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PubMed2003

Highly resistant metabolically deficient dwarf mutant of Escherichia coli is the cause of a chronic urinary tract infection.

We present the case of a 68-year-old diabetic woman who has been suffering from chronic urinary tract infections, recurring over a period of at least 5 years, caused by a slowly growing metabolically deficient dwarf mutant (MDD) of Escherichia coli. This MDD strain was auxotrophic for histidine, was resistant to multiple antibiotics, and showed atypical growth behavior. Colonies were tiny on routine media but were able to revert to normal growth after extended incubation. This strain was identified as E. coli by 16S ribosomal DNA sequencing, and virulence factor profiles were determined by PCR. Seven MDD isolates collected over the 5-year period were grown from midstream urine to significant colony counts and shown to belong to the same clonal group by pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR. These MDDs were repeatedly misidentified by biochemical methods due to their slow growth and atypical colony morphology. This case highlights the importance of recognizing MDDs of Enterobacteriaceae in patients with chronic infections. To our knowledge this is the first report of an MDD of E. coli causing a chronic urinary tract infection.

Aged↗
PubMed1992

An improved intra-oral enamel demineralization test model for the study of dental caries.

The intra-oral enamel demineralization test (IEDT) was introduced by Brudevold et al. (1984). This caries model involves human subjects wearing palatal appliances each holding eight bovine enamel blocks covered by a bacterial cell layer prepared by the harvesting of cultures of Streptococcus mutants (test plaque). The original model used the iodide permeability test for assessment of the extent of demineralization of bovine enamel blocks resulting from acid production by the test plaque after dietary substrate challenge. The IEDT model has been expanded and improved by us in the following ways: (1) Based on encouraging findings from an in vitro study (Zero et al., 1990), the surface microhardness test has been adopted to measure the extent of demineralization occurring at three sites on the enamel blocks corresponding to an area over which the effective plaque thickness is 0.5, 1.5, and 2.5 mm; (2) intra-oral pH of the test plaque is measured by means of a Beetrode miniature pH electrode at baseline, then at five, 10, 15, 30, and 45 min after the start of a test; (3) plaque samples are collected at the end of a test and analyzed for organic acid content by means of HPLC; (4) the bacterial test challenge has been expanded to include different cariogenic bacteria which are grown under various growth conditions. The improved model has the capability of studying fundamental aspects of the caries process, namely, the relationships among dietary substrate challenge, plaque pH change, plaque organic acid profiles, microbial virulence properties, and enamel demineralization. Furthermore, the model has the potential for use in more applied research on caries-preventive agents such as fluoride.

Adult↗
PubMed2000

Atypical Actinobacillus pleuropneumoniae isolates that share antigenic determinants with both serotypes 1 and 7.

In the present study, the characterization of 3 atypical isolates of Actinobacillus pleuropneumoniae is presented. Two isolates (1B and 27E) showed positive reactions in coagglutination, immunodiffusion, and indirect hemagglutination tests for serotypes 1 and 7, whereas the third isolate (26B) reacted with antisera to serotypes 1, 4, and 7. These atypical isolates of A. pleuropneumoniae possessed a capsular polysaccharide (CPS) antigenically related to serotype 1 as well as an O-chain lipopolysaccharide antigenically related to serotype 7 or to serotypes 4 and 7, as shown by the use of monoclonal antibodies. Results of toxin profile and virulence assays for mice and pigs showed them to be more related to A. pleuropneumoniae serotype 7 field isolates. All 3 isolates induced antibodies mainly against serotype 7/4 O-long-chain lipopolysaccharide (LC-LPS) and, to a lesser extent, to the CPS of serotype 1, in experimentally infected pigs. Diagnostic laboratories that use a LC-LPS-based enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of A. pleuropneumoniae infection in swine would probably diagnose herds infected with these atypical isolates as being infected by A. pleuropneumoniae serotypes 7 or 4, whereas those that use a CPS-based ELISA would probably consider them as infected by A. pleuropneumoniae serotype 1.

Actinobacillus Infections↗
PubMed2026

Whole-genome profiling of antimicrobial resistance and virulence determinants in extensively-drug resistant Pseudomonas aeruginosa isolates causing ventilator associated pneumonia in Egypt.

Among critically ill ICU patients under prolonged mechanical ventilation, Pseudomonas aeruginosa is one of the most common cause of ventilator-associated pneumonia (VAP), with antimicrobial pressure leading to emergence of multidrug, extensively drug and pandrug-resistant (PDR) strains. In Egypt, very little genomic data exist on P. aeruginosa associated with VAP. This study aimed at characterizing the antimicrobial resistance (AMR) determinants, virulence repertoire, MGEs, and sequence types of two highly drug-resistant Pseudomonas aeruginosa isolates, including one pandrug-resistant colistin-resistant isolate and one extensively drug-resistant colistin-susceptible isolate from respiratory tract of Egyptian ICU patients suffering from VAP. The two isolates were identified conventionally and confirmed to the species level using MALDI-TOF MS. Antibiotic susceptibility was assessed using the VITEK-2 Compact system and the broth microdilution method. Genome analysis was performed using PATRIC, ResFinder, CARD, and Mobile Element Finder. For both isolates, resistance was found to all antibiotics routinely tested, however, one isolate had high-level colistin resistance (MIC > 64 µg/mL), while the other isolate was still colistin susceptible. Whole-genome sequencing identified two rare sequence types, ST2023 and ST2685, both 6.5-7.6 Mb in size with a 66% GC content. The presence of 21 MGEs in the SRR36105565 genome shows that it has high genomic flexibility, including a broader resistome than other strains, such as blaVIM-2 and OXA variants, aminoglycoside-modifying enzymes, crpP, and disinfectant-resistance markers. Both isolates retained large virulence determinants including Type III and Type VI secretion systems, alginate regulation genes, quorum-sensing networks, and siderophore biosynthesis clusters. It also represents one of the first genomic studies of VAP associated PDR P. aeruginosa from Egypt. The combination of widespread AMR with intact virulence supports the potential value of future genomic surveillance efforts and improved antimicrobial stewardship in local ICUs.

Pneumonia, Ventilator-Associated↗
PubMed1992

Acid-induced autoagglutination found in chicken pathogenic Escherichia coli strain.

Chicken pathogenic Escherichia coli strains were found to autoagglutinate in a static culture of trypticase soy broth (TSB). One strain, designated PDI-386, was further studied for its autoagglutinating property. Acidity in the cultured medium caused by glucose degradation induced the autoagglutination. The bacterial cells grown in a glucose-free L-broth could be aggregated by adding acid, which suggests a potentiality of autoagglutination of the strain grown in the L-broth. The autoagglutinating parent (Agg) formed small colonies with irregular edges like rough colonies on the TS agar, whereas its non-autoagglutinating variant (Nag) formed larger smooth colonies with a perfectly round edge. The Nag colony was easily generated from the Agg colony on the TS agar. The autoagglutinating property was very unstable when the bacteria was passed in the TSB, but rather stable in the L-broth. Under electron microscope, the Agg were found to possess pili of more than 20 microns in length. However, the phenotypic expression of autoagglutination did not correlate with that of mannose-sensitive hemagglutination against guinea pig erythrocytes. Incubation of the Nag in the L-broth at room temperature for more than 10 days provoked the reversion of the autoagglutination. There was no difference between the Agg and the Nag in terms of surface hydrophobicity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of membrane proteins and LPS, and plasmid profiles. The virulence of the Agg was higher than that of the Nag. The autoagglutination property is, however, so unstable that the pathogenicity of E. coli isolates from chickens should be carefully evaluated.

Agglutination↗
PubMed2002

[Urinary infection].

The term urinary infection defines those processes located in the urinary tract due to the presence of microorganisms in the urine, particularly bacteria. Regardless the sex or age of the patient, they represent the second most frequent type of infection, after respiratory tract infections, not only in outpatients but also in hospitalized patients. There is an ongoing interest about the etiology and pathogenesis of urinary infections in order to identify the virulence-factor profiles of bacterial isolates and the characteristics of the infection-prone individuals. Urinary infections are caused by a wide range of pathogens, specially gramnegative rods (E. coli, P. mirabilis, Klebsiella). However, there is an increasing evidence of the role as pathogens of some microorganisms that have long been regarded as apathogenic like E. faecalis and the Staphylococcus group. The treatment of such infections represents a challenge for the future mainly in the fields of control of antimicrobial resistance and vaccine development.

Bacteriuria↗
PubMed1991

Occurrence of Plesiomonas shigelloides--associated diarrhoea in Calabar, Nigeria.

The prevalence, clinical profiles and virulence factors of Plesiomonas shigelloides were determined in patients attending the University of Calabar Teaching Hospital, Microbiology Laboratory. P. shigelloides was isolated from 12 (1.4%) of 880 patients with diarrhoea and from none of the controls (P less than 0.05). Isolates were mostly from febrile children less than or equal to 10 years with most of the infections occurring during the rainy months. Although our findings suggest the significance of Plesiomonas in acute diarrhoea in this environment, our isolates did not seem to show any of the proxy indicators of virulence usually associated with other enteric pathogens.

Adolescent↗
PubMedDate not supplied

Bacterial colonization and in vivo expression of F1 (type 1) fimbrial antigens in chickens experimentally infected with pathogenic Escherichia coli.

Escherichia coli strains that cause septicemia of poultry often possess F1 (type 1) fimbriae (encoded by pil [fim] homologous gene clusters) and/or P fimbriae (encoded by pap homologous gene clusters). These fimbriae are thought to be involved in infection and colonization. To study the dynamics of infection due to E. coli with different virulence determinant profiles and to examine the expression of these fimbriae in vivo, three pathogenic E. coli isolates--O1 (pil+/pap+), O2 (pil+/pap), and O78 (pil+/pap+)--were administered intratracheally to 1.5-week-old chickens. Chickens were euthanatized from 3 to 144 hr after infection. The three isolates caused lesions in 30 to 55% of birds. Colonization rates of the trachea, lungs, internal organs, and pericardial fluid were similar for all three isolates, whereas significant differences among isolates were observed in colonization of the air sacs and blood. Bacteria appeared rapidly in the blood, liver, and spleen, whereas presence in the pericardial fluid generally occurred only after 24 hr postinoculation. The dynamics of colonization of the air sacs varied among isolates. Immunofluorescence of frozen tissue sections demonstrated F1 fimbriae (pil expressed) but not P fimbriae on all three isolates colonizing the trachea and on the O1 and O78 isolates colonizing the air sacs. Results suggest that F1 fimbriae are involved in the early stages of development of colisepticemia by promoting association of pathogenic E. coli with the trachea and air sacs of chickens.

Animals↗
PubMed2006

Serotypes, virulence genes, intimin types and PFGE profiles of Escherichia coli isolated from piglets with diarrhoea in Slovakia.

Two hundred and fifty Escherichia coli isolates from diarrhoeic and healthy piglets were serotyped and tested for the presence of virulence genes for fimbriae, intimin, heat-labile (LT) and heat-stable (STa and STb) enterotoxins, Stx toxins, and enteroaggregative heat-stable 1 (EAST1) enterotoxin by polymerase chain reaction (PCR). Although 220 isolates from diarrhoeic piglets belonged to 43 O serogroups and 77 O:H serotypes, 60% were of one of the 10 serogroups O2, O8, O15, O54, O84, O101, O141, O147, O149 and O157, and 60% belonged to only 10 serotypes (O8:H-, O54:H-, O84:H7, O101:H-, O141:H-, O141:H4, O147:H-, O149:H10, O163:H-, and ONT:H-). PCR showed that 79% of 220 isolates carried genes for at least one of the virulence factors tested. The gene encoding for EAST1 was the most prevalent (65%) followed by those encoding for STb (49%), LT (42%), STa (13%), and Stx2e (4%). Eighty-three (38%) of the 220 E. coli isolates carried the gene for F4 (K88), whereas genes for F18, F5 (K99), F41, F6 (P987), F17, and intimin (eae) were detected in 9%, 3%, 3%, 3%, 1%, and 3%, respectively. Seropathotype O149:H10:F4:LT/STb/EAST1 (70 isolates) was the most common, representing 32% of isolates. Pulsed-field gel electrophoresis (PFGE) analysis with XbaI of 15 O149:H10 representative isolates from diarrhoeic piglets distinguished 14 types. The 15 isolates exhibited a wide variability of distinct restriction patterns though all belonged to the same serotype (O149:H10), and all but one showed identical virulence determinants (F4, LT, STb, and EAST1). Among 30 isolates from healthy piglets only two virulence genes were detected: EAST1 (26%) and eae (17%). In total, 12 isolates were positives for the eae gene: five isolates had intimin beta1, four possessed intimin theta and three showed intimin type xiB. This is believed to be the first study describing the presence of intimin type xiB in E. coli of porcine origin.

Adhesins, Bacterial↗
PubMed2002

Global molecular epidemiology of the O15:K52:H1 extraintestinal pathogenic Escherichia coli clonal group: evidence of distribution beyond Europe.

Escherichia coli O15:K52:H1 is a significant extraintestinal pathogen in Europe (G. Prats et al., J. Clin. Microbiol. 38:201-209, 2000). To search for evidence of this clonal group outside of Europe, 75 non-European E. coli isolates of serogroup O15 were compared with five members of the O15:K52:H1 clonal group from Barcelona, Spain, according to genomic background, virulence genotypes, and antimicrobial resistance profiles. Amplification phylotyping showed that 16 (21%) of the 75 non-European O15 isolates corresponded with the O15:K52:H1 clonal group. The 16 non-European O15:K52:H1 clonal group members represented diverse geographic locales. They were isolated almost exclusively from humans with extraintestinal infections and accounted for 50% of all O15 isolates from five human clinical collections studied. Most non-European clonal group members exhibited a consensus virulence factor profile that included the F16 or F7-2 papA alleles (P fimbrial structural subunit), papG allele II (P fimbrial adhesin), iha (putative adhesin siderophore), and iutA (aerobactin receptor). This resembles the virulence profiles of (i) European representatives of the O15:K52:H1 clonal group and (ii) phylogenetically related "clonal group A," a recently recognized significant contributor to trimethoprim-sulfamethoxazole resistance in the United States (A. R. Manges et al., N. Engl. J. Med. 345:1007-1013, 2001). Antimicrobial resistance profiles were variable, and resistance was inconsistently transferred by conjugation. These findings indicate that the O15:K52:H1 clonal group is broadly distributed beyond Europe, exhibits previously unrecognized phenotypic and genotypic diversity, and contributes significantly to extraintestinal infections in humans.

Animals↗
PubMed1999

Virulence-associated factors of uropathogenic Escherichia coli strains isolated from pigs.

Thirty one Escherichia coli strains isolated from pigs with urinary tract infections were investigated for presence of virulence factors and plasmid DNA profile. The most frequent virulence factors presented by these strains were mannose-resistant fimbriae, including P. fimbriae (54.8%) and aerobactin production (45.2%). The pap) operon, detected by PCR, was found in 54.8% of the strains, which is similar to its frequency in human strains. Other characteristics such as the presence of mannose-sensitive hemagglutinin (16.1%), indicative of type 1 pili, and production of hemolysin (25.8%), colicin (38.7%) and toxins (22.6% for LT and for VT) were less frequent. No strains were positive for STa production. Plasmid profiles were variable among isolates from either the same or different farms.

Animals↗
PubMed1995

Salmonella typhimurium DT 193: differentiation of an epidemic phage type by antibiogram, plasmid profile, plasmid fingerprint and salmonella plasmid virulence (spv) gene probe.

Of over 2000 isolates of Salmonella typhimurium DT 193 from humans examined in the 2 year period 1991-92, 93% were antibiotic-resistant with the most common R-types being ASSuT (38%) and T (29%). Fourteen plasmid profiles were identified in DT 193 R-type ASSuT with the majority of isolates being characterized by a single plasmid of 80 MDa (pDEP 34) which in addition to coding for ASSuT, also hybridized with a spv gene probe prepared from the 50 MDa Salm. dublin serovar-specific plasmid. On the basis of restriction fragment length polymorphisms, two variant lines of pDEP 34-like plasmids were identified and a third line which had lost the genes coding for resistance to ampicillin, streptomycin and sulphonamides, was recognized. Although 18 plasmid profile types were identified in DT 193 R-type T, all isolates carried a high mol. wt plasmid which coded for tetracycline resistance only. Further discrimination was achieved on the basis of hybridization of tetracycline resistance plasmids with the spv gene probe and restriction enzyme fingerprinting. These results demonstrate that Salm. typhimurium DT 193 can be rapidly subdivided by antibiogram and that further subdivision can be achieved on the basis of plasmid profile, plasmid fingerprint and hybridization with a spv gene probe.

Animals↗
PubMed2006

Escherichia coli isolates associated with uncomplicated and complicated cystitis and asymptomatic bacteriuria possess similar phylogenies, virulence genes, and O-serogroup profiles.

The genetic and serological characteristics of Escherichia coli isolates from patients with uncomplicated cystitis (UC), complicated cystitis (CC), and complicated asymptomatic bacteriuria (CASB) were determined. Phylogenetic group B2 was predominant in all categories. The prevalences of 14 out of 18 virulence factor genes were similar among the three categories, while pap, iha, ompT, and PAI were more frequently seen in isolates associated with UC than CC or CASB.

Bacteriuria↗
PubMed2002

Uropathogenic Escherichia coli as agents of diverse non-urinary tract extraintestinal infections.

Escherichia coli isolates from 3 consecutively encountered patients with serious, invasive, non-urinary tract extraintestinal infections (pneumonia, deep surgical wound infection, and vertebral osteomyelitis with associated epidural/psoas/iliacus abscesses) were characterized, using molecular methods, as to extended virulence genotype and phylogenetic background. All 3 isolates exhibited virulence genotypes and genomic profiles characteristic of specific familiar virulent clones of extraintestinal pathogenic E. coli (ExPEC), which traditionally have been regarded primarily as uropathogenic or as associated with meningitis. These included E. coli O1/O2:K1:H7, E. coli O18:K1:H7, and a recently described E. coli O11/O17/O77:K52:H18 clonal group (clonal group A). These findings demonstrate the extraintestinal pathogenic versatility of ExPEC clones, which supports the use of an inclusive designation for such strains and suggests the possibility of cross-syndrome protective interventions. They also provide novel evidence that multidrug-resistant epidemic clonal group A can cause extraintestinal infections other than uncomplicated urinary tract infections and can cause them in hosts other than young women.

Aged↗
PubMed2006

Proinflammatory and immunomodulatory cytokine mRNA time course profiles in hamsters infected with a virulent variant of Leptospira interrogans.

In order to quantify in vivo the mRNAs of cytokines which play important roles in leptospirosis, we have developed quantitative real-time PCR assays for interleukin-2 (IL-2), IL-4, IL-10, IL-12p40, tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), transforming growth factor beta, and two housekeeping genes (encoding beta-actin and hypoxanthine phosphoribosyltransferase). We used a lethal hamster model reflecting severe leptospirosis in humans. The LightCycler system was used to quantify the gene expression levels with the SYBR green I detection format using external standard curves for each target. We compared the expression levels of cytokine mRNA in the peripheral blood mononuclear cells of both control (uninfected) hamsters and Leptospira interrogans-inoculated hamsters from 1 to 24 h and then 1 to 4 days postinfection. In this kinetic study, there was pronounced expression of Th1 cytokine mRNA (TNF-alpha, IFN-gamma, and IL-12), with transcripts being detected as early as 1 h postinfection. Expression of anti-inflammatory cytokines, such as IL-4 and IL-10, was prominent in delayed samples from 1 to 4 days postinfection in response to infection with Leptospira interrogans. Our data are the first to establish that pathogenic leptospires can stimulate in vivo the production of type 1 cytokines involved in cellular immunity by using this informative animal model. Measuring and assessing cytokine profiles may provide a useful method for accurate study of the mechanisms of anti-Leptospira immunity, indications of prognosis factors, and prospective evaluation of leptospirosis vaccine efficacy in humans.

Animals↗
PubMed1997

Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids, outer membrane protein profiles and siderophore production.

The virulence of 18 strains of Vibrio anguillarum serogroup O1 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only strains that carried the 67 kbp virulence plasmid or derivatives of it produced the outer membrane protein, OM2. All virulent strains harboured the 67 kbp plasmid or derivatives of it, indicating its importance for virulence. However, some strains carrying the virulence plasmid or a derivative of it, produced siderophores as well as OM2 but were non-pathogenic to fish. Likewise, among the virulent strains, considerable variation in LD50 values was recorded. Plasmid profiling and restriction analysis showed that the virulence plasmid existed in various molecular weights from 26 to 80 kbp, with 65-67 kbp being the most common, and that this plasmid displayed various restriction profiles. The presence of other plasmids did not seem to affect the pathogenic properties.

Animals↗
PubMed2004

Phenotypic and genotypic analyses of enterohemorrhagic Escherichia coli O145 strains from patients in Germany.

Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O145 are emerging as causes of diarrhea and the hemolytic-uremic syndrome. However, there have been few genetic analyses of this EHEC group. We investigated the serotypes, virulence genes, plasmid profiles, pulsed-field gel electrophoresis (PFGE) patterns, and genetic variability of the fliC and eae genes in 120 EHEC O145 strains isolated from cases of hemolytic-uremic syndrome (n = 24) or diarrhea (n = 96) in Germany between 1996 and 2002. Three isolates belonged to serotype O145:H28, one to serotype O145:H25, and 116 were nonmotile (O145:H(-)). One hundred fourteen of the nonmotile strains shared fliC restriction fragment length polymorphism (RFLP) patterns identical to that of the O145:H28 strains. The remaining two nonmotile strains displayed a fliC-RFLP pattern identical to that of the O145:H25 strain. Each of the 117 strains with the fliC-RFLP(H28) pattern harbored eae gamma, whereas the three strains with the fliC-RFLP(H25) pattern possessed eae beta. Five different stx genotypes, six combinations of plasmid-encoded putative virulence genes, 29 plasmid profiles, and 47 PFGE types were identified. Strains within some of the PFGE types could be further subtyped by means of distinct plasmid profiles. These data demonstrate that the EHEC O145 serogroup is comprised of two different serotypes that possess distinct eae types. The heterogeneity of EHEC O145 strains at the chromosomal and plasmid level, in particular the high diversity in PFGE patterns, provides a basis for molecular subtyping of these pathogens.

Electrophoresis, Gel, Pulsed-Field↗
PubMed2001

Measure of molecular diversity within the Trypanosoma brucei subspecies Trypanosoma brucei brucei and Trypanosoma brucei gambiense as revealed by genotypic characterization.

We have evaluated whether sequence polymorphisms in the rRNA intergenic spacer region can be used to study the relatedness of two subspecies of Trypanosoma brucei. Thirteen T. brucei isolates made up of 6 T. b. brucei and 7 T. b. gambiense were analyzed using restriction fragment length polymorphism (RFLP). By PCR-based restriction mapping of the ITS1-5.8S-ITS2 ribosomal repeat unit, we found a fingerprint pattern that separately identifies each of the two subspecies analyzed, with unique restriction fragments observed in all but 1 of the T. b. gambiense "human" isolates. Interestingly, the restriction profile for a virulent group 2 T. b. gambiense human isolate revealed an unusual RFLP pattern different from the profile of other human isolates. Sequencing data from four representatives of each of the two subspecies indicated that the intergenic spacer region had a conserved ITS-1 and a variable 5.8S with unique transversions, insertions, or deletions. The ITS-2 regions contained a single repeated element at similar positions in all isolates examined, but not in 2 of the human isolates. A unique 4-bp [C(3)A] sequence was found within the 5.8S region of human T. b. gambiense isolates. Phylogenetic analysis of the data suggests that their common ancestor was a nonhuman animal pathogen and that human pathogenicity might have evolved secondarily. Our data show that cryptic species within the T. brucei group can be distinguished by differences in the PCR-RFLP profile of the rDNA repeat.

Animals↗