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PubMed2000

Peritoneal culture alters Streptococcus pneumoniae protein profiles and virulence properties.

We have examined the properties of Streptococcus pneumoniae cultured in the murine peritoneal cavity and compared its virulence-associated characteristics to those of cultures grown in vitro. Analysis of mRNA levels for specific virulence factors demonstrated a 2.8-fold increase in ply expression and a 2.2-fold increase in capA3 expression during murine peritoneal culture (MPC). Two-dimensional gels and immunoblots using convalescent-phase patient sera and murine sera revealed distinct differences in protein production in vivo (MPC). MPC-grown pneumococci adhered to A549 epithelial cell lines at levels 10-fold greater than those cultured in vitro.

Animals↗
PubMed1980

Purification of Theiler's murine encephalomyelitis virus and analysis of the structural virion polypeptides: correlation of the polypeptide profile with virulence.

Theiler's murine encephalomyelitis viruses (TMEV) are separable into two groups based on their biological behavior: those highly virulent isolates which are unable to cause persistent infection and the less virulent isolates which regularly produce persistent central nervous system infection in mice. Two highly virulent and five less virulent TMEV were found to have the same buoyant density (1.34 g/ml) on isopycnic centrifugation and virion structure by electron microscopy. Negatively stained virus particles purified in Cs(2)SO(4) gradients appeared to have icosahedral symmetry and measured 28 nm in diameter. Mature virions were found to possess three major structural polypeptides, VP1, VP2 and VP3, in the range of 25,000 to 35,000 daltons, and a smaller fourth major polypeptide, VP4, of 6,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The precursor of VP2 and VP4, VP0, which is a minor polypeptide of mature picornavirus particles, was also identified. However, a slight but consistent difference in several of the capsid polypeptides between the highly virulent and less virulent TMEV was found. VP1 was slightly larger (34,000 versus 33,500 daltons) and VP2 was slightly smaller (31,000 versus 32,000 daltons) for the highly virulent strains compared to the same polypeptide species in the less virulent viruses. VP0 was also slightly smaller (35,500 versus 36,000 daltons) for the highly virulent isolates compared to their less virulent counterparts. Finally, trypsin which was used initially in our purification procedure resulted in preferential cleavage of a 2,000-molecular-weight fragment or fragments from VP1 of only the less virulent isolates.

Centrifugation, Density Gradient↗
PubMed2002

Prevalence and genetic profiling of virulence determinants of non-O157 Shiga toxin-producing Escherichia coli isolated from cattle, beef, and humans, Calcutta, India.

We investigated the prevalence of Shiga toxin-producing Escherichia coli (STEC) in hospitalized diarrhea patients in Calcutta, India, as well as in healthy domestic cattle and raw beef samples collected from the city's abattoir. Multiplex polymerase chain reaction using primers specific for stx1 and stx2 detected STEC in 18% of cow stool samples, 50% of raw beef samples, and 1.4% and 0.6% of bloody and watery stool samples, respectively, from hospitalized diarrhea patients. Various virulence genes in the STEC isolates indicated that stx1 allele predominated. Plasmid-borne markers, namely, hlyA, katP, espP, and etpD, were also identified. Bead enzyme-linked immunosorbent assay and Vero cell assay were performed to detect and evaluate the cytotoxic effect of the Shiga toxins produced by the strains. STEC is not an important cause of diarrhea in India; however, its presence in domestic cattle and beef samples suggests that this enteropathogen may become a major public health problem in the future.

Adolescent↗
PubMed2006

Differentiation of Escherichia coli pathotypes by oligonucleotide spotted array.

To accurately determine the pathotypes of Escherichia coli strains, a comprehensive assessment of each strain that targets multiple genes is required. A new approach to the identification and characterization of E. coli pathotypes was developed by constructing gene-specific probes (70-mers) for not only the virulence genes associated with each E. coli pathotype but also the O157-, CFT073-, and K-12-specific and common genes of each pathotype. Analysis of oligonucleotide probes with reference and clinical isolates of E. coli pathotypes indicated that the array could differentiate the pathotypes on the basis of their virulence and specific gene patterns. Probes targeting common genes of E. coli were present in all the reference and clinical strains. Salmonella enterica subsp. enterica-specific genes and Salmonella core genes were used as negative controls. The entire E. coli pathotype showed reactivity to only 4 of the 81 Salmonella-specific gene probes. Characterization of the genetic and virulence profiles of a single strain by using probes for virulence factors and specific and common genes in the spotted array is an ideal diagnostic tool for determination of E. coli pathotypes and could also have a significant impact on the epidemiological analysis of E. coli infections.

DNA, Bacterial↗
PubMed2006

Virulence-associated gene profiling of Streptococcus suis isolates by PCR.

Definition of virulent Streptococcus suis strains is controversial. One successful approach for identification of virulent European strains is differentiation of capsular serotypes (or the corresponding cps types) and subsequent detection of virulence-associated factors, namely the extracellular factor (EF, epf), the muramidase-released protein (MRP, mrp) and the hemolysin suilysin (SLY, sly). In this work we present a novel multiplex PCR (MP-PCR) and an mrp variant PCR for identification and characterization of virulent S. suis strains. These new methods were used to identify association of disease with particular profiles of virulence-associated genes. The MP-PCR allowed identification of S. suis through detection of the housekeeping gene gdh, differentiation of four cps types (1, 2, 7 and 9), and detection of epf, mrp, sly and arcA (arginine deiminase from S. suis). Furthermore, this study describes the first PCR assay for differentiation of at least six mrp variants. Expression of the corresponding size variants of MRP was shown for four of the six mrp variants, but was undetectable for the two larger mrp variants in the particular strains investigated. The results of this study suggest that cps7 strains are associated with pneumonia and that variation of mrp is very pronounced among these strains. Gene profiles of invasive, pneumonia and carrier S. suis isolates by combination of PCR assays allowed differentiation of 24 different genotypes among cps1, 2, 7 and 9 strains. Forty-five percent of the invasive S. suis diseases investigated in this study were caused by only two of these genotypes, namely cps2/mrp+/epf+/sly+ and cps9/mrp(*)/epf-/sly+. Thus, this study demonstrates for the first time a uniform profile of the particular virulence-associated genes for the vast majority of the investigated invasive cps9 strains.

Animals↗
PubMed1997

Escherichia coli virulence factors and serotypes in acute bacterial prostatitis.

BACKGROUND: Escherichia coli is the most frequent pathogen in both acute bacterial prostatitis and acute uncomplicated urinary infections. To assess the virulence profiles of E. coli in acute prostatitis, the serotypes and virulence factor (VF) genotypes were determined. METHODS: We studied 107 E. coli isolates from cases of acute bacterial prostatitis, 76 isolates from acute pyelonephritis, 194 isolates from acute Cystitis and 80 fecal isolates from healthy people. All pyelonephritis and cystitis isolates were from women. Seven urovirulence determinants were analyzed by DNA colony hybridization, including the genes for type 1 fimbria (pil), P fimbria (pap), S fimbria (sfa), afimbrial adhesin AFA-I (afaI), alpha-hemolysin (hly), cytotoxic necrotizing factor 1 (cnf1) and aerobactin (aer). O:H:K serotypes were also determined. RESULTS: With the exception of pil and afaI, all VFs were significantly more often associated with prostatitis, pyelonephritis and cystitis isolates than with the fecal isolates. The prevalence of sfa, hly and cnf1 was higher in prostatitis isolates than in pyelonephritis and cystitis isolates, and the pap+sfa+hly+cnf1+ genotype was dominant among prostatitis isolates (48.8%). Nine O serotype (O1, O2, O4, O6, O16, O18, O22, O25 and O75) accounted for 79.4%, 73.7% and 78.4% of the prostatitis, pyelonephritis and cystitis strains, respectively. There was an apparent correlation between serotype and genotype in uropathogenic E. coli. CONCLUSION: The predominance of O serotypes in female urinary tract infections and a high percentage of multiple VFs among the prostatitis isolates suggested that VFs play important roles in the pathogenesis of acute bacterial prostatitis.

Acute Disease↗
PubMed2003

Extraintestinal pathogenic Escherichia coli as a cause of invasive nonurinary infections.

Multiple Escherichia coli isolates from four adults with extraintestinal infections underwent molecular phylotyping and virulence profiling. A patient with secondary peritonitis had two low-virulence E. coli strains from phylogenetic groups A and D. In contrast, three patients with invasive extraurinary infections (septic arthritis/pyomyositis, nontraumatic meningitis/hematogenous osteomyelitis, and pneumonia) each had a single high-virulence phylogenetic group B2 strain resembling typical isolates causing urinary infection and/or sepsis, i.e., extraintestinal pathogenic E. coli.

Aged↗
PubMed2003

Atypical enteropathogenic Escherichia coli strains: phenotypic and genetic profiling reveals a strong association between enteroaggregative E. coli heat-stable enterotoxin and diarrhea.

The virulence profiles of most atypical enteropathogenic Escherichia coli (EPEC) strains are unknown. A total of 118 typical and atypical strains of EPEC serotypes and non-EPEC serogroups isolated from children with or without acute diarrhea who were from different cities in Brazil were examined for virulence-associated markers and adherence to HEp-2 cells, and also had random amplified polymorphic DNA (RAPD) analysis performed. Atypical strains were identical to typical strains with regard to the virulence factors encoded on the locus of enterocyte effacement (LEE). In contrast with typical EPEC strains, none of the atypical strains reacted with the bfpA probe, and half of the strains hybridized with the perA probe. Most atypical strains presented Tir sequences that correlated with enteropathogenic or enterohemorrhagic E. coli (98%), had LEE inserted in either selC or pheU (88%), and presented a typeable intimin (52%). Eighteen new serotypes were found in the EPEC strains. Atypical and typical EPEC strains belonged to different RAPD clusters. Most atypical strains showed a localized-like adherence pattern (61.5%). Of the non-LEE-encoded virulence factors, enteroaggregative E. coli heat-stable enterotoxin was noted most frequently (45%) and was significantly associated with diarrhea (P=.01). Thus, this virulence marker may be used as an additional tool for the diagnosis of truly atypical pathogenic strains.

Bacterial Toxins↗
PubMed2001

Virulence of Leishmania infantum is expressed as a clonal and dominant phenotype in experimental infections.

Human Leishmania infantum infection results in a spectrum of clinical expressions ranging from cutaneous to either asymptomatic or fatal visceral disease. In this context, characterization of parasite virulence appears to be relevant as a biological marker of intrinsic parasitic factors that can affect the pathology of leishmaniasis. Since parasite populations in naturally infected hosts are likely to be composed of multiclonal associations, we first explored the biodiversity of parasite virulence at the intrastrain level in vitro and in vivo by using 11 clones isolated from three strains previously known to express different virulence phenotypes in mice. Subsequently, we studied the course of infection in mice inoculated simultaneously or successively with strains or clones showing various virulence phenotypes. Analysis of in vitro growth characteristics showed no differences among clones from the different parental strains. By contrast, in vivo experiments evidenced a marked intrastrain heterogeneity of virulence to mice. One out of five clones obtained from a virulent strain showed a typical virulence phenotype, while the remaining four clones had low-virulence profiles, as did the six clones isolated from two low-virulence strains. In mixed multiclonal infections, the virulence phenotype was expressed as a dominant character over the associated low-virulence clones. After a challenge with either a homologous or a heterologous strain or clone, virulence phenotypes were conserved and expressed as in naive mice independently from the preexisting population. These results strongly suggest that parasite virulence in L. infantum visceral leishmaniasis is clonal and dominant in nature.

Animals↗
PubMed1990

Role of lipopolysaccharides in adherence of Actinobacillus pleuropneumoniae to porcine tracheal rings.

The ability of 17 Actinobacillus pleuropneumoniae isolates, representing serotypes 1, 2, 5, and 7, to adhere to tracheal rings maintained in culture was examined. Porcine tracheal rings were infected, and 8 h after inoculation, adherent bacterial cells were evaluated. A. pleuropneumoniae adhered to tracheal rings, and marked variations were observed between and even within serotypes, suggesting that adherence of this microorganism is not primarily related to the serotype of the isolate. No relationship was found between adherence to porcine tracheal rings and plasmid profiles, virulence in mice, hemagglutination, capsular material thickness, or whole-cell protein profiles. On the other hand, we observed that all isolates of serotypes 1 and 5 had a semirough-type lipopolysaccharide (LPS), whereas isolates of serotypes 2 and 7 had a smooth-type LPS (75%) or a semirough-type LPS (25%). Results showed that 83% of isolates with a smooth-type LPS adhered in large numbers to tracheal rings, whereas 80% of isolates with a semirough-type LPS adhered poorly (P less than 0.007). Our data indicated that the degree of adherence of A. pleuropneumoniae to porcine tracheal rings appeared to be related, at least in part, to LPS profiles. Furthermore, LPS seemed to be the adhesin of A. pleuropneumoniae, since purified LPS blocked adherence of this microorganism to porcine tracheal rings.

Actinobacillus↗
PubMed1989

Congo red-mediated regulation of levels of Shigella flexneri 2a membrane proteins.

The ability of Shigella spp. to bind Congo red from agar medium is generally correlated with their virulence properties. We used a metabolically active culture of Shigella flexneri 2a to determine the effect of Congo red on its membrane protein profiles. Virulent S. flexneri grown in the presence of Congo red at 37 degrees C showed increased levels of three proteins with Mrs of 43,000, 58,000, and 63,000 (43K, 58K, and 63K proteins) in the Sarkosyl-soluble membrane fractions. The observed phenomenon was temperature dependent. At 30 or 42 degrees C the protein levels remained unaffected by the presence of Congo red. Similar regulation of the levels of the 43K, 58K, and 63K membrane proteins was also observed with Shigella dysenteriae 1 and enteroinvasive Escherichia coli, but not with enteropathogenic E. coli. The cellular uptake of Congo red seemed to be essential, but not sufficient, for regulation. All three proteins reacted with human convalescent-phase sera in immunoblots of S. flexneri 2a Sarkosyl-soluble membrane fractions. Using the 43K-specific antiserum as the primary antibody, by indirect immunofluorescence studies, we detected an increase in the level of the 43K protein in S. flexneri which had invaded epithelial cells. These observations strongly indicate that the 43K, 58K, and 63K proteins are virulence associated. We propose that the observed regulatory effect of Congo red on membrane proteins of S. flexneri is mediated through induction. Since the same regulatory effect was also observed during the invasion of epithelial cells by S. flexneri, it is suggested that Congo red mimics some host tissue factor in vitro.

Adult↗
PubMed1979

Differentiation of vaccine strains and field isolates of pseudorabies (Aujeszky's disease) virus: thermal sensitivity and rabbit virulence markers.

Eleven cloned North American pseudorabies virus (PRV) strains and the European vaccine strains K and BUK were characterized by their thermal sensitivity and rabbit virulence markers. Heat sensitivity of the strains and isolates ranged from the highly heat resistant strain K, to the extremely heat labile strain BUK and isolate Be. The inactivation curves of each virus were transformed to the logarithmic scale and their standardized slopes and predicted virus survival values at 30 minutes were plotted against each other. The result was a distribution of points that represented a thermal sensitivity spectrum (TSS). Virus strains were subsequently categorized into 1 of 3 groups according to their position in the TSS. Viruses were also categorized into three groups according to their ability to clinically infect rabbits, their ability to produce pruritus and the time required to kill. When individual strains were described according to their marker profiles, 5 of 9 possible marker combinations were revealed. The 2 vaccine strains were each described by separate profiles. Virulent field isolates were characterized by 1 of 3 different profiles.

Animals↗
PubMed2000

Detection of virulence associated genes, haemolysin and protease amongst Vibrio cholerae isolated in Malaysia.

Eighty-four strains of Vibrio cholerae O1, O139 and non-O1/non-O139 from clinical and environmental sources were investigated for the presence of the toxin co-regulated pilus gene, tcpA, the virulence cassette genes ctxA, zot, ace and cep and also for their ability to elaborate haemolysin and protease. The ctxA and zot genes were detected using DNA-DNA hybridization while the ace, cep and tcpA genes were detected using PCR. Production of haemolysin and protease was detected using mammalian erythrocytes and an agar diffusion assay respectively. Analysis of their virulence profiles showed six different groups designated Type I to Type VI and the major distinguishing factor among these profiles was in the in vitro production of haemolysin and/or protease. Clinical O1, O139 and environmental O1 strains were similar with regard to presence of the virulence cassette genes. All environmental O1 strains with the exception of one were found to possess ctxA, zot and ace giving rise to the probability that these strains may actually be of clinical origin. One strain which had only cep but none of the toxin genes may be a true environmental isolate. The virulence cassette and colonization factor genes were absent in all non-O1/non-O139 environmental strains but production of both the haemolysin and protease was present, indicating that these may be putative virulence factors. These findings suggest that with regard to its pathogenic potential, only strains of the O1 and O139 serogroup that possess the tcpA gene which encodes the phage receptor, have the potential to acquire the CTX genetic element and become choleragenic.

Cholera↗
PubMed2006

Virulence attenuation and phenotypic variation of Paracoccidioides brasiliensis isolates obtained from armadillos and patients.

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, the most important systemic mycosis in Latin America. The virulence profiles of five isolates of P. brasiliensis were studied in two different moments and correlated with some colonial phenotypic aspects. We observed a significant decrease in the virulence and an intense phenotypic variation in the mycelial colony. The recognition of all ranges of phenotypic and virulence variation of P. brasiliensis, as well as its physiological and genetic basis, will be important for a better comprehension of its pathogenic and epidemiological features.

Animals↗
PubMed2006

Capsular serotype K1 or K2, rather than magA and rmpA, is a major virulence determinant for Klebsiella pneumoniae liver abscess in Singapore and Taiwan.

Capsular serotypes, magA, and rmpA have been documented in high prevalence for Klebsiella pneumoniae liver abscess. To investigate the regional difference and the correlation of capsular serotype, magA, and rmpA with virulence, 73 isolates were collected in Singapore and Taiwan. Capsular serotypes were determined by countercurrent immunoelectrophoresis, the presence of magA and rmpA was determined by PCR, and virulence was determined by phagocytosis and mouse inoculation. Isolates from Singapore were similar to those from Taiwan in genomic heterogeneity, prevalence of serotype, and the presence of magA and rmpA. The most common serotype was K1 (34/73; 46.6%), followed by K2 (15/73; 20.5%). magA was restricted to serotype K1. All K1 or K2 isolates and 66.7% (16/24) of isolates that were neither serotype K1 nor serotype K2 (non-K1/K2) carried rmpA. Serotype K1 or K2 isolates demonstrated significantly more phagocytic resistance and virulence than did rmpA-positive and -negative groups of non-K1/K2 isolates. In the non-K1/K2 group, the virulence profiles of rmpA-positive strains from Taiwan and Singapore were different by phagocytosis assay and in the mouse model, indicating that factors other than rmpA contributed to virulence. The characteristics of K. pneumoniae liver abscess in Singapore and Taiwan are similar. Capsular serotype K1 or K2 plays a more important role than magA and rmpA in determining virulence in K. pneumoniae liver abscess.

Animals↗
PubMed2005

Fluoroquinolone-resistant Escherichia coli, Indonesia.

In a recent, population-based survey of 3,996 persons in Indonesia, fluoroquinolone (FQ)-resistant Escherichia coli was prevalent in the fecal flora of 6% of patients at hospital admission and 23% of patients at discharge, but not among healthy relatives or patients visiting primary healthcare centers (2%). Molecular typing showed extensive genetic diversity with only limited clonality among isolates. This finding suggests that independent selection of resistant mutants occurs frequently. FQ-resistant isolates exhibited a higher rate of spontaneous mutation, but sparser virulence profiles, than FQ-susceptible isolates from the same population. The resistant isolates belonged predominantly to phylogenetic groups A (57%) and B1 (22%) but also to the moderately virulent group D (20%). Hypervirulent strains from the B2 cluster were underrepresented (1%). Because FQ-resistant E. coli can cause disease, especially nosocomial infections in immunocompromised patients, spread of such strains must be stopped.

Anti-Infective Agents↗
PubMed2026

Ascites Bactericidal Capacity in Patients With Decompensated Cirrhosis.

BACKGROUND & AIMS: Patients with decompensated cirrhosis are at high risk of developing bacterial infections. The most common infection is spontaneous bacterial peritonitis in ascites fluid, caused by gut bacterial translocation and facilitated by cirrhosis associated immune dysfunction. Common causative agents of spontaneous bacterial peritonitis are thought to represent pathobionts that increase in abundance in the gut with cirrhosis severity, rather than opportunistic commensals. Despite this there have been few studies describing the characteristics of spontaneous bacterial peritonitis pathogens and immune responses to them. METHODS: We investigated the bactericidal capacity of cell-free ascites fluid and ascites macrophages. RESULTS: Nonpathogenic Escherichia coli K12 was rapidly killed in cell-free ascites fluid, whereas extraintestinal pathogenic E coli and Klebsiella pneumoniae strains, representative of the most common causes of spontaneous bacterial peritonitis, were resistant. A transposon-based genome-wide screen identified a requirement for genes involved in O-antigen biosynthesis for survival of both organisms in cell-free ascites fluid, including the transcription factor RfaH, which controls expression of surface polysaccharides. Although extraintestinal pathogenic E coli were resistant to fluid-mediated killing, ascites macrophages, especially VSIG4Hi cells, phagocytosed and killed extraintestinal pathogenic E coli and nonpathogenic E coli K-12 with similar efficacy. Mice with toxin-induced cirrhosis had higher bacterial burdens in the liver compared with control mice when infected with extraintestinal pathogenic E coli, but rapidly cleared an rfaH mutant attenuated for capsule and O-antigen biosynthesis. CONCLUSIONS: These data highlight the importance of understanding bacterial virulence profiles in addition to taxonomy in cirrhosis dysbiosis and identify critical roles for capsule and O-antigen in host evasion by spontaneous bacterial peritonitis-associated pathogens. Targeting bacterial virulence factors and/or host macrophage functions may reduce the risk of spontaneous bacterial peritonitis in cirrhosis.

Animals↗
PubMed2004

Virulence characteristics and phylogenetic background of multidrug-resistant and antimicrobial-susceptible clinical isolates of Escherichia coli from across the United States, 2000-2001.

BACKGROUND: Increases in antimicrobial resistance in Escherichia coli have been paralleled by an increasing incidence of E. coli sepsis, suggesting a possible link between resistance and virulence. METHODS: All 76 multidrug-resistant (MDR) E. coli isolates (i.e., those resistant to > or =3 antimicrobial agents, including ampicillin, ceftazidime, trimethoprim-sulfamethoxazole, gentamicin, and ciprofloxacin) reported to the Tracking Resistance in the United States Today studies during 2000-2001 and 76 closely matched pansusceptible control isolates were studied. Extended virulence profiles and E. coli phylogenetic group (A, B1, B2, or D) were compared between groups. RESULTS: The MDR isolates, which represented predominantly non-B2 phylogenetic groups (91%), exhibited significantly reduced molecular virulence, compared with the predominantly group B2-derived control isolates (58%). Only 30% of MDR isolates, compared with 61% of control isolates (P<.001), qualified as extraintestinal pathogenic E. coli (ExPEC), and even these isolates exhibited significantly lower virulence scores than did susceptible ExPEC (7.25 vs. 9.0; P=.001). Phylogenetic differences accounted for the apparent virulence differences between MDR and control isolates. CONCLUSIONS: These findings argue against a direct link between virulence traits and antimicrobial resistance in E. coli. Instead, they call into question why non-B2 strains are more commonly MDR, with differential exposure to selection pressure (including in agriculture) as one possible explanation.

Adhesins, Escherichia coli↗