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PubMed1997

Whole-cell and periplasmic protein banding patterns of environmental and human Bacteroides fragilis strains.

In order to investigate the relationship among virulent and avirulent Bacteroides fragilis strains, SDS-PAGE of whole-cell proteins (WP) and periplasmic proteins (PP) were used to establish a protein profile of strains isolated from human infections, fecal flora and environmental water. Despite different sources of the strains, no significant differences were observed as determined by the WP SDS-PAGE analysis. In contrast, the proteins obtained from the bacterial periplasm showed differences in the electrophoretic protein profile. Two distinct PP profile patterns were obtained. Pattern A included 6 out of the 8 virulent strains and pattern B, 6 out of 8 avirulent strains. Interestingly, an environmental strain that was capable of inducing abscesses in mice, had a PP profile highly similar to that of the virulent strains from human infections. These data indicate that PP from B. fragilis may be useful to characterize differences among virulent and avirulent strains. Moreover, strains isolated from environmental water may also be a source of exogenous infections by B. fragilis.

Animals↗
PubMed1997

Comparison of field and vaccine strains of Australian fowlpox viruses.

The mild fowlpox vaccine, FPV M, widely used in Australia is composed of two predominant genotypes based upon differences identifiable in restriction enzyme analyses of plaque purified derivatives of this vaccine. The differences, where identifiable, were in the end fragments of the genomes. Five field isolates of FPV from chickens in New South Wales showed restriction enzyme profiles closely related to the more virulent (standard) vaccine strain, FPV S. The FPV S strain differs from FPV M in both terminal genome fragments and in the presence of a PstI fragment of approximately 10kb (this fragment was also present in PstI digests of all of the field isolates). Plaque purified derivatives of FPV M showed similar lesion development upon inoculation into the wing web of chickens. The field isolates showed significantly higher virulence in day-old and three-week-old chickens in comparison with FPV M. One field isolate was similar to the FPV S vaccine. Two isolates had slowly developing wing web lesions, caused significant secondary lesions in three-week-old chickens and generalised poxvirus infection when inoculated into day-old chickens. For two isolates, the primary wing web lesion took even longer to develop and resolve although these isolates did not cause generalised poxvirus infection. It was possible to identify four virulence/pathogenicity types amongst these vaccine and field isolates of FPV. These strains may allow the characterisation of FPV encoded virulence factors. The field strains with higher virulence may be suitable as parent strains for the construction of FPV recombinants with enhanced immune responses to co-expressed vaccine antigens when compared with current FPV M strain based recombinants.

Animals↗
PubMed2006

Effect of fish skin mucus on the soluble proteome of Vibrio salmonicida analysed by 2-D gel electrophoresis and tandem mass spectrometry.

Vibrio salmonicida is the causative agent of cold-water vibriosis in farmed marine fish species. Adherence of pathogenic bacteria to mucosal surfaces is considered to be the first steps in the infective processes, and proteins involved are regarded as virulence factors. The global protein expression profile of V. salmonicida, grown with and without the presence of fish skin mucus in the synthetic media, was compared. Increased levels of proteins involved in motility, oxidative stress responses, and general stress responses were demonstrated as an effect of growth in the presence of mucus compared to non-mucus containing media. Enhanced levels of the flagellar proteins FlaC, FlaD and FlaE indicate increased motility capacity, while enhanced levels of the heat shock protein DnaK and the chaperonin GroEL indicate a general stress response. In addition, we observed that peroxidases, TPx.Grx and AhpC, involved in the oxidative stress responses, were induced by mucus proteins. The addition of mucus to the culture medium did not significantly alter the growth rate of V. salmonicida. An analysis of mucus proteins suggests that the mucus layer harbours a protein species that potentially possesses catalytic activity against DNA, and a protein with iron chelating activity. This study represents the first V. salmonicida proteomic analysis, and provides specific insight into the proteins necessary for the bacteria to challenge the skin mucus barrier of the fish.

Aliivibrio salmonicida↗
PubMed1998

Inter- and intra-phage type differentiation of Salmonella enterica subsp. enterica serovar enteritidis isolates using molecular typing methods.

Seventy-six Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) isolates which represented 14 phage types and RDNC isolates were investigated by several independent molecular methods. Eleven different plasmid profiles were detected. Plasmid-encoded virulence genes of the spv gene cluster were shown by hybridization with a spvB/C gene probe to be located on plasmids of either 55 kbp or 95 kbp. Ribotyping as performed with gene probes that recognized either the entire rrn operon or 16S rRNA genes allowed no differentiation between the S. Enteritidis isolates. Three different hybridization patterns could be observed by IS200-typing. Macrorestriction analysis of XbaI-, SpeI- and NotI-digested whole cell DNA proved to be the most discriminatory method as confirmed by the calculation of discriminatory indices. Based on the data obtained from plasmid analysis and macrorestriction analyses with three suitable restriction endonucleases, the 76 S. Enteritidis isolates were assigned to 26 different genomic groups. The observation that several genomic groups included isolates of different phage types has suggested that a combination of suitable molecular methods and phage typing is most useful for tracing S. Enteritidis isolates in epidemiological investigations.

Animals↗
PubMed1993

Genetic variation and phylogenetic relationships among worldwide collections of the Russian wheat aphid, Diuraphis noxia (Mordvilko), inferred from allozyme and RAPD-PCR markers.

Genetic analyses were conducted on Diuraphis noxia (Mordvilko) populations collected from wheat, barley and other grasses from various countries throughout the world. These collections had been found to contain clones that differed in virulence from various cultivars, cuticular hydrocarbon profiles and life cycle characters. Discrete genetic markers analysed in this study included allozymes and arbitrary regions of the genome amplified by the polymerase chain reaction (RAPD-PCR). In all, 23 enzymes were evaluated; 17 of these enzymes representing 20 isozyme loci, were judged suitable for allozyme analysis. Polymorphisms were detected at three (15 per cent) loci: beta-esterase (beta-EST), phosphoglucose isomerase (PGI), and 6-phosphogluconate dehydrogenase (6-PGDH). The average expected heterozygosity amongst these loci was 4.9 per cent in the worldwide collection. Allozyme variation was absent within most populations, particularly within those countries where the species was recently introduced. Much greater genetic variation was detected when populations were analysed with RAPD-PCR. Populations were analysed with 69 polymorphic bands amplified by seven primers. All populations could be distinguished with this method. Cluster analyses indicated strong similarities between U.S.A. populations and collections from South Africa, Mexico, France and Turkey. The most variation was detected among populations from the Middle East and southern Russia.

Animals↗
PubMed1998

Heterogeneity of phenotypic and genotypic traits including organic-acid resistance in Escherichia coli O157 isolates.

We undertook an epidemiologic study for the sensitivity of both Shiga-like toxin (Slt)-producing Escherichia coli (STEC) O157 and non-STEC O157 strains isolated from different patients with diarrhea to hydrochloric acid (HCl) and organic acids such as acetate, propionate, butyrate and lactate, and other pathogenic factors. The E. coli O157 isolates examined showed a wide variety of organic-acid susceptibility patterns. E. coli O157 isolates resistant to HCl or acetate were found more frequently than those resistant to other organic acids. These isolates also showed diverse pathogenicity patterns for the presence of the virulence genes, antibiotic susceptibility and plasmid profile.

Acids, Acyclic↗
PubMed2006

Emergent properties of reduced-genome Escherichia coli.

With the use of synthetic biology, we reduced the Escherichia coli K-12 genome by making planned, precise deletions. The multiple-deletion series (MDS) strains, with genome reductions up to 15%, were designed by identifying nonessential genes and sequences for elimination, including recombinogenic or mobile DNA and cryptic virulence genes, while preserving good growth profiles and protein production. Genome reduction also led to unanticipated beneficial properties: high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Eradication of stress-induced transposition evidently stabilized the MDS genomes and provided some of the new properties.

DNA Transposable Elements↗
PubMed2026

Molecular epidemiology and antimicrobial resistance of human Streptococcus suis isolates in Guangxi, China, 2015-2021.

BACKGROUND: Streptococcus suis (S. suis) is an important zoonotic pathogen and a common colonizer of the upper respiratory tract of pigs. Human infections have been reported in several regions of China, including Guangxi, but genomic and antimicrobial resistance data from this region remain limited. This study investigated the molecular epidemiology, antimicrobial susceptibility, and genomic characteristics of human S. suis isolates collected in Baise City, Guangxi, from 2015 to 2021. METHODS: This retrospective study included 39 non-duplicate clinical isolates confirmed as S. suis by whole-genome analysis. Antimicrobial susceptibility testing was performed using a broth microdilution-based system and interpreted according to the Clinical and Laboratory Standards Institute guidelines. Serotypes were determined by agglutination using type-specific antisera. Whole-genome sequencing was used for species confirmation, multilocus sequence typing, detection of antimicrobial resistance and virulence-associated genes, and core-protein phylogenetic analysis. RESULTS: The median patient age was 55 years, and 36/39 (92.3%) patients were male. Meningitis was documented in 34/39 (87.2%) patients, and hearing impairment occurred in 21/39 (53.8%). Pig- or pork-related exposure was recorded in 15/39 (38.5%) patients. Resistance was highest to tetracycline (38/39, 97.4%), followed by erythromycin and clindamycin (26/39, 66.7% each). Four isolates (10.3%) showed intermediate susceptibility to penicillin, but none were resistant. All isolates remained susceptible to ampicillin, ceftriaxone, levofloxacin, linezolid, vancomycin, and meropenem. Serotype 2 predominated (32/39, 82.1%), followed by serotype 14 (7/39, 17.9%), while ST1 (29/39, 74.4%) and ST7 (7/39, 17.9%) were the two major sequence types. Resistance genes were mainly associated with tetracyclines, macrolides, lincosamides, and aminoglycosides. All ST1 isolates carried mrp and lacked tet(40), while all ST7 isolates showed the reverse pattern. CONCLUSION: Serotype 2 and ST1 predominated among the human S. suis isolates collected at this center. Resistance to tetracycline, erythromycin, and clindamycin was common, while susceptibility to the β-lactams tested was largely preserved. Differences in virulence- and resistance-associated gene profiles were also observed between the major lineages, indicating distinct genetic characteristics among the locally circulating isolates.

Streptococcus suis↗
PubMed2005

An immune response directed to proteinase and adhesin functional epitopes protects against Porphyromonas gingivalis-induced periodontal bone loss.

Porphyromonas gingivalis, a pathogen associated with periodontitis, bound to fibrinogen, fibronectin, hemoglobin, and collagen type V with a similar profile to that of its major virulence factor, the cell surface RgpA-Kgp proteinase-adhesin complex. Using peptide-specific, purified Abs in competitive inhibition ELISAs and epitope mapping assays, we have identified potential adhesin binding motifs (ABMs) of the RgpA-Kgp complex responsible for binding to host proteins. The RgpA-Kgp complex and synthetic ABM and proteinase active site peptides conjugated to diphtheria toxoid, when used as vaccines, protected against P. gingivalis-induced periodontal bone loss in the murine periodontitis model. The most efficacious peptide and protein vaccines were found to induce a high-titer IgG1 Ab response. Furthermore, mice protected in the lesion and periodontitis models had a predominant P. gingivalis-specific IL-4 response, whereas mice with disease had a predominant IFN-gamma response. The peptide-specific Abs directed to the ABM2 sequence (EGLATATTFEEDGVA) protected against periodontal bone loss and inhibited binding of the RgpA-Kgp complex to fibrinogen, fibronectin, and collagen type V. Furthermore, the peptide-specific Abs directed to the ABM3 sequence (GTPNPNPNPNPNPNPGT) protected against periodontal bone loss and inhibited binding to hemoglobin. However, the most protective Abs were those directed to the active sites of the RgpA and Kgp proteinases. The results suggest that when the RgpA-Kgp complex, or functional binding motif or active site peptides are used as a vaccine, they induce a Th2 response that blocks function of the RgpA-Kgp complex and protects against periodontal bone loss.

Adhesins, Bacterial↗
PubMedDate not supplied

Comparative study of different methods for detection of toxic and other enzymatic factors in Vibrio cholerae strains.

The purpose of this work was to characterize the toxin profile and the presence of other virulence factors involved in the pathogenesis and biology of 13 V. cholerae O1 (11 clinical cases and 2 waters) and 6 V. cholerae non O1 strains (4 clinical cases and 2 waters) using genetic (PCR), immunological (RPLA), biochemical (NAD degradation, haemolysis, Kanagawa phenomenon, caseinase, lecithinase, mucinase, amylase, esculine hydrolysis) and cell culture (Vero E6, HEp-2) assays. The results indicated a concordance between PCR-RPLA (84%), PCR-NAD (73%) and RPLA-NAD (84%) methods. The sensitivity of RPLA and NAD degradation methods were comparable to PCR in detecting CT in Vibrio cholerae O1 strains. Although NAD degradation method was not exclusively specific for the CT detection, it proved its usefulness in screening certain virulent, CT-negative clones of V. cholerae. The cytotoxic effect on Vero E6 cells, enzyme production (Kanagawa haemolysins, lecithinase, caseinase, esculine hydrolysis) as well as adherence ability on inert substrate proved to be much more constant in V. cholerae non O1 (CT- negative) than in V. cholerae O1 (CT-positive). All V. cholerae non O1 strains isolated in diarrheal cases were Kanagawa positive. This complex of virulence factors detected in V. cholerae non O1 strains could probably contribute during interepidemic periods to human-to-human transmission and to greater resistance as compared to O1 strains in the environment.

Animals↗
PubMed1989

Virulence of Escherichia coli strains in relation to their hemolysin formation, mannose-resistant hemagglutination, hydroxamate production, K 1-antigen and the plasmid profile in patients with chronic pyelonephritis.

Microorganisms initiating infections of the kidneys and the upper urinary tract are characterized by specific properties. In 96 patients with non-obstructive chronic pyelonephritis, Escherichia coli strains were isolated and their virulence properties studied. Most frequently hemolysin formation and mannose-resistant hemagglutination (n = 39/40%), less frequently the ability to produce hydroxamate/aerobactin (n = 18/19%) and the K 1-antigen (n = 13/14%) were detected. In 34 E. coli strains (35%) two and more virulence factors were found. A statistically significant correlation exists between mannose-resistant hemagglutination (evidence of P- and F7-F14 fimbriae) and hemolysin formation on the one hand and hydroxamate production and K 1-antigen on the other (confidence intervals are 0.56-0.75 and 0.75-0.89, respectively). Disease activity and pathogenicity of E. coli strains showed a statistically significant association (chi 2-value, DF 1: hemolysin 29.5, mannose-resistant hemagglutination 17.1, K 1-antigen 4.8, hydroxamate 0.5). The analysis of the plasmid profile yielded species in molecular weight range from 0-170 Md. A correlation between single virulence features and certain plasmid combinations failed to be demonstrated. The results revealed a close connection between certain virulence properties of E. coli strains and a specific uropathogenicity. The clinical significance of this observation should be further elucidated.

Adolescent↗
PubMed2005

Genetic analysis of enteropathogenic and enterohemorrhagic Escherichia coli serogroup O103 strains by molecular typing of virulence and housekeeping genes and pulsed-field gel electrophoresis.

We investigated the genetic relationships of 54 Escherichia coli O103 strains from humans, animals, and meat by molecular typing of housekeeping and virulence genes and by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) of seven housekeeping genes revealed seven profiles, I through VII. MLST profiles I plus III cover 45 Shiga toxin-producing E. coli (STEC) O103:H2 strains from Australia, Canada, France, Germany, and Northern Ireland that are characterized by the intimin (eae) epsilon gene and carry enterohemorrhagic E. coli (EHEC) virulence plasmids. MLST profile II groups five human and animal enteropathogenic E. coli (EPEC) O103:H2 strains that were positive for intimin (eae) beta. Although strains belonging to MLST groups II and I plus III are closely related to each other (92.6% identity), major differences were found in the housekeeping icdA gene and in the virulence-associated genes eae and escD. E. coli O103 strains with MLST patterns IV to VII are genetically distant from MLST I, II, and III strains, as are the non-O103 E. coli strains EDL933 (O157), MG1655 (K-12), and CFT073 (O6). Comparison of MLST results with those of PFGE and virulence typing demonstrated that E. coli O103 STEC and EPEC have recently acquired different virulence genes and DNA rearrangements, causing alterations in their PFGE patterns. PFGE typing was very useful for identification of genetically closely related subgroups among MLST I strains, such as Stx2-producing STEC O103 strains from patients with hemolytic uremic syndrome. Analysis of virulence genes contributed to grouping of E. coli O103 strains into EPEC and STEC. Novel virulence markers, such as efa (EHEC factor for adherence), paa (porcine adherence factor), and cif (cell cycle-inhibiting factor), were found widely associated with E. coli O103 EPEC and STEC strains.

Adhesins, Bacterial↗
PubMed2006

Design of a seven-genome Escherichia coli microarray for comparative genomic profiling.

We describe the design and evaluate the use of a high-density oligonucleotide microarray covering seven sequenced Escherichia coli genomes in addition to several sequenced E. coli plasmids, bacteriophages, pathogenicity islands, and virulence genes. Its utility is demonstrated for comparative genomic profiling of two unsequenced strains, O175:H16 D1 and O157:H7 3538 (Deltastx(2)::cat) as well as two well-known control strains, K-12 W3110 and O157:H7 EDL933. By using fluorescently labeled genomic DNA to query the microarrays and subsequently analyze common virulence genes and phage elements and perform whole-genome comparisons, we observed that O175:H16 D1 is a K-12-like strain and confirmed that its phi3538 (Deltastx(2)::cat) phage element originated from the E. coli 3538 (Deltastx(2)::cat) strain, with which it shares a substantial proportion of phage elements. Moreover, a number of genes involved in DNA transfer and recombination was identified in both new strains, providing a likely explanation for their capability to transfer phi3538 (Deltastx(2)::cat) between them. Analyses of control samples demonstrated that results using our custom-designed microarray were representative of the true biology, e.g., by confirming the presence of all known chromosomal phage elements as well as 98.8 and 97.7% of queried chromosomal genes for the two control strains. Finally, we demonstrate that use of spatial information, in terms of the physical chromosomal locations of probes, improves the analysis.

Coliphages↗
PubMed2005

Iron salts perturb biofilm formation and disrupt existing biofilms of Pseudomonas aeruginosa.

Bacterial biofilms are thought to aid in the survivability of a variety of intractable infections in humans. Specifically, biofilm production in Pseudomonas aeruginosa has been shown to play a significant role in chronic infection of cystic fibrosis (CF) patients. Unfortunately, no clinically effective inhibitors of biofilm formation are available. A rapid screen of 4509 compounds for nonantibiotic biofilm inhibitors in Pseudomonas aeruginosa PA14 was executed in 384-well plates. Among those compounds, ferric ammonium citrate inhibited biofilm formation in a dose-dependent manner; other iron salts functioned similarly. In addition to biofilm inhibition in static culture, pregrown biofilms could be disrupted and cleared by switching to iron-rich media in flow-chamber experiments. Furthermore, P. aeruginosa strains taken from the sputum of 20 CF patients showed a similar response to elevated iron levels. Previous expression-profiling analyses demonstrated that high levels of iron repress the expression of genes whose products are essential for scavenging iron and that expression of these genes is critical for virulence. Our results, combined with existing transcriptional-profiling data, now indicate that elevated iron concentrations repress the expression of certain genes essential for biofilm production in P. aeruginosa.

Biofilms↗
PubMed2000

Molecular analysis of the accessory gene regulator (agr) locus and balance of virulence factor expression in epidemic methicillin-resistant Staphylococcus aureus.

Potential relationships between virulence factor expression and transmissibility were assessed in epidemic methicillin-resistant Staphylococcus aureus (MRSA) clones CMRSA-1 and CMRSA-3. A major subtype of CMRSA-1 exhibited normal transcription of RNAIII, which facilitates the induction of secreted virulence factors and repression of colonization factor expression at high cell density. However, these isolates characteristically did not express alpha-toxin or protease and displayed a limited profile of secreted proteins. CMRSA-1 also expressed a novel cell surface glycoprotein and exhibited a unique polymorphism within the accessory gene regulator (agr) locus. CMRSA-3 displayed attenuated activation of RNAIII transcription, which was consistent with its higher fibronectin-binding and coagulase activity relative to sporadic MRSA or CMRSA-1 (P=.05), low protease activity, and limited profile of secreted proteins. Thus, the balance of virulence factor expression in CMRSA-1 and CMRSA-3 favors the colonization phase of infection, and CMRSA-1 possesses unique genotypic and phenotypic traits.

Bacterial Proteins↗
PubMed1991

Plasmid associated virulence properties of environmental isolates of Aeromonas hydrophila.

The plasmid profiles, and their association with antimicrobial resistance, of 60 strains of Aeromonas hydrophila isolated from fish, shellfish and water were investigated. Only two strains were susceptible to all the antimicrobial agents tested; the highest incidences of resistance were to tetracycline (96.7%), prystanamycin (93.3%), ampicillin (91.7%) and cephalothin (91.7%). Forty strains harboured one or more plasmids and the plasmid profile most frequently detected (15%) was the association of three small plasmids of 4.2, 3.2 and 2.8 Mda. Curing experiments indicated that the plasmid-free derivative strains simultaneously lost their resistance determinants to tobramycin, neomycin, gentamicin and kanamycin. More than 90% of the strains tested produced siderophores and displayed haemolytic activity. However, the relationship between these virulence characters and the presence of plasmids was different; in 74.5% of the strains there was siderophore production and plasmids were detectable, whereas only 60% of the strains simultaneously possessed plasmids and haemolytic activity.

Aeromonas hydrophila↗
PubMed2006

Comparative proteomic profiles and the potential markers between Burkholderia pseudomallei and Burkholderia thailandensis.

Burkholderia pseudomallei is a bacterial pathogen causing the melioidosis disease, which is predominantly found in tropical areas of Southeast Asia and Northern Australia. Burkholderia thailandensis is a closely related species to B. pseudomallei but it is non-pathogenic species. In this study, we have constructed a proteome reference map of B. pseudomallei at the stationary phase of growth by using two-dimensional gel electrophoresis with a pH 4-7 immobilized pH gradient combined with matrix-assisted laser desorption ionization time of flight mass spectrometry. Approximately 550 spots could be detected by Coomassie brilliant blue G-250 staining, and 88 spots representing 77 unique proteins were identified. Eleven of the gene products were found in multiple spots indicating as isoforms. In attempt to detect distinctive expressed proteins between a virulent and a non-virulent species, the use of comparative proteomic profiles under the same condition were performed. We could identify more than 20 different spots. Twelve out of 14 spots are detected in B. pseudomallei and six proteins have been identified and indicated that they are involved in virulent characters of bacteria. Two hypothetical proteins were expressed and found only in B. pseudomallei. These proteins are potential markers to distinguish between these two species. Our study also provides a useful information of global intracellular protein expression and is a valuable starting point for analyzing a proteomic pathogenicity of the bacterial pathogen.

Amino Acid Sequence↗
PubMed2006

Haemophilus somnus (Histophilus somni) in bighorn sheep.

Respiratory disease and poor lamb recruitment have been identified as limiting factors for bighorn-sheep populations. Haemophilus somnus (recently reclassified as Histophilus somni) is associated with respiratory disease in American bison, domestic sheep, and cattle. It is also harbored in their reproductive tracts and has been associated with reproductive failure in domestic sheep and cattle. Therefore, reproductive tract and lung samples from bighorn sheep were evaluated for the presence of this organism. Organisms identified as H. somnus were isolated from 6 of 62 vaginal but none of 12 preputial swab samples. Antigen specific to H. somnus was detected by immunohistochemical study in 4 of 12 formalin-fixed lung tissue samples of bighorn sheep that died with evidence of pneumonia. Notably, H. somnus was found in alveolar debris in areas of inflammation. The 6 vaginal isolates and 2 H. somnus isolates previously cultured from pneumonic lungs of bighorn sheep were compared with 3 representative isolates from domestic sheep and 2 from cattle. The profiles of major outer membrane proteins and antigens for all of the isolates were predominantly similar, although differences that may be associated with the host-parasite relationship and virulence were detected. The DNA restriction fragment length profiles of the bighorn-sheep isolates had similarities not shared with the other isolates, suggesting distinct phylogenetic lines. All of the isolates had similar antimicrobial profiles, but the isolates from the bighorn sheep produced less pigment than those from the domestic livestock, and growth of the former was not enhanced by CO2. Wildlife biologists and diagnosticians should be aware of the potential of these organisms to cause disease in bighorn sheep and of growth characteristics that may hinder laboratory detection.

Animals↗