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.     

Chromosome-mediated resistance of Yersinia enterocolitica serotype O9 to intracellular killing by mouse peritoneal macrophages

Abstract The survival of Yersinia enterocolitica serotype O9 within mouse peritoneal macrophages was investigated. To evaluate the role of the virulence plasmid in the resistance to intracellular killing, an isogenic pair of virulent (plasmid-bearing) and avirulent (plasmid-less) O9 strains was used. The virulent strain was able to express plasmid-encoded outer membrane proteins and to colonize the Peyer's patches of orally infected mice. When mice were infected intraperitoneally, both strains were recovered at similar rates and over the same time from the peritoneal cavity. When in vitro assays were performed, both strains showed similar resistance to intracellular killing by monolayers of resident and inflammatory peritoneal macrophages. Previous opsonization of bacteria did not modify their survival within macrophage monolayers. We concluded that serotype O9 strains display a chromosome-mediated resistance to intracellular killing by mouse peritoneal macrophages. Moreover, macrophage resistance does not seem to be of importance for virulence of serotype O9 strains in mice.     

Mutations affecting expression and maintenance of genes encoding the serotype b capsule of Haemophilus influenzae.

Deletion mutagenesis analysis of a duplicated gene necessary for Haemophilus influenzae serotype b capsule expression showed that only one functional copy of this gene is required for capsule production and for virulence in infant rats. Mutant strains generated in this study differed from each other and from the parental strain in their ability to maintain the large tandem duplication which contains the genes involved in serotype b capsule expression.     

Toxic and reactogenic properties of pertussis bacteria (experimental and clinical studies).

The results of the study revealed correlation between the reactogenicity of pertussis vaccines in epidemiological observations and the toxic properties of pertussis bacteria in experiment. Quantitative and qualitative differences were found between the toxic factors in pertussis bacteria depending on their type-specific serological activity. Serotype 1.0.3 microbes exhibit more pronounced toxicity which accounts for the greater reactogenicity of vaccines prepared from this serotype as compared with the preparation produced from the serotype 1.2.3. The obtained results suggest the necessity of considering the greater toxicity of the serotype 1.0.3 in the preparation of pertussis vaccines.     

Interrelation of virulent and avirulent (vaccinal) strains of Shigella flexneri 2A in long-term bacterial carriage in gnotobiotic rats

The ability of S. flexneri 2a virulent and avirulent (vaccine) strains No. 516 and No. 516 m to displace one another during long-term carrier state in germ-free rats has been studied. The long-term persistence of the vaccine strain in the intestine of the rats has been shown to produce colonization resistance to subsequent infection with the virulent culture of these bacteria. During carrier state in rats progressive S--R dissociation of the bacteria occurs, type II antigen is lost with simultaneous retention of group 3, 4 antigens and the resulting transformation of S. flexneri, serotype 2a, into variant y; the virulence of Shigella S-forms is also lost.     

Changes in capsular serotype alter the surface exposure of pneumococcal adhesins and impact virulence

We examined the contribution of serotype on Streptococcus pneumoniae adhesion and virulence during respiratory tract infection using a panel of isogenic TIGR4 (serotype 4) mutants expressing the capsule types 6A (+6A), 7F (+7F) and 23F (+23F) as well as a deleted and restored serotype 4 (+4) control strain. Immunoblots, bacterial capture assays with immobilized antibody, and measurement of mean fluorescent intensity by flow cytometry following incubation of bacteria with antibody, all determined that the surface accessibility, but not total protein levels, of the virulence determinants Pneumococcal surface protein A (PspA), Choline binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP) changed with serotype. In vitro, bacterial adhesion to Detroit 562 pharyngeal or A549 lung epithelial cells was modestly but significantly altered for +6A, +7F and +23F. In a mouse model of nasopharyngeal colonization, the number of +6A, +7F, and +23F pneumococci in the nasopharynx was reduced 10 to 100-fold versus +4; notably, only mice challenged with +4 developed bacteremia. Intratracheal challenge of mice confirmed that capsule switch strains were highly attenuated for virulence. Compared to +4, the +6A, +7F, and +23F strains were rapidly cleared from the lungs and were not detected in the blood. In mice challenged intraperitoneally, a marked reduction in bacterial blood titers was observed for those challenged with +6A and +7F versus +4 and +23F was undetectable. These findings show that serotype impacts the accessibility of surface adhesins and, in particular, affects virulence within the respiratory tract. They highlight the complex interplay between capsule and protein virulence determinants.     

The NLRP3 inflammasome is differentially activated by pneumolysin variants and contributes to host defense in pneumococcal pneumonia

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1β. This IL-1β production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1β production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.     

The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi-Spa type III secretion apparatus and the secreted IpaA-D proteins, all of which are encoded by a virulence plasmid. In wild-type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N-terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.     

Biological activity and identification of a peptide inhibitor of LuxS from Streptococcus suis serotype 2

The virulence of bacterial communities may be regulated by mechanisms involving the synthesis of the quorum-sensing signal autoinducer 2 (AI-2), which allows both intra- and interspecies communication. AI-2 is produced in bacteria that express the gene luxS . In the present study, expressed and purified LuxS from Streptococcus suis serotype 2 (SS2) was used to catalyze the substrate S -ribosylhomocysteine in a reaction that leads to the production of AI-2. The biological activity of the in vitro synthesized AI-2 was demonstrated in a Vibrio harveyi strain BB170 bioassay; real-time PCR results showed that biosynthesis of AI-2 can increase the virulence of SS2. Phage-encoded peptides that specifically interact with the LuxS enzyme were selected following three rounds of phage display. One such peptide inhibitor (TNRHNPHHLHHV) of LuxS was shown to partially inhibit the activity of the enzyme. Furthermore, 14 peptides containing the consensus sequence HSIR showed high affinity with LuxS. The selected and characterized specific inhibitor as well as the high-affinity ligands may facilitate the identification of new vaccination targets, opening up new approaches to the development of therapeutic drugs.     

Development of a multiplex PCR approach for the identification of Shiga toxin-producing Escherichia coli strains and their major virulence factor genes

AIMS: To develop and evaluate a multiplex PCR (mPCR) system for rapid and specific identification of Shiga toxin-producing Escherichia coli (STEC) and their main virulence marker genes. METHODS AND RESULTS: A series of mPCR assays were developed using primer pairs that identify the sequences of Shiga toxins 1 and 2 (stx1 and stx2, including the stx2c, stx2d, stx2e and stx2f variants), intimin (eaeA), and enterohaemorrhagic E. coli enterohaemolysin (ehlyA). Moreover, two additional genes (rfb O157 and fliC H7), providing the genotypic identification of the O157:H7 E. coli serotype, were detected. As an internal positive control, primers designated to amplify the E. coli 16S rRNA were included in each mPCR. All the amplified genes in the E. coli reference strains were sucessfully identified by this procedure. The method was then used for the examination of 202 E. coli isolates recovered from cattle and children. Among them, 25 (12.4%) were stx positive including the strains of O157:H7 serotype (six isolates) and O157:NM serogroup (four strains). Moreover, 20 STEC strains possessed the eaeA (intimin) and ehlyA (enterohaemolysin) genes. CONCLUSIONS: The developed mPCR-based system enabled specific detection of STEC bacteria and identification of their main virulence marker genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify STEC bacteria and the majority of their virulence gene markers, including four variants of Shiga toxin, as well as the differentiation of O157:H7 from non-O157 isolates represents a considerable advancement over other PCR-based methods for rapid characterization of STEC.     

胸膜肺炎放线杆菌萘啶酸抗性菌株的选育和信号标签突变株的构建

胸膜肺炎放线杆菌(APP)是重要的猪呼吸道病原菌,给世界养猪业造成严重的经济损失.信号标签突变(STM)技术是在宿主动物体内鉴定病原菌毒力因子的高通量方法.通过体外传代选育出APP血清1型和3型萘啶酸抗性菌株,再以萘啶酸抗性菌株为受体菌,以携带mini-Tn10的标签质粒(pLOF/TAG1-48)的E.coli CC118 λ pir或S17-1λpir为供体菌,在或不在E.coli DH5α(pRK2073)的辅助下,进行三亲本或两亲本接合,通过抗性筛选、PCR和Southern杂交鉴定转座突变株.结果表明:体外萘啶酸加压传代很容易选育出萘啶酸抗性APP菌株,该抗性的产生与DNA促旋酶A亚基基因gyrA的突变有关.在APP与E. coli接合实验中,两亲本接合比三亲本接合操作更简单,效率也较高;APP不同菌株在接合和转座效率上存在很大差异,血清1型菌株高于血清3型菌株,3型标准菌株高于地方分离株JL03-R.本研究为APP STM突变体库的构建与毒力基因的鉴定奠定了基础.     

Evaluation of Virulence Test Procedures for Yersinia enterocolitica Recovered from Foods

Recently a heat stable toxin (ST) and the vw factors of the plague bacteria were identified in Yersinia enterocolitica recovered from human infections. The vw factors were reported to be associated with a plasmid of 42-46 M Daltons in size and were essential for the expression of virulence. With this knowledge virulence tests were developed which allowed us to assess the virulence potential of food-borne Y. enterocolitica regardless of biotypes or serotypes. The tests evaluated were: (1) rapid presumptive test for the virulence plasmid; (2) gel electrophoretic confirmation of the virulence plasmid; (3) Laird's qualitative oral feeding test with thirst stressed mice; (4) quantitative LD₅₀ determination by i.p. injection of the mouse lethal ( i.e. serotype 0:8) strains in saline; (5) quantitative LD₅₀ determination of mouse non-lethal ( i.e. serotype 0:3) strains by i.p. injection of these strains suspended in 1 ml of 10% iron dextran saline solution for virulence enhancement. These tests were evaluated with the serotypes 0:3 and 0:8 strains associated with human infections with and without the virulence plasmid with reproducible results. Then the virulence tests procedures were applied to 79 food isolates. The virulence plasmid was detected only in the Nilehn biotype 2, 3 and 4 strains, but it was absent in Nilehn biotype 1 or the atypical strains that ferment rhamnose. The virulence of food and clinical isolates of Y. enterocolitica can be assayed fairly accurately with the above tests.     

Characterization of the RpoS status of clinical isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, sigma(S) (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella: We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the sigma(S) protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of sigma(S), showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

Antibody inhibition of HeLa cell invasion by Yersinia enterocolitica

Antisera were prepared in rabbits against formalized and heat-killed bacteria of Yersinia enterocolitica serotype O:5,27 and against formalized bacteria of serotype O:8. Both strains used for immunization demonstrated adhesion to and invasion of HeLa cells. Coating of the bacteria with antibody did not greatly alter adhesion (i.e., extracellular attachment) to HeLa cells; however, antibody against formalized bacteria of both serotypes inhibited HeLa cell invasion by the homologous and heterologous strains. The Fab fragments from purified immunoglobulins also demonstrated cross-reacting inhibition of HeLa cell invasion. Antibody against heat-killed bacteria of serotype O:5,27 had no inhibitory activity. Adsorption of the antiserum against formalized bacteria of serotype O:5,27 with lipopolysaccharide from the homologous strain removed anti-lipopolysaccharide antibody but did not remove the inhibitory activity. The antiserum against formalized bacteria of serotype O:8 showed no antibody against lipopolysaccharide from serotype O:5,27 and no agglutinins against heat-killed bacteria of this strain. From these results, it is tentatively suggested that protein structures are important in mediating epithelial cell invasion by Y. enterocolitica.     

The role of toxins and virulence factors in hetero trophic bacteria present in drinking water supplies

Heterotrophic plate count (HPC) bacteria are ubiquitous in both man-made and natural water systems and consist of several bacterial species which rely on carbon molecules, generally pre-formed by other organisms, as a nutrient source. Previous studies have suggested that heterotrophs may be significant to human health if present in drinking water supplies and may be responsible for incidences of gastro intestinal disturbance.
Bacteria which are associated with gastrointestinal (GI) illness may be classed as enterotoxigenic (ETEC), entero invasive (EIEC), enteropathogenic (EPEC) and/or entero haemorrhagic (EHEC). An overview of the literature, discussing both the proposed role of heterotrophs in causing GI symptoms and the known factors associated with entero toxicity, e.g. cytotoxic and cytotonic activity, invasiveness and haemolysin activity will be presented.
The results of a recent UK study will also be discussed. In this study isolates were obtained from both pre-flush and post-flush drinking water samples taken from both houses and multi-occupancy buildings from several geographically unrelated sites. Studies to determine the occurrence of toxigenic/virulence factors in the heterotrophs isolated were undertaken, by assessing their potential to cause GI upset, by determination of their invasiveness and cytotonic and cytotoxic effects using three different cell lines, together with haemolysin activity.
The results of this study concur with others and suggest that the frequency of isolation of heterotrophic species with the capability to cause GI symptoms is too low to be of significant concern to health authorities or water companies. Furthermore, where a positive result was obtained with respect to the possession of virulence characteristics, the bacteria in question usually displayed more than one type of virulence factor.     

Phenotypic characterization and putative virulence factors of human,animal and environmental isolates of <Emphasis Type="Italic">Plesiomonas shigelloides</Emphasis>

Plesiomonas shigelloides (a bacterium widely distributed in aquatic ecosystems causing both intestinal and extra-intestinal diseases) shows a host of putative virulence markers, such as hemolysins, cytotoxins, production of exoenzymes associated with pathogenicity, adhesive ability and vacuolation of cell lines in vitro. Technical difficulties in detecting some of these virulence factors together with scantiness of epidemiological information, due to the lack of routine analysis for P. shigelloides as etiological agent of gastroenteritis, lead to sporadic and occasional finding of these bacteria. All this casts doubt on the real virulence potential of P. shigelloides and fuels a debate about assignment of these bacteria to the list of human pathogens. Here we demonstrated the phenotypic diversity and the putative virulence markers by examining serotype biochemical and virulence properties of 60 strains of P. shigelloides isolated from human, animal and environmental samples in different countries, which showed the unpredictable occurrence of the above properties depending on various locations and diverse sources.     

Lipase Activity from Strains of Pasteurella multocida

Thirteen clinical isolates of Pasteurella multocida from a variety of different animals and humans were examined for their ability to produce lipase. Lipase substrates used included Tween 20, Tween 40, Tween 80, and Tween 85. Lipase activity was detected in the filtrates of organisms grown to the exponential phase in Roswell Park Memorial Institute-1640 defined media (RPMI-1640), but activity increased in the filtrates when the cultures were allowed to proceed to the stationary phase. All strains examined (except for serotype 2) showed lipase activity against at least one of the Tweens. Tween 40 was the best substrate to demonstrate lipase activity. Pasteurella multocida serotype 8 produced the most active lipase against Tween 40 (3,561.7 units of activity/μg of protein). This activity continued to increase after P. multocida entered a stationary growth phase. P. multocida lipase activity was optimal at pH 8.0. Lipase activity of P. multocida serotype 8 was eluted from a Sepharose 2B column at several points, indicating that several lipases may be produced in vitro by this organism. These data demonstrate that clinical isolates of P. multocida produce lipase; therefore, this enzyme should be considered a potential virulence factors for this organism. Received: 16 September 1999 / Accepted: 22 November 1999     

Intestinal carriage of Yersinia pseudotuberculosis by wild birds and mammals in Japan

Fecal specimens were obtained from wild birds and mammals in the eastern part of Shimane Prefecture, Japan, an area where serotype 1b, 2b, 3, and 4b strains of Yersinia pseudotuberculosis were found to be prevalent in humans. Each of 869 animals, including 259 wild birds and 610 wild mammals, was screened for yersiniae. A total of 37 strains of Y. pseudotuberculosis were isolated from 34 (5.6%) mammals, including 23 raccoon dogs (Nyctereutes procyonoides), eight deer (Cervus nippon), two hares (Lepus brachyurus), and one marten (Martes melampus), and from two (0.8%) birds, including one eastern spot-billed duck (Anas poecilorhyncha) and one wigeon (Anas penelope). The Y. pseudotuberculosis isolates collected belonged to virulence plasmid-positive (serotypes 1b, 2b, 3, 4b, and 6) and virulence plasmid-negative (serotype 5a) strains, the most predominant serotype being 4b. The close relationship between the regional distributions of Y. pseudotuberculosis in wild animals and humans suggests that wild animals are an important source of infection.     

Intestinal carriage of Yersinia pseudotuberculosis by wild birds and mammals in Japan.

Fecal specimens were obtained from wild birds and mammals in the eastern part of Shimane Prefecture, Japan, an area where serotype 1b, 2b, 3, and 4b strains of Yersinia pseudotuberculosis were found to be prevalent in humans. Each of 869 animals, including 259 wild birds and 610 wild mammals, was screened for yersiniae. A total of 37 strains of Y. pseudotuberculosis were isolated from 34 (5.6%) mammals, including 23 raccoon dogs (Nyctereutes procyonoides), eight deer (Cervus nippon), two hares (Lepus brachyurus), and one marten (Martes melampus), and from two (0.8%) birds, including one eastern spot-billed duck (Anas poecilorhyncha) and one wigeon (Anas penelope). The Y. pseudotuberculosis isolates collected belonged to virulence plasmid-positive (serotypes 1b, 2b, 3, 4b, and 6) and virulence plasmid-negative (serotype 5a) strains, the most predominant serotype being 4b. The close relationship between the regional distributions of Y. pseudotuberculosis in wild animals and humans suggests that wild animals are an important source of infection.     

Heterogeneity in Induction Level,Infection Ability,and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are foodborne pathogens responsible for diarrhea and hemolytic-uremic syndrome (HUS). Shiga toxin, the main STEC virulence factor, is encoded by the stx gene located in the genome of a bacteriophage inserted into the bacterial chromosome. The O26:H11 serotype is considered to be the second-most-significant HUS-causing serotype worldwide after O157:H7. STEC O26:H11 bacteria and their stx-negative counterparts have been detected in dairy products. They may convert from the one form to the other by loss or acquisition of Stx phages, potentially confounding food microbiological diagnostic methods based on stx gene detection. Here we investigated the diversity and mobility of Stx phages from human and dairy STEC O26:H11 strains. Evaluation of their rate of in vitro induction, occurring either spontaneously or in the presence of mitomycin C, showed that the Stx2 phages were more inducible overall than Stx1 phages. However, no correlation was found between the Stx phage levels produced and the origin of the strains tested or the phage insertion sites. Morphological analysis by electron microscopy showed that Stx phages from STEC O26:H11 displayed various shapes that were unrelated to Stx1 or Stx2 types. Finally, the levels of sensitivity of stx-negative E. coli O26:H11 to six Stx phages differed among the 17 strains tested and our attempts to convert them into STEC were unsuccessful, indicating that their lysogenization was a rare event.