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1.  Pyrosequencing of the DNA gyrase gene in Neisseria species: effective indicator of ciprofloxacin resistance in Neisseria gonorrhoeae 
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.
PMCID: PMC2953271  PMID: 17207083
Neisseria gonorrhoeae; Neisseria species; ciprofloxacin; gyrA; species verification
2.  Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing 
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.
PMCID: PMC2768773  PMID: 16274961
DNA sequencing; Ciprofloxacin resistance; Neisseria gonorrhoeae; Pre-programmed DNA sequencing; Pyrosequencing technology
3.  Molecular characterization of Neisseria gonorrhoeae identifies transmission and resistance of one ciprofloxacin-resistant strain 
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.
PMCID: PMC2769514  PMID: 17367469
Neisseria gonorrhoeae; ciprofloxacin resistance; molecular epidemiology; porB gene; NG-MAST
4.  Comparison of Mismatch Amplification Mutation Assay with DNA Sequencing for Characterization of Fluoroquinolone Resistance in Neisseria gonorrhoeae 
Journal of Clinical Microbiology  2004;42(2):591-594.
A mismatch amplification mutation assay (MAMA) was developed for identification of point mutations in quinolone resistance-determining region (QRDR) of gyrA at codons 91 and 95. MAMA PCR was used to detect mutations at codons 91 and 95 of gyrA in 117 Neisseria gonorrhoeae isolates (with ciprofloxacin MICs of 0.004 to >32 μg/ml) from Bangladesh during 1997 to 2001. The QRDR regions of the gyrA genes from 31 randomly selected isolates were sequenced, and the results were compared with those of MAMA PCR. Using mismatch PCR, a mutation at Ser91 could be detected in all 27 (resistant and intermediate) isolates, and an Asp95-to-Gly95 mutation could be detected in all 15 isolates, as detected by sequencing. MAMA PCR offers a simple, inexpensive, rapid, and easier alternative for detection of point mutations in fluoroquinolone resistance in N. gonorrhoeae.
PMCID: PMC344476  PMID: 14766821
7.  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).
PMCID: PMC89751  PMID: 10681343
8.  Neutralization of Macrophage Inflammatory Protein 2 (MIP-2) and MIP-1α Attenuates Neutrophil Recruitment in the Central Nervous System during Experimental Bacterial Meningitis 
Infection and Immunity  1999;67(5):2590-2601.
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.
PMCID: PMC116008  PMID: 10225925
9.  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. One 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.
PMCID: PMC89232  PMID: 10103206
10.  Rapid Emergence of High-Level Resistance to Quinolones in Campylobacter jejuni Associated with Mutational Changes in gyrA and parC 
Antimicrobial Agents and Chemotherapy  1998;42(12):3276-3278.
Quinolone resistance in clinical isolates of Campylobacter jejuni in Sweden increased more than 20-fold at the beginning of the 1990s. Resistance to 125 μg 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.
PMCID: PMC106034  PMID: 9835526
11.  Synergistic Effect of Combinations of Sulfamethoxazole, Trimethoprim, and Colistin Against Pseudomonas maltophilia and Pseudomonas cepacia 
Eighteen strains of Pseudomonas maltophilia and eight strains of P. cepacia were tested for susceptibility to sulfamethoxazole, trimethoprim, and colistin in different combinations. A synergistic effect was found against most of the strains.
PMCID: PMC444683  PMID: 4143759
12.  Separation of Two Hemolysins from Aeromonas hydrophila by Isoelectric Focusing 
Infection and Immunity  1971;4(4):503-505.
Two hemolytic proteins (hemolysin I and II) with isoelectric points of 5.5 and 4.3 were purified by isoelectric focusing from the culture supernatant fluid of Aeromonas hydrophila. The purified hemolysins were unstable after dialysis and upon heating at 37 C and were inactivated by proteolytic enzymes. The two hemolysins were lethal for mice in a dose of 10 μg (hemolysin I) and 3 μg (hemolysin II) and showed similar hemolytic spectra on red cells of different species. Both were also cytotoxic for HeLa cells and human fibroblasts and gave dermonecrosis in rabbits.
PMCID: PMC416338  PMID: 5154892

Results 1-12 (12)