An enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies to different parts of the gram-negative lipopolysaccharide core region.

Bovine serum albumin was complexed with the core antigens of either Escherichia coli J5 LPS, Salmonella minnesota R595 LPS or E. coli lipid A. These core-BSA complexes were used for solid-phase coating in ELISAs for anti-core antibodies. Antibodies, binding to various parts of the core region were easily quantified in a single experimental set-up, which was hitherto not possible. The ELISA has only 3 incubation steps and is not costly as only moderate amounts of the core antigens (i.e., 1 microgram per test) were needed for coating. The sensitivity proved to be excellent and the complexes were biologically fully active (compared to native, smooth LPS), which make them suitable for the screening (after fusion) of monoclonal anti-core antibodies. Another possible application is the large-scale screening of blood-bank sera in order to find samples with a high anti-core antibody content.

Murine ascitic fluids contain varying amounts of an inhibitor that interferes with complement-mediated effector functions of monoclonal antibodies.

The ability of murine monoclonal antibodies (mAbs), directed to the inner core of Gram-negative bacterial lipopolysaccharide (LPS, endotoxin), to enhance complement-mediated killing of bacteria, was investigated. The mAbs were tested as present in ascitic fluid. It was found that ascites contains an factor that inhibited the activity of complement. This effect was evident in assays for complement-mediated lysis of antibody-coated Gram-negative bacteria (bacterial killing) or of opsonised red blood cells. Moreover, the amount of inhibitor was found to vary from one ascites to another and spanned a 60-fold range. Thus, in vitro or in vivo experiments where complement is known to play a determining role may yield incorrect results when ascites is used as a source of antibody; the use of ascites prepared from irrelevant antibody as a negative control does not eliminate this problem.

Application of chromosomal restriction endonuclease digest analysis for use as typing method for Clostridium difficile.

The usefulness of restriction endonuclease analysis of chromosomal DNA as a typing method for Clostridium difficile was tested. Over four months all faecal samples were routinely cultured for C difficile. DNA of all isolated strains was isolated and tested with the restriction endonuclease Hind III. The patterns obtained after electrophoresis in agarose gels seemed to be strain specific. Antibiotic susceptibility profiles agreed with the results of the restriction endonuclease analysis, though they were much less discriminating. Analysis of the results indicated that restriction endonuclease analysis is a suitable typing method for C difficile, which may be very valuable in epidemiological studies where a highly discriminating typing method is needed.

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.

Killing of Escherichia coli by human polymorphonuclear leucocytes in the presence of Bacteroides fragilis.

The inhibitory effect of Bacteroides fragilis on the in vitro killing of Escherichia coli by polymorphonuclear leucocytes was studied with two pairs of E coli and B fragilis isolated from human wound infections. Both B fragilis strains behaved similarly: they inhibited the killing of one E coli strain, while the killing of the other E coli strain was not affected. The different behaviour of the two E coli strains depended on their need for fresh serum in the killing by the polymorphonuclear leucocytes. The inhibitory effect of the B fragilis strains could be completely accounted for by their effect on complement.

Interactions between polymorphonuclear leucocytes, Bacteroides sp, and Escherichia coli: their role in the pathogenesis of mixed infection.

Five Bacteroides fragilis strains and five Bacteroides vulgatus strains were compared with regard to their ability to consume complement and to fix C3, their killing by polymorphonuclear leucocytes, and their ability to inhibit the bactericidal effect of serum and polymorphs on Escherichia coli strains. Complement consumption was positively related to C3 fixation, but no relation was observed between these variables and the killing of the anaerobes. Greatest inhibition of the killing of E coli by serum and polymorphs was achieved with the bacteroides strains that fixed most complement. The greater virulence of B fragilis in mixed infections with E coli was not reflected either by a greater ability to inhibit the killing of E coli or a greater resistance of the anaerobes themselves to the bactericidal effect of serum and polymorphs.

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.

Purification and partial characterization of an iron regulated outer membrane protein of B. fragilis under non-denaturing conditions.

The CHAPS-PAGE gelsystem we applied gave a good separation of the proteins of Bacteroides fragilis under non-denaturing conditions. We succeeded with preparative CHAPS-PAGE in purifying an iron regulated outer membrane protein (a 44 kDa polypeptide on SDS-PAGE) of B. fragilis. This integral membrane protein proved to be a lipopolysaccharide binding protein with an isoelectric point of approximately pH 5.5. This method of purifying membrane proteins could be an important step in research into the function of membrane proteins.

Early events after intra-abdominal infection with Bacteroides fragilis and Escherichia coli.

Growth of Bacteroides fragilis and Escherichia coli was monitored during early stages of single (mono-) and mixed intra-abdominal infection in a rat fibrin clot model. When B. fragilis and E. coli were together involved in the infection, B. fragilis numbers increased about 6 h after an initial decline. This increase was not found with B. fragilis mono-infections. The numbers of E. coli increased rapidly in both mono- and mixed infections and stayed high for several days, but only mixed infection resulted in abscesses that persisted for more than 7 days. Macrophages, the main component of the peritoneal cellular defence mechanism, were outnumbered by polymorphonuclear leucocytes during the first 6 h of infection. Further characterisation of the macrophage population by means of monoclonal antibodies showed a shift from resident to exudate macrophages as the result of influx of the latter.

Uropathogenic properties of Proteus mirabilis and Proteus vulgaris.

A group of faecal isolates of Proteus vulgaris and P. mirabilis was studied for the presence of possible virulence factors such as growth rates in urine and broth, haemolysin production, hydrophobicity, sensitivity to the bactericidal activity of human serum and cell invasiveness. Differences were found in haemolysin production, cell invasiveness and experimental virulence in a mouse model. These differences might explain why P. mirabilis is much more common in human urinary-tract infections than P. vulgaris.

Novobiocin resistance and virulence of strains of Staphylococcus saprophyticus isolated from urine and skin.

A method was developed to study virulence of coagulase-negative staphylococci. Our results showed that coagulase-negative staphylococci injected into adult mice by the intracerebral route did not give rise to lethal infections, whereas mice aged 2 days were much more susceptible. Novobiocin-resistant strains of Staphylococcus saprophyticus were more virulent than strains of Staphylococcus epidermidis. Strains of S. saprophyticus biotype 3 of Baird-Parker's classification varied in virulence according to novobiocin sensitivity. In the classification of Kloos and Schleifer, S. saprophyticus biotype 3 can be subdivided into four distinct staphylococcal species, namely S. saprophyticus , S.cohnii, S.haemolyticus and S.warneri. S. chonii and S. saprophyticus were equally virulent for mice aged 2 days, but novobiocinsensitive S. haemolyticus was less virulent. On epidemiological grounds, however, it would seem that S. saprophyticus has some undefined advantage in invading the urinary tract.

Pathogenic synergy between Escherichia coli and Bacteroides fragilis: studies in an experimental mouse model.

An animal model is described for quantitative evaluation of pathogenic synergy between Escherichia coli and Bacteroides fragilis in which adjuvants were not required for abscess formation. Two sets of strains of E. coli and B. fragilis isolated from human wound infections were tested. Pathogenic synergy was observed in only one of the two combinations and was dependent on properties of E. coli.

Chemiluminescence of human leukocytes stimulated by clinical isolates of Klebsiella.

Chemiluminescence (CL) of human polymorphonuclear leukocytes was determined after stimulation with 89 clinical isolates of Klebsiella which differed in serotype and in their virulence for mice. With K1, K2, K4 and K5 strains, a significantly lower CL response was observed than with K3, K6 and K greater than 6 strains. These results correlated well with virulence: greater virulence could be explained by greater resistance to phagocytosis.

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.

Pathogenic synergy between Escherichia coli and Bacteroides fragilis or B. vulgatus in experimental infections: a non-specific phenomenon.

The virulence of Bacteroides fragilis and B. vulgatus for mice was compared in a skin-infection model. These strains were also tested for pathogenic synergy in mixed infections with Escherichia coli. Strains of B. fragilis were generally more virulent than strains of B. vulgatus and, with one exception, the effect of Bacteroides strains in mixed infections merely reflected their inherent virulence.

The role of K antigens as virulence factors in Klebsiella.

The importance of K antigen of Klebsiella as a virulence factor was studied in nine pairs of K+ and K- strains, each pair isogenic apart from the presence of K antigen. Loss of K antigen by nine K+ strains resulted in the reduced virulence of their K- variants in a mouse-skin model. This reduced virulence of K- strains for mice may be explained in all strains by a higher degree of phagocytosis as measured by chemiluminescence response of human polymorphonuclear leukocytes (PMNL) and in most strains by enhanced killing by either human PMNL or human serum or both. Although the protective role of the K antigen in serum-induced killing and killing by PMNL was generally evident, our results also suggested that other virulence factors were sometimes involved.

Effect of Bacteroides fragilis cellular components on chemotactic activity of polymorphonuclear leukocytes towards Escherichia coli.

Chemotaxis of polymorphonuclear leukocytes (PMNL) in response to cell components of Bacteroides fragilis alone or in combination with Escherichia coli was evaluated. E. coli produced much more powerful chemotactic factors than B. fragilis. The culture filtrate (CF), outer membrane (OM) preparation, and lipopolysaccharide (LPS) of B. fragilis slightly stimulated chemotactic activity of PMNL. The culture filtrate and OM preparation were capable of inhibiting the chemotaxis of PMNL in response to the chemotactic factors of E. coli but LPS of B. fragilis was not able to do so. Reduction by B. fragilis of PMNL chemotaxis in response to E. coli was not specific for B. fragilis but also occurred in the presence of facultative bacteria. In parallel with chemotaxis, lysozyme release, but not beta-glucuronidase release, by PMNL was significantly stimulated by E. coli but not by B. fragilis.

Polymorphonuclear leukocyte chemotaxis by mixed anaerobic and aerobic bacteria.

The induction of chemotactic activity of polymorphonuclear leukocytes (PMNL) by anaerobic and aerobic bacteria alone or in combination was evaluated. Washed cells as well as the supernate of Proteus mirabilis were chemotactic for leukocytes. The supernate of cultures of two strains of Bacteroides fragilis contained small amounts of chemotactic factors. No chemotactic factors were released from the non-fragilis Bacteroides strains. The supernates of cultures of anaerobic bacteria were capable of inhibiting chemotaxis of leukocytes to the chemotactic factors of P. mirabilis. P. mirabilis and two strains of B. fragilis generated chemotactic factors in serum but none of the other Bacteroides spp. tested were able to induce serum chemotactic factors.

Haemolysis by urinary Escherichia coli and virulence in mice.

The influence of haemolysin production on virulence was studied in an experimental mouse model. Urinary strains of Escherichia coli can be divided into three virulence groups by determining their kinetics in the mouse kidney after intravenous injection. Virulent strains of groups II and III were more often haemolytic than avirulent group-I strains. Haemolytic virulent strains often caused haemoglobinuria in the mice, and killed the mice more rapidly than did non-haemolytic virulent strains. No relationship was found between alpha-haemolytic activity and virulence in wild-type haemolytic strains. When haemolysin production was reduced or eliminated by treatment with actinomycin-D or rifampicin, six out of seven group-II strains tested gave the same results as avirulent group-I strains. However, the kinetics in the mouse kidney of four haemolytic group-III strains tested was not changed after reduction or elimination of haemolysin production; only a small decrease in toxicity was observed. It is concluded that haemolysin production by E. coli is a decisive virulence factor in most of the mouse-nephropathogenic group-II strains, but not in the virulent group-III strains.

Virulence of Klebsiella strains in experimentally induced skin lesions in the mouse.

The virulence of 93 clinical isolates of Klebsiella was compared in a mouse model by subcutaneous injection. Skin pathogenicity was measured by estimating the number of viable bacteria in the lesions 24 h after infection with a dose of 10(7) bacteria. Strains of serotypes K1-6 were compared with strains of serotypes higher than K6. All K1 and K5, and some K2 and K4 strains were more virulent for mice than strains with a serotype higher than K6. The K3 strains were significantly less virulent than the strains with a serotype higher than K6. The bacteriological findings were confirmed by histological examination with some strains. No differences in virulence were observed between strains of the same serotype isolated from patients with cystitis or from those with pyelonephritis, nor between strains of the same serotype isolated from the blood of patients with septicaemia or from other sites. The mouse model has been found satisfactory for observing differences in virulence between Klebsiella isolates.