albino mice and investigated the interdependence between virulence, growth rate, and neuraminidase production. The minimal number of germs, lethal to mice (LD100) varied among the strains between 10(1) and mor ethan 10(7) per animal. The course of the disease was also very different. Some strains killed the mice rapidly within 3 days. In other strains, however, even a very high number of bacteria caused death only 7 days after infection or later, in which cases the disease became manifest long in advance by ruffled fur and glutinous eyes. A significant correlation was found between the average neuraminidase production in different media and the virulence (p equal to 99%; Fig. 2). We found a less marked dependence between growth rate and virulence (Fig. 3). Neuraminidase activity and growth rate seem to influence virulence rather independently. This is concluded from the fact, that some virulent strains are characterized by quick growth and by producing either no neuraminidase or in small quantities only. On the other hand, neuraminidase production cannot be separated completely from growth rate. An induction of neuraminidas activity in vivo was not observed, even when the bacteria persisted in the mice over a long period of time. We therefore conclude that a short term enzyme induction is not probable.

Detection of coagulase activity in Erysipelothrix rhusiopathiae.

Coagulase activity was detected in 99% of 225 strains of Erysipelothrix rhusiopathiae. These strains included isolates from a variety of animal and environmental sources. Activity could be detected by the tube or slide technique, with the tube reactions being easier to interpret. Coagulation of rabbit and/or bovine plasma was observed, with most strains reacting in both. The activity appeared to be a common characteristic of the species and may be useful in differentiating E. rhusiopathiae from Listeria and Corynebacterium species, which fail to demonstrate the activity. There was no correlation noted between coagulase activity and the serotype, source, geographic origin, or virulence (as detected by mouse pathogenicity tests) of the isolates.

[Biochemical research on the neuraminidase of Erysipelothrix rhusiopathiae]. / Biokhimichni prouchvaniia na neuraminidaza ot Erysipelothrix rhusiopath

Purified neuraminidase preparation from Erysipelothrix rhusiopathiae is studied biochemically. The main physicochemical characteristics of the enzyme are determined: molecular mass, isoelectric point, electrophoretic mobility, chemical nature, UV spectrum, substrate specificity, etc. The obtained data are compared to those for other bacterial neuraminidases.

Analysis of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum by multilocus enzyme electrophoresis.

The genetic diversity of 74 Australian field isolates of Erysipelothrix rhusiopathiae and 22 reference strains for serovars of E. rhusiopathiae or Erysipelothrix tonsillarum was examined by multilocus enzyme electrophoresis. Four serovar reference strains of E. tonsillarum (strains KS 20 A, Wittling, Lengyel-P, and Bano 107 for serovars 25, 3, 10, and 22, respectively) were genetically distinct from E. rhusiopathiae. However, the E. tonsillarum reference strain for serovar 14 (Iszap-4) and the reference strain for serovar 13 (Pecs-56), which has been said to represent a new genomic species, were found to cluster with typical isolates and reference strains of E. rhusiopathiae. Our reference strain for serovar 7 (Rotzunge) was also genetically typical of E. rhusiopathiae, thus indicating that these serotype reactivities cannot be relied upon as a means of identifying isolates as E. tonsillarum. Australian field isolates of E. rhusiopathiae were genetically diverse. Those recovered from sheep or birds were more diverse than those isolated from pigs, and isolates of serovar 1 were more diverse than those of serovar 2. The diversity found among isolates of the same serovar and the presence of isolates of different serovars in the same electrophoretic types (ETs) indicated that serotyping of E. rhusiopathiae was unreliable for use as an epidemiological tool. Some ETs contained isolates recovered from different animal species. ET 41 contained 32.2% of the field isolates and two reference strains, indicating that this clone of E. rhusiopathiae is both widespread and commonly associated with disease in various species of animals.

[Neuraminidase neutralizing antibodies in pigs with chronic Erysipelothrix rhusiopathiae infection (author's transl)]. / Uber das Vorkommen Neuraminidase-neutralisierender Antiköper bei chronisch rotlaufkranken Schweinen

Investigating sera from pigs with chronic E. rhusiopathiae infection relative gamma-globulin values, agglutination titers, and neuraminidase-neutralizing humoral antibodies were determined at the 12th and 15th, 11th and 14th, and 3rd and 6th month after infection. We found increasing gammaglobulin values and increasing neuraminidase neutralizing antibodies but decreasing agglutination titers. Few animals don't have antibodies against neuraminidase and died during the observation period. Furthermore immunodiffusion test demonstrated precipitating lines of antibodies against living E. rhusiopathiae cells and active neuraminidase. No or very weak bands were observed against heat killed cells and heat-inactivated enzyme.

[Investigations of the enzyme spectrum of Erysipelothrix rhusiopathiae (author's transl)]. / Untersuchungen zum Enzymspektrum von Erysipelothrix rhusiopathiiase.

The enzyme spectrum of non proliferating cells of Erysipelothrix rhusiopathiae was investigated by means of different low molecular synthetic substrates. Activities of aminopeptidases were found directed against compounds of L-alanine, L-arginine, L-aspartic acid, glycine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-tryptophane, and L-tyrosine, but not against compounds of l-cystine, L-glutaminic acid, L-histidine, L-hydroxyproline, and L-valine (Table 1). The pH optimum of the investigated aminopeptidases ranges from neutral to alkaline reaction (Table 2). Trypsin, chymotrypsin, or chymotrypsin-like proteases were not detected. E. rhusiopathiae possess esterase activity splitting esters of lower carboxylic acids, i. e. acetic acid, propionic acid, butyric acid, caproic acid, and caprylic acid, but no lipase activity. Under the provoked glycosidases only alpha- and beta-D-galactosidase and glucosaminidase were positive. Weak activities of phosphatases and arylsulfatase were found also (Table 3).

[Inhibition of bacterial neuraminidases by different anions (author's transl)]. / Uber die Hemmung bakterieller Neuraminidasen durch verschiedene Anionen

It was shown that neuraminidase of Vibrio comma is inactivated by Ca..-binding anions like citrate, EDTA, oxalate, phosphate or tartrate. There is, however, no inhibition of the newly described enzymes of Erysipelothrix insidiosa and Streptococcus viridans. Pyruvate and, to a lesser extent, also citrate inactivate all the neuraminidases investigated independently of their activation by Ca..ions.