Role of bacterial virulence factors and host factors in the outcome of Escherichia coli bacteraemia.

In a study of the role of virulence factors in the outcome of Escherichia coli bacteraemia, blood isolates from 30 hospitalised patients were characterised with regard to O and K antigens, P and type 1 fimbriae, haemolysin production, cytonecrotising factor 1 production, serum resistance, ability to activate neutrophils and resistance to killing. Patients were analysed to identify host factors contributing to morbidity and mortality. In univariate analyses the presence of a K antigen, type 1 fimbriae, absence of haemolysin production, serum resistance and resistance to killing were associated with morbidity and mortality. In multivariate analyses only the absence of haemolysin production was associated with morbidity and mortality, after taking host factors into account. These preliminary findings suggest that host factors override bacterial virulence factors in determining the course of Escherichia coli bacteraemia. The negative association between haemolysin production and clinical deterioration during Escherichia coli bacteraemia might indicate predominance of less virulent strains in patients with other risk factors for morbidity and mortality or inactivation of neutrophil products needed for host defence.

Serotyping and genotyping of genital Chlamydia trachomatis isolates reveal variants of serovars Ba, G, and J as confirmed by omp1 nucleotide sequence analysis.

Urogenital isolates (n = 93) of Chlamydia trachomatis were differentiated into serovars and variants by serotyping with monoclonal antibodies and genotyping by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified omp1 gene, respectively. The types of 87 of the 93 isolates (94%) were identical, as determined by both methods. Among these 87 isolates, 3 isolates were identified as the recently described new serovariant Ga/IOL-238 by omp1 nucleotide sequence analysis of the variable domains. Of the remaining six isolates, three isolates serotyped as both L2 and Ba but were identified as Ba/A-7 by genotyping by RFLP analysis of omp1. The omp1 nucleotide sequences of variable domains VD1, VD2, and VD4 of these urogenital Ba strains were identical to the sequences of the variable domains of Ba/J160, an ocular Ba type. The three remaining isolates were serotyped as J, but the patterns obtained by RFLP analysis of omp1, which were identical for the three isolates, differed from that of prototype serovar J/UW36. omp1 nucleotide sequence analysis revealed that these strains are genovariants of serovar J/UW36. Nucleotide sequence differences between serovar J/UW36 and this J genovariant, designated Jv, were found in both variable and constant domains. In conclusion, this study shows that the PCR-based genotyping of clinical C. trachomatis isolates by RFLP analysis of omp1 has a higher discriminatory power and is more convenient than serotyping. Variants of C. trachomatis serovars Ba, G, and J were identified and characterized.

Improvement of growth of Chlamydia pneumoniae on HEp-2 cells by pretreatment with polyethylene glycol in combination with additional centrifugation and extension of culture time.

The following adaptations led to improved growth of Chlamydia pneumoniae on HEp-2 cells compared to that by the standard method: monolayer preincubation with 7% polyethylene glycol (PEG), extension of incubation time to 7 days, and extension of incubation to 7 days in combination with centrifugation on days 3, 4, and 5. These adaptations resulted in approximate increases in numbers of inclusion-forming units (IFU) of 2-, 5-, and 69-fold, respectively. A combination of preincubation with PEG, prolonged incubation, and centrifugation on days 3, 4, and 5 increased the numbers of IFU >300-fold. This is therefore recommended as the optimal method for culturing C. pneumoniae.

Effect of an acidic environment on the susceptibility of Helicobacter pylori to trospectomycin and other antimicrobial agents.

The susceptibility of 30 clinical isolates of Helicobacter pylori to trospectomycin, ampicillin, metronidazole, clarithromycin, azithromycin and clindamycin under varying pH conditions was evaluated. An acidic environment was shown to affect unfavourably the activity of all the antimicrobial agents tested. This pH effect was most marked for the two macrolides and for clindamycin.

Diversity in lipid A binding ligands: comparison of lipid A monoclonal antibodies with rBPI23.

1. Mabs with a high affinity for free lipid A do not bind when it is covalently linked, i.e. in the form of LPS. 2. Lipid A-binding Mabs may be divided into three categories: I. Monoreactive Mabs that bind to the hydrophillic backbone of lipid A II. Polyreactive Kdo Mabs III. Polyreactive Mabs that bind by hydrophobic interactions 3. rBPI23 binds either free or covalently linked lipid A.

Comparative in-vitro activity of piperacillin-tazobactam against recent clinical isolates, a Dutch national multicentre study.

In this Dutch national survey piperacillin-tazobactam had a MIC90 of < or = 8 mg/L for Enterobacteriaceae (Enterobacter cloacae excluded) and Pseudomonas aeruginosa, 64 mg/L for E. cloacae, and 4 mg/L for Acinetobacter spp. The corresponding MIC90 values for piperacillin alone were < or = 256 mg/L, 256 mg/L, 16 mg/L and 32 mg/L. Only 15 of 93 piperacillin-tazobactam resistant E. cloacae strains were sensitive for ceftazidime, whereas 41 of 93 ceftazidime-resistant E. cloacae were sensitive to piperacillin-tazobactam.

Utilization of haem from the haptoglobin-haemoglobin complex by Bacteroides fragilis.

Possession of specialized iron acquisition systems is a prerequisite for the survival of pathogenic bacteria in their host. The purpose of this study was to determine whether Bacteroides fragilis, a clinically important Gram-negative anaerobic bacterium, possesses a specific haem-uptake system. Growth studies indicated that this microorganism can utilize haem from either haemoglobin or haptoglobin-haemoglobin as its sole source of iron. Iron-repressible haem-binding protein complexes (HBP complexes), involved in the uptake of haem from haptoglobin-haemoglobin were detected by means of lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE). Four polypeptides of approximately 60, 58, 49 and 35 kDa, which are part of these HBP complexes, were identified as haem-binding proteins. A 44 kDa iron-repressible outer-membrane protein is needed for a functional HBP complex, but the exact role of this protein in the uptake of haem is still unknown.

[Successful culture of Chlamydia pneumoniae in 2 asymptomatic patients]. / Succesvolle kweek van Chlamydia pneumoniae bij twee asymptomatische patiënten.

In two women, partners of 41 and 36 years old, positive cultures of Chlamydia pneumoniae were repeatedly obtained from the throat. Both individuals were asymptomatic during the period described. Eradication was not achieved despite treatment with doxycycline. C. pneumoniae on the basis of serological data is considered a common cause of respiratory tract infections. However, this micro-organism has only been isolated in the Netherlands once. Carrier status has been described in the literature, although its frequency is unknown.

Recombinant human bactericidal/permeability-increasing protein (rBPI23) is a universal lipopolysaccharide-binding ligand.

A recombinant 23-kDa protein (rBPI23) derived from human bactericidal/permeability-increasing protein (BPI) possesses potent endotoxin-neutralizing abilities in vitro and in vivo. Binding of rBPI23 to those endotoxins (lipopolysaccharides [LPSs]) encountered clinically would be a prerequisite for efficacy in decreasing mortality among patients suffering from gram-negative sepsis and shock, a disease state in which an etiological role for LPS has been implicated. rBPI23 binds well to lipid A (n = 7), to rough-mutant O-chain-deficient LPS (n = 18, Re to Ra chemotypes), to lipid A-core covalently linked to the O chain, to LPSs from clinically relevant serotypes (n = 100), and to bacterial cells (n = 88) of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, the species most often implicated in clinical gram-negative sepsis and shock. Significant binding of rBPI23 to these antigens took place at rBPI23 concentrations of 1 to 500 ng/ml (median, 16 to 32 ng/ml). Binding did not involve 3-deoxy-D-manno-octulosonate of the inner core. Determining the exact epitope recognized by rBPI23 would require further studies with synthetic lipid A substructures. The demonstrated ability of rBPI23 to universally bind LPS provides a sound basis for further testing of its endotoxin-neutralizing abilities, including clinical trials.

A multicenter survey of resistance in The Netherlands using the Etest.

To obtain data about the prevalence of resistance in bacterial isolates causing serious infections in the Netherlands, a multicenter survey was carried out using the Etest for quantitative susceptibility testing. More than 6000 isolates belonging to ten species were tested against eight antibiotics. Moreover, the Etest was validated against the agar dilution method and the reproducibility of the Etest was studied. In spite of pH differences between the agar plates used for Etesting and agar dilution testing, a good correlation (that is 86%-97% within log2 dilution steps) was found between both methods. Comparison of Etest values of the participating laboratories and the reference laboratory showed > 80% conformity within 1 log2 dilution step and 90% within 2 log2 dilution steps, indicating a sufficient reproducibility of the Etest. Resistance percentages were low for most species and antibiotics, relatively high percentages (10%-20%) indicating natural insusceptibility rather than development or increase of resistance.

The role of neuraminidase in haemagglutination and adherence to colon WiDr cells by Bacteroides fragilis.

The role of neuraminidase in haemagglutination and adherence to colon WiDr cells by eight strains of Bacteroides fragilis and four strains of oral black-pigmented gram-negative anaerobes was studied. Neuraminidase treatment resulted in a very small increase of haemagglutination by some of the strains but had no effect on adherence to WiDr cells by all bacterial strains tested except one strain of Prevotella intermedia (HG 110). Inhibition of neuraminidase had no effect on haemagglutination or adherence, nor was any correlation found between haemagglutinating ability and neuraminidase activity in the B. fragilis strain. The results indicated that haemagglutination and adherence of B. fragilis to WiDr cells were not mediated by neuraminidase.

Lactoferrin is a lipid A-binding protein.

Lactoferrin (LF), a cationic 80-kDa protein present in polymorphonuclear leukocytes and in mucosal secretions, is known to have antibacterial effects on gram-negative bacteria, with a concomitant release of lipopolysaccharides (LPS, endotoxin). In addition, LF is known to decrease LPS-induced cytokine release by monocytes and LPS priming of polymorphonuclear leukocytes. Its mechanism of action is incompletely understood. We have now demonstrated by in vitro-binding studies that LF binds directly to isolated lipid A and intact LPS of clinically relevant serotypes of the species which most frequently cause bacteremia (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa), as well as to lipid A and LPS of mucosal pathogens (among others, Neisseria meningitides and Haemophilus influenzae). Binding to LPS was inhibitable by lipid A and polymyxin B but not by KDO (3-deoxy-D-manno-octulosonate), a glycoside residue present in the inner core of LPS. Binding of LF to lipid A was saturable, and an affinity constant of 2 x 10(9) M-1 was calculated for the LF-lipid A interaction. Our data may explain, in part, the mechanism whereby LF exerts its antibacterial and anti-endotoxic effects. Further studies on the ability of LF to block the detrimental effects of LPS, both in vitro and in vivo, are warranted.

Frequencies of lipopolysaccharide core types in Escherichia coli strains from bacteraemic patients.

We have investigated the distribution of the various core types (R1, R2, R3, R4 and K-12) in 138 Escherichia coli isolates obtained from positive blood cultures. Rabbit antisera, raised against five rough strains expressing the respective core types, were made monospecific by extensive absorption. The reactivity of the antisera was tested in ELISA with bacterial cells that had been autoclaved for full exposure of core epitopes. One hundred and thirty strains could be typed directly, while eight strains required prior digestion with proteinase K for removal of cross-reactions. Ninety-four of the strains (68%) expressed the R1 type, and 9 (6.5%), 12 (8.7%), 7 (5.1%) and 3 (2.2%) strains expressed the R2, R3, R4 and K-12 core types, respectively. An R1R4 mixed core type, hitherto not yet described, was found in 13 (9.4%) strains. Results obtained with polyclonal antisera were in agreement with those obtained with monoclonal antibodies to the R1, R2 and R3 core types. Core typing may serve as an additional serological marker next to conventional typing of O-, H- and K-antigens.

Binding studies of a monoclonal antibody specific for 3-deoxy-D-manno-octulosonic acid with a panel of Klebsiella pneumoniae lipopolysaccharides representing all of the O serotypes.

A monoclonal antibody (MAb) raised against Salmonella minnesota R595 and specific for alpha-3-deoxy-D-manno-octulosonic acid (alpha-Kdo) of the inner core was tested for binding to lipopolysaccharides (LPS) of Klebsiella pneumoniae. The MAb was tested in several assay systems (enzyme-linked immunosorbent assay, passive hemolysis, and inhibition of passive hemolysis) with a large panel (n = 23) of K. pneumoniae LPS representing all nine currently known O serotypes. MAb 20 showed reactivity with almost all O serotypes of K. pneumoniae LPS, and this reactivity could be inhibited by synthetic Kdo. This suggests an epitope in the cores of these Klebsiella LPS much like that in the inner core of LPS of S. minnesota. Large differences in reactivity between LPS of different strains belonging to the same O serotype were observed. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LPS followed by immunoblotting, reactivity of MAb 20 was observed only with the fast-moving fraction possibly representing the nonsubstituted core. No binding was seen with the high-molecular-weight fraction that contained core material substituted with several units of O-antigen building blocks. The chemical basis for these differences in reactivity remains to be established. As far as we know, this is the first report containing comprehensive immunochemical data on the LPS core of K. pneumoniae.

Direct PCR enables detection of Mycoplasma pneumoniae in patients with respiratory tract infections.

The sensitivities of three methods of detection of Mycoplasma pneumoniae by a 16S rDNA PCR were compared by using a serial dilution of M. pneumoniae. These methods consisted of a PCR performed directly on cells after a proteinase K pretreatment (direct PCR), a PCR after purification of nucleic acids (DNA-PCR), and a PCR with rRNA sequences as the target after reverse transcription. The direct PCR and the reverse transcription PCR had a sensitivity of 1.5 CFU (approximately 250 genomes). By purification, a 10-fold loss of target DNA occurred, as shown by a 10-fold decrease in sensitivity (15 CFU) of the DNA-PCR. The presence of an excess of human background DNA did not influence the sensitivity of either PCR. The direct PCR was evaluated on samples from patients with respiratory complaints. Direct PCR amplification was possible in 94.9% of the samples, which were tested by amplification of a 326-bp fragment of the beta-globin gene, which was performed to test for the suitability of amplification. Nucleic acid purification was performed on the beta-globin-negative samples, after which only 2% remained negative. A positive correlation between the direct M. pneumoniae PCR and serology, as tested by the microparticle agglutination assay (MAG assay), was found in 88.1% of the cases. A positive MAG assay result was found for samples from 10 (17%) of the patients; samples from 6 (10.2%) of these patients were also positive by PCR. Samples from three patients were found to be positive by the M. pneumoniae PCR and negative by the MAG assay. Persistence of M. pneumoniae, as detected by PCR was observed in three patients. These results indicate that the direct PCR with 16S rDNA could prove to be useful in the detection of M. pneumoniae in respiratory tract samples, although more studies are needed to evaluate the correlation between clinical symptoms and positive test results.

Comparison of Vitek and Cobas Micro systems with a semiautomated conventional microsystem for identification and susceptibility testing of gram negative bacilli.

AIMS: To compare the sensitivity and specificity of two semiautomated systems against a conventional (MIC 2000) test system for the identification and antibiotic susceptibility of Gram negative bacteria. METHODS: Clinical isolates of Gram negative bacilli (188 urinary and 229 non-urinary strains) were identified and tested for antibiotic susceptibility in the Cobas Micro and MIC 2000 systems. Of these, 359 strains were then tested in the Vitek and MIC 2000 systems. Two hundred and forty three strains were tested in all three systems immediately after isolation. Forty three were also tested only in the Vitek and MIC 2000 systems immediately after isolation. The remaining 174 strains were tested after storage at -20 degrees C for several months. RESULTS: The Cobas Micro and MIC 2000 systems agreed on the identification of 310 of the 417 (74.3%) strains; the Vitek and MIC 2000 systems agreed on 338 of the 359 (94.2%) strains. The Cobas Micro system correctly identified 86.8% of strains tested after storage and 65.4% of those immediately after isolation. Organism-antibiotic combinations (non-urinary isolates) were tested in the Cobas Micro and MIC 2000 systems (n = 2335), in the Vitek and MIC 2000 systems (n = 999). Essential correlation (complete agreement plus minor errors) was observed in 98% (with 90% complete agreement) in the former and in 97% (with 86% complete agreement) in the latter. For the urinary isolates, 1949 organism-antibiotic combinations were analysed in the Cobas Micro and MIC 2000 systems where complete agreement was observed in 92% (with 3% very major discrepancies), for 1382 urinary organism-antibiotic combinations tested in the Vitek and MIC 2000 systems, the figures were 95% and 2%, respectively. CONCLUSIONS: The Vitek system is highly accurate in the identification and antibiotic susceptibility testing of Gram negative bacteria. The Cobas Micro system has many shortcomings in its identification of Gram negative rods, especially freshly isolated strains, but it is comparable with the Vitek system in antibiotic susceptibility testing.

Antigenic and immunogenic differences in lipopolysaccharides of Escherichia coli J5 vaccine strains of different origins.

Escherichia coli strain J5 mutants of various origins have often been used as vaccines for induction of cross-reactive, cross-protective antibodies directed against the lipopolysaccharide (LPS) core region. The antigenic composition of LPS from J5 strains of different origin, i.e. strains J5(U), J5(UK), J5(2877) and J5(a), was investigated using monoclonal antibodies (mAbs) reactive only with LPS of a given chemotype, i.e. one specific for the incomplete E. coli core of the Rc chemotype, a second mAb reactive only with the E. coli R3 complete core, and a third specific for the O-antigen of E. coli serovar O111. The LPS of strains J5(U) and J5(a) is almost exclusively composed of LPS of the Rc chemotype, LPS of the J5(UK) strain is composed of Rc LPS and R3 complete core, while LPS of the J5(2877) strain contains Rc, R3 complete core and O-antigen. Growth of the bacteria in medium supplemented with galactose led to increased expression of complete core. The immune responses to the various strains were investigated. Antiserum to the J5 strain expressing the largest amount of R3 core [J5(UK)] had much higher anti-R3 LPS antibody titres compared to antiserum to the other strains. mAb 53, representative of the anti-R3 response to J5 strains containing complete core, bound to those E. coli LPS expressing the R3 core. Thus, the R3 LPS, present in some J5 vaccine strains is at least partially responsible for some of the cross-reactivities exhibited by some anti-J5 antisera.(ABSTRACT TRUNCATED AT 250 WORDS)

Migration of rat peritoneal cells after intra-abdominal infection with Bacteroides fragilis and Escherichia coli.

A fibrin clot model for intra-abdominal abscess formation was used to study the migratory properties of peritoneal cells from rats during the early stages of infection. Peritoneal cells and fibrin clot remnant were harvested 6 h after implantation of a sterile, singly infected (Escherichia coli or Bacteroides fragilis) or mixed infected (E. coli and B. fragilis) fibrin clot. Histological study of fibrin clots, removed 6 h after implantation, showed a deeper infiltration by host cells of B. fragilis infected clots compared to the others. This difference in infiltration by peritoneal cells was not due to differences in fibrinolytic activity of the bacterial strains. Differential cell counts of the peritoneal cells from rats implanted with sterile, singly and mixed infected fibrin clots showed distribution over subpopulations to be independent of the bacterial content of the infected clots used. In vitro migration assays showed no significant differences in migration by peritoneal cells from rats implanted with clots containing a different bacterial composition. Since B. fragilis infected fibrin clots were more deeply infiltrated by host defence cells than the other clots, and only mixed infected clots led to persistent abscesses in this model, we conclude that local conditions within the fibrin matrix rather than intrinsic cellular capacities of the host cells are important for the process of abscess formation.