Kinetics of infection induced byYersinia

Yersinia enterocolitica of different serotypes andY. intermedia, Y. frederiksenii, andY. kristensenii, in a total of nine strains, were inoculated intragastrically and intravenously into Swiss mice. The animals were observed daily to check for clinical alterations. Groups of five were killed intermittently at 6-h, and 3-, 6-, 10-, 15-,and 21-day periods or more after the inoculation; possible macroscopic alterations of the organs and tissues were checked. Development of infection at these periods was followed by performing viable bacterial counts on homogenates of selected tissues and the kinetics of infection was established. Clinical and pathologic alterations occurred only in the animals inoculated with the human strains ofY. enterocolitica 0:3 and 0:8, independent of the route of infection. After intragastric inoculation, theY. enterocolitica strains considered to be adapted to man were isolated from all organs and tissues, with the exception of the blood, from which only serotype 0:8 was isolated; otherYersinia strains were found only in the cecal content. After intravenous challenge, all the strains infected the organs and tissues at different times and in varied intensity, with exception of Peyer's patches and mesenteric lymph nodes, which were not infected by all theYersinia strains.     

Molecular characterization of Yersinia enterocolitica by pulsed-field gel electrophoresis and hybridization of DNA fragments to ail and pYV probes.

Sixty strains of Yersinia enterocolitica from five serogroups (O:3; O:9; O:8; O:5; and O:5,27) and eight non-Y. enterocolitica strains, recovered from diverse sources (humans, animals, food, and the environment) in Europe, Argentina, and the United States, were examined by the pulsed-field gel electrophoresis (PFGE) technique of contour clamped homogeneous electric field electrophoresis (CHEF) by using NotI and XbaI as restriction enzymes. NotI and XbaI generated 36 and 33 restriction endonuclease digestion profiles (REDP), respectively. By combining the results of both enzymes, 42 unique genomic groups were differentiated. DNA fragments were transferred to nylon membranes and hybridized with digoxigenin-labelled oligonucleotide probes to the ail gene and virulence plasmid to determine hybridization patterns and the potential virulence of the strains. The strains were tested for the presence of the plasmid by PFGE-CHEF and phenotypic characteristics encoded for by the virulence plasmid. Thirty of the 60 Y. enterocolitica strains tested harbored the virulence plasmid. The specificity of the ail and pYV probes was 100% when tested with 68 Yersinia strains and 19 different non-Yersinia strains. Sixteen selected Y. enterocolitica strains were tested for their virulence by lethality in iron- and desferrioxamine-sensitized mice. No correlation between REDP and the virulence of the strains was observed. The observed REDP and the hybridization patterns were very homogeneous within a serogroup and independent of the source of isolation. In addition, PFGE-CHEF was shown to be valuable in identifying and confirming serogroups. Principal component analysis of Dice similarity indices from REDP was an excellent tool for determining genetic relatedness among strains.     

Virulence of Escherichia coli Serotypes for Mice

A study was undertaken to determine whether virulence in mice could be used to assess the pathogenicity of a variety of Escherichia coli serotypes. Sixty-one E. coli strains isolated from animals, poultry, or humans were serotyped to determine their O, K, and H antigens, and were administered to mice via the intraperitoneal route with and without a mucin adjuvant. The ld(50) dose was then determined for each serotype. The results indicated that the source of the serotype may be associated with virulence for mice. Serotypes isolated from nonenteric, systemic sources showed a greater virulence for mice inoculated intraperitoneally than did the enteric and the nonenteric, nonsystemic (localized) isolates. It was observed that not all serotypes belonging to a specific serogroup were virulent for mice and that the presence or absence of a K antigen had no effect on the virulence of strains of one serotype.     

Use of a marker plasmid to examine differential rates of growth and death between clinical and environmental strains of Vibrio vulnificus in experimentally infected mice

Vibrio vulnificus is Gram-negative bacterium that contaminates oysters, causing highly lethal sepsis after consumption of raw oysters and wound infection. We previously described two sets of V. vulnificus strains with different levels of virulence in subcutaneously inoculated iron dextran-treated mice. Both virulent, clinical strains and attenuated, environmental strains could be recovered in high numbers from skin lesions and livers; however, the attenuated environmental strains required significantly higher numbers of colony-forming units (cfu) in the inoculum to produce lethal infection. Using some of these strains and an additional clinical strain, we presently asked if the different abilities to cause infection between the clinical and environmental strains were due to differences in rates of growth or death of the bacteria in the mouse host. We therefore constructed a marker plasmid, pGTR902, that functions as a replicon only in the presence of arabinose, which is not present in significant levels in animal tissues. V. vulnificus strains containing pGTR902 were inoculated into iron dextran-treated and untreated mice. Measuring the proportion of bacteria that had maintained the marker plasmid recovered from mice enabled us to monitor the number of in vivo divisions, hence growth rate; whereas measuring the number of marker plasmid-containing bacteria recovered enabled the measurement of death of the vibrios in the mice. The numbers of bacterial divisions in vivo for all of the strains over a 12-15 h infection period were not significantly different in iron dextran-treated mice; however, the rate of death of one environmental strain was significantly higher compared with the clinical strains. Infection of non-iron dextran-treated mice with clinical strains demonstrated that the greatest effect of iron dextran-treatment was increased growth rate, while one clinical strain also experienced increased death in untreated mice. V. vulnificus inoculated into iron dextran-treated mice replicated extremely rapidly over the first 4 h of infection with doubling times of approximately 15-28 min. In contrast, one of the environmental strains exhibited a reduced early growth rate. These results demonstrate that differences in virulence among naturally occurring V. vulnificus can be explained by diverse abilities to replicate rapidly in or resist defences of the host. The marker plasmid pGTR902 should be useful for examining virulence of bacteria in terms of differentiating growth verses death in animal hosts for most Gram-negative bacteria.     

Characteristics of Yersinia pseudotuberculosis isolated from animals in Brazil

Strains (105) of Yersinia pseudotuberculosis isolated in Brazil between 1982 and 1990 were bio-serotyped. They were also studied for plasmid profile, autoagglutination and calcium dependence at 37 °C, Congo red uptake, pyrazinamidase activity, esculin hydrolysis, salicin fermentation and drug sensitivity: 95·24% were biotype 2, serogroup O:3; 2·86% were biotype 1, serogroup O:1; and 1·90% were biotype 2, non-agglutinable. Plasmids were found in 77·14% of the strains (one in each strain). There was total correlation between the presence of the virulence plasmid and autoagglutination, calcium dependence at 37 °C and Congo red uptake. The esculin, salicin and pyrazinamidase tests were not efficient in differentiating pathogenic from non-pathogenic Y. pseudotuberculosis isolates. All strains were highly sensitive to the drugs used. These results indicate that Y. pseudotuberculosis is a potential pathogen for humans in Brazil, especially because the bio-serogroups detected among animals are those most frequently associated with human diseases.     

Ribotyping and virulence markers of Yersinia pseudotuberculosis strains isolated from animals in Brazil

Ribotyping and virulence markers has been used to investigate 68 Yersinia pseudotuberculosis strains of serogroups O:1a and O:3. The strains were isolated from clinical material obtained from healthy and sick animals in the Southern region of Brazil. Ribotypes were identified by double digestion of extracted DNA with the restriction endonucleases SmaI and PstI, separation by electrophoresis and hybridization with a digoxigenin-labeled cDNA probe. The presence of the chromosomal virulence marker genes inv, irp1, irp2, psn, ybtE, ybtP-ybtQ, and ybtX-ybtS, of the IS100 insertion sequence, and of the plasmid gene lcrF was detected by polymerase chain reaction. The strains were grouped into four distinct ribotypes, all of them comprising several strains. Ribotypes 1 and 4 presented distinct profiles, with 57.3% genetic similarity, ribotypes 2 and 3 presented 52.5% genetic similarity, and genetic similarity was 45% between these two groups (1/4 and 2/3). All strains possessed the inv, irp1, and irp2 genes. Additionally, strains of serogroup O:1a carried psn, ybtE, ybtP-ybtQ, ybtX-ybtS, and IS100. As expected lcrF was only detected in strains harboring the virulence plasmid. These data demonstrate the presence of Y. pseudotuberculosis strains harboring genotypic virulence markers in the livestock from Southern Brazil and that the dissemination of these bacteria may occur between herds.     

Evidence for the existence of distinct populations of Vibrio anguillarum serogroup O1 based on plasmid contents and ribotypes.

A total of 103 Vibrio anguillarum serogroup O1 strains displaying 15 different plasmid profiles were characterized with respect to biochemical properties and ribotypes. The results confirmed that V. anguillarum O1 is a biochemically homogeneous group. The 103 strains could be allocated to three main clusters with high similarity coefficients. None of the biochemical properties were connected with the presence of plasmids. In total, 12 different ribotypes were demonstrated, with HindIII being used as the restriction enzyme. Forty of the strains were isolated from the same Danish fish farm, some from the kidneys of diseased fish and some from the environment, and some strains were isolated from the mucus, gills, and feces of healthy fish. Nineteen of these isolates possessed the 67-kb virulence plasmid alone or in combination with other plasmids, while 21 had no plasmids. All strains isolated from the kidneys of diseased fish on this farm had plasmids. Irrespective of their origin (kidneys, gills, or mucus), all 19 strains carrying the 67-kb virulence plasmid had the same ribotype, profile 1, while isolates without plasmids belonged to five different profiles, all different from profile 1. These results suggest that pathogenic V. anguillarum O1 strains possessing a virulence plasmid and nonpathogenic strains without plasmids from a small geographical area and even from the same fish may constitute two essentially distinct populations. Thus, it may be suggested that an exchange of virulence plasmids among strains is unlikely to occur in vivo.     

Persistence of Shiga toxin-producing Escherichia coli O26 in cow slurry

AIMS: The main objective of this study was to evaluate the growth and survival of Shiga toxin-producing Escherichia coli (STEC) O26 in cow slurry; this serogroup is regarded as an important cause of STEC-associated diseases. METHODS AND RESULTS: Four STEC were examined by polymerase chain reaction (PCR) to determine whether they harbour key virulence determinants and also by pulsed-field gel electrophoresis (PFGE) to obtain overview fingerprints of their genomes. They were transformed with the pGFPuv plasmid and were separately inoculated at a level of 10(6) CFU ml(-1) in 15 l of cow slurry. All STEC O26 strains could be detected for at least 3 months in cow slurry without any genetic changes. The moisture content of the slurry decreased over time to reach a final value of 75% while the pH increased from 8.5 to 9.5 units during the last 50 days. CONCLUSION: STEC O26 strains were able to survive in cow slurry for an extended period. SIGNIFICANCE AND IMPACT OF THE STUDY: Long-term storage of waste slurry should be required to reduce the pathogen load and to limit environmental contamination by STEC O26.     

Role of pili in the pathogenesis of Pseudomonas aeruginosa burn infection

The present study using three isogenic mutants (F+P-, F-P+, F-P-) of Pseudomonas aeruginosa indicates that the presence of pili enhances the virulence of the organisms in experimental P. aeruginosa burn infection of mice. The 50% lethal dose (LD50) value for burned mice inoculated with non-piliated (P-) mutant was at least ten times higher than those inoculated with piliated (P+) bacteria. Meanwhile the LD50 value for burned mice inoculated with non-flagellated (F-) mutant was at least 10(5) times higher than those inoculated with flagellated (F+) bacteria. At 24 hr after inoculation, the bacterial counts in burned skin of mice inoculated with P+ bacteria were ten times higher than those inoculated with P- bacteria; and at 48 hr the bacterial counts became a hundred times higher in the former mice than the latter. At 24 hr after inoculation, P+ bacteria were isolated from blood, liver (F+P+), lung (F+P+), and kidney, while P- bacteria were not present in these tissues. And at 48 hr after inoculation, P+ bacteria were isolated from all tissues, while P- bacteria were isolated from some sites only. These results suggested that pili and flagella each play an important role as virulence factors independently, and that pili-mediated enhancement of virulence of P. aeruginosa was attributed to pili-mediated enhanced colonization of the organisms at the burned skin surfaces.     

Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids,outer membrane protein profiles and siderophore production

The virulence of 18 strains of Vibrio anguillarum serogroup O1 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only strains that carried the 67 kbp virulence plasmid or derivatives of it produced the outer membrane protein, OM2. All virulent strains harboured the 67 kbp plasmid or derivatives of it, indicating its importance for virulence. However, some strains carrying the virulence plasmid or a derivative of it, produced siderophores as well as OM2 but were non-pathogenic to fish. Likewise, among the virulent strains, considerable variation in LD50 values was recorded. Plasmid profiling and restriction analysis showed that the virulence plasmid existed in various molecular weights from 26 to 80 kbp, with 65-67 kbp being the most common, and that this plasmid displayed various restriction profiles. The presence of other plasmids did not seem to affect the pathogenic properties.     

Experimental Infection of Mice with Yersinia enterocolitica Serotype O9 by Oral and Parenteral Routes: Spreading and Enterotropism of Virulent Yersiniae

An isogenic pair of Yersinia enterocolitica serotype O9 strains, with and without virulence plasmid, was used to study the plasmid role in the infection of BALB/c mice by oral, intraperitoneal, and intravenous routes. The plasmid-bearing strain, but not its plasmid-less derivative, caused enteric infection after challenge by all three routes. The virulence plasmid did not influence the peritoneal clearance of yersiniae, but only the plasmid-bearing yersiniae were able to move from the peritoneal cavity to the bloodstream, and thus they spread to spleen and liver. Moreover, plasmid-bearing yersiniae were able to move from the liver to the gallbladder, and they shed in bile into the intestine. Western blot analysis of antibody responses to chromosomally encoded outer membrane proteins revealed similar patterns with sera from mice challenged with each one of the two strains by intraperitoneal route. In contrast, only the plasmid-bearing strain elicited an antibody response to these antigens in mice challenged by oral route. Although mice experimentally infected with plasmid-bearing O9 yersiniae developed an enteric infection, irrespective of the inoculation route, differences between the first steps in infection by oral and parenteral routes may be important, especially when the infection model is used as an approach to study the yersinia-host interactions. Received: 31 August 1998 / Accepted: 13 November 1998     

Differences in virulence for mice between disease and carrier strains of Neisseria meningitidis

The virulence of 11 prototype strains of Neisseria meningitidis, which had been used in the development of the serotyping scheme for serogroup B meningococci, were examined in mice treated with iron dextran. These strains, together with those previously examined, allowed for a good assessment of the virulence differences between carrier and disease strains. All of a total of 17 disease strains displayed virulence for mice (60% with high virulence), whereas only approximately 50% of 13 carrier strains possessed virulence (only 15% with high virulence). Because the ability to initiate infection in mice is independent of exogenous iron, this model system for infection appears particularly suited to studies of the physiological bases for virulence in N. meningitidis.     

01群与非01群0139型霍乱弧菌毒力的对比研究

非０１群０１３９型霍乱弧菌是近年引起南亚次大陆霍乱流行的新型病原体,将其与０１群霍乱弧菌的毒力特性进行对比研究对于了解其特性及研制相关的菌苗具有重要意义。本文报告了４株０１３９型霍乱弧菌与０１群霍乱弧菌菌株的对比测定结果。发现０１３９型霍乱弧菌与０１群霍乱弧菌有所不同,呈不透明的菌落形态,光学显微镜及电子显微镜检显示有荚膜的表型,在体内具有较高的繁殖能力并产生肠毒素,体内侵袭试验结果表明所有４株０     

Note: Isolation, characterization and epidemiology of Yersinia enterocolitica from humans and animals

During an 11-year period (1983 to 1994), 51 strains of Yersinia enterocolitica were isolated from humans and animals. Specimens were collected from a total of 3601 sources consisting of 956 patients with enteritis, 300 patients with urinary tract infection, 1564 healthy humans, 510 swine, 38 guinea-pigs, 118 rats and 115 rabbits. Five strains of Y. enterocolitica , bio/serogroups 2/O:9 and 4/O:3, virulence positive, were recovered from patients. Forty-two variants of Y. enterocolitica belonging to pathogenic serogroup O:3, Voges-Proskauer-negative biogroup 3 were recovered from swine, rats and rabbits. The rate of isolation of Y. enterocolitica from diarrhoeal swine was apparently greater than those from healthy swine. The incidence of human infections due to Y. enterocolitica was very low and bioserogroups of isolates were different from the strains which were isolated from animals. There was no evidence to suggest that swine were the source of Y. enterocolitica in humans.     

Evaluation of the usefulness of selected virulence markers for identification of virulent Yersinia enterocolitica strains. II. Genotypic markers associated with the pYV plasmid

Pathogenic strains of Yersinia enterocolitica bear virulence associated plasmid pYV. Unfortunately plasmid pYV is easily lost by these bacteria incubated at elevated temperatures (37 degrees C) or long stored at room temperatures. This sometimes makes difficult the detection of the virulence plasmid, especially by its isolation or biochemical tests. On the other hand, observations done by some authors suggest that polymerase chain reaction (PCR) could be useful for demonstration of the pYV plasmid of Yersinia strains. Accordingly to this observation the aim of the presented study was to check the usefulness of plasmid-localised genes virF and yadA, detected by PCR, for the identification of the virulent strains of Y. enterocolitica. In the presented study one hundred and fifty two clinical strains of Y. enterocolitica belonging to serogroup O3 were investigated by the PCR for the presence of genes virF and yadA. Bacterial strains were first tested for the presence of pYV plasmid. In addition the phenotypic features: calcium dependence, Congo red binding and autoagglutination were determined. In this way the virulence plasmid was found in 130 of 152 examined strains. For PCR studies also forty plasmid-cured strains of Y. enterocolitica and 32 non-Y. enterocolitica, Enterobacteriaceae strains were included. The obtained results show that the tested genes were present only in Yersinia strains possessing the pYV plasmid and no one non-specific PCR product was observed. The detection level of these genes in nested PCR permits to detect pathogenic Y. enterocolitica in suspension composed of 1 x 10(3) CFU/ml of pYV+ bacilli and 3 x 10(9) CFU/ml plasmid-cured, isogenic bacteria. In the study it was shown that genes virF and yadA were useful virulence markers, which could be helpful in clinical studies for the detection of the virulence plasmid in Y. enterocolitica strains long stored or incubated at elevated temperatures.     

Experimental colonization of mice by fresh clinical isolates of Helicobacter pylori is not influenced by the cagA status and the vacA genotype

Developing murine models of infection by Helicobacter pylori is quite useful but not all the strains are able to colonize the mouse. In order to study the influence of the two main virulence factors, CagA and VacA, on the establishment of H. pylori in mice, we have inoculated C57BL/6 mice with 15 strains randomly chosen among clinical strains freshly isolated from biopsy specimens of infected patients and five reference strains. Only six of the clinical strains and two of the reference strains could infect the animals regardless of the cagA status and the vacA genotype. We concluded that 40% of the H. pylori strains are able to infect mice and that the capacity of colonization is not influenced by the cagA status and the vacA genotype. These factors cannot be used to predict the success of an experimental infection.     

Role of the Salmonella abortusovis virulence plasmid in the infection of BALB/c mice

Following oral inoculation of BALB/c mice, Salmonella abortusovis strain SS44 was recovered in lower numbers from the Peyer's patches and mesenteric lymph nodes compared with S. typhimurium strain SL1344, whereas splenic infections were equivalent between the two serovars. SS44 was cured of its virulence plasmid or subjected to mutagenesis of the spv genes, and the Spv(-) derivatives were tested for virulence in mice. Plasmid-cured S. abortusovis SU40 retained virulence in BALB/c mice when inoculated intraperitoneally. On the other hand, mice infected orally with SU40 had greatly reduced splenic infection compared to those infected with wild-type SS44. Similar results were obtained after Tn5 insertion mutagenesis of the spvR gene or deletion of the spvABCD locus. These results suggest that in the gut-associated lymphoid tissues S. abortusovis may replicate less than S. typhimurium and that the S. abortusovis virulence plasmid primarily affects systemic infection after oral inoculation but not after intraperitoneal administration in the mouse model.     

Clonality of Vibrio anguillarum strains isolated from fish from the Scandinavian countries, Sweden, Finland and Denmark

In order to investigate whether outbreaks of vibriosis in the Baltic region were caused by the spread of certain pathogenic clones, 291 Vibrio anguillarum isolates from Finland (n = 156), Sweden (n = 88) and Denmark (n = 47) were studied with respect to serogroup, ribotype, plasmid content, and biochemical phenotypes as expressed with the PhenePlate (PhP) typing system. For comparison, 54 V. anguillarum serogroup O1 from other countries worldwide were included. Most isolates from Finland, Sweden and Denmark belonged to serogroup O1 (255), followed by O2 (30). Four Finnish isolates cross-reacted strongly with antisera against two new serogroups VaNT2 and VaNT4, whereas two strains were non-typeable. The serogroup O1 isolates displayed ten different ribotype patterns, whereas the other strains were considerably more diverse with respect to ribotypes. Most of the O1 isolates carried the 67 kb virulence plasmid and a group of Finnish isolates, in addition, carried an 86 kb plasmid. Additional plasmids with molecular weights of 63, 76, 135 or 260-290 kb were found in single O1 isolates. With few exceptions, strains of serogroup O2 either had no plasmids or carried one or two small plasmids. PhenePlate typing revealed considerable diversity within the species, serogroup O1 being the most homogeneous. A few PhP types were dominant, whereas other types were observed only in one to four isolates. The prevalence of the different types changed significantly from one year to another but in Finland, one clonal lineage became increasingly important from 1992 (20% of isolates) to 1996 (80%). Remaining clones were mostly restricted to specific geographic areas. By cluster analysis, it was demonstrated that most of the isolates from Finland, Sweden and Denmark belonged to two clusters, and most of the strains from Southern Europe fell into two other, distinct clusters. Most isolates from the UK, North America, Chile and Tasmania grouped together in a distinct cluster. For the typing of V. anguillarum, O-serotyping should be the primary method. For isolates belonging to serogroups other than O1, plasmid profiling in combination with ribotyping gives a very good discrimination between strains, whereas for serogroup O1, another method is required. It is concluded that PhP typing is a tool that provides a good discrimination between O1 isolates.     

Comparison of extraintestinal pathogenic Escherichia coli strains from human and avian sources reveals a mixed subset representing potential zoonotic pathogens

Since extraintestinal pathogenic Escherichia coli (ExPEC) strains from human and avian hosts encounter similar challenges in establishing infection in extraintestinal locations, they may share similar contents of virulence genes and capacities to cause disease. In the present study, 1,074 ExPEC isolates were classified by phylogenetic group and possession of 67 other traits, including virulence-associated genes and plasmid replicon types. These ExPEC isolates included 452 avian pathogenic E. coli strains from avian colibacillosis, 91 neonatal meningitis E. coli (NMEC) strains causing human neonatal meningitis, and 531 uropathogenic E. coli strains from human urinary tract infections. Cluster analysis of the data revealed that most members of each subpathotype represent a genetically distinct group and have distinguishing characteristics. However, a genotyping cluster containing 108 ExPEC isolates was identified, heavily mixed with regard to subpathotype, in which there was substantial trait overlap. Many of the isolates within this cluster belonged to the O1, O2, or O18 serogroup. Also, 58% belonged to the ST95 multilocus sequence typing group, and over 90% of them were assigned to the B2 phylogenetic group typical of human ExPEC strains. This cluster contained strains with a high number of both chromosome- and plasmid-associated ExPEC genes. Further characterization of this ExPEC subset with zoonotic potential urges future studies exploring the potential for the transmission of certain ExPEC strains between humans and animals. Also, the widespread occurrence of plasmids among NMEC strains and members of the mixed cluster suggests that plasmid-mediated virulence in these pathotypes warrants further attention.     

多重PCR方法检测霍乱弧菌的研究

霍乱弧菌是霍乱的病原体,可以分为O1群、O139群和非O1/非O139群。O1群和O139群霍乱弧菌产生的霍乱肠毒素(也称霍乱毒素)是产生霍乱的主要原因,也只有O1群和O139群霍乱弧菌可引起霍乱。其他群的霍乱弧菌毒性不高,但在食品中也不允许被检出。实验以霍乱胶原酶基因和霍乱毒素基因为目的基因,试图建立一种PCR方法对霍乱弧菌进行检测研究,结果表明此方法可以用于食品中的霍乱弧菌检测。