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PubMed2006

Identification of MglA-regulated genes reveals novel virulence factors in Francisella tularensis.

The facultative intracellular bacterium Francisella tularensis causes the zoonotic disease tularemia. F. tularensis resides within host macrophages in vivo, and this ability is essential for pathogenesis. The transcription factor MglA is required for the expression of several Francisella genes that are necessary for replication in macrophages and for virulence in mice. We hypothesized that the identification of MglA-regulated genes in the Francisella genome by transcriptional profiling of wild-type and mglA mutant bacteria would lead to the discovery of new virulence factors utilized by F. tularensis. A total of 102 MglA-regulated genes were identified, the majority of which were positively regulated, including all of the Francisella pathogenicity island (FPI) genes. We mutated novel MglA-regulated genes and tested the mutants for their ability to replicate and induce cytotoxicity in macrophages and to grow in mice. Mutations in MglA-regulated genes within the FPI (pdpB and cds2) as well as outside the FPI (FTT0989, oppB, and FTT1209c) were either attenuated or hypervirulent in macrophages compared to the wild-type strain. All of these mutants exhibited decreased fitness in vivo in competition experiments with wild-type bacteria. We have identified five new Francisella virulence genes, and our results suggest that characterizations of additional MglA-regulated genes will yield further insights into the pathogenesis of this bacterium.

Animals↗
PubMed2002

Prevalence and molecular characterization of Escherichia coli O157:H7 in bulk tank milk and fecal samples from cull cows: a 12-month survey of dairy farms in east Tennessee.

A study on the prevalence of Escherichia coli O157:H7 was conducted on 30 dairy farms in east Tennessee between May 2000 and April 2001. This pathogen was isolated from 8 of 30 (26.7%) dairy farms at various sampling times. A total of 415 fecal samples from cull dairy cows and 268 bulk tank milk samples were analyzed. Overall, 10 of 683 (1.46%) samples (2 of 268 [0.75%] milk samples and 8 of 415 [1.93%] fecal samples) tested positive for E. coli O157:H7. Food and Drug Administration Bacteriological Analytical Manual protocols were used for the conventional isolation and confirmation of E. coli O157:H7. Samples were shake cultured (150 rpm) at 42 degrees C for 24 h in tryptic soy broth containing 2 mg of novobiocin per liter. White colonies isolated on cefixime-tellurite sorbitol MacConkey agar plates were evaluated for fluorescence on sorbitol MacConkey agar supplemented with 0.025 g of methylumbelliferyl-beta-D-glucuronide per liter. Nonfluorescing white colonies were biochemically typed and serologically confirmed. Multiplex polymerase chain reaction profiles of E. coli O157:H7 isolates indicated the presence of common virulence factors (Shiga toxin, enterohemolysin, and intimin) of Shiga toxin-producing E. coli, suggesting the potential human pathogenicity of bacterial isolates. Pulsed-field gel electrophoresis profiles of SpeI and XbaI restriction enzyme-digested genomic DNA were used to establish relatedness among bacterial isolates. Data from this study indicate that both cull dairy cows and bulk tank milk pose a potential hazard with regard to human foodborne illness. It is therefore imperative to develop on-farm and preharvest pathogen reduction programs to control the carriage of E. coli O157:H7 pathogens.

Animals↗
PubMed2001

Salmonella serotype Virchow causing salmonellosis in a Spanish region. Characterization and survey of clones by DNA fingerprinting, phage typing and antimicrobial resistance.

The diversity of Salmonella serotype Virchow organisms causing human salmonellosis in a Spanish region over 1990-1996 was studied by genetic and phenotypic procedures. Isolates showing identical DNA fingerprintings (ribotypes, RAPD-, REP- and ERIC-types) were clustered into the same lineage. Eight lineages were defined, of which only one caused diseases throughout the studied period. Eleven phage types (PTs) were represented, the most frequent being PTs 8, 19, 31, throughout the study period, and PT4a only during 1994. Class I integrons with variable regions of 1000-, 1600-, and 2300-bp in size were respectively present in 24, 3 and 5 multiresistant isolates; 43.5% of isolates were susceptible to antimicrobials, the rest were grouped into 17 R-profiles, including from one up to eight resistances. Plasmids could be recovered from 71.5% of isolates and grouped into 25 plasmid profiles (with 1-7 plasmids each); a 3.6 kb cryptic-plasmid and a 60 kb virulence-plasmid were those most frequently found. Phage type, presence and size of integrons, and resistance profile were used to differentiate 39 clones. During the period studied 135 cases of Virchow salmonellosis were identified; 93 were apparently sporadic whereas the remainder were associated with four outbreaks. Infants under 1 year constituted the most frequent age group, with 30 gastroenteritis and two septicaemia episodes. In the four outbreaks, different clones falling into the prevalent lineage were implicated but each clone was involved in only one outbreak.

Adolescent↗
PubMedDate not supplied

Antiorthostatic suspension stimulates profiles of macrophage activation in mice.

The antiorthostatic suspension model simulates certain physiological effects of spaceflight. We have previously reported BDF1 mice suspended by the tail in the antiorthostatic orientation for 4 days express high levels of resistance to virulent Listeria monocytogenesinfection. In the present study, we examined whether the increased resistance to this organism correlates with profiles of macrophage activation, given the role of the macrophage in killing this pathogen in vivo. We infected BDF1 mice with a lethal dose of virulent L. monocytogenes on day 4 of antiorthostatic suspension and 24 h later constructed profiles of macrophage activation. Viable listeria could not be detected in mice suspended in the antiorthostatic orientation 24 h after infection. Flow cytometric analysis revealed the numbers of granulocytes and mononuclear phagocytes in the spleen of infected mice were not significantly altered as a result of antiorthostatic suspension. Splenocytes from antiorthostatically suspended infected mice produced increased titers of IL-1. Serum levels of neopterin, a nucleotide metabolite secreted by activated macrophages, were enhanced in mice infected during antiorthostatic suspension, but not in antiorthostatically suspended naive mice. Splenic macrophages from mice infected on day 4 of suspension produced enhanced levels of lysozyme. In contrast to the results from antiorthostatically suspended infected mice, macrophages from antiorthostatically suspended uninfected mice did not express enhanced bactericidal activities. The collective results indicate that antiorthostatic suspension can stimulate profiles of macrophage activation which correlate with increased resistance to infection by certain classes of pathogenic bacteria.

Aerospace Medicine↗
PubMed1996

Virulence and genotype stability of Salmonella enterica serovar Berta during a natural outbreak.

Strains of Salmonella enterica serotype Berta, collected over a period of 6 years from a well documented natural outbreak in Denmark, have been characterized in order to assess the stability of chromosomal typing systems and virulence properties. Outbreak strains were identical in Pvu II and PSTI IS200 profiles, all but two strains showed the same Sma I ribotype, and all but one strain showed the same Not I pulsed field gel electrophoretic pattern, indicating that these molecular markers remained almost constant during the outbreak. In general, strains of S. Berta were found to be of moderate to low virulence; log VC10 values were found to vary between 3.0 and 4.4 after i.p. challenge of mice, and maximum CFU in internal organs of day-old chicks varied between 2 and 4 log10 units following oral challenge. The minor differences observed between strains in vivo did not correlate with differences in in vitro invasion into cultured MDCK cells, nor with in vitro growth characteristics. A succession of different plasmid profile types was observed during the outbreak but a hierarchical selection of clones based on differences in virulence was unlikely to have caused the succession of types of S. Berta during this outbreak.

Animals↗
PubMed2004

Computer-based analysis of Haemophilus parasuis protein fingerprints.

The present study aimed to compare the whole-cell protein profiles of Haemophilus parasuis field isolates by using a computer-based analysis, and evaluate the relationship between polyacrylamide gel electrophoresis (PAGE) type and virulence potential based on isolation site. A dendrogram clustering isolates with similar protein profiles was generated. Haemophilus parasuis isolates were grouped into 2 major PAGE type groups. The PAGE type II isolates were characterized by the presence of major proteins with molecular weights varying from between 36 and 38 kDa and included 90.7% of the isolates recovered from systemic sites, such as pleura, pericardium, peritoneum, lymph nodes, joints, and brain. Isolates classified as PAGE type I were characterized by the absence of this group of proteins and included 83.4% of the isolates recovered from the upper respiratory tract of healthy animals. The present study further corroborates the existence of a unique group of major proteins in potentially virulent H. parasuis isolates.

Animals↗
PubMed1987

Plasmids of Salmonella muenster and their relation to virulence.

The plasmid content of 104 strains of Salmonella muenster collected from bovine and human sources in Ontario from 1982 to 1984 was determined. The strains were classified into 17 different groups on the basis of the sizes of the plasmids they contained. Further division was made on the basis of the fragments obtained following digestion of the plasmids with restriction endonucleases BglI and BglII. Representative strains from each plasmid profile group were compared for resistance to serum and for virulence in mice. No differences in serum resistance or in LD50 by the oral or intraperitoneal route in mice could be associated with the presence of plasmids. Although the strains of S. muenster were recovered from clinical cases in cattle and humans, they were of low virulence for mice: the LD50 values were approximately 10,000-fold greater than that of Salmonella typhimurium.

Animals↗
PubMed1989

Lipopolysaccharide alteration mediated by the virulence plasmid of Salmonella.

Lipopolysaccharide (LPS) of Salmonella dublin, S. enteritidis, S. typhimurium, S. choleraesuis and their derivative strains was analysed to investigate the correlation between LPS and virulence plasmid of Salmonella. All wild-type strains had smooth type LPS, i.e. LPS with long O-specific polysaccharide. The virulence plasmid-cured strain of S. dublin, C524, exhibited a shorter O-specific chain than its parent strain, 5240. No distinct ladder bands were observed at the high molecular weight region on the SDS-PAGE gel for C524 LPS. By chemical analysis the number of O-repeating unit of C524 LPS was shown to be approximately one. The chain length of O-specific polysaccharide was restored by reintroduction of the virulence plasmid. The alteration of LPS by curing and reintroduction of the virulence plasmid was not observed when other wild-type strains of S. dublin were used. In the case of S. enteritidis, S. typhimurium, and S. choleraesuis, alteration of neither chemical composition nor electrophoretical profile of LPS was detected by curing and reintroduction of the virulence plasmids. Those results suggest that certain factor for regulation of the chain length of O-specific polysaccharide is encoded on the virulence plasmid of S. dublin.

Lipopolysaccharides↗
PubMed2006

Transcription Profiling of the mgrA Regulon in Staphylococcus aureus.

MgrA has been shown to affect multiple Staphylococcus aureus genes involved in virulence and antibiotic resistance. To comprehensively identify the target genes regulated by mgrA, we employed a microarray method to analyze the transcription profiles of S. aureus Newman, its isogeneic mgrA mutant, and an MgrA-overproducing derivative. We compared genes that were differentially expressed at exponential or early stationary growth phases. Our results showed that MgrA affected an impressive number of genes, 175 of which were positively regulated and 180 of which were negatively regulated in an mgrA-specific manner. The target genes included all functional categories. The microarray results were validated by real-time reverse transcription-PCR quantitation of a set of selected genes from different functional categories. Our data also indicate that mgrA regulates virulence factors in a fashion analogous to that of the accessory gene regulatory locus (agr). Accordingly, exoproteins are upregulated and surface proteins are downregulated by the regulator, suggesting that mgrA may function in concert with agr. The fact that a large number of genes are regulated by mgrA implies that MgrA is a major global regulator in S. aureus.

Bacterial Proteins↗
PubMed2006

Nontyphoidal Salmonella causing focal infections in patients admitted at a Spanish general hospital during an 11-year period (1991-2001).

In focal infections (FI) caused by nontyphoidal Salmonella serotypes and recorded at a Spanish hospital 1991-2001, clinical and microbiological features were analyzed. Thirty-five revised episodes were related to infections of the digestive (10), urinary (10), pulmonar (4), vascular (4), osteoarticular (3) and central nervous (3) systems, and with a submaxillary lymph node. At least 16 episodes were associated with previous or concomitant gastroenteritis, 19 with primary or secondary bacteremia, and 18 with underlying diseases of different severity. Eighteen patients were male and 14 female (data were not available for three patients), while 1, 4, 12 and 15 patients were, respectively, categorized as children, young adults, senior adults and elderly. Sources of Salmonella strains were urine (13), blood (11), purulent abscess (8), cerebrospinal fluid (3), peritoneal fluid, pleural fluid, wound exudates, aneurism (2 of each), ascitic fluid, sputum, tracheal aspirate, needle aspirate, bone and lymph node (1 of each) samples. Only 28 Salmonella strains involved in FIs were available for further analysis. They were discriminated into 6 serotypes, and into 13 XbaI macrorestriction, 6 virulence, 11 antimicrobial resistance, 5 integron and 10 plasmid profiles. Broadly, the pattern of serotype distribution of salmonellas involved in FIs matched that of those causing gastroenteritis, with the pandemic Enteritidis and Typhimurium (18 and 6 strains, respectively) being clearly predominant. Within serotype, the same lineages (as revealed by XbaI-macrorestriction analysis as well as R- and V-profiles) were represented in both disease groups, with host-related factors apparently playing a more critical role than the individual strain in the outcome of the disease.

Adolescent↗
PubMed2026

Alleviation of Helicobacter pylori-Induced Pathogenicity and Gastric Inflammation by Majonoside-R2- and Ginsenoside Rg1-Rich Fractions From Panax vietnamensis Ha Et Grushv.: A Metabolomics-Guided Investigation.

Helicobacter pylori infection remains a major global health concern due to its association with gastric inflammation, ulceration, and gastric malignancies. This study evaluated the effects of Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) root fractions on H. pylori virulence and host inflammatory responses. UHPLC-MS/MS-based metabolomic profiling coupled with feature-based molecular networking was employed to characterize the chemical profiles of different solvent fractions, identifying the dichloromethane (DCM) fraction as enriched in ginsenosides, particularly the ocotillol-type saponin majonoside R2 (MR2). In vitro assays showed that, despite minimal direct antibacterial activity, the DCM fraction at sub-inhibitory concentrations significantly reduced urease activity, acid tolerance, biofilm formation, and the expression of major virulence genes, including vacA and cagA. In H. pylori-infected AGS gastric epithelial cells, the DCM fraction and MR2 decreased VacA and CagA translocation, suppressed pro-inflammatory signaling and cytokine production, restored antioxidant defenses, and alleviated mitochondrial apoptosis. By contrast, ginsenoside Rg1 selectively modulated host inflammatory and oxidative stress responses without affecting bacterial virulence gene expression. These results demonstrate that Ngoc Linh ginseng root fractions mitigate H. pylori-induced pathogenic effects primarily through anti-virulence and host-directed mechanisms, highlighting their potential relevance for the development of gastric health-promoting functional products.

Helicobacter pylori↗
PubMedDate not supplied

Virulence factors of salmonella ser. enteritidis strains isolated in Italy from food-borne outbreaks.

Twelve Salmonella ser. Enteritidis strains phage type 4 isolated in Italy from different food-borne outbreaks were characterized for the expression of different virulence traits, for antibiotic resistance, and for plasmid DNA profile. All the twelve S. Enteritidis strains were able to invade and multiply within HeLa cell monolayers, even if at a lower efficiency if compared to an invasive Shigella flexneri strain. The strains were not hemolytic and produced only a moderate-level cytotoxic effect on HeLa cell monolayers. Moreover, all the strains examined produced mannose-sensitive hemagglutination with chicken erythrocytes but were not able to adhere to tissue culture cells. The strains did not produce the hydroxamate-type siderophore aerobactin or the specific ferric-aerobactin receptor. The S. Enteritidis strains were resistant only to spectinomycin, and eleven strains harbored a 38 MDa non-conjugative plasmid, while one strain harbored a 64 MDa conjugative plasmid which carried a colicinogenic activity-encoding locus. The uniformity of antibiotic resistance pattern, of the plasmid DNA content, and of the virulence factors produced indicated that the S. Enteritidis clinical isolates examined are clonally-related.

Journal Article↗
PubMed2006

Comparative genomics of host-specific virulence in Pseudomonas syringae.

While much study has gone into characterizing virulence factors that play a general role in disease, less work has been directed at identifying pathogen factors that act in a host-specific manner. Understanding these factors will help reveal the variety of mechanisms used by pathogens to suppress or avoid host defenses. We identified candidate Pseudomonas syringae host-specific virulence genes by searching for genes whose distribution among natural P. syringae isolates was statistically associated with hosts of isolation. We analyzed 91 strains isolated from 39 plant hosts by DNA microarray-based comparative genomic hybridization against an array containing 353 virulence-associated (VA) genes, including 53 type III secretion system effectors (T3SEs). We identified individual genes and gene profiles that were significantly associated with strains isolated from cauliflower, Chinese cabbage, soybean, rice, and tomato. We also identified specific horizontal gene acquisition events associated with host shifts by mapping the array data onto the core genome phylogeny of the species. This study provides the largest suite of candidate host-specificity factors from any pathogen, suggests that there are multiple ways in which P. syringae isolates can adapt to the same host, and provides insight into the evolutionary mechanisms underlying host adaptation.

Gene Expression Profiling↗
PubMed2006

Transcriptional profiling of a Staphylococcus aureus clinical isolate and its isogenic agr and sarA mutants reveals global differences in comparison to the laboratory strain RN6390.

The production of Staphylococcus aureus virulence factors is under the control of complex regulatory circuits. Most studies aimed at defining these regulatory networks have focused on derivatives of the strain NCTC 8325, most notably RN6390. However, all NCTC 8325 derivatives, including RN6390, possess an 11 bp deletion in rsbU. This deletion renders NCTC 8325 derivatives naturally sigma-factor-B deficient. Recent studies have shown that RN6390 is also deficient, in comparison to clinical isolates, with respect to biofilm formation, a process which is important for both pathogenesis and antimicrobial resistance. Based on these considerations, the authors carried out genome-scale transcriptional profiling, comparing RN6390 with the virulent rsbU-positive clinical isolate UAMS-1. The results revealed significant genome-wide differences in expression patterns between RN6390 and UAMS-1, and suggested that the overall transcriptional profile of UAMS-1 is geared toward expression of factors that promote colonization and biofilm formation. In contrast, the transcriptional profile of RN6390 was heavily influenced by RNAIII expression, resulting in a phenotype characterized by increased production of exoproteins, and decreased capacity to form a biofilm. The greater influence of agr in RN6390 relative to UAMS-1 was also evident when the transcriptional profile of UAMS-1 was compared with that of its isogenic sarA and agr mutants. Specifically, the results indicate that, in contrast to NCTC 8325 derivatives, agr plays a limited role in overall regulation of gene expression in UAMS-1, when compared with sarA. Furthermore, by defining the sarA regulon in a biofilm-positive clinical isolate, and comparing the results with transcriptional profiling experiments defining biofilm-associated gene expression patterns in the same strain, the authors identified a sarA-regulated operon (alsSD) that is also induced in biofilms, and demonstrated that mutation of alsSD results in reduced capacity to form a biofilm.

Adaptation, Physiological↗
PubMed1984

Diagnostic value of plasmid analyses and assays for virulence in Yersinia enterocolitica.

The possession of a 42- to 48-megadalton plasmid alone does not appear to be predictive of virulence in Yersinia species. Twelve of 100 Yersinia enterocolitica strains contained a 42 to 48-megadalton plasmid, and 4 of 30 Y. enterocolitica-like strains contained a 42- to 48-megadalton plasmid. Seven strains of Y. enterocolitica contained the 42- to 48-megadalton plasmid plus an 82-megadalton plasmid, and these were the only study strains lethal for mice. Based on restriction endonuclease digestion, the 42- to 48-megadalton plasmid DNA from these seven strains were similar and were not similar to the 42- to 48-megadalton plasmids present in the other nine strains. The ability to invade guinea pig eye tissues, calcium dependency, autoagglutination, and colonial morphology at 37 degrees C were also associated with plasmid DNA, but the relationships were either variable or not reciprocal. Neither tissue culture invasiveness nor heat-stable toxin production was associated with plasmid DNA. It was concluded that biochemical speciation and a total plasmid profile in combination with enzyme digests are predictive of virulence in Y. enterocolitica as it is measured by mouse lethality.

Animals↗
PubMed2006

The Escherichia coli BarA-UvrY two-component system is a virulence determinant in the urinary tract.

BACKGROUND: The Salmonella enterica BarA-SirA, the Erwinia carotovora ExpS-ExpA, the Vibrio cholerae BarA-VarA and the Pseudomonas spp GacS-GacA all belong to the same orthologous family of two-component systems as the Escherichia coli BarA-UvrY. In the first four species it has been demonstrated that disruption of this two-component system leads to a clear reduction in virulence of the bacteria. Our aim was to determine if the Escherichia coli BarA-UvrY two-component system is connected with virulence using a monkey cystitis model. RESULTS: Cystitis was generated in Macaque fascularis monkeys by infecting the bladder with a 1:1 mixture of the uropathogenic Escherichia coli isolate DS17 and a derivative where the uvrY gene had been disrupted with a kanamycin resistance gene. Urine was collected through bladder punctuation at subsequent time intervals and the relative amount of uvrY mutant was determined. This showed that inactivation of the UvrY response regulator leads to a reduced fitness. In similar competitions in culture flasks with Luria Broth (LB) the uvrY mutant rather had a higher fitness than the wild type. When the competitions were done in flasks with human urine the uvrY mutant initially had a lower fitness. This was followed by a fluctuation in the level of mutant in the long-term culture, with a pattern that was specific for the individual urines that were tested. Addition of LB to the different urine competition cultures however clearly led to a consistently higher fitness of the uvrY mutant. CONCLUSION: This paper demonstrates that the BarA-UvrY two-component system is a determinant for virulence in a monkey cystitis model. The observed competition profiles strengthen our previous hypothesis that disruption of the BarA-UvrY two-component system impairs the ability of the bacteria to switch between different carbon sources. The urine in the bladder contains several different carbon sources and its composition changes over time. Inability to efficiently switch between the carbon sources may thus provide an explanation to the reduced fitness of the uvrY mutant in the cystitis model.

Animals↗
PubMed1993

[Virulence of plasmids of diarrhea-causing strains of Salmonellae wien and Escherichia coli].

Use of molecular genetic methods in the study of sources and ways of spreading the infection provides precise data on the mutual clonal origin of the strains, on the basis of the determination of plasmid DNK maps. Results of the investigation of Salmonellae Wien strain show the same type of resistance to antimicrobial drugs and the impact of resistotypization as an epidemiologic marker. The analysis of the plasmid profile in these strains has confirmed the presence of FIme virulence plasmid of 80 megadalton in size and resistance plasmid of 60 megadalton which was by conjugation transferred to the recipient strain (in vitro experiment). The presence of antigen factors of colonization in escherichial coli, a cause of diarrhea, was confirmed: CFA-I in the serogroup 078, and CFA-II in the serogroup 06.

Antigens, Bacterial↗
PubMed2003

Mutation of luxS of Streptococcus pneumoniae affects virulence in a mouse model.

The LuxS protein is required for the biosynthesis of the type 2 autoinducer (AI-2), which is involved in quorum sensing in a wide range of bacterial species. We have determined the effects of a defined luxS mutation on the virulence of Streptococcus pneumoniae. Although the luxS mutant displayed reduced virulence relative to its wild-type parent, the type 2 strain D39, it was by no means avirulent in a mouse model. After intranasal administration, the luxS mutant was able to colonize the nasopharynx of the mouse as efficiently as the wild type. However, it was less able to spread from the nasopharynx to the lungs or the blood. Intraperitoneal coadministration studies indicated that the luxS mutant was less fit and was readily outcompeted by wild-type D39. However, when administered on its own by this route, the mutant was able to proliferate and cause fatal systemic disease, albeit at a lower rate than the wild type. Western blot analysis of whole-cell lysates of the mutant and its parent did not reveal any differences in the levels of several well-characterized virulence proteins. However, analysis of Coomassie blue-stained protein profiles after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that mutation of luxS had pleiotropic effects on protein expression in all cellular compartments. This is consistent with the product of luxS having a regulatory role in S. pneumoniae. This is the first report of a direct role for luxS (and by inference, AI-2) in the virulence of a gram-positive pathogen. However, the fact that mutagenesis of luxS does not completely attenuate S. pneumoniae has implications for the possible use of AI-2 antagonists for treatment of pneumococcal infections.

Animals↗