A novel gyrB mutation in a fluoroquinolone-resistant clinical isolate of Salmonella typhimurium.

In order to study the role of gyrB in antibiotic resistance in post-ciprofloxacin therapy fluoroquinolone-resistant clinical isolates of Salmonella typhimurium, plasmid pBP548, which contains the Escherichia coli gyrB gene, was used in complementation studies. In a heterodiploid strain, the wild-type (quinolone sensitive) allele is dominant over the resistant allele therefore, eleven clinical isolates were complemented with gyrB encoded on pBP548. Only one transformant, L18pBP548, exhibited increased susceptibility to the quinolones nalidixic acid, ciprofloxacin and sparfloxacin. The amino acid sequence of the gyrase B protein from a wild-type and the pre-therapy S. typhimurium (deduced from the nucleotide sequence) was identical to that of E. coli from codons 436 to 470; however, a point mutation was identified in codon 463 of gyrB of the quinolone-resistant post-therapy isolate L18, giving rise to an amino acid substitution of serine to tyrosine.

A DNA expression array to detect toxic stress response in European flounder (Platichthys flesus).

As a first stage in developing a DNA array-based approach to investigating the effects of pollutants on an environmentally relevant European fish species, we have constructed a 160-gene custom microarray for European flounder. Degenerate primers were used to amplify 110 different fragments of stress-related and other genes from European flounder cDNA and genomic DNA. Additionally, 22 fragments were obtained by suppressive subtractive hybridisation (SSH). These fragments were cloned and sequenced, then, with additional control genes, used to create a cDNA microarray for flounder. After optimisation of the arraying process, hepatic mRNA was isolated from flounder caught in the polluted Tyne and relatively unpolluted Alde estuaries. Fluorescent cDNA probes were synthesised from the mRNA and used in dual-colour hybridisations to the microarray. A number of transcripts were differentially expressed between Tyne and Alde female flounder but these changes were not significant, due to high inter-individual variation. However, in comparisons between Tyne and Alde male flounder, 11 transcripts were found to significantly differ in expression (P<0.05). Seven transcripts were more highly expressed in the Tyne male fish (CYP1A, UDPGT, alpha-2HS-glycoprotein, dihydropyrimidine dehydrogenase, Cu/Zn SOD, aldehyde dehydrogenase and paraoxonase). Four transcripts (Elongation factor 1 (EF1), EF2, Int-6 and complement component C3) were found to be significantly less abundant in the Tyne male fish. Selected genes were assayed by real-time PCR, then normalised to alpha-tubulin. These assays confirmed the significance of the array results for CYP1A, UDPGT and EF1, but not for Cu/Zn SOD. This study provides a link between traditional single-gene biomarker studies and the emerging field of eco-toxicogenomics, demonstrating the utility of microarray studies on environmentally sampled, non-model organisms.

Mutations in gyrA gene of quinolone-resistant Salmonella serotypes isolated from humans and animals.

The quinolone resistance-determining regions (QRDRs) of the gyrA genes of quinolone-resistant clinical and veterinary salmonella isolates were sequenced. Substitutions analogous to a substitution of a Ser to a Phe at position 83 (Ser83-->Phe) and Asp87-->Gly or Tyr in Escherichia coli were found, as was a single novel mutation outside of the QRDR resulting in Ala119-->Glu. The data suggest that gyrA mutations are associated with quinolone resistance in veterinary and clinical salmonella isolates and that the limits of the QRDR may require revision.

Siderophore-specific induction of iron uptake in Pseudomonas aeruginosa.

Pseudomonas aeruginosa has two siderophore-based high-affinity iron-uptake systems utilizing pyoverdin and pyochelin. Using strain IA1, a mutant deficient in production of both siderophores, we have shown that addition of purified siderophore to the growth medium induces expression of specific iron-regulated outer-membrane proteins and increases 55Fe-siderophore transport. Addition of pyoverdin from the parent strain PAO1 or from a clinical strain 0:12 induced expression of an 85 kDa IROMP and increased the rate of 55Fe-pyoverdin transport. Transport rates for 55Fe-PAO1 pyoverdin increased from 1.27 to 3.57 pmol Fe min-1 per 10(9) cells. Addition of purified pyochelin induced expression of a 75 kDa IROMP accompanied with increased 55Fe-pyochelin uptake without affecting 55Fe-pyoverdin transport. 55Fe-pyochelin transport increased from 0.3 to 10.6 pmol min-1 per 10(9) cells. Addition of pyoverdin from the parent strain or a chromatographically distinct pyoverdin caused increased reactivity with an anti-85 kDa mAb in Western blotting, indicating that the same receptor is being induced. These results suggest that P. aeruginosa can respond specifically to the presence of siderophore and moreover that not only can the pyoverdin receptor transport its cognate ferri-pyoverdin but also different ferri-pyoverdins, albeit at a reduced rate.

Subtilisin-related serine proteases in the mammalian constitutive secretory pathway.

Proteolytic cleavage is a key step in the activation of many secreted proteins. Since the identification of the prototype yeast enzyme Kex2, seven subtilisin-related serine proteases, together with a number of isoforms, have been identified in mammalian cells, five of which act within the constitutive secretory pathway. Overlapping expression patterns and substrate specificities complicate analysis but individual roles are beginning to emerge for some members of the group.

Evidence for an efflux pump mediating multiple antibiotic resistance in Salmonella enterica serovar Typhimurium.

The mechanism of multiple antibiotic resistance in six isolates of Salmonella enterica serovar Typhimurium recovered from a patient treated with ciprofloxacin was studied to investigate the role of efflux in the resistance phenotype. Compared to the patient's pretherapy isolate (L3), five of six isolates accumulated less ciprofloxacin, three of six isolates accumulated less chloramphenicol, and all six accumulated less tetracycline. The accumulation of one or more antibiotics was increased by carbonyl cyanide m-chlorophenylhydrazone to concentrations similar to those accumulated by L3 for all isolates except one, in which accumulation of all three agents remained approximately half that of L3. All isolates had the published wild-type sequences of marO and marR. No increased expression of marA, tolC, or soxS was observed by Northern blotting; however, three isolates showed increased expression of acrB, which was confirmed by quantitative competitive reverse transcription-PCR. However, there were no mutations within acrR or the promoter region of acrAB in any of the isolates.

Uptake of BRL 41897A, a C(7) alpha-formamido substituted cephalosporin, via the ferri-pyochelin transport system of Pseudomonas aeruginosa.

Several catechol-substituted cephalosporin antibiotics are transported into Gram-negative organisms via components of the iron uptake system. We provide evidence that the C(7) alpha-formamido substituted cephalosporin BRL 41897A enters Pseudomonas aeruginosa via the high molecular weight pyochelin transport system. No MIC could be recorded when cells were grown under iron-replete conditions, but when grown in iron-depleted succinate medium strain PAO1 the MIC was 4 mg/L. A derivative of PAO1 that is deficient in the biosynthesis of pyochelin and pyoverdin, IA1, was more resistant, having MIC of 16 mg/L. This was reduced to 4 mg/L by growing the cells in the presence of pyochelin, which specifically induces the pyochelin uptake system. Growth in the presence of pyochelin also increased the rate and extent of uptake of a radiolabelled iron-antibiotic complex. Growth in the presence of 1/8 of the MIC resulted in significant reduction in expression of the ferripyochelin receptor in the outer membrane, coupled with decreased sensitivity to the agent.

The pyocin Sa receptor of Pseudomonas aeruginosa is associated with ferripyoverdin uptake.

We have used Tn5 mutagenesis to obtain a mutant resistant to pyocin Sa. When grown in iron-deficient succinate medium this mutant lacked an 85-kDa iron-regulated outer membrane protein (IROMP), and expression of a 75-kDa IROMP was increased compared with that in the parent strain. The mutant was deficient in pyoverdin biosynthesis and showed a 95% decrease in transport of ferripyoverdin purified from the parent strain, suggesting that the 85-kDa IROMP is the specific receptor for ferripyoverdin and pyocin Sa. The mutant compensated for the deficiency in pyoverdin biosynthesis and transport by exhibiting a fourfold increase in ferripyochelin transport. The low-level transport of ferripyoverdin in the Sa-resistant mutant, which extended to heterologous pyoverdins from other strains, suggests that Pseudomonas aeruginosa has a second ferripyoverdin uptake system of lower affinity and broader specificity.