| 1. |
- Andrésen, Cecilia, et al.
(författare)
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Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance
- 2010
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Ingår i: Protein Science. - : Cold Spring Harbor Laboratory Press. - 0961-8368 .- 1469-896X. ; 19:4, s. 680-692
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Tidskriftsartikel (refereegranskat)abstract
- The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.
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| 2. |
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| 3. |
- Diab, Asim, et al.
(författare)
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Neutralization of macrophage inflammatory protein 2 (MIP-2) and MIP-1α attenuates neutrophil recruitment in the central nervous system during experimental bacterial meningitis
- 1999
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 67:5, s. 2590-2601
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Tidskriftsartikel (refereegranskat)abstract
- 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-1α, 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-1α, 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-1α, 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-1α 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-1α-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-1α bioactivity in vivo results in decreased neutrophil influx. These data are also the first demonstration that the C-C chemokine MIP-1α is involved in neutrophil recruitment in vivo.
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| 4. |
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| 5. |
- Gharizadeh, Baback, et al.
(författare)
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Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing
- 2005
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Ingår i: International Journal of Antimicrobial Agents. - : Elsevier BV. - 0924-8579 .- 1872-7913. ; 26:6, s. 486-490
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Tidskriftsartikel (refereegranskat)abstract
- Quinolone resistance is rapidly increasing in Neisseria gonorrhoeae and is posing a significant public health threat that requires constant surveillance. A rapid and reliable mutation detection assay has been developed. The assay is based on pre-programmed short DNA sequencing and is designed to detect point mutations in the gyrA gene that are highly related to ciprofloxacin resistance, i.e. in codons 91 and 95. By developing an assay based on pyrosequencing and exploiting the pre-programmed nucleotide dispensation capability of this technology, the sequence comprising the mutations will be analysed and promptly reveal whether the N. gonorrhoeae pathogen carries resistance to ciprofloxacin. A panel of 40 N. gonorrhoeae clinical isolates, of which 27 phenotypically displayed decreased susceptibility or resistance to ciprofloxacin, was used in the present study. All point mutations in the short stretch of the N. gonorrhoeae gyrA gene were easily discriminated, and the genotypic results obtained by pre-programmed sequencing were mainly in agreement with the phenotypically identified decreased susceptibility or resistance to ciprofloxacin. The new method used in the present study has the potential for rapid and reliable identification of known as well as previously unknown drug resistance mutations.
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| 6. |
- Kugelberg, Elisabeth, et al.
(författare)
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Reduction of the fitness burden of quinolone resistance in Pseudomonas aeruginosa
- 2005
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Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press (OUP). - 0305-7453 .- 1460-2091. ; 55:1, s. 22-30
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: Quinolone resistance in the opportunistic pathogen Pseudomonas aeruginosa is commonly caused by mutations that alter the target molecules DNAgyrase/topoisomerase IV, or cause activation of various efflux systems.We have analysed the effect of quinolone resistance caused by DNA gyrase/topoisomerase IV mutations on bacterial fitness. Methods: Norfloxacin-resistant mutants were isolated and by DNA sequencing the mutations conferring resistance were identified. Mutant fitnesswas determined by measuring growth rates in vitro. Mutants with reducedgrowth rates were serially passaged to obtain growth-compensatedmutants. The level of DNA supercoiling was determined by isolatingplasmid DNA from the susceptible, resistant and compensated mutants andcomparing the topoisomer distribution patterns by gel electrophoresis inthe presence of chloroquine. Results: Low-level resistance (4-48 mg/L) was caused by single mutations in gyrA or gyrB. Among these strains, three out of eight mutants showed lower fitness,whereas high-level resistant (>256 mg/L) mutants with doublemutations in gyrA and parC, parE, nfxB or unknown genes all showed areduced fitness. Slow-growing resistantmutants with a gyrA mutation had decreased DNA supercoiling. Afterserial passage in laboratory medium, mutant fitnesswas increased by compensatory mutation(s) that restored supercoiling tonormal levels. The compensatory mutation(s) was not located in any ofthe genes (gyrAB, topA, parCE, hupB, fis, hupN, himAD or PA5348) thatwere expected to affect supercoiling. Conclusions: Our results show that 'no cost' and compensatory mutations are common in quinolone-resistant P. aeruginosa.
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| 7. |
- Lindbäck, Emma, et al.
(författare)
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Pyrosequencing of the DNA gyrase gene in Neisseria species : effective indicator of ciprofloxacin resistance in Neisseria gonorrhoeae
- 2006
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Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley. - 0903-4641 .- 1600-0463. ; 114:12, s. 837-841
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Tidskriftsartikel (refereegranskat)abstract
- The quinolone resistance determining region (QRDR) of the gyrA gene in ciprofloxacin-susceptible strains (n=53) and strains of Neisseria spp. with reduced susceptibility (n=70) was determined by the pyrosequencing method. Results showed that the QRDR of the gyrA gene is an effective molecular indicator of resistance to ciprofloxacin in Neisseria gonorrhoeae, and presumably in Neisseria meningitidis, but not in all other Neisseria spp. This sequence was not unique for N. gonorrhoeae and seems unsuitable for species verification of N. gonorrhoeae. However, whether it is also possible to use this region for verification depends on the specificity of the primary screening method used.
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| 8. |
- Lindbäck, Emma, et al.
(författare)
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Transformation of ciprofloxacin-resistant Neisseria gonorrhoeae gyrA, parE and porB1b genes
- 2006
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Ingår i: International Journal of Antimicrobial Agents. - : Elsevier BV. - 0924-8579 .- 1872-7913. ; 28:3, s. 206-211
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Tidskriftsartikel (refereegranskat)abstract
- In several transformation experiments, we have shown that introduction of an alteration in GyrA at position 95 of a ciprofloxacin-susceptible Neisseria gonorrhoeae strain (minimum inhibitory concentration (MIC) 0.008 mg/L) increases the MIC to 0.064 mg/L. Two alterations (positions 91 and 95) increase the MIC to 0.125-0.25 mg/L. Transformants with ciprofloxacin MICs of 0.5-16 mg/L were obtained from a moderately ciprofloxacin-resistant strain (MIC 0.25 mg/L). These transformants had alterations in the gene for PorB1b and probably other genes. In one transformant, an alteration in ParE was also introduced, which probably contributed to ciprofloxacin resistance. The ciprofloxacin-resistant transformants had the donor porB1b sequence, and most of them also had altered serovars. We conclude that alterations in N. gonorrhoeae PorB1b could be involved in ciprofloxacin resistance.
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| 9. |
- Lundbäck, David, et al.
(författare)
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Molecular epidemiology of Neisseria gonorrhoeae- identification of the first presumed Swedish transmission chain of an azithromycin-resistant strain
- 2006
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Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley. - 0903-4641 .- 1600-0463. ; 114:1, s. 67-71
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Tidskriftsartikel (refereegranskat)abstract
- In the present study, 10 azithromycin-resistant Neisseria gonorrhoeae isolates from 6 Swedish male patients in 2004, 3 sporadic Swedish azithromycin-resistant N. gonorrhoeae isolates from recent years and one Swedish N. gonorrhoeae isolate from 2003 that was susceptible to azithromycin but assigned the same serological variant (serovar), i.e. IB-37, as the isolates from 2004 were included. The isolates were characterized phenotypically using antibiograms and serovar determination and genetically with pulsed-field gel electrophoresis (PFGE), entire porB gene sequencing and N. gonorrhoeae multiantigen sequence typing (NG-MAST). The epidemiological information and the results of the thorough phenotypic characterisation and genetic characterisation identified the first presumed domestic transmission of one azithromycin-resistant N. gonorrhoeae strain in Sweden in 2004. This stresses the need for continuous surveillance of the antibiotic susceptibility of N. gonorrhoeae in order to identify emergence of new resistance, monitor the changing patterns of the susceptibility, and be able to update treatment recommendations on a regular basis.
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| 10. |
- Nilsson, Elin, 1979-, et al.
(författare)
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Pseudomonas aeruginosa infections are prevented in cystic fibrosis patients by avian antibodies binding Pseudomonas aeruginosa flagellin : Pseudomonas aeruginosa infections are prevented in cystic fibrosis patients by avian antibodies binding Pseudomonas aeruginosa flagellin
- 2007
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Ingår i: Journal of chromatography. B. - : Elsevier BV. - 1570-0232 .- 1873-376X. ; 856:1-2, s. 75-80
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Tidskriftsartikel (refereegranskat)abstract
- Pseudomonas aeruginosa (PA) is the main cause of morbidity and mortality in cystic fibrosis (CF) patients. CF patients with chronic PA infections have a more rapid deterioration of their lung function and the bacteria become impossible to eradicate from the lungs. Antibiotic resistance among PA strains in CF patients is steadily increasing. Specific chicken (IgY) antibodies against PA have been shown to have potential to prevent PA infections in CF. Anti-Pseudomonas IgY reduces PA adhesion to epithelia, but the mechanism has not been fully elucidated. To gain further insight into the prophylactic effect of these antibodies, the immunoreactivity was investigated by 2D electrophoresis of PA strains, immunoblotting and MALDI-TOF-MS. To confirm the identity of the proteins, the tryptic peptides were analyzed by MALDI-TOF-MS to accurately measure their monoisotopic masses as well as determine their amino acid sequences. In order to facilitate fragmentation of the peptides they were N-terminally or C-terminally labeled. Several strains were investigated and anti-Pseudomonas IgY was immunoreactive against all of these strains, which strengthens its potential as a prophylactic treatment against PA. Flagellin was identified as the major antigen. Flagellin is the main protein of the flagella and is crucial for establishing infections in hosts as well as being involved in PA chemotaxis, motility, adhesion and inflammation. Furthermore, secreted flagellin elicits an inflammatory response. In conclusion, anti-Pseudomonas IgY binds flagellin, which may prevent PA infections in CF patients by hindering host invasion.
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| 11. |
- Unemo, Magnus, 1970-
(författare)
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Genotypic and phenotypic characterisation of Neisseria gonorrhoeae
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The bacterium Neisseria gonorrhoeae (the gonococcus) is the aetiological agent of gonorrhoea, which remains a major sexually transmitted infection/disease (STI/STD) worldwide. The incidence of gonorrhoea was previously high in many countries, Sweden included. The incidence in Sweden culminated in 1970 with 487 cases per 100,000 inhabitants. After that, the incidence declined almost every year until an all-time low of 2.4 was observed in 1996. ln 1997 the incidence of gonorrhoea began to significantly increase in Sweden, due mainly to a larger number of domestic cases of young heterosexuals of both sexes and homosexual men. This observation, in combination with the widespread use of suboptimal methods for characterisation and, in some countries, for diagnosis of the bacterium, as well as the rapid increase of resistance to several antibiotics, has intensified the research in the field of N. gonorrhoeae.In the present thesis (paper 1), a high prevalence of decreased susceptibility or resistance to several of the traditionally used gonorrhoea antibiotics was identified and correlated to the geographic area of exposure, especially Asia. Nevertheless, effective antibiotics for treating gonorrhoea are at hand. A substantial genetic heterogeneity within identical serological variants (serovars), i.e. intraserovar, as well as interserovar of N. gonorrhoeae strains circulating within the community was revealed, emphasising the importance of using highly discriminative (DNA-based) epidemiological characterisation methods for the bacteria (papers II-V). Effective DNA-based characterisation methods, i.e. pulsed-field gel electrophmesis (PFGE) of genomic DNA digested with rarely cutting restriction endonucleases and porB gene sequencing, were adapted, optimised and evaluated against conventional phenotypic methods, as epidemiological tools on Swedish N. gonorrhoeae isolates. These molecular techniques showed a significantly higher discriminatory ability, reproducibility, and objectivity than the serovar determinations using the Genetic Systems (OS) or the Pharmacia panel (Ph) ofmonoclonal antibodies (MAbs) (papers II & III). According to a comparison of serologic and genetic parB-based typing of N. gonorrhoeae, the precise amino acid residues of porB, critical for the reactivity of several of the GS MAbs, were difficult to identity (paper IV). In papers IV & V, a determination of genetic group (genogroup) and genetic variant (genovar) was developed based on real-time PCR of the entire porB gene with subsequent sequence analysis in real-time by synthesis, i.e. pyrosequencing technology, of short, highly polymorphic porB gene segments. This method provides a rapid, high-throughput, objective, highly discriminative, typeable, portable for interlaboratory comparisons, and reproducible molecular characterisation for N. gonorrhoeae. Genogroup and genovar determination can complement or even replace the internationally established serovar determination in routine use for following the transmission of individual strains in the community and confirming presumed epidemiological connections or discriminating isolates of suspected clusters of gonorrhoea cases.
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| 12. |
- Unemo, Magnus, et al.
(författare)
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Molecular characterization of Neisseria gonorrhoeae identifies transmission and resistance of one ciprofloxacin-resistant strain
- 2007
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Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley. - 0903-4641 .- 1600-0463. ; 115:3, s. 231-240
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Tidskriftsartikel (refereegranskat)abstract
- A highly discriminative and objective genetic characterization of N. gonorrhoeae, which increases our knowledge of strain populations in different geographic areas, is crucial for the development of improved control measures. In the present study, conventional phenotypic characterization and genetic characterization by means of pulsed-field gel electrophoresis (PFGE), sequencing of the entire porB gene, N. gonorrhoeae multiantigen sequence typing (NG-MAST), and pyrosequencing of a quinolone resistance determining region (QRDR) of the gyrA gene of Swedish ciprofloxacin-resistant N. gonorrhoeae serovar IB-10 isolates (n=45) were performed. The genetic characterization identified one widely spread ciprofloxacin-resistant N. gonorrhoeae ST147 strain. In addition, isolates with slightly different genetic characteristics, which presumably reflect the ongoing evolution only, were also identified. All the isolates contained single nucleotide polymorphisms in QRDR of the gyrA gene that are highly correlated with ciprofloxacin resistance. Consequently, comprehensive characterization identified the first confirmed large domestic transmission, mainly among young heterosexuals, of one ciprofloxacin-resistant N. gonorrhoeae strain in Swedish society during 2002-2003. In conclusion, a precise, i. e. genetic, characterization for identification of individual strains is a very valuable support to the crucial active surveillance of the epidemiological characteristics and the antibiotic susceptibility of N. gonorrhoeae in the effective treatment of gonorrhoea.
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