Bengt Wretlind

Molecular Mechanisms of Fluoroquinolone Resistance in Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

ABSTRACT Twenty P. aeruginosa isolates were collected from six cystic fibrosis (CF) patients, aged 27 to 33, in 1994 (9 isolates) and 1997 (11 isolates) at the CF Center, Copenhagen, Denmark, and were typed by pulse-field gel electrophoresis (PFGE) or ribotyping. Five of the patients had isolates with the same PFGE or ribotyping patterns in 1997 as in 1994, and ciprofloxacin had a two- to fourfold higher MIC for the isolates collected in 1997 than those from 1994. Genomic DNA was amplified for gyrA, parC, mexR, and nfxB by PCR and sequenced. Eleven isolates had mutations in gyrA, seven isolates had mutations at codon 83 (Thr to Ile), and four isolates had mutations at codon 87 (Asp to Asn or Tyr). Sixteen isolates had mutations in nfxB at codon 82 (Arg to Leu). Increased amounts of OprN were found in six isolates and OprJ in eight isolates as determined by immunoblotting. No isolates had mutations in parC or mexR. Six isolates had mutations in efflux pumps without gyrA mutations. The average number of mutations was higher in isolates from 1997 than in those from 1994. The results also suggested that efflux resistance mechanisms are more common in isolates from CF patients than in strains from urine and wounds from non-CF patients, in which mutations ingyrA and parC dominate (S. Jalal and B. Wretlind, Microb. Drug Resist. 4:257–261, 1998).

Age at Acquisition of Helicobacter pylori Infection: Comparison of a High and a Low Prevalence Country

Helicobacter pylori (HP) is now generally accepted as the main aetiological agent in chronic active gastritis and peptic ulcer. Infection with HP is widespread, but the routes of transmission are still unclear. Several studies have shown increasing prevalence of antibodies against HP with age. In developing countries, age at peak incidence of seroconversion is probably considerably lower than in developed countries. We performed a cross-sectional study to determine the age at maximum incidence of seroconversion to HP in a high-prevalence country (Ethiopia) and in a low-prevalence country (Sweden). Sera from 242 Ethiopian children, aged 2-14 years and from 295 Swedish children aged 1-15 years were analysed using an enzyme linked immunosorbent assay (ELISA) for detecting immunoglobulin G (IgG) antibodies. In Ethiopia, a comparison was made of a local and a reference strain for preparation of the antigen, but there was little difference in outcome. A comparison between antigen prepared from the reference strain and the pooled antigen used in the Swedish study also showed little difference. The sharpest rise in seroprevalence was found in the age range 2-4 years. Among 4-year-olds, some 60% had already seroconverted, and among 12-year-olds almost 100% had done so. In Sweden, the sharpest rise appeared between the ages of 9 and 10 years. Above 10 years of age seroprevalence was around 20%. Infection with HP is acquired in early childhood in Ethiopia, but somewhat later, although still before the teens, in Sweden. To determine properly the risk factors for infection with HP, possible exposure must be assessed around the age of seroconversion, since seropositivity may remain for a long time but environmental factors may have changed since primary infection.

Carbapenem resistance mechanisms in Pseudomonas aeruginosa: alterations of porin OprD and efflux proteins do not fully explain resistance patterns observed in clinical isolates.

Imipenem resistance in Pseudomonas aeruginosa is considered to be associated with loss of the porin OprD combined with activity of chromosomal β‐lactamase (AmpC), while overexpression of multidrug efflux pumps is considered to confer meropenem resistance. Carbapenem resistance can also result from production of metallo‐β‐lactamases. Transcription of oprD and efflux pump genes mexB, mexY and mexF was analysed in 23 clinical isolates of P. aeruginosa by quantitative RT‐PCR. oprD was sequenced in all, and mexR, regulator of efflux pump MexAB‐OprM, in selected isolates. Four isolates that were imipenem susceptible had significant reduction of oprD mRNA and presence of oprD mutations causing frameshift or translational stop. In strains only resistant to imipenem no significant difference in transcription of oprD was observed between low‐level and high‐level resistant isolates. The differences could not be explained by either pattern of oprD mutations. Increased transcription of mexB generally correlated well with meropenem resistance. One high‐level meropenem‐resistant isolate showed no signicant change in mexB mRNA, but sequencing confirmed presence of a nalB mutation. Furthermore, one meropenem‐susceptible isolate showed significant increase in mexB transcription, but no mexR mutations. In summary, our findings indicate that the resistance patterns observed cannot be fully explained by the currently described carbapenem resistance mechanisms.

Diagnostic and prognostic value of interleukin-6 and C-reactive protein in community-acquired pneumonia.

The diagnostic and prognostic value of admission serum levels of interleukin-6 (IL-6) and C-reactive protein (CRP) was investigated in 203 hospital-treated patients with community-acquired pneumonia (CAP). In serum samples obtained during the first 24 h after admission, IL-6 was detectable in 198 patients (98%), with a median value of 50 ng/l. Ten % of the patients had IL-6 values of 1000 ng/l. A clear positive correlation between IL-6 and CRP was found (r = 0.29, p < 0.0001). Patients with high IL-6 or CRP levels had longer duration of fever, longer hospital stay, and had less often recovered clinically or radiographically on follow-up weeks after discharge. A high IL-6, but not a high CRP, also seemed to be associated with a higher mortality. Bacteremic pneumococcal pneumonia had the highest levels of IL-6 (mean 2852 and median 420 ng/l) and CRP (mean 292 and median 285 mg/l). High IL-6 values were also seen in patients with non-bacteremic pneumococcal pneumonia, while all patients with pneumonia due to other bacterial, or viral, aetiology had IL-6 levels of < or = 300 ng/l. In conclusion, IL-6 and CRP are promising diagnostic and prognostic tools in the management of CAP. Further studies are needed to establish the usefulness of repeated measurements early in the hospital course of the disease.

Role of efflux pumps and mutations in genes for topoisomerases II and IV in fluoroquinolone-resistant Pseudomonas aeruginosa strains.

We have investigated the occurrence of mutations in topoisomerase II (DNA gyrase) subunit B(gyrB) and topoisomerase IV subunit E(parE) and the hyperexpression of genes for four efflux pump proteins in 20 previously described, fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa. Amino acid alterations were found in GyrB in five strains and in ParE in three strains with MIC of norfloxacin > or = 8 mg/L, and it is likely that some of the alterations contribute to the quinolone resistance exhibited by these strains. Seventeen of the 20 strains overproduced mRNA for one or more pump proteins (MexB, MexD, MexF, or MexY), which caused multidrug resistance phenotype in more than half of strains. Two strains were hypermutable and one of them was highly resistant, but the other strain was only moderately resistant.

Diagnostic use of cerebral and extracerebral oxysterols

Abstract Background: 24S-Hydroxycholesterol (24OHC) and 27-hydroxycholesterol (27OHC) are two structurally similar oxysterols of different origins–the former almost exclusively formed in the brain and the latter formed to a lesser extent in the brain than in most other organs. Hypothesis to be tested: Neuronal damage and/or demyelination causes increased flux of 24OHC from the brain into the cerebrospinal fluid (CSF), whereas a defect blood-brain barrier causes increased flux of 27OHC from the circulation into the CSF. Methods: Isotope dilution-mass spectrometry was used to assay the two oxysterols in CSF and plasma from more than 250 patients with different neurological and geriatric diseases. Results: The CSF-levels of the two oxysterols were much more affected by the different diseases than the plasma levels. Patients with active demyelinating diseases had increased levels of 24OHC in CSF with a relatively high 24OHC/27OHC ratio. Patients with meningitis in general had high levels of both steroids with a low 24OHC/27OHC ratio. Patients with Alzheimers disease had slightly increased levels of 24OHC in CSF with less increase in 27OHC. Patients with multiple sclerosis had a tendency to have higher levels of 24OHC during active periods with a high 24OHC/27OHC ratio. Conclusions: Measurements of the two oxysterols in CSF and plasma may add significantly to existing biochemical methods for evaluation of neurological diseases.

Chromosomal mechanisms of aminoglycoside resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients

In total, 40 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients were included in this study. Twenty of these were collected in 1994 and 1997, from six CF patients, and the rest were collected from different CF patients in 2000 and 2001. The relative expression of mRNA for the efflux pump protein MexY was determined by real-time PCR and correlated with susceptibilities to amikacin and tobramycin. The chromosomal genes mexZ, rplY, galU, PA5471 and nuoG, which were found to have a role in the gradual increase in MICs of aminoglycoside antibiotics in laboratory mutants of P. aeruginosa, were analysed. MexY mRNA overproduction was found in 17/20 isolates collected in 1994 and 1997, and was correlated with decreased susceptibility to aminoglycosides. Alteration of the MexXY-OprM efflux system has been the main mechanism of resistance to aminoglycoside antibiotics in CF P. aeruginosa isolates over the 3-year period. In several isolates, expression of the PA5471 gene product might have some effect on elevated MICs of aminoglycosides. Inactivation of rplY, galU and/or nuoG may explain the gradual increase in MICs of aminoglycosides in laboratory mutants but probably not in the CF environment, as rplY and galU were unaltered in all isolates, and nuoG was not expressed in only one isolate. No 16S rRNA A-site mutations were found in any of the four copies of the gene in 13 investigated isolates.

Interleukin-6 and interleukin-8 in serum and urine in patients with acute pyelonephritis in relation to bacterial-virulence-associated traits and renal function

Urine and serum concentrations of interleukin (IL)-6 and IL-8 were determined in 43 women with acute pyelonephritis caused by Escherichia coli. Urine and serum samples were also collected 2 weeks after the infection and during a subsequent episode of cystitis (n = 8) or asymptomatic bacteriuria (n = 8). Concentrations of IL-6 and IL-8 were related to the expression of 5 virulence markers of E. coli and glomerular filtration rate (GFR) after pyelonephritis. Patients with acute pyelonephritis had elevated urine and serum IL-6 and IL-8 levels as compared to 37 healthy women (IL-6: p < 0.001 in both cases, and IL-8: p < 0.001 in both cases). Patients infected with E. coli producing hemolysin and/or cytotoxic necrotizing factor (CNF) had significantly higher IL-6 levels in serum during acute pyelonephritis as compared to patients infected with strains without the ability to produce these factors (p = 0.0025 and p = 0.0154, respectively). Patients who had high concentrations of IL-8 in urine during acute pyelonephritis had lower GFR at follow-up as compared to patients with lower levels of IL-8 in urine (r = -0.48, p = 0.0123). In conclusion, acute pyelonephritis is accompanied by elevated urinary and serum IL-6 and IL-8 levels. Bacteria producing hemolysin and CNF seem to induce higher concentrations of IL-6 in serum. The secretion of IL-8 from renal cells may participate in the initiation and maintenance of renal inflammation which in turn may influence renal function.

Bacteriology of maxillary sinusitis in relation to quality of the retained secretion.

The bacteriological findings in 200 patients with acute maxillary sinusitis are reported. It is concluded that the sampling technique--by antral aspiration--is highly significant in the evaluation of the bacteriological background of sinusitis, whereas the anerobic transport of the sample seems to be of less importance. By aspiration the purulent secretion can be properly separated from the non-purulent secretion. Pneumococci, H. influenzae and anaerobic bacteria can be isolated in about 90% of the patients with true sinus empyema, while sterile conditions are rare.

Tumor necrosis factor-α, interleukin-1β, and interleukin-1 receptor antagonist in dialysate and serum from patients on continuous ambulatory peritoneal dialysis

Abstract Dialysate and serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-1ra were investigated in 20 patients on continuous ambulatory peritoneal dialysis (CAPD), who altogether had 30 episodes of peritonitis. Bacterial growth was found in 25 (83%) of the dialysate samples. Staphylococcus epidermidis was the single most common microorganism, found in 44% of the culture-verified peritonitis. Samples from dialysate bags were obtained during the first month of dialysis and during peritonitis from the first three bags on day 1 (the day of admittance) and from nightbags on days 3 and 10. Serum samples were drawn on days 1 and 10. The peak concentrations of cytokines occurred on the first day of infection. In dialysates, TNF-α was elevated in 96% of the patients, with a peak median concentration of 160 pg/mL (range, P P P