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Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA.

Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1+/2+) and avirulent (pXO1+/2-, pXO1-/2+ and pXO1-/2-) strains of B. anthracis and distinguished "anthrax-like' strains from other B. cereus group bacteria.

Bacillus anthracis↗

Identification of virulence associated markers in the cell wall of pigeon Streptococcus gallolyticus strains.

The cell wall protein profiles of 56 isolates of Streptococcus gallolyticus of differing virulence for pigeons were compared by SDS-PAGE. Additionally, Western blot analysis was performed on the cell wall proteins of 14 strains using sera of pigeons, experimentally infected with A(+)T1 or A(-)T2 strains of S. gallolyticus. The profile of silver stained gels exhibited a complex array of 20-50 bands ranging from less than 6.5-210kDa. A band with molecular mass of 114kDa was only observed in isolates that belonged to the highly virulent A(+)T1, A(+)T2, A(+)T3 and A(-)T1 culture supernatant groups. A band with a slightly higher molecular mass (115kDa) as well as a 207kDa band were only detected in isolates that belonged to the moderately A(-)T3 or low A(-)T2 virulent culture supernatant groups. The 114 and 115kDa band were recognised by all homologous and heterologous pigeon sera used whereas the 207kDa band was only recognised by sera of pigeons infected with a A(-)T2 strain. These findings may indicate that the 114, 115 and 207kDa bands are useful as additional virulence associated markers for pigeon S. gallolyticus strains.

Animals↗

Lipid composition of Paracoccidioides brasiliensis: possible correlation with virulence of different strains.

The lipid content and composition of four strains of Paracoccidioides brasiliensis were analysed to determine any possible correlation with their virulence for hamsters and mice. Two strains, Pb168 and Pb141, were equal in virulence, Pb9 was slightly virulent and Pb140 was avirulent under the experimental conditions. No correlation was observed between virulence and the total lipid or phospholipid content of the strains. The lipid yield was highest in Pb9 and lowest in Pb168. Polar lipids were highest in Pb9 and least in Pb140. Phosphatidylcholine was the dominant phospholipid in all strains but its percentage was lower in the avirulent strain Pb140. Diphosphatidylglycerol, the least saturated lipid in all strains, was less abundant in Pb140 than in the virulent strains Pb168 and Pb141. In all four strains, neutral lipids constituted the major fraction of total lipids and triglycerides were the predominant individual lipid class, being more abundant in the avirulent and slightly virulent strains that in the virulent strains. The fatty acid profiles of total lipids and individual lipid classes of neutral and polar lipids obtained from the four strains were similar; however, the individual lipid classes showed patterns of preferential distribution of these fatty acids.

Animals↗

Patterns of virulence gene expression differ between biofilm and tissue communities of Streptococcus pyogenes.

The ability of Streptococcus pyogenes to form biofilm-like bacterial communities during infection of soft tissue has suggested that the capacity to produce biofilm may be important for pathogenesis. To examine this relationship, a panel of mutants was evaluated for their ability to form biofilm on abiotic surfaces in several assays. Several established virulence factors were crucial for biofilm formation, including the M protein, required for initial cell-surface interactions, and the hyaluronic acid capsule, required for subsequent maturation into a three-dimensional structure. Mutants lacking the transcription regulators Mga and CovR (CsrR) also failed to form biofilm. Comparison of transcriptional profiles revealed differential regulation of approximately 25% of the genome upon adaptation to biofilm. During infection of zebrafish, several virulence factors (notably cysteine protease and streptokinase) were regulated in a biofilm-like manner. However, the overall profile of virulence factor expression indicated that tissue communities have a pattern of gene expression different from biofilm. Taken together, these data show that while biofilm and tissue communities have many characteristics in common, that biofilm reproduces only a subset of the myriad cues used by tissue communities for regulation of virulence.

Animals↗

Characterization of Salmonella enteritidis strains.

A study was conducted to characterize 318 Salmonella enteritidis strains that were mainly isolated from poultry and their environment in Canada. Biotype, phagetype (PT), plasmid profile (PP), hybridization with a plasmid-derived virulence sequence probe, antibiotic resistance, outer membrane proteins (OMPs), and lipopolysaccharide (LPS) profiles were determined. Relationships of these properties to one another, and their diagnostic and pathogenic significance were assessed. Biotyping indicated that failure to ferment rhamnose was sometimes useful as a marker for epidemiologically related strains. Phagetyping was the most effective method for subdividing S. enteritidis; it distinguished 12 PTs. Phagetype 13 was occasionally associated with septicemia and mortality in chickens. The strains belonged to 15 PPs. A 36 megadalton (MDa) plasmid was found in 97% of the strains. Only the 36 MDa plasmid hybridized with the probe. Seventeen percent of the strains were drug resistant; all strains were sensitive to ciprofloxacin. Thirty-five of 36 strains possessed the same OMP profile, and 36 of 41 strains contained smooth LPS.

Animals↗

Immunological analysis of extracellular products and cell surface components of motile Aeromonas isolated from fish.

The present work describes the characterization of antigens present in the extracellular products (ECP) and cell wall of strains of motile Aeromonas isolated from rainbow trout culture systems. The relationships among virulence for fish, O-serogroup and profile of LPS were also examined. The slide agglutination test showed that most of the virulent strains of motile Aeromonas (72%) were included in the serotypes O3, O6, O11 and O19 (Guinée and Jansen System). However, there were also non-pathogenic strains within these groups. Electrophoretic analysis of lipopolysaccharides (LPS) and proteins from cell envelope and ECP showed heterogeneity not only among the different serogroups but also within the same serotype. Immunoblot assays of cell envelope components, and of LPS present in the ECP demonstrated a close relationship among Aeromonas strains from the same serotype, while strains from different serotypes were not immunologically related. Moreover, this assay showed that motile Aeromonas belonging to distinct serotypes produced extracellular proteins immunologically related. On the other hand, antigenic cross reactivity was observed between the LPS obtained from cell envelope and those obtained from the ECP. The present results point out the need to include strains representative of each of the serotypes which predominates in a particular area and their ECPs in the formation of vaccines against motile Aeromonas septicaemia.

Aeromonas↗

Whole-Genome Sequencing Reveals Virulence and Antimicrobial Resistance Determinants of Lactococcus garvieae Causing Lactococcosis in Cage-Cultured Nile Tilapia (Oreochromis niloticus) in Thailand.

Lactococcosis is an important bacterial disease affecting farmed fish worldwide and is primarily associated with Lactococcus garvieae, Lactococcus petauri, and Lactococcus formosensis. In Thailand, information on L. garvieae infection in tilapia remains limited, particularly regarding genome-based identification, virulence determinants, and antimicrobial resistance profiles. This study characterized two L. garvieae isolates, AAHM-LG2501 and AAHM-LG2509, recovered from a lactococcosis outbreak in cage-cultured Nile tilapia (Oreochromis niloticus) in Ubon Ratchathani province, Thailand. Both isolates exhibited typical phenotypic characteristics of L. garvieae, including Gram-positive cocci, alpha hemolysis, positive capsule staining, and positive carbohydrate fermentation. Whole-genome sequencing confirmed both isolates as L. garvieae, with genome sizes of approximately 1.95 Mb and a G + C content of 38.9%. Genome-based taxonomic analysis supported species identification based on dDDH and ANI values, and both isolates were assigned to sequence type ST95 and serotype I. Virulence factor analysis identified 288 virulence-associated genes representing 97 virulence factors across 14 functional categories. Capsule-associated genes were prominent, together with genes involved in heme uptake, adhesion, hemolysis, stress survival, biofilm formation, and host adaptation. Ten capsule biosynthesis genes, including cpsABCFGKO, cps4A, and cps4I, as well as LPxTG cell wall anchor protein genes, were detected. Antimicrobial susceptibility testing showed resistance to nalidixic acid, oxolinic acid, and oxacillin, while reduced inhibition zones were observed for enrofloxacin and sulfamethoxazole-trimethoprim. Genome analysis identified predicted antimicrobial resistance determinants, including lsaD, vanT, vanY, and mdtA. Resistance-associated protein variants were detected in gyrA and gyrB, suggesting that target alteration may contribute to fluoroquinolone resistance. Overall, this study provides genome-level evidence of virulence and antimicrobial resistance determinants in L. garvieae from Thai tilapia and highlights the importance of whole-genome sequencing for accurate diagnosis, epidemiological surveillance, and disease management in aquaculture.

Animals↗

Virulence factors and genetic relatedness of Escherichia coli strains isolated from pigs with post-weaning diarrhea.

Forty-six Escherichia coli strains isolated from post-weaning diarrhea of pigs were analysed for their phenotypic and genotypic properties. The isolates were of serogroups O138, O139, and O141 and most of them possessed hemolytic activities. PCR analysis showed that 34 of the isolates harboured the genes for shiga toxin 2e and 32 strains possessed the genes for heat-stable enterotoxins I and II. Ten strains had the fedA gene of F18 fimbriae. The genetic relationships among all isolates were tested by random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) PCR analyses. Using the RAPD test with two different primers, six fingerprints were distinguished whereas the ERIC analysis revealed only three DNA patterns. Some strains possessing identical phenotypic and genotypic virulence determinants exhibited distinct RAPD profiles and some isolates with different pathogenic markers showed the same RAPD and ERIC pictures. Thus, RAPD, and to a less extent ERIC techniques, revealed intra- and interserogroup genotypic variations among the E. coli strains analyzed.

Agglutination Tests↗

Density profile of group B streptococci, type III, and its possible relation to enhanced virulence.

The buoyant densities of virulent and colonizing group B streptococci, type III, were determined by centrifugation of bacteria on a linear, hypotonic density gradient. A total of 28 strains were investigated. Eleven strains were obtained from blood cultures of babies with early-onset disease, and eight strains were isolated from the cerebrospinal fluid of babies with late-onset septicemia and meningitis. Nine colonizing strains were genital isolates from pregnant women subsequently giving birth to healthy children. In each strain the buoyant density was determined before and after neuraminidase treatment. All strains showed an increase in the buoyant density after enzymatic removal of sialic acid, and the density differences before and after desialylation were calculated. The mean values of these differences for blood, cerebrospinal fluid, and colonizing isolates were 23.4, 25.3, and 10.6 mg/ml, respectively. The mean value for the colonizing strains differed significantly from the mean value for each group of virulent strains. All colonizing strains banded singly in the gradient, whereas five of the virulent strains divided into two density populations. Extracts of the low-density cells produced markedly more dense immunoprecipitates with type antiserum than did extracts of the high-density bacteria. One double-banding strain was positive for R protein. After separation of the two density populations, this antigen was detected only in the low-density population. The results indicate that bacterial buoyant density is inversely related to the amount of capsular polysaccharide enveloping the cell and that a determination of the density profile of the bacteria may be used for discriminating strains with an increased pathogenic potential.

Antigens, Bacterial↗

Ribosomal ribonucleic acid synthesis by virulent Treponema pallidum.

Ribosomal ribonucleic acid (rRNA) synthesis by virulent Treponema pallidum was monitored by incorporation of [(3)H]uridine into trichloroacetic acid-precipitable counts and examination of radiolabeled rRNA on polyacrylamide gels. Verification that rRNA synthesis originated with T. pallidum was based upon co-electrophoresis with Escherichia coli rRNA, proportionate reductions in the amount of rRNA synthesized when numbers of treponemes were decreased, and inclusion of appropriate animal cell controls. The rate of treponemal rRNA synthesis was greater at temperatures of 37 and 39 degrees C than at 33 degrees C; rRNA synthesis was inhibited at 4 and 42 degrees C and was effectively inhibited by actinomycin D. Kinetic experiments indicated that the majority of rRNA synthesis occurred early after extraction of treponemes from infected rabbit testicular tissue. Polyacrylamide gel profiles demonstrated the capacity of virulent T. pallidum to synthesize and process RNA to 23s, 16s, and 4 to 5s classes. Although motility of T. pallidum appeared unaffected during longer periods of incubation, pulselabeling experiments confirmed significant reductions in the rate of rRNA synthesis. When the effect of various environmental conditions upon rRNA synthesis was investigated, optimal synthesis was found to occur in an atmosphere of 20% oxygen whereas virtually no synthesis was observed under anaerobic or low-oxygen conditions.

Animals↗

Cryptococcus neoformans methionine synthase: expression analysis and requirement for virulence.

This paper describes (i) the expression profile of the methionine synthase gene (MET6) in the human pathogenic fungus Cryptococcus neoformans and (ii) the phenotypes of a C. neoformans met6 mutant. In contrast to the MET3 gene, which showed no significant change in expression in any environmental condition tested, the MET6 gene showed a substantial induction in response to methionine and a dramatic transcriptional induction in response to homocysteine. Like a met3 mutant, the met6 mutant was a methionine auxotroph. However, relative to a met3 mutant, the met6 mutant grew very slowly and was less heat-shock resistant. In contrast to a met3 mutant, the met6 mutant lost viability when starved of methionine, and it was deficient in capsule formation. Like a met3 mutant, the met6 mutant was avirulent. In contrast to a met3 mutant, the met6 mutant was hypersensitive to fluconazole and to the calcineurin inhibitors FK506 and cyclosporin A. A synergistic fungicidal effect was also found between each of these drugs and met6. The phenotypic differences between the met3 and met6 mutants may be due to the accumulation in met6 mutants of homocysteine, a toxic metabolic intermediate that inhibits sterol biosynthesis.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

Schizodeme analysis of Trypanosoma cruzi Colombian strain clones isolated from the acute phase of murine infection.

Colombian strain of Trypanosoma cruzi, biodeme Type III (T. cruzi I), has been cloned by micromanipulation at two phases of the acute infection: early (10 days ) and advanced (30 days). Twelve clones were obtained therefrom. Characterization by their biological and biochemical behavior showed an identity among the several clones and their parental strain, albeit with different degrees of virulence. Molecular characterization of the kinetoplast DNA (kDNA) after amplification by polymerase chain reaction revealed identical profiles of the bands from the kDNA minicircle by the analysis of restriction fragment length polymorphism for the isolated clones, their parental strain, and to the clones isolated at two different phases of the infection. Results suggest the predominance of a "principal clone", in the composition of the Colombian strain, responsible for the biological and biochemical behavior. However, no relationship was detected between the molecular profile of kDNA and the degree of virulence presented by the several clones.

Acute Disease↗

Association between a large plasmid and 15- to 17-kilodalton antigens in virulent Rhodococcus equi.

Rhodococcus equi strains showing 15- to 17-kDa antigens in immunoblots were found to be virulent in mice. To study the genes specific to these antigens in virulent R. equi, we compared plasmid profiles, immunoblot profiles, and murine pathogenicity profiles of 10 strains of R. equi. All the strains showing 15- to 17-kDa antigens contained a large plasmid of approximately 85 kbp and were virulent in mice; however, the remaining strains lacked both the antigens and the large plasmid and were avirulent in mice. Mutants of virulent strains ATCC 33701 and L1, which were cured of the large plasmid by repeated passage at 38 degrees C, lacked the 15- to 17-kDa antigens and showed a dramatic decrease in lethality in mice. These results suggested that the presence of an 85-kbp plasmid may be essential for virulence and expression of 15- to 17-kDa antigens of R. equi and offered support for earlier observations that freshly isolated strains of R. equi killed mice, whereas laboratory-adapted strains did not.

Actinomycetales Infections↗

Aeromonas hydrophila strains with moderate virulence.

A group of Aeromonas hydrophila strains was obtained from very different origins which showed very similar characteristics (lipopolysaccharide, outer-membrane protein profile, toxin production, bacteriophage sensitivity and moderate virulence), besides the heterogenicity observed among the A. hydrophila strains. This group of A. hydrophila strains with moderate virulence can be easily recognized by their bacteriophage sensitivity pattern.

Aeromonas↗

Prevalence and virulence properties of Vibrio cholerae non-O1, Aeromonas spp. and Plesiomonas shigelloides isolated from Cambé Stream (State of Paraná, Brazil).

The incidence of Vibrio cholerae, Aeromonas spp. and Plesiomonas shigelloides was determined in water samples from Cambé Stream. The samples were collected from seven different sites. The serogroups, virulence markers and drug resistance profiles were also evaluated. Twelve Aer. hydrophila, 12Aer. caviae, eight Aer. sobria, seven Ple. shigelloides and two V. cholerae non-O1 were isolated. They belonged to different serogroups and all produced haemolysis in different assays. Five of the Aeromonas strains and one of V cholerae non-O1 were positive for enterotoxin activity. Haemagglutination and its inhibition, using erythrocytes of different origins, was variable for Aeromonas spp. and V. cholerae, while none of the Ple. shigelloides haemagglutinated in association with any type of erythrocyte. All isolates exhibited multiple drug resistance. These results indicate that the occurrence of V. cholerae non-O1, Aeromonas spp. and Ple. shigelloides, in water used for vegetable irrigation, human recreation and animal consumption, among others, represents a potential risk for humans.

Aeromonas↗

Genomic and virulence characteristics of Staphylococcus aureus isolates from foodborne outbreak cases.

This study aimed to investigate the genomic characteristics, enterotoxin production, and antimicrobial resistance profiles of Staphylococcus aureus isolates associated with foodborne outbreaks. A total of 19 bacterial isolates were collected from foodborne outbreaks in Guizhou Province, China between 2014 and 2023. Following biochemical identification, all isolates were confirmed as S. aureus. Phylogenetic analysis divided the 19 strains into seven branches. Enterotoxin production was detected using standard microbiological techniques and immunoassays. Antimicrobial susceptibility was evaluated using the broth microdilution method. Whole-genome sequencing and subsequent bioinformatic analyses were conducted to characterize virulence genes, antimicrobial resistance genes, multilocus sequence typing (MLST) genotypes, and phylogenetic relationships among the isolates. This study found that all strains produced classical staphylococcal enterotoxins, with staphylococcal enterotoxin (SEA) showing the highest detection rate (63.16%). Virulence gene profiling revealed widespread presence of hlb, hlgA, nuc, clfB, spa, and set genes. All strains were resistant to penicillin, with high resistance rates for erythromycin and cefoxitin. Multidrug resistance occurred in 11 of the 19 strains, and 22 resistance genes were identified. MLST analysis showed that ST6 and ST59 were the dominant types, with ST59 methicillin-resistant S. aureus (MRSA) strains displaying stronger resistance and more virulence determinants. These findings provide insights into the virulence, resistance, and molecular epidemiology of S. aureus strains involved in foodborne outbreaks, and may provide useful information for future surveillance and risk assessment.

Staphylococcus aureus↗

Transcriptional profiling of target of RNAIII-activating protein, a master regulator of staphylococcal virulence.

Staphylococcus aureus is a gram-positive bacterium that is part of the normal healthy flora but that can become virulent and cause infections by producing biofilms and toxins. The production of virulence factors is regulated by cell-cell communication (quorum sensing) through the histidine phosphorylation of target of RNAIII-activating protein (TRAP), which is a 21-kDa protein that is highly conserved among staphylococci. Using microarray analysis, we show here that the expression and phosphorylation of TRAP upregulate the expression of most, if not all, toxins known to date, as well as their global regulator agr. In addition, we show here that the expression and phosphorylation of TRAP are also necessary for the expression of genes known to be necessary for the survival of the bacteria in a biofilm, like arc, pyr, and ure. TRAP is thus demonstrated to be a master regulator of staphylococcal pathogenesis.

Adaptor Proteins, Signal Transducing↗

In silico genome mining and characterization of putative horse feces-derived bacterial phytases as potential monogastric animal feed additive candidates.

Phytic acid exerts a significant antinutritional effect in poultry, swine, and fish, which can be mitigated by supplementing monogastric feeds with efficient microbial phytases. Accordingly, mining bacterial genomes for novel phytases represents a strategic computational approach to identifying candidates for improving monogastric animal nutrition. In this study, 162 bacterial genomes associated with horse feces were systematically mined using an in silico pipeline to identify and characterize putative phytases.A total of 69 non-redundant sequences were identified and classified as histidine acid phytase (HAPhy) or protein tyrosine phosphatase-like phytase (PTPLPhy). HAPhys were detected in the genomes of Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Acinetobacter baumannii, and Cutibacterium equinum, whereas PTPLPhys were found in K. pneumoniae, Limosilactobacillus reuteri, Pediococcus acidilactici, Bifidobacterium pseudolongum, and Prescottella equi. Principal component analysis identified glucose-1-phosphatase (CAJ1242485.1) and bifunctional acid phosphatase (NHR17779.1) as the HAPhy candidates exhibiting the most favorable predicted physicochemical properties for potential feed applications. Similarly, among the PTPLPhys, protein tyrosine phosphatase (UNQ40438.1) and a hypothetical protein (CAJ1246072.1) showed the most favorable computational profiles. Biosafety analysis identified potential virulence factors, indicating that sources should be screened prior to feed application. High-quality AlphaFold2 models were obtained for these phytases (90.9-97.2). Molecular docking analysis showed that NHR17779.1 exhibited the strongest binding to phytic acid, whereas CAJ1246072.1 demonstrated the weakest interaction. Overall, this study identifies the horse fecal microbiota as a diverse source of putative phytases that may serve as promising targets for genetic and protein engineering; however, further in vitro and in vivo studies are essential to validate the enzymatic activity and industrial efficacy of these computational candidates.

Bacterial phytase↗