Mechanisms of action and resistance 4 S. mitis isolates (9.7%) harbouring a mef gene. Nevertheless, their MICs of ERY were similar to mef(−) MSVS isolates (range: 0.05−0.5 mg/mL). The genetic environment study suggests mef gene might be included in a genetic element similar to MEGA. When we performed transformation experiments, we got mef(+) S. pneumoniae R6 transformants. Nevertheless, these transformants were ERY-resistant, with ERY MICs similar to mef(+), MRVS isolates. Conclusions: mef gene may be not enough for determining ERYresistance in VS clinical isolates. Nevertheless, their DNA seems to be able to confer ERY-resistance to pneumococci. R2119 Carbapenem resistance of Escherichia coli strains R. Schaumann, D. Adler, A.C. Rodloff (Leipzig, DE) Objectives: Carbapenem (CP) resistance of Escherichia coli strains is extremely rare and seldomly discussed. The aim of the present study was to investigate the resistance mechanisms of three recent clinical E. coli isolates that were CP resistant. Methods: MICs for imipenem (IMP), meropenem (MER), and ertapenem (ERT) as well as ESBL or MBL production were determined by Etest. Furthermore, the three strains were analysed for blaTEM, blaSHV, blaampC, blaACC, blaVIM, and blaIMP by PCR and isoelectric focusing and visualisation by nitroceﬁn and coomassie blue was carried out. In addition, the strains were serially passaged on antibiotic free columbia agar plates and MICs re-determined. Conversely the three strains and – for control purposes – eight susceptible E. coli strains were incubated on solid media containing IMP, MER, and ERT, respectively, at twice their MICs. After incubation growing bacteria were harvested and incubated at four times MICs. This procedure was repeated with increasing antibiotic concentrations. The resulting MICs were conﬁrmed by Etest. Results: The MICs for the three resistant strains ranged from 4 to >32 mg/L. The other E. coli strains showed MICs between 0.004 and 0.38 mg/L. After passages on antibiotic free medium, the MICs for the three strains decreased to 0.38 to 16 mg/L. However, after passages on antibiotic containing agar plates the MICs were >32 mg/L for all three CPs tested. The resistant strains contained blaampC and blaTEM. Isoelectric focusing demonstrated b-lactamases of various isoelectric points while the MBL and ESBL tests were negative or not calculable except for one strain. Furthermore, elevated MICs were inducible with ERT and IMP in the susceptible strains but not with MER. Conclusion: E. coli strains may posses various mechanisms such as blaampC and blaTEM that in combination cause CP resistance. They may loose their phenotypic resistance after several passages on antibiotic free medium. Conversely, employing passages on antibiotic containing medium led to reappearance of the resistant phenotype. R2120 Characterisation of unusual patterns of MLS resistance among Streptococcus pyogenes clinical isolates L. Vitali, S. Rombini, D. Petrelli, S. D’Ercole, G. Pasquantonio, C. Ripa, M. Prenna (Camerino, Rome, Ancona, IT) Objectives: In Gram-positive cocci, macrolide, lincosamide, and streptogramin B (MLS) resistance can be expressed either constitutively (cMLS) or inducibly (iMLS) and is mediated by the presence of erm genes. The present work sought to investigate the iMLS and cMLS phenotypes of a group of erm(B)-positive Streptococcus pyogenes clinical isolates showing non-standard behaviours in the erythromycin (ERY)–clindamycin (CLI) double disk diffusion testing. Methods: Eight clinical isolates coming from patients with pharyngitis and two reference strains were analysed. Their ERY resistance phenotype was determined by the double disk diffusion testing and by growth in liquid medium using cells induced by preincubation with ERY (0.1 mg/L) followed by challenge with 50 mg/L of CLI. Inoculum preparation was accomplished by the two alternative procedures suggested by CLSI. Muller–Hinton II (MH) and Brain-Heart-Infusion (BHI), both supplemented with 5% sheep blood, were used as test media.
S613 Cultures were incubated at 35ºC in an atmosphere of 5% CO2 for a maximum of 24 h. Results: Double disk diffusion testing, with the inoculum prepared by direct colony suspension, assigned 50% of the strains to the cMLS phenotype. Within this group three strains showed an inner hazy Dshaped inhibition zone around the CLI disk (HD). Among the four iMLS strains, three presented small colonies growing proximal to CLI disk in otherwise essentially clear zone (D+). Induction experiments in liquid medium showed that HD strains responded to some extent to induction but only during the logarithmic phase of growth, while in the D+ group even non-induced samples became resistant when cells reached the stationary phase. To test weather the efﬁciency in the control of the iMLS resistance was growth rate dependent, MH was replaced with BHI and the inoculum prepared following the growth method. In these conditions, D+ strains reverted to a plain iMLS and HD to a cMLS, both in agar and in liquid. These effects were even more evident after an incubation time of 6−8 h. Conclusion: These results indicate that double disk diffusion testing for MLS phenotype determination in S. pyogenes should be performed starting with an inoculum prepared by the growth method and results read also during the course of the experiment (e.g. at 6−8 h). The use of BHI instead of MH further diminish difﬁculties in the assignment of some S. pyogenes strains to the correct MLS phenotype.
R2121 In vitro activity of tigecycline and comparators against Acinetobacter baumannii isolated from severe infections in Italy M. Mezzatesta, M. Trovato, V. Nicolosi, D. Nicolosi, G. Nicoletti, A. Cassone, G. Fadda, G. Schito, S. Stefani (Catania, Rome, Genoa, IT) Objectives: In a recent multi-centre survey (2003–2004), conducted in 45 laboratories throughout Italy with the aim of monitoring microrganisms responsible for severe infections and their antibiotic resistance, Acinetobacter baumannii was isolated from the ICU of 12 centres as the third most frequent pathogen. Due to the increasing importance of this microrganism in hospital epidemiology, which is partly due to its success in acquiring resistance to carbapenem or showing a multi-drug-resistance (MDR) phenotype, it is mandatory to look at the activity of new antimicrobial agents. Methods: Included in the study were 88 clinically signiﬁcant strains of A. baumannii. Tigecycline and comparators were tested by MICs following the CLSI guidelines. For the carbapenem resistant strains, a preliminary phenotypic screening for the presence of metallo-enzymes (MBL) was performed by Etest, and PCR was used to investigate the presence of blaOXA, blaIMP and blaVIM genes. PFGE was performed to test clonality. Results: A. baumannii was a frequent cause of ventilator-associated pneumonia and bacteraemia. Resistance to ceftazidime, ciproﬂoxacin and aztreonam was widespread in almost 90% of strains; resistance to imipenem was 51% and resistance to meropenem was 64%, amikacin and gentamicin were active against 30% of strains and colistin was very active. Tigecycline had a MIC90 value of 2 mg/L and our strains showed a unimodal distribution of susceptibility, demonstrating that this new drug is more active than other tetracyclines. Among the 45 imipenemresistant isolates (MIC gt; 16 mg/L) only 17 strains showed a reduction in the imipenem MIC with EDTA indicating MBL activity, but no PCR products for blaIMP and blaVIM were obtained from the strains analysed. Further studies are in progress for the characterisation of the blaOXA resistance determinants. PFGE analyses showed the existence of a multiresistant A. baumannii clone, widespread in different hospitals. Conclusion: In conclusion, tigecycline showed a potent activity against the MDR A. baumannii strains maintaining the same MIC90 of 2 mg/L against the majority of carbapenem-resistant strains. The use of additional molecular techniques to ﬁngerprint isolates will provide further information on these clinically important MDR A. baumannii.