Biomedical subjects

B Wretlind

Publications and source records attributed to B Wretlind.

At least 19 recordsLinked to original sources

PubMed2005

Meropenem susceptibility breakpoint for Pseudomonas aeruginosa strains hyperproducing mexB mRNA.

Twenty-five isolates of Pseudomonas aeruginosa with different meropenem susceptibilities were subjected to quantitative RT-PCR for analysis of transcription levels of oprD, mexB and mexD, and, in selected isolates, PA3720, which is hyper-expressed in nalC efflux mutants. Regulator genes of efflux pump MexAB-OprM, mexR and PA3721 (putative) were sequenced in selected isolates. The potential for mathematical reconstruction of the ideal susceptible population using normalised resistance interpretation (NRI) was also studied. In three isolates with intermediate susceptibility to meropenem (according to Swedish breakpoints), a reduction in MIC from 4 to 2 mg/L was observed with efflux inhibitor MC-207,110. These isolates would be considered susceptible according to British Society for Antimicrobial Chemotherapy and NCCLS breakpoints. These three isolates had between 4.6- and 5.0-fold increases in mexB transcription. None of these isolates had significant nalB mutations, but an Ala145-->Val mutation was observed in PA3721 in two of the isolates. However, these isolates had moderately increased production of PA3720 only. Single-strain regression analysis did not detect any major biological differences between the different groups. Using NRI, a disk-diffusion susceptibility breakpoint of >/= 28 mm was generated. Isolates with intermediate susceptibility to meropenem, which are considered fully susceptible in many countries, displayed possible low-grade meropenem resistance mechanisms, implying that the susceptibility breakpoint should be reconsidered. The increased transcription of mexB mRNA in such isolates seems unrelated to nalB or nalC mutations.

Anti-Bacterial Agents↗

PubMed2004

Viridans streptococcal septicaemia in neutropenic patients: role of proinflammatory cytokines.

The immunostimulatory activity of viridans streptococcal strains isolated from neutropenic patients with severe sepsis (n=9) or uncomplicated bacteraemia (n=10) was compared. Peripheral blood mononuclear cells from healthy individuals were stimulated with heat-killed bacteria or culture supernatants, and cytokine production assessed. All strains were potent inducers of IL1beta, IL8, and TNFalpha production. Heat-killed bacteria induced consistently higher IL1beta and TNFalpha production than did the cell-free bacterial supernatants (P<0.01). The strains did not induce any proliferative response, nor any significant TNFbeta or IFNgamma production. No difference in cytokine-inducing capacity could be detected between the cohorts of severe and nonsevere isolates. Comparison of strains causing severe and nonsevere episodes in the same patient (n=2) revealed a significantly higher induction of IL1beta by the severe episodes associated isolates as compared to the nonsevere (P<0.04). The study underscores the importance of the host-pathogen interplay in determining the level of inflammation, and hence the severity of disease.

Bacteremia↗

PubMed2004

Expression of the MexXY efflux pump in amikacin-resistant isolates of Pseudomonas aeruginosa.

The MexZ-MexX-MexY multidrug efflux system in Pseudomonas aeruginosa was studied to determine its contribution to aminoglycoside resistance. Amikacin-resistant (AR) mutants were generated from P. aeruginosa strain PAO1, and clinical isolates of P. aeruginosa were collected from cystic fibrosis patients. The regulatory gene mexZ and the intergenic region (mexOZ) between mexZ and mexX were investigated for mutation by PCR and DNA sequence analysis. The results showed that 14 of 15 AR clinical isolates and one of ten laboratory mutants had at least one mutation in mexZ and/or mexOZ. To study the effect of mexZ and mexOZ mutations, the production of MexY mRNA was investigated quantitatively by real-time PCR. Seven of ten AR mutants (MIC 4-8 mg/L) produced 8-21-fold more MexY mRNA than PAO1. These isolates were sensitive to fluoroquinolones, carbapenems and ceftazidime. One AR mutant (MIC 64 mg/L) that produced > 200-fold more MexY mRNA than PAO1 was also resistant to fluoroquinolones, carbapenems and ceftazidime. Thirteen of 15 AR clinical isolates produced 3.4-727-fold more MexY mRNA. No evidence was found for the aminoglycoside-modifying enzymes 6'-N-acetyltransferase type Ib, 4'-O-nucleotidyltransferase type IIb or aminoglycoside 3'-phosphotransferase IIps in these strains. Nine AR mutants overproduced MexY without mutations in mexZ or mexOZ, suggesting that MexXY efflux is also regulated by gene(s) other than mexZ.

Amikacin↗

PubMed2003

Will triclosan in toothpaste select for resistant oral streptococci?

The aims of the study were to determine the in vitro sensitivity of triclosan against viridans streptococci and the impact of triclosan on the normal oral microflora. Nine subjects used a triclosan-containing toothpaste for 2 weeks. Saliva samples were collected on days 0 and 14 and were analyzed quantitatively. The minimum inhibitory concentrations of triclosan and of several antimicrobial agents were determined for the streptococci isolated on days 0 and 14. No major changes occurred in the normal oral microflora during the study period. There were no differences in susceptibility between streptococcal strains collected at days 0 and 14 against triclosan or antimicrobial agents. Short-term use of triclosan has no major impact on normal oral microflora or on streptococcal susceptibility of antimicrobial agents. The effects of long-term use should be evaluated.

Anti-Infective Agents, Local↗

PubMed2001

Mitogenic action of tumour necrosis factor-alpha and interleukin-8 on explants of human duodenal mucosa.

Our aim is to examine whether tumour necrosis factor-alpha (TNF-alpha) and interleukin affect the mitotic activity in explants of human duodenal mucosa and to estimate the release of cytokines from explants incubated with TNF-alpha. Biopsy specimens of normal duodenal mucosa were taken from 19 subjects that underwent upper endoscopy for investigation of dyspeptic symptoms or chronic gastrointestinal bleeding. The specimens were processed following guidelines for organ culture technique. Paired biopsy specimens from 12 subjects were cultured for 23 h to achieve steady state and thereafter the explants were incubated 25 h with 10(-13)-10(-9) M of TNF-alpha or IL-8. Mitoses were arrested in the metaphase by adding vincristine sulphate for the last three hours. The explants were then fixed and processed for microdissection. Fifteen crypts were microdissected and the total number of metaphases was determined using the whole crypt as reference volume. The number of metaphases per crypt was also estimated in explants incubated with 10(-10) M TNF-alpha in the presence of anti-IL-8 antibodies. Additional duodenal explants from seven subjects were incubated with 10(-10) M TNF-alpha for 25 h. Thereafter the release of IL-1-beta, IL-6, IL-8 and interferon gamma (IFN-gamma) into the culture medium was measured by enzyme immunoassay and expressed as pg/mg protein. TNF-alpha and IL-8 significantly increased the number of metaphases/crypts (P<0.0001). The addition of anti-IL-8 slightly reduced the number of metaphases/crypt compared to the values observed in the explants incubated with 10(-10) M TNF-alpha alone (P<0.0001). The number of metaphases/crypt in the explants incubated with 10(-10) M TNF-alpha in the presence of anti-IL-8 antibodies was, however, markedly and significantly higher than that of the controls (P<0.000). TNF-alpha induced the release of IL-8 (P<0.01) and IL-6 (P<0.05) from the duodenal explants. TNF-alpha and IL-8 are potent mitogens to human small intestinal crypts. The mitogenic action of TNF-alpha is primarily a direct effect of the cytokine and only to a minor extent mediated by a secondary production of IL-8 in the duodenal explant. Our findings indicate that TNF-alpha and IL-8 may participate in the regulation of cell proliferation in the human small intestinal epithelium.

Cell Division↗

PubMed2001

Mechanisms of resistance to imipenem in imipenem-resistant, ampicillin-sensitive Enterococcus faecium.

Enterococcus faecium has six penicillin-binding proteins (PBP), where PBP5 seems to be the main target for beta-lactam antibiotics. The PBP profiles of three imipenem-resistant, ampicillin-sensitive E. faecium strains, isolated from the same patient, were studied using biotinylated ampicillin and chemiluminescence detection. Imipenem resistance in these strains was found to be associated with hyperproduction of PBP5 compared to the ampicillin- and imipenem-susceptible strain ATCC 19434. PBP5 in the imipenem-resistant strains (S1, B2) exhibited a selectively decreased affinity for imipenem. An 854 bp DNA fragment, corresponding to the penicillin-binding domain of pbp5fm, was studied in the resistant strains and the reference strain. Four amino acid substitutions were observed in the resistant strains compared to the susceptible one. The contribution of these substitutions to the increased production of PBP5 in these strains is unclear since the substitution was observed also in a strain without increased production of PBP5. Our results suggest that the moderate imipenem resistance observed in these strains is associated with increased production of PBP5 with relatively decreased affinity for imipenem, and that evolution of imipenem resistance in E. faecium is dinstinct from that of the other beta-lactams such as ampicillin.

Ampicillin↗

PubMed2000

IL-6 and IL-1 receptor antagonist in stable angina pectoris and relation of IL-6 to clinical findings in acute myocardial infarction.

OBJECTIVES: To determine if increased inflammatory activity, as reflected by interleukin-6 (IL-6) and interleukin-1 receptor antagonist (IL-1ra) levels, is present in patients with stable angina pectoris and if IL-6 levels on admission to the coronary care unit in patients with acute myocardial infarction (AMI) are related to heart failure and fever response. SUBJECTS AND METHODS: We studied 28 patients with stable angina pectoris enrolled for coronary angiography, and compared them with sex- and age-matched controls. Thirty-four patients with AMI were studied and samples for determination of IL-6 levels were taken on admission within 36 h of onset of symptoms. IL-6 and IL-1ra were determined in serum by enzyme immunoassay. RESULTS: Levels of IL-6 and IL-1ra were higher in patients with stable angina pectoris than in controls (mean 4.6 +/- 3.6 vs. 3.0 +/- 2.9 ng L-1, P < 0.03, and 774 +/- 509 vs. 490 +/- 511 ng L-1, P < 0.01, respectively). IL-6 and IL-1ra levels were not related to angiographic findings. IL-6 levels were high in patients with AMI (38.9 +/- 75.6 ng L-1). Patients with prolonged fever (duration > 4 days) had higher IL-6 levels (94.7 +/- 138.2 vs. 21.7 +/- 29.7 ng L-1, P < 0.05). IL-6 levels were not related to heart failure. CONCLUSIONS: Our results indicate that increased inflammatory activity is present not only in acute coronary syndromes, but also in a chronic form of ischaemic heart disease, giving further evidence for a central role of inflammatory processes in coronary artery disease. With regard to AMI, we found increased inflammatory activity in patients with prolonged fever.

Aged↗

PubMed2000

TNF-alpha and IL-8 in consecutive sputum samples from cystic fibrosis patients during antibiotic treatment.

Proinflammatory cytokines in sputum are useful markers of the activity of lung disease in cystic fibrosis (CF). Tumour necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) concentrations in sputum of 10 CF patients were determined during exacerbation and IL-8 in sputum of 48 patients at a yearly follow-up when patients were in optimal clinical condition. In 9 patients of the former group, TNF-alpha levels were increased during exacerbation. In 4 patients, the peak occurred within 2 d (median value > 1500 ng/l), whereas the remaining 5 had peak values on days 3-6 (median value 720 ng/l). IL-8 levels were > 800 microg/l in all 10 patients, and in 9 cases there was a positive correlation between IL-8 and TNF-alpha. Baseline IL-8 levels of 48 patients showed considerable variation (median 207 microg/l, range 1.5-392). There was a significant correlation between IL-8 concentrations and current colonization with either Pseudomonas aeruginosa or Staphylococcus aureus in the lower airways (p = 0.002), immunoglobulin G levels (p = 0.02) and the severity of the pathological findings shown by chest X-ray (p = 0.008). High IL-8 and TNF-alpha values correlated with symptoms of deterioration. IL-8 levels seemed to be markers of both current bacterial colonization and the degree of lung damage.

Acute Disease↗

PubMed2000

Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients.

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 in gyrA and parC dominate (S. Jalal and B. Wretlind, Microb. Drug Resist. 4:257-261, 1998).

Adult↗

PubMed1999

Rapid identification of mutations in a multidrug efflux pump in Pseudomonas aeruginosa.

The gene mexR regulates negatively the expression of the MexA-MexB-OprM efflux pump in Pseudomonas aeruginosa, and mutations in mexR cause a multiple antibiotic resistance phenotype. Five hundred and forty resistant clones of P. aeruginosa PAO503 were isolated after selection for resistance to chloramphenicol or tetracycline. All isolates showed similar phenotypes and were resistant to tetracycline, chloramphenicol and norfloxacin. Nineteen randomly selected isolates were analyzed. Since mutational analysis by direct sequencing of all regions of interest in several strains is time-consuming and expensive, a screening method, Non-Isotopic RNase Cleavage Assay (NIRCA), was applied to identify mutant genes so that they could be targeted for DNA sequencing. NIRCA is a simple but rapid method for mutational analysis and can be performed in 3-4 h. Results of NIRCA analysis were compared with DNA sequencing. Both NIRCA and DNA sequencing analysis showed mexR gene mutations in 11 of 19 isolates but no alterations in 8 strains. An immunoblot assay showed overexpression of OprN, a component of another multidrug efflux pump, MexE-MexF-OprN, in those eight isolates. Nucleotide sequencing of quinolone resistance-determining regions of DNA gyrase (gyrA) or topoisomerase IV (parC) showed no alterations in any of the 19 mutants. The data indicate that two efflux pump systems, MexA-MexB-OprM and MexE-MexF-OprN, were involved in multidrug resistance including quinolones and that NIRCA is a sensitive method for screening mutations.

Bacterial Outer Membrane Proteins↗

PubMed1999

Dialysis culture for determination of MIC and MBC of benzylpenicillin against Borrelia burgdorferi.

Minimal inhibitory concentration (MIC) of benzylpenicillin (Pen G) against strains of Borrelia burgdorferi has earlier been determined by a recently developed dialysis culture technique which provides a constant concentration of Pen G. To further investigate this method, both MIC and minimal bactericidal concentration (MBC) of Pen G against B. burgdorferi were determined and the results were compared with those obtained by other authors using different methods. The incubation period was 7 days and subcultures for MBC were observed for a further 2 weeks. The study showed results of MIC and MBC similar to those of broth microdilution, indicating sensitivity, but lower values than the majority of MIC and MBC results reported by other authors using broth macrodilution. It is essential for the results of antibiotic sensitivity testing in vitro against slowly growing bacteria like Borrelia burgdorferi that the concentration of antibiotics such as Pen G, which are unstable in solution, is constant during the incubation period.

Borrelia burgdorferi Group↗

PubMed1999

Alterations in GyrA and ParC associated with fluoroquinolone resistance in Enterococcus faecium.

High-level quinolone resistance in Enterococcus faecium was associated with mutations in both gyrA and parC genes in 10 of 11 resistant strains. On low-level resistant strain without such mutations may instead possess an efflux mechanism or alterations in the other subunits of the gyrase or topoisomerase IV genes. These findings are similar to those for other gram-positive bacteria, such as Enterococcus faecalis.

Amino Acid Sequence↗

PubMed1999

Neutralization of macrophage inflammatory protein 2 (MIP-2) and MIP-1alpha attenuates neutrophil recruitment in the central nervous system during experimental bacterial meningitis.

Chemokines are low-molecular-weight chemotactic cytokines that have been shown to play a central role in the perivascular transmigration and accumulation of specific subsets of leukocytes at sites of tissue damage. Using in situ hybridization (ISH), we investigated the mRNA induction of macrophage inflammatory protein 2 (MIP-2), MIP-1alpha, monocyte chemoattractant protein 1 (MCP-1), and RANTES. Challenge of infant rats' brains with Haemophilus influenzae type b intraperitoneally resulted in the time-dependent expression of MIP-2, MIP-1alpha, MCP-1, and RANTES, which was maximal 24 to 48 h postinoculation. Immunohistochemistry showed significant increases in neutrophils and macrophages infiltrating the meninges, the ventricular system, and the periventricular area. The kinetics of MIP-2, MIP-1alpha, MCP-1, and RANTES mRNA expression paralleled those of the recruitment of inflammatory cells and disease severity. Administration of anti-MIP-2 or anti-MIP-1alpha antibodies (Abs) resulted in significant reduction of neutrophils. Administration of anti-MCP-1 Abs significantly decreased macrophage infiltration. Combined studies of ISH and immunohistochemistry showed that MIP-2- and MIP-1alpha-positive cells were neutrophils and macrophages. MCP-1-positive cells were neutrophils, macrophages, and astrocytes. Expression of RANTES was localized predominantly to resident astrocytes and microglia. The present study indicates that blocking of MIP-2 or MIP-1alpha bioactivity in vivo results in decreased neutrophil influx. These data are also the first demonstration that the C-C chemokine MIP-1alpha is involved in neutrophil recruitment in vivo.

Animals↗

PubMed1998

Age-related prevalence of Shigella and Salmonella antibodies and their association with diarrhoeal diseases in Peruvian children.

Shigella and Salmonella antibodies in relation to diarrhoea were studied in a cohort of 413 children between 2 and 27 months of age in peri-urban Lima, Peru. Blood samples were obtained at 2, 3 and 12 months of age. Antibody titres against lipopolysaccharide from Shigella flexneri serotype Y, Shigella dysenteriae serotype 1, Shigella sonnei, Salmonella serogroups AO, BO, DO, and Shigella Ipa and Salmonella typhi Vi antigens were measured by enzyme immunoassay. IgG titres against S. flexneri and Shigella Ipa were higher at 2 than at 3 or 12 months of age (p=0.001), while the changes in IgG titres against S. dysenteriae, S. sonnei and Salmonella were not pronounced. IgA and IgM titres against S. flexneri, Shigella Ipa, S. dysenteriae, S. sonnei and Salmonella were significantly higher at 12 than at 2 or 3 months of age (p=0.001). Stool samples were obtained from children in 64% of all diarrhoeal episodes. Shigella spp. were isolated from 20% of the children during the first 2 y of life and Salmonella in 3%. Most isolates were from children at 13-24 months of age (78%). IgG antibodies at 12 months of age did not protect against shigellosis during the second year of life.

Age Distribution↗

PubMed1998

Mechanisms of quinolone resistance in clinical strains of Pseudomonas aeruginosa.

Principal mechanisms of bacterial resistance to quinolones are modification of target enzymes, DNA gyrase (gyrA) and topoisomerase IV (parC), or reduction of intracellular concentration due to mutations in the regulatory genes for efflux systems, such as mexR and nfxB. We have examined gyrA, parC, mexR, and nfxB genes from 16 quinolone-resistant clinical isolates of Pseudomonas aeruginosa to determine the relation between mutations in DNA replicating enzymes or regulatory genes for efflux systems and to correlate the mutations with minimal inhibitory concentrations (MICs). The quinolone resistance-determining regions (QRDR) of these genes were amplified by PCR and sequenced by capillary electrophoresis. Fourteen of 16 isolates had mutations in gyrA, and 13/14 strains with MIC to norfloxacin > or = 8 mg/L had threonine at position 83 changed to isoleucine. Seven of 8 strains with MIC > or = 32 mg/L had mutations in parC. One of these strains showed a parC mutation at position 74 without any mutation in gyrA. Four strains had mexR and two strains nfxB mutations. The data indicate that gyrA mutation is the most important component of quinolone resistance, and simultaneous presence of parC mutations is associated with high-level resistance. parC mutation alone may contribute to resistance, and gyrA mutation may not be a prerequisite for parC mutation to express resistance. mexR and nfxB mutations were found mostly in strains with high-level resistance.

Anti-Infective Agents↗

PubMed1998

Rapid emergence of high-level resistance to quinolones in Campylobacter jejuni associated with mutational changes in gyrA and parC.

Quinolone resistance in clinical isolates of Campylobacter jejuni in Sweden increased more than 20-fold at the beginning of the 1990s. Resistance to 125 microgram of ciprofloxacin per ml in clinical isolates was associated with chromosomal mutations in C. jejuni leading to a Thr-86-Ile substitution in the gyrA product and a Arg-139-Gln substitution in the parC product.

4-Quinolones↗

PubMed1997

Pseudomonas aeruginosa adherence to external auditory canal epithelium.

BACKGROUND: Pseudomonas aeruginosa is an important causative agent for external otitis. The specific bacterium-host reaction has not been investigated. It is therefore unknown whether adhesion of the external otitis strain to the external auditory canal epithelium is increased compared with strains isolated from other infections. DESIGN: A cohort study was designed to outline adhesion of P aeruginosa to the external auditory canal epithelium, cultured in vitro, of the guinea pig. Factors important for pathogenesis were also studied. PATIENTS: Pseudomonas aeruginosa strains from nonhospitalized patients were collected consecutively at the bacteriological laboratories at Karolinska Hospital, Stockholm, and Huddinge Hospital, Huddinge, Sweden. External otitis strains were compared with strains from leg ulcers, urinary tract infections, and cystic fibrosis. METHODS: Adhesion to the external auditory canal epithelium cultured in vitro was measured and compared groupwise with the mean profile of pathogenic factors. RESULTS: Adhesion to the epithelium was significantly increased for external otitis strains. These strains also had a significantly increased deoxyribonuclease production and a significantly decreased production of pyocyanin and alginate. CONCLUSIONS: The significantly increased ability of P aeruginosa, isolated from external otitis, to adhere to external auditory canal epithelium was combined with a significant production of pathogenic factors. The P aeruginosa that causes external otitis could therefore be considered a particular phenotype. The enzyme profile for external otitis strains was similar to that of the control groups except for the strains from cystic fibrosis. Adhesion to guinea pig vs human epithelium must be compared, and the effects of extracellular proteins on adhesion should be studied to further understand how P aeruginosa adheres to the external auditory canal.

Animals↗

PubMed1997

Cytokine mRNA profiles during the course of experimental Haemophilus influenzae bacterial meningitis.

Intraperitoneal inoculation of Haemophilus influenzae type b (Hib) to 3-week-old Sprague-Dawley rats resulted in nonlethal meningitis with high levels of leukocytes in the cerebrospinal fluid (CSF) and positive bacterial culture. Using in situ hybridization, levels of cytokine mRNA-expressing cells were determined in the brain, CSF, and spleen from Hib-inoculated and uninfected control rats. IFN-gamma, IL-1 beta, IL-4, IL-6, IL-10, IL-12, and TNF-alpha mRNA levels were elevated at 12 hr postinoculation (pi) in spleen and CSF. At this time point, strong expression of IL-6 and TGF-beta was detected in the brain, and also of IL-10 at 48 hr while IFN-gamma and IL-12 were expressed at very low levels throughout the observation time. Delayed cytokine induction occurred in CSF compared to spleen and brain. TGF-beta was high in CSF at 48 hr, and some elevation of IL-1 beta, IL-6, IL-10, TNF-alpha, IFN-gamma, and IL-12 was evident at 72 hr pi. This may suggest measures that promote production of TGF-beta and/or IL-10 should be evaluated in treatment of bacterial meningitis.

Animals↗