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Aim:To investigate the action mechanism of 1-benzyl-1,4-diazepane (1-BD) as efflux pump inhibitor (EPI) in Escherichia colimutants: ΔacrABor overexpressing AcrAB and AcrEF efflux pumps. Materials & methods:Effect of 1-BD on: antibiotic potentiation, by microdilution method; membrane functionality, by fluorimetric assays; ethidium bromide accumulation, by fluorometric real-time efflux assay; AcrB expression, by quantitative photoactivated localization microscopy. Results:1-BD decreases the minimal inhibitory concentration of levofloxacin and other antibiotics and increase ethidium bromide accumulation in E. colioverexpressing efflux pumps but not in the ΔacrABstrain. 1-BD increases membranes permeability, without sensibly affecting inner membrane polarity and decreases acrABtranscription. Conclusion:1-BD acts as an EPI in E. coliwith a mixed mechanism, different from that of major reference EPIs.

ABSTRACTThe strain diversity and the population structure of nosocomial Acinetobacterisolated from patients admitted to different hospitals in Florence, Italy, during a 3-year surveillance program, were investigated by amplified fragment length polymorphism (AFLP). The majority of isolates (84.5%) were identified as A. baumannii, confirming this species as the most common hospital Acinetobacter. Three very distinct A. baumanniiclonal groups (A1, A2, and A3) were defined. The A1 isolates appeared to be genetically related to the well-characterized European EU II clone. A2 was responsible for three outbreaks which occurred in two intensive care units. Space/time population dynamic analysis showed that A1 and A2 were successful nosocomial clones. Most of the A. baumannniiisolates were imipenem resistant. The genetic determinants of carbapenem resistance were investigated by multiplex PCR, showing that resistance, independently of hospital origin, period of isolation, or clonal group, was associated with the presence of a blaOXA-58-likegene and with ISAba2and ISAba3elements flanking this gene. blaOXA-58appeared to be horizontally transferred. This study showed that the high discriminatory power of AFLP is useful for identification and typing of nosocomial Acinetobacterisolates. Moreover the use of AFLP in a real-time surveillance program allowed us the recognition of clinically relevant and widespread clones and their monitoring in hospital settings. The correlation between clone diffusion, imipenem resistance, and the presence of the blaOXA-58-likegene is discussed.

ABSTRACTIn this study we report the results of analysis of 253 isolates of Staphylococcus aureus(132 methicillin [meticillin]-resistant S. aureus[MRSA] isolates and 121 methicillin-susceptible S. aureus[MSSA] isolates) from 209 patients admitted to 18 high-risk wards of six hospitals located in Florence, Italy, over an 8-month period during which a program of epidemiological surveillance of hospital-acquired infections was conducted. The majority (69%) of the 87 reported S. aureusinfections were caused by MRSA. No outbreak events have been reported. All the isolates were typed by amplified fragment length polymorphism (AFLP), and AFLP profiles were analyzed in order to define similarity groups. The discriminatory power of AFLP is very high with MSSA (Simpson index of diversity [D], 95.9%), whereas its resolution capability with MRSA (D, 44.7%) is hampered by the well-known high clonality of these populations (the main MRSA group accounted for 74% of the MRSA isolates). Combining AFLP, improved by visual inspection of polymorphisms, with multiplex PCR greatly increases MRSA resolution (D, 85.5%), resolving the MRSA population to a level that is one of the highest reported in the literature. Widespread and sporadic clones of MSSA and MRSA were identified, and their diffusion in the different hospitals and wards over the surveillance period was studied. The understanding of MSSA and MRSA population structures should be the starting point for the design of a more rational surveillance program for S. aureusspecies, maximizing benefits and reducing the cost of infection control strategies.

We report the epidemiological investigation of an outbreak of Pseudomonas aeruginosainfection in 6 patients who shared, during different periods, the same 2 rooms of a bone marrow transplantation unit. Phenotypic and molecular analysis of isolates from patients and from the environment strongly suggested a single, environmental source of infection.

The functional role of three conserved amino acid residues in Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1) has been investigated by site-directed mutagenesis. Crystallographic analyses indicated that Glu65, Ser103 and Glu104 are in hydrogen-bonding distance of the N-terminal amino group of the γ-glutamyl moiety of the co-substrate, GSH. Glu65 was mutated to either aspartic acid or leucine, and Ser103 and Glu104 were both mutated to alanine. Glu65 mutants (Glu65→Asp and Glu65→Leu) lost all enzyme activity, and a drastic decrease in catalytic efficiency was observed for Ser103→Ala and Glu104→Ala mutants toward both 1-chloro-2,4-dinitrobenzene and GSH. On the other hand, all mutants displayed similar intrinsic fluorescence, CD spectra and thermal stability, indicating that the mutations did not affect the structural integrity of the enzyme. Taken together, these results indicate that Ser103 and Glu104 are significantly involved in the interaction with GSH at the active site of PmGST B1-1, whereas Glu65 is crucial for catalysis.

Glutathione S-transferases (GSTs) normally use hydroxy-group-containing residues in the N-terminal domain of the enzyme for stabilizing the activated form of the co-substrate, glutathione. However, previous mutagenesis studies have shown that this is not true for Beta class GSTs and thus the origin of the stabilization remains a mystery. The recently determined crystal structure of Proteus mirabilis GST B1-1 (PmGST B1-1) suggested that the stabilizing role might be fulfilled in Beta class GSTs by one or more residues in the C-terminal domain of the enzyme. To test this hypothesis we mutated His106 and Lys107 of PmGST B1-1 to investigate their possible role in the enzyme's catalytic activity. His106 was mutated to Ala, Asn and Phe, and Lys107 to Ala and Arg. The effects of the replacement on the activity, thermal stability and antibiotic-binding capacity of the enzyme were examined. The results are consistent with the involvement of His106 and Lys107 in interacting with glutathione at the active site but these residues do not contribute significantly to catalysis, folding or antibiotic binding.

The function of the open reading frame (ORF) YOR108w of Saccharomyces cerevisiae has been analysed. The deletion of this ORF from chromosome XV did not give an identifiable phenotype. A mutant in which both ORF YOR108w and LEU4 gene have been deleted proved to be leucine auxotrophic and α-isopropylmalate synthase (α-IPMS)-negative. This mutant recovered α-IPMS activity and a Leu⁺ phenotype when transformed with a plasmid copy of YOR108w. These data and the sequence homology indicated that YOR108w is the structural gene for α-IPMS II, responsible for the residual α-IPMS activity found in a leu4Δ strain. The leu4Δ strain appeared to be very sensitive to the leucine analogue trifluoroleucine. In the absence of leucine, its growth was not much impaired in glucose but more on non-fermentable carbon sources. Copyright © 2000 John Wiley & Sons, Ltd.

The function of the open reading frame (ORF) YOR108w of Saccharomyces cerevisiaehas been analysed. The deletion of this ORF from chromosome XV did not give an identifiable phenotype. A mutant in which both ORF YOR108w and LEU4gene have been deleted proved to be leucine auxotrophic and α‐isopropylmalate synthase (α‐IPMS)‐negative. This mutant recovered α‐IPMS activity and a Leu+phenotype when transformed with a plasmid copy of YOR108w. These data and the sequence homology indicated that YOR108w is the structural gene for α‐IPMS II, responsible for the residual α‐IPMS activity found in a leu4Δstrain. The leu4Δstrain appeared to be very sensitive to the leucine analogue trifluoroleucine. In the absence of leucine, its growth was not much impaired in glucose but more on non‐fermentable carbon sources. Copyright © 2000 John Wiley & Sons, Ltd.

The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported. A PCR‐based strategy was used to construct disruption cassettes in which the kanMX4dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994). The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN.PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4. The correctness of the cassette integration was tested by PCR. After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin‐resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed phenotypic analysis of the non‐lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor differences between parental and mutant strains were found for some deletant haploids. Copyright © 1999 John Wiley & Sons, Ltd.

The resistance to 5,5,5‐trifluoro‐DL‐leucine, encoded by the dominant allele LEU4‐1, was used as a selectable marker to transform laboratory and natural Saccharomyces cerevisiaestrains by the lithium acetate procedure. Results of transformation of S. cerevisiaelaboratory and wine natural strains showed that trifluoroleucine resistance is a very effective selection marker and can be widely used to transform prototrophic S. cerevisiaestrains. The LEU4‐1gene could also be exploited to improve wine flavour, as indicated by the higher isoamyl alcohol content of the transformants compared to the parental strains.

The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported. A PCR-based strategy was used to construct disruption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994). The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN.PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4. The correctness of the cassette integration was tested by PCR. After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin-resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed phenotypic analysis of the non-lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor differences between parental and mutant strains were found for some deletant haploids. Copyright © 1999 John Wiley & Sons, Ltd.

The structural gene of the Proteus mirabilis glutathione transferase GSTB1-1 (gstB) has been isolated from genomic DNA. A nucleotide sequence determination of gstB predicted a translational product of 203 amino acid residues, perfectly matching the sequence of the previously purified protein [Mignogna, Allocati, Aceto, Piccolomini, Di Ilio, Barra and Martini (1993) Eur. J. Biochem. 211, 421–425]. The P. mirabilis GST sequence revealed 56% identity with the Escherichia coli GST at DNA level and 54% amino acid identity. Similarity has been revealed also with the translation products of the recently cloned gene bphH from Haemophilus influenzae (28% identity) and ORF3 of Burkholderia cepacia (27% identity). Putative promoter sequences with high similarity to the E. coli σ70 consensus promoter and to promoters of P. mirabiliscat and glnA genes preceded the ATG of the gstB open reading frame (ORF). gstB was brought under control of the tac promoter and overexpressed in E. coli by induction with isopropyl-β-d-thiogalactopyranoside and growth at 37 °C. The physicochemical and catalytic properties of overexpressed protein were indistinguishable from those of the enzyme purified from P. mirabilis extract. Unlike the GST belonging to Mu and Theta classes, GSTB1-1 was unable to metabolize dichloromethane. The study of the interaction of cloned GSTB1-1 with a number of antibiotics indicates that this enzyme actively participates in the binding of tetracyclines and rifamycin.

Total glutathione content, glutathione peroxidase, glutathione transferase and glutathione reductase activities have been measured in 12 species of yeasts. All the strains tested contained glutathione, though in different amounts, as well as the above mentioned enzymes. To discriminate between the selenium-dependent and the selenium-independent form, glutathione peroxidase activity has been measured with both H2O2 and cumene hydroperoxide. Rhodotorula glutinis appeared to be the only strain in which the selenium-dependent form was not found, but this yeast exhibited the highest level of selenium-independent glutathione peroxidase activity as compared to the other strains.

The role of the evolutionarily conserved residue Pro‐53 in Proteus mirabilisglutathione transferase B1‐1 has been examined by replacing it with a serine residue using site‐directed mutagenesis. The effect of the replacement on the activity, thermal stability and antibiotic binding capacity of the enzyme was examined. The results presented support the view that Pro‐53 participates in the maintenance of the proper conformation of the enzyme fold rather than playing a direct role in the catalytic reaction. Furthermore, this residue appears to be an important determinant of the antibiotic binding to the enzyme. Experiments with wild type and mutated enzymes provide evidence that glutathione transferases may play an important role in antibiotic resistance exhibited by bacteria.

Growth inhibition and cell killing caused by sulphite were reduced in seven Saccharomyces cerevisiae sulphite-resistant independent mutants, compared to their parental strains. Genetic analysis showed that in the seven mutants resistance was inherited as a single-gene dominant mutation and that all the analyzed mutations were allelic, thus identifying a major gene responsible for sulphite resistance in S. cerevisiae. Two of the mutants, MBS20-9 and MBS30, were further characterized. 35S-sulphite uptake experiments showed that the ability to accumulate sulphite was markedly reduced in the two resistant strains. No difference between resistant and sensitive strains with respect to glyceraldehyde-3-phosphate dehydrogenase sensitivity to sulphite, or to intracellular glutathione content, were revealed. In contrast, the extracellular acetaldehyde concentration was higher in the resistant mutants, both in the presence and in the absence of sulphite.

In order to investigate the roles of near N‐terminus Tyr, Cys, and Ser residues in the activity of bacterial glutathione transferase (GSTB1‐1) site‐directed mutagenesis was used to replace the following residues: Tyr‐4, Tyr‐5, Ser‐9, Cys‐10, Ser‐11, and Ser‐13. The results presented here show that, unlike all other alpha, mu, pi, theta and sigma classes of glutathione transferases so far investigated, GSTB1‐1 does not utilise any Tyr, Ser or Cys residue to activate glutathione. These results also suggest that the bacterial glutathione transferases may require classification into their own class.

In order to investigate the roles of near N-terminus Tyr, Cys, and Ser residues in the activity of bacterial glutathione transferase (GSTB1-1) site-directed mutagenesis was used to replace the following residues: Tyr-4, Tyr-5, Ser-9, Cys-10, Ser-11, and Ser-13. The results presented here show that, unlike all other alpha, mu, pi, theta and sigma classes of glutathione transferases so far investigated, GSTB1-1 does not utilise any Tyr, Ser or Cys residue to activate glutathione. These results also suggest that the bacterial glutathione transferases may require classification into their own class.

The resistance to 5,5,5-trifluoro-dl-leucine, encoded by the dominant allele LEU4-1, was used as a selectable marker to transform laboratory and natural Saccharomyces cerevisiae strains by the lithium acetate procedure. Results of transformation of S. cerevisiae laboratory and wine natural strains showed that trifluoroleucine resistance is a very effective selection marker and can be widely used to transform prototrophic S. cerevisiae strains. The LEU4-1 gene could also be exploited to improve wine flavour, as indicated by the higher isoamyl alcohol content of the transformants compared to the parental strains.