Clinical isolates. A total of 38 non duplicate clinical isolates of K. pneumoniae resistant to carbapenems [i.e. imipenem (IPM), meropenem (MEM), ertapenem (ERT)] were included in this study. The isolates were collected in a period of four months (September to December 2011) from separate patients who were hospitalized in the teaching hospital Policlinico of Bari, Bari, Italy. Microorganisms were isolated from multiple infection sites, including blood (n = 10), urine (n = 11), bronchial aspirate (n = 10), rectal swabs (n = 3), throat swab (n = 1), sputum (n = 2), and bile (n = 1) specimens. K. pneumoniae (n = 4), Pseudomonas aeruginosa (n = 2), Acinetobacter spp. (n = 2), and Escherichia coli (n = 2) with no known resistance were included as negative control.
Antimicrobial susceptibility determination and carbapenemases assays. Detailed antimicrobial susceptibility was carried out automatically and interpreted according to the recommendations of European Committee for Antimicrobial Susceptibility testing (EUCAST) (Vading et al. 2011). All K. pneumoniae strains were identified as possible KPC producers by MicroScan Walkaway System using 43 MS GNC panels (Siemens, New York, NY) on the basis of resistance to IPM, MEM and ERT. The resistance level of K. pneumoniae isolates to IPM was confirmed by Etest (bioMérieux, France) according to the manufacturers’ instructions. All isolates were screened for the production of carbapenemases, using MHT which is a phenotypic test used to determine if resistance to carbapenems is mediated by a carbapenemase enzyme (Carvalhaes et al. 2010). This test was performed using both MEM and IPM 10 μg disks. The presence of a distorted inhibition zone after overnight incubation was interpreted as a positive test result. The isolates were further investigated by combined disk test with IPM and IPM plus phenylboronic acid (PBA) or ethylenediaminetetraacetic acid (EDTA) as inhibitors of KPC or MBLs, respectively (Tsakris et al. 2009). The stock solution of PBA was prepared by dissolving PBA (benzeneboronic acid; Sigma-Aldrich, Steinheim, Germany) in dimethylsulfoxide and water at a concentration of 20 mg/mL (Coudron 2005). From this solution 20 μL was dispensed onto IPM disks. The combined disk IPM/EDTA was purchased from Biolife, Italy. Inhibition tests were performed for the detection of ESBLs and therefore stock solution of PBA was also dispensed onto disks containing ceftazidime (CAZ) or cefotaxime (CTX) with and without clavulanate (CA) at the same final amount (i.e. 400 μg). All the antibiotic disks were commercially available. The tests were performed by inoculating Mueller Hinton agar plates with the standard disk diffusion method; an increase in the growth-inhibitory zone around the disk containing the added beta-lactamase inhibitor was observed. The test was considered positive for KPC or MBLs when the growth inhibitory zone around either the IPM/PBA or the IPM/EDTA disk was 5 mm or greater of the growth inhibitory zone diameter around the disk containing IPM alone. With regard to the detection of ESBLs, when the zone diameter of either CTX-CA or CAZ-CA disk tested in combination with PBA (CTX-CA-PBA or CAZ-CA-PBA, respectively) was 5 mm or greater of the zone diameter of CTX or CAZ containing PBA (CTX-PBA or CAZ-PBA, respectively) the test was considered positive. The presence of AmpC beta-lactamase was phenotypically tested by determining IPM MICs in agar with and without 200 μg/ml cloxacillin and by using the AmpC detection Etest strips (bioMérieux, France).
Detection of bla KPC gene by molecular methods. All K. pneumoniae isolates were also investigated by nucleic acid sequence-based amplification, NASBA™, NucliSens EasyQ KPC (bioMérieux, France), for the detection of the blaKPC gene. This molecular method couples nucleic acid sequence-based amplification (NASBA) with real-time procedure assay. NASBA is a sensitive, isothermal, transcription-based amplification system designed specifically for the detection of KPC RNA target in real time mode. Nucleic acid amplification uses primers that are specific for KPC RNA sequences and for the synthetic KPC internal control RNA. Any KPC RNA present in the sample is co-amplified along with the internal control otherwise other nucleic acid sequences will not be amplified. According to the manufacturer’s instructions, a 0.5 McFarland suspension of each isolate was prepared from an overnight non selective culture plate and heated at 95°C for 5 min. The reaction mixture was prepared in the tube strip by transferring 2.5 μl of internal control solution, 2.5 μl of heated bacterial suspension, 10 μl of primers solution (including both primers, molecular beacon probes and nucleotides) and incubated for 2 minutes at 65°C and 2 minutes at 41°C. After addition of 5 μL of enzyme mix (AMV-RT, RNase H and T7 RNA polymerase, bovine serum albumin), amplification was followed for 90 minutes at 41°C in NucliSENSE EasyQ Analyzer (bioMérieux, France) according to the assay protocol. Results were validated for each isolate according to the amplification of the internal control.
In order to compare the results obtained by Nuclisens EasyQ KPC method with a gold standard molecular test all the 38/38 isolates, along with the negative control strains, were further investigated by PCR assay using the fol-lowing primers: bla KPC For: TGTCACTGTATCGCCGTC; e bla KPC Rev: CTCAGTGCTCTACAGAAAACC (Yigit et al. 2001). The amplification protocol consisted of a denaturation step at 95°C (5 min) followed by 35 cycles at 95°C for 60 sec, 55°C for 40 sec, 72°C for 90 sec; was also included a cycle of extension at 72°C for 10 min.