RT-PCR was carried out using a SmartCycler®

II apparatus (Cepheid®, Sunnyvale); the results were analyzed using the rest 2009 software available at http://www1.qiagen.com/Products/REST2009Software.aspx#Tabs=t0 (Pfaffl et al., 2002). For comparative purposes, an SHV-1-producing K. pneumoniae strain I118 with a natural pattern of susceptibility to β-lactams (Table 1) (Livermore, 1995) from the collection of the hospital in Plzeň was used. The experiments were repeated three times. Six C-NS K. pneumoniae isolates from different patients were identified during the study period; from three of these patients, C-S isolates were also retained and were available for the analysis (Table 2). The investigation NVP-BKM120 solubility dmso of the clinical and microbiological data revealed that five of the six patients (patients P1–P5) had been colonized or infected with AmpC- or ESBL-producing C-S K. pneumoniae strains before the identification of the C-NS isolates (Table 2); however,

these C-S strains had not been stored. These five patients received long therapies with carbapenems, mostly with meropenem. Because of other infections, all of the patients were treated with a variety of antimicrobials overall. Regarding the C-NS K. pneumoniae, only patient P5 presented Alpelisib datasheet with symptoms of an infection caused by this organism (urinary tract infection), and was treated successfully with amikacin for this disease. The remaining patients were only colonized with the C-NS K. pneumoniae; therefore, they were not treated with antibiotics against these organisms. In all but one of these patients the C-NS isolates were not observed in further examinations performed 1–2 times weekly. The repeated C-NS isolates were only collected from the patient Racecadotril P4, who was severely ill with

a poor prognosis and eventually died because of organ failure in sepsis (not caused by K. pneumoniae). In three of the patients (patients P2, P3, and P6), the C-S K. pneumoniae isolates were identified within several weeks after the episodes with the C-NS isolates (Table 2), and these isolates were included in this study. The MICs are shown in Table 1. The MICs of carbapenems for the C-NS isolates varied from 2 to 32 μg mL−1, whereas the C-S isolates had MICs of ≤0.12 μg mL−1. Almost all of the isolates exhibited uniform resistance to other β-lactams tested, including penicillin–inhibitor combinations and expanded-spectrum cephalosporins. Two C-S isolates (P2/I177971 and P3/C154247) were susceptible to cefepime (MIC, 0.5 μg mL−1) and one of these (P3/C154247) had a low-level resistance to cefotaxime and ceftazidime (MICs, 2 and 8 μg mL−1, respectively). Except for ciprofloxacin, to which all of the isolates were resistant, the MICs of non-β-lactams varied; of note was the resistance to colistin in one C-NS isolate (P5/C163243; MIC, 16 μg mL−1).