A. Brauner

Interleukin-6 and interleukin-8 in the urine of children with acute pyelonephritis

Interleukin-6 (IL-6) and interleukin-8 (IL-8) are important mediators of the inflammatory response in serious bacterial infections. We studied the levels of these two cytokines (standardised for urinary creatinine) in the urine of infants and children during and 6 weeks after acute pyelonephritis and in non-renal febrile controls and healthy children without apparent infection. IL-6 was detected in the urine of 52% of children with pyelonephritis compared with 15% of other children (P<0.001). The median urinary IL-6 level in acute pyelonephritis was 4 pg/μmol compared with undetectable levels in the control group (P<0.001). IL-8 was detected in 98% of children with pyelonephritis and 42% of other children (P<0.001). The median concentration of IL-8 was 188 pg/μmol in pyelonephritis; it was undetectable in controls (P<0.001). IL-8 levels were higher in children less than 1 year of age (P<0.001).

Bacteriuria, Bacterial Virulence and Host Factors in Diabetic Patients

The prevalence of bacteriuria as well as bacterial virulence and host factors were studied in 514 diabetic outpatients and 405 nondiabetic controls. The prevalence of bacteriuria was not significantly higher in diabetic women (15/239, 6.3 %) than in age‐matched nondiabetic women (8/236, 3.4 %). In diabetic and nondiabetic men, the prevalence was also similar but lower than in women. E. coli was found in 55 % of urine cultures with significant growth from diabetic patients, while in 91 % of positive cultures from nondiabetic controls. Most E. coli strains lacked ability of P‐fimbriae‐mediated adhesion and aerobactin‐mediated iron uptake, indicating low bacterial virulence. Long‐term metabolic control (HbA1c), prevalence of retinopathy, neuropathy and previous foot ulcers were similar in bacteriuric and nonbacteriuric diabetic patients, matched according to gender, age, and duration of diabetes. Renal function was also similar, though the frequency of proteinuria and elevated blood pressure tended to be higher in the bacteriuric than in the noninfected group. Eight‐three percent of the bacteriuric patients reported previous urinary tract infections but only 61 % of nonbacteriuric patients (p = 0.07). As compared to non‐diabetic women, diabetic women reported significantly more previous urinary tract infections (p < 0.01). In conclusion, the prevalence of bacteriuria in diabetic outpatients was not significantly higher than in non‐diabetic outpatients or healthy volunteers. No studied host factor was clearly associated with bacteriuria in diabetic patients, although proteinuria and hypertension tended to be more common. The infecting E. coli strains were of low virulence.

PRODUCTION OF CYTOTOXIC NECROTIZING FACTOR, VEROCYTOTOXIN AND HAEMOLYSIN BY PYELONEPHRITOGENIC ESCHERICHIA COLI

Two hundred and thirty-two strains ofEscherichia coli isolated from children with non-obstructive acute pyelonephritis (n=65), women with non-obstructive acute pyelonephritis (n=63) and the faecal flora of healthy children (n=33) and adults (n=71) were examined for cytotoxic necrotizing factor production, haemolysin synthesis, verocytotoxin production and expression of mannose-resistant haemagglutination of human erythrocytes. Forty-eight per cent of the pyelonephritogenicEscherichia coli strains produced cytotoxic necrotizing factor and 61 % produced haemolysin compared to 25 % and 27 % of faecal control strains (p<0.001 and p<0.001 respectively). Cytotoxic necrotizing factor production did not occur among the non-haemolyticEscherichia coli strains which confirms the close association between these two toxic factors. The bacterial phenotypes producing both haemolysin and cytotoxic necrotizing factor, and the phenotype expressing both these toxic factors and mannose-resistant haemagglutination occurred significantly more often in pyelonephritogenic strains than in faecal isolates (p<0.001). Haemolytic strains without the ability to produce cytotoxic necrotizing factor were more common in faecal isolates than in uropathogenic strains (p=0.05). Strains lacking the ability to synthesize both these toxins were also over-represented in faecal isolates (p<0.01).

Serum resistance in Escherichia coli strains causing acute pyelonephritis and bacteraemia.

The capacity of Escherichia coli to resist the bactericidal action of serum was examined in 367 clinical isolates obtained from children with acute pyelonephritis (n = 57), adults with acute pyelonephritis (n = 55), non‐diabetic patients with bacteraemia (n = 101), diabetic patients with bacteraemia (n = 65) and from the faecal flora of healthy controls (n = 89). The incidence of serum‐resistant E. coli strains was significantly higher in pyelonephritogenic strains from children and adults (93% and 82%) as compared to faecal control strains (57%, p < 0.001 and p< 0.005 respectively). Strains causing bacteraemia in non‐diabetic and diabetic patients were more often serum resistant (72% and 80%) as compared to control strains (p < 0.05 and p < 0.001 respectively). The frequency of serum‐sensitive strains was similar in diabetic patients with decreased renal fuction or proteinuria compared to those with normal renal function. There were no significant correlations between serum resistance of E. coli and expression of P fimbriae, type I fimbriae or mannose‐resistant haemagglutination, cell surface hydrophobic properties, production of aerobactin, haemolysin or cytotoxic necrotizing factor in 53 pyelonephritogenic strains from adult patients.

Relative importance of eight virulence characteristics of pyelonephritogenic Escherichia coli strains assessed by multivariate statistical analysis.

We have previously reported univariate statistical analysis of the prevalences of putative virulence determinants in Escherichia coli isolated from children and adults with acute pyelonephritis. The expression of P-fimbriae, cell surface hydrophobicity, mannose resistant haemagglutination, haemolysin synthesis, cytotoxic necrotizing factor production and aerobactin mediated iron uptake occurred more often in a collection of 115 Escherichia coli strains isolated from children and women with acute non-obstructive pyelonephritis compared to 96 strains isolated from the commensal fecal flora. With the aim to study which of these virulence markers were independently associated with strains causing infection we performed a multivariate statistical analysis with the data from these strains. The previously proposed virulence factors, expression of type 1 fimbriae and adhesion to HeLa cells were also included in the analysis. P-fimbriae, mannose resistant haemagglutination and the production of haemolysin were, in the multivariate analysis, associated with strains isolated from patients with acute pyelonephritis.

P-fimbriation and haemolysin production are the most important virulence factors in diabetic patients with Escherichia coli bacteraemia: A multivariate statistical analysis of seven bacterial virulence factors

Diabetic patients, as compared to non-diabetic subjects, run an increased risk of acquiring Gram-negative bacteraemia. We therefore studied the prevalence and coexpression of seven bacterial virulence markers of 69 Escherichia coli strains isolated from 64 bacteraemic patients with diabetes mellitus and 67 E. coli strains from faeces of healthy controls. The strains were analyzed for haemolysin (HLY) production, aerobactin-mediated iron uptake (AMI), cytotoxic necrotizing factor (CNF) production, expression of cell surface hydrophobicity, P-fimbriae, mannose-resistant haemagglutination (MRHA) and mannose-sensitive haemagglutination (MSHA). All bacterial properties were significantly more common among the bacteraemic strains (P < 0.02 vs. controls). Correlations between HLY and CNF (P < 0.0004) and between P-fimbriae and MRHA (P < 0.0001), MSHA (P < 0.0002) or AMI (P < 0.05), as well as between MRHA and MSHA (P < 0.0005) were observed. In patients with proteinuria, as sign of diabetic complications in the urinary tract, HLY-negative strains, P-fimbriae-negative strains, and strains which were both HLY-/CNF-negative, were more common (P = 0.04, P < 0.01 and P = 0.048, respectively). Using a multivariate statistical analysis, production of HLY and the expression of P-fimbriae were the two virulence factors with the highest discrimination between bacteraemic and control strains. In conclusion, all virulence factors studied were more prevalent in bacteraemic than in control strains, although HLY and P-fimbriae were shown to be of greatest and independent importance. Low virulent strains (P-fimbriae-, HLY- and CNF-negative) were more prevalent in diabetic patients with signs of renal complications.

Immunoglobulin G antibodies toPseudomonas aeruginosa lipopolysaccharides and exotoxin A in patients with cystic fibrosis or bacteremia

IgG antibodies to ninePseudomonas aeruginosa lipopolysaccharides (LPS) and exotoxin A in sera from 11 patients with bacteremia and 51 patients with cystic fibrosis (CF) were analyzed. The methods used were enzyme immunoassay (EIA) and immunoblotting. Nine of the 11 bacteremic patients were infected with strains expressing an LPS serotype identical to one of the test antigens. In sera from six of these nine patients, antibody homologous to the serotype of the infecting strain was observed. An antibody response to heterologousPseudomonas aeruginosa LPS antigens was observed in nine patients. Eight of the bacteremic patients mounted an antibody response to exotoxin A. Thirty-five CF patients chronically colonized withPseudomonas aeruginosa possessed significantly higher levels of antibody to all of the test antigens than 16 patients with intermittent or no colonization (p<0.001). For exotoxin A and serotype 3 the sensitivity was 91 % and 94 %, and the specificity 94 % and 88 % respectively. When the results for exotoxin A and serotype 3 were combined, the sensitivity was 91 % while the specificity was 81 %. The pronounced antibody response to heterologous LPS antigens, as measured by the EIA and immunoblot, suggests expression of a common antigen determinant. A simplified serological assay utilizing exotoxin A and serotype 3 as test antigens may be useful for detectingPseudomonas aeruginosa infections in patients with CF and chronic colonization and in bacteremic patients from whom cultures are not available.

Hydrophobic properties of Escherichia coli causing acute pyelonephritis

Cell surface hydrophobic properties and expression of P-fimbriae were examined in 130 strains of Escherichia coli derived from women (n = 66) and children (n = 64) with acute non-obstructive pyelonephritis and in 170 faecal strains of E. coli from healthy adults (n = 103) and children (n = 67) by use of the salt aggregation test and the P-fimbriae-specific particle agglutination test. The strains of E. coli isolated were aggregated in salt solutions of varying molarity (0.001-1.6 M final concentration). Patients with predisposing medical or urological conditions in the urinary tract were excluded. Pyelonephritic strains of E. coli from the women and children had a higher degree of cell surface hydrophobicity (80 and 98% respectively) than faecal strains from healthy adults and children (57 and 82% respectively, P less than 0.01 and P less than 0.01). Both pyelonephritic and faecal strains of E. coli from the children were more often salt aggregation positive (hydrophobic) than faecal strains of E. coli from healthy adults (P less than 0.01 and P less than 0.001, respectively). Pyelonephritic strains of E. coli from women and children were more often P-fimbriated (79 and 84% respectively) than faecal control strains from women and children (15 and 33%, P less than 0.001 and P less than 0.001, respectively) but there was no significant correlation between expression of P-fimbriae and cell surface hydrophobicity.

CELL SURFACE HYDROPHOBICITY, ADHERENCE TO HELA CELL CULTURES AND HAEMAGGLUTINATION PATTERN OF PYELONEPHRITOGENIC ESCHERICHIA COLI STRAINS

Cell surface hydrophobicity, haemagglutination pattern and adherence to HeLa cells were examined in 230 strains of Escherichia coli collected from women (n = 61 strains) and children (n = 65 strains) with non-obstructive acute pyelonephritis and in 104 faecal control strains of E. coli from healthy adults (n = 71 strains) and children (n = 33 strains). Pyelonephritogenic E. coli strains showed a significantly increased incidence of hydrophobic properties (90%) and mannose resistant haemagglutination (MRHA) of human erythrocytes (83%) than faecal control strains (64 and 23% respectively, P less than 0.001 in both cases). Mannose sensitive haemagglutination (MSHA) was observed in 48% of the pyelonephritogenic E. coli strains and in 50% of the faecal control strains (NS). The incidence of adherence to HeLa cells was low both in pyelonephritogenic and faecal control strains, 6 and 7% respectively (NS). The bacterial phenotypes MRHA + MSHA + and MRHA + MSHA- appeared significantly more often in pyelonephritogenic E. coli strains (35 and 48% respectively) than in faecal control strains (5 and 17% respectively, P less than 0.001 in both cases). The phenotype MRHA- MSHA + occurred significantly more often in control strains (45%) than in pyelonephritogenic strains (13%, P less than 0.001). Eighty-three per cent of the pyelonephritogenic E. coli strains expressing hydrophobic properties showed MRHA and 50% of the hydrophobic strains showed MSHA. There were no significant correlations between cell surface hydrophobic properties and haemagglutination pattern or adherence to HeLa cells in pyelonephritogenic E. coli strains nor in faecal control strains.

False-positive treponemal serology in patients with diabetes mellitus

In sera from 476 diabetic outpatients, positive reaction in the Fluorescent Treponemal Antibody-Absorption (FTA-Abs) test, a commonly used serological test for syphilis, was found in 36 of the patients. None of 100 healthy control subjects were positive in the FTA-Abs test. Additional treponemal and nontreponemal tests confirmed the diagnosis of syphilis in only three of the diabetic patients. In 10 of the 36 patients, the positive FTA-Abs reactivity appeared to be due to cross-reactivity between the treponemal antigen and Borrelia burgdorferi, which causes Lyme borreliosis. In the remaining 23 patients (5%), no other explanation for a false-positive FTA-Abs reactivity was found besides diabetes. Diabetic patients with false-positive FTA-Abs reactivity had similar degree of long-term metabolic control and prevalence of islet cell antibodies (ICA) as well as late diabetic complications as FTA-Abs negative diabetic patients, matched regarding to sex, age, type, and duration of diabetes. In conclusion, false-positive FTA-Abs reactivity is not rare in diabetic patients. The reason for this phenomenon is unknown, but could be a sign of autoimmunity of its own. Hence, in diabetic patients with FTA-Abs test indicating syphilis, the diagnosis must be verified with a combination of other tests.