The bottom layer consisted of Mueller Hinton agar containing the antibiotic at Cmin, which was allowed to harden with the plate slanted sufficiently to cover the entire bottom. The top layer, added to the dish in the Sunitinib ic50 normal position, contained antibiotics at Cmax. An inoculum of 1010 CFU/mL of each strain was homogenously spread onto each plate and incubated for 48 hrs at 37°C. After incubation, colonies grown at the highest drug concentration were sampled, checked for purity, and re-plated on a new antibiotic-containing agar plates. A total of 10 consecutive passages on antibiotic containing plates were followed by 10 passages on antibiotic-free plates in order to evaluate stability of acquired

resistance. MIC values were determined after 1, 5 and 10 passages on antibiotic containing plates and after 5 and 10 passages in antibiotic free medium in order to evaluate stability of acquired resistance. Acquisition of resistance was defined as a MIC value higher than resistance breakpoint. Characterization of acquired resistance To determine whether E. coli mutants that had acquired stable resistance to quinolones had alterations in topoisomerase IV or Palbociclib DNA gyrase, parC, parE, gyrA, and gyrB were amplified by PCR and sequenced as described previously [35]. Amplification products were purified with the QIAquick PCR purification kit (Qiagen Inc., Milan Italy)

using the manufacturer’s instructions. Sequencing was performed on an ABI PRISM 310 genetic analyzer (Applied Biosystems, Monza, Italy). Only mutations known to be associated with resistance to fluoroquinolones were considered (Ser83, Asp87 and Ala93 in GyrA, Ser80 and Glu84 in ParC) [36]. References 1. Luzzaro F, Viganò EF, Fossato D, Grossi A, Sala A, Sturla C, Saudelli M, Toniolo MRIP A, AMCLI Lombardia Hospital Infectious Study Group: Prevalence and drug susceptibility of pathogens causing bloodstream infections in northern Italy: a two years study in 16 hospitals. Eur J Clin

Microbiol Infect Dis 2002, 21:849–855.PubMed 2. Kang CI, Kim SH, Bang JW, Kim HB, Kim NJ, Kim EC, Oh MD, Choe KW: Community-acquired versus nosocomial Klebsiella pneumoniae bacteriemia: clinical features, treatment outcomes, and clinical implication of antimicrobial resistance. J Korean Med Sci 2006, 21:816–822.PubMedCrossRef 3. Gobernado M, Valdes L, Alos JI, García Rey C, Dal-Ré Saavedra R, García de Lomas J: Quinolone resistance in female outpatient urinary tract isolates of Escherichia coli : age-related differences. Rev Esp Quimioterap 2007, 20:206–210. 4. Andreu A, Alos JI, Gobernado M, Marco F, de la Rosa M, García-Rodríguez JA, García-Rodríguez JA, Grupo Cooperativo Español para el Estudio de la Sensibilidad Antimicrobiana de los Patógenos Urinarios: Etiology and antimicrobial susceptibility among uropathogens causing community-acquired urinary tract infections: a nationwide surveillance study. Enferm Infec Microbiol Clin 2005, 23:4–9.CrossRef 5.