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P882 Biological cost of resistance to fosfomycin in Escherichia coli isolates
S228 Methods: Both the PAO1deltamutS and the wild-type PAO1 P. aeruginosa strains (5×109−1×1010 cfu/mL) were exposed to agar plates containing different antibiotic concentrations (range 0.25–256 mg/L) of tobramycin (TOB), ceftazidime (CAZ), imipenem (IMP), ciprofloxacin (CIP), and levofloxacin (LEV). Sixty five mutants recovered from plates immediately below the mutant prevention concentration were selected and susceptibility (standard CLSI agar dilution) to different antibiotics was determined. Topoisomerase (II and IV) mutations, expression of efflux pumps (MexAB-OprM, MexCD-OprJ, and MexXY) and AmpC b-lactamase, and OprD deficiency were analyzed in selected mutants. Results: The original PAO1deltamutS strain was less susceptible than its parental wild-type PAO1 strain for CAZ (8 and 1 mg/L, respectively) and TOB (2 and 0.5 mg/L). Mutants derived form the PAO1deltamutS strain were obtained in plates with higher CAZ, IMP, or TOB antibiotic concentrations (1−2 log dilutions) than those permitting the growth of mutants originated in normo-mutable PAO1 strain. Indeed mutants obtained from PAO1deltamutS strain were regularly less susceptible (1−4 log dilutions) than those selected form the wild-type PAO1 strain. Mutants selected on CAZ and IMP have reduced susceptibility restricted to b-lactam antibiotics. In these mutants, the MICs and enzymatic activity were compatible with a higher production of chromosomal AmpC enzyme in the case of CAZ selected mutants, whereas AmpC hyper-production superimposed with OprD reduction was observed in the case of IMP. In mutants selected with CIP or LEV, overproduction of efflux pumps was mainly observed with PAO1 strain and overproduction of pumps superimposed with gyrA mutations with PAO1deltamutS strain. Conclusions: The higher antibiotic resistance levels of mutants derived from dense populations of PAO1deltamutS P. aeruginosa when compared with those from the corresponding normo-mutable PAO1 wild-type strain might result from the selection of cells carrying double mutations. Hyper-mutable strains contribute to the single-step selection of variants with higher levels of antibiotic resistance.
P881 Altered outer membrane protein profiles of Pseudomonas aeruginosa highly susceptible to the efflux pump inhibitor Phe-Arg-b-naphthylamide A.B. Campo-Esquisabel, S. Mart´ı, J.M. Sierra, L. Martinez-Martinez, J. Vila (Santander, Barcelona, ES) Objective: Multidrug effllux systems play an important role in resistance of P. aeruginosa (Pae) to several antibiotics. Phe-Arg-b-naphthylamide (PAN) is usually able to inhibit these systems. When evaluating the relevance of efflux in some clinical isolates of Pae we noticed that some of them were inhibited by low concentration of PAN alone. We have evaluated OMP expression in Pae highly susceptible to PAN. Methods: Three Pae clinical isolates (HUMV1, HUMV2 and HUMV3) for which MIC of FEP was at least 2 times higher than that of CAZ were selected among a collection of 20 isolates with a similar phenotype. MICs of PAN and of FEP in the absence and in the presence of 20 mg/l of PAN were determined by reference microdilution (CLSI guidelines). The study of clonal relation between the isolates was performed by pulsed-field gel electrophoresis (PFGE) digested with SpeI. Fractions of enriched cell envelope proteins from the three organisms grown in media without or with subMIC of PAN profiles were characterised by SDSPAGE. Proteins were further analysed by MALDI-TOF mass spectometry after in-gel trypsin digestion. Results: MICs (mg/l) of FEP for HUMV1, HUMV2 and HUMV3 were 64, 32 and 16 mg/l, respectively. MIC of FEP+PAN for HUMV3 was 4 mg/l, but HUMV1 and HUMV2 did not grow in the presence of 20 mg/l of PAN. In fact, MICs of PAN alone for HUMV1 and HUMV2 were 4 and 2 mg/l, respectively. The PFGE profile for HUMV1 and HUMV3 was identical, and their corresponding protein profiles differed in three proteins identified as: major outer membrane lipoprotein I (present in HUMV3, absent in HUMV1), 30S ribosomal protein S16 (reduced expression in HUMV1) and lisozyme (present in HUMV1, absent in HUMV3). Major outer membrane lipoprotein I was also absent in the PAN-susceptible HUMV2 isolate.
17th ECCMID / 25th ICC, Posters Conclusions: Hypersusceptibility of two P. aeruginosa strains to the efflux pump inhibitor Phe-Arg-b-naphthylamide was linked to loss of major outer membrane lipoprotein I. P882 Biological cost of resistance to fosfomycin in Escherichia coli isolates J.I. Al´os, P. Garc´ıa-Pe˜na, J. Tamayo (M´ostoles, ES) Fosfomycin is a bactericidal antibiotic that acts by inhibiting the cell wall, and which is used mainly in the treatment of uncomplicated urinary tract infections (UTI). Resistance to fosfomycin develops rapidly in experimental conditions, although despite its frequent use in UTI, resistance in E. coli, the main uropathogen, is very low (1−3%), and has remained so for many years. The objective of this study was to ascertain whether E. coli fosfomycin-resistant strains have less fitness than those that are fosfomycin-sensitive in competing, and would therefore tend to disappear in their competition with fosfomycin-sensitive strains in the absence of antibiotics. Methods: Fosfomycin-resistant strains (n = 8) with different phenotypes of resistance to other antibiotics. All but one were lactose (+). Fosfomycin-resistant strains (n = 13) that had same phenotypes of resistance to other antibiotics as the resistant strains and which furthermore had the opposite pattern of lactose fermentation. Thirty-three (33) competition experiments by pairs of strains were conducted (Fosfomycin-R versus Fosfomycin-S with the rest of resistance determinants being equal and with a different lactose fermentation capacity to perform the differential counts in MacConkey agar). The experiments were performed in nutrient broth (NB). Equal amounts of the strains were challenged (approx. 50% and approx. 50%) for 4 days, with a daily change to a new medium. Five differential counts were performed (days 0, 1, 2, 3, and 4). The 33 experiments were performed in duplicate, and the mean of both results are presented below. Results: In 20 experiments (60.6%) there was a relative increase in the fosfomycin-sensitive strain that translated into a count of >60% (between 65%-99%) on the fourth day. In 6 experiments (18.2%) there was a relative increase in the fosfomycin-resistant strain that translated into a count of >60% (between 65%-75%) on the fourth day. In 7 experiments (21.2%), on the fourth day none of the strains reached 60%. When the data of the 26 (20+6) experiments in which there were changes were analysed by the Chi2 test there was an statistically significant difference (P = 0.044). Conclusions: • Resistance to fosfomycin entails a biological cost (less fitness) for the majority of the E. coli strains assayed. • This biological cost hinders their competition with fosfomycinsensitive strains in the normal intestinal flora, which would make them less likely to cause UTI. P883 Antibiotic resistance of enterococci isolated from blood cultures during 2003–2006 U. Arslan, B. Feyzioglu, E.B. Uysal, E.I. Tuncer, D. Findik (Konya, TR) Objectives: Enterecocci have become a significant problem due to their aetiologic role in bacterial infections. An alarming problem is the increasing rate of enterococci resistant to vancomycin (VRE) and therefore the source and spreading of these strains are very important epidemiological problems. The aim of the study was to evaluate antibiotic resistance and monitoring of VRE isolated from blood cultures during July 2003 – October 2006 in our hospital. Methods: A total of 138 strains of enterococci were isolated from blood cultures. The identification of the isolated bacteria was performed by conventional methods and bioM´erieux API 20 STREP test (Marcy I’Etoile, France). The susceptibility testing was carried out by discdiffusion method and Etest. Determination of glycopeptide resistance genotypes (van A, van B, van C1, van C2/3) of VRE was performed by GenoType Enterococcus assay (Hain Lifescience GmbH, Nehren, Germany).
P881 Altered outer membrane protein profiles of Pseudomonas aeruginosa highly susceptible to the efflux pump inhibitor Phe-Arg-b-naphthylamide A.B. Campo-Esquisabel, S. Mart´ı, J.M. Sierra, L. Martinez-Martinez, J. Vila (Santander, Barcelona, ES) Objective: Multidrug effllux systems play an important role in resistance of P. aeruginosa (Pae) to several antibiotics. Phe-Arg-b-naphthylamide (PAN) is usually able to inhibit these systems. When evaluating the relevance of efflux in some clinical isolates of Pae we noticed that some of them were inhibited by low concentration of PAN alone. We have evaluated OMP expression in Pae highly susceptible to PAN. Methods: Three Pae clinical isolates (HUMV1, HUMV2 and HUMV3) for which MIC of FEP was at least 2 times higher than that of CAZ were selected among a collection of 20 isolates with a similar phenotype. MICs of PAN and of FEP in the absence and in the presence of 20 mg/l of PAN were determined by reference microdilution (CLSI guidelines). The study of clonal relation between the isolates was performed by pulsed-field gel electrophoresis (PFGE) digested with SpeI. Fractions of enriched cell envelope proteins from the three organisms grown in media without or with subMIC of PAN profiles were characterised by SDSPAGE. Proteins were further analysed by MALDI-TOF mass spectometry after in-gel trypsin digestion. Results: MICs (mg/l) of FEP for HUMV1, HUMV2 and HUMV3 were 64, 32 and 16 mg/l, respectively. MIC of FEP+PAN for HUMV3 was 4 mg/l, but HUMV1 and HUMV2 did not grow in the presence of 20 mg/l of PAN. In fact, MICs of PAN alone for HUMV1 and HUMV2 were 4 and 2 mg/l, respectively. The PFGE profile for HUMV1 and HUMV3 was identical, and their corresponding protein profiles differed in three proteins identified as: major outer membrane lipoprotein I (present in HUMV3, absent in HUMV1), 30S ribosomal protein S16 (reduced expression in HUMV1) and lisozyme (present in HUMV1, absent in HUMV3). Major outer membrane lipoprotein I was also absent in the PAN-susceptible HUMV2 isolate.
17th ECCMID / 25th ICC, Posters Conclusions: Hypersusceptibility of two P. aeruginosa strains to the efflux pump inhibitor Phe-Arg-b-naphthylamide was linked to loss of major outer membrane lipoprotein I. P882 Biological cost of resistance to fosfomycin in Escherichia coli isolates J.I. Al´os, P. Garc´ıa-Pe˜na, J. Tamayo (M´ostoles, ES) Fosfomycin is a bactericidal antibiotic that acts by inhibiting the cell wall, and which is used mainly in the treatment of uncomplicated urinary tract infections (UTI). Resistance to fosfomycin develops rapidly in experimental conditions, although despite its frequent use in UTI, resistance in E. coli, the main uropathogen, is very low (1−3%), and has remained so for many years. The objective of this study was to ascertain whether E. coli fosfomycin-resistant strains have less fitness than those that are fosfomycin-sensitive in competing, and would therefore tend to disappear in their competition with fosfomycin-sensitive strains in the absence of antibiotics. Methods: Fosfomycin-resistant strains (n = 8) with different phenotypes of resistance to other antibiotics. All but one were lactose (+). Fosfomycin-resistant strains (n = 13) that had same phenotypes of resistance to other antibiotics as the resistant strains and which furthermore had the opposite pattern of lactose fermentation. Thirty-three (33) competition experiments by pairs of strains were conducted (Fosfomycin-R versus Fosfomycin-S with the rest of resistance determinants being equal and with a different lactose fermentation capacity to perform the differential counts in MacConkey agar). The experiments were performed in nutrient broth (NB). Equal amounts of the strains were challenged (approx. 50% and approx. 50%) for 4 days, with a daily change to a new medium. Five differential counts were performed (days 0, 1, 2, 3, and 4). The 33 experiments were performed in duplicate, and the mean of both results are presented below. Results: In 20 experiments (60.6%) there was a relative increase in the fosfomycin-sensitive strain that translated into a count of >60% (between 65%-99%) on the fourth day. In 6 experiments (18.2%) there was a relative increase in the fosfomycin-resistant strain that translated into a count of >60% (between 65%-75%) on the fourth day. In 7 experiments (21.2%), on the fourth day none of the strains reached 60%. When the data of the 26 (20+6) experiments in which there were changes were analysed by the Chi2 test there was an statistically significant difference (P = 0.044). Conclusions: • Resistance to fosfomycin entails a biological cost (less fitness) for the majority of the E. coli strains assayed. • This biological cost hinders their competition with fosfomycinsensitive strains in the normal intestinal flora, which would make them less likely to cause UTI. P883 Antibiotic resistance of enterococci isolated from blood cultures during 2003–2006 U. Arslan, B. Feyzioglu, E.B. Uysal, E.I. Tuncer, D. Findik (Konya, TR) Objectives: Enterecocci have become a significant problem due to their aetiologic role in bacterial infections. An alarming problem is the increasing rate of enterococci resistant to vancomycin (VRE) and therefore the source and spreading of these strains are very important epidemiological problems. The aim of the study was to evaluate antibiotic resistance and monitoring of VRE isolated from blood cultures during July 2003 – October 2006 in our hospital. Methods: A total of 138 strains of enterococci were isolated from blood cultures. The identification of the isolated bacteria was performed by conventional methods and bioM´erieux API 20 STREP test (Marcy I’Etoile, France). The susceptibility testing was carried out by discdiffusion method and Etest. Determination of glycopeptide resistance genotypes (van A, van B, van C1, van C2/3) of VRE was performed by GenoType Enterococcus assay (Hain Lifescience GmbH, Nehren, Germany).