Specimen collection
The study was conducted in a 10 bedded ICU from September 2011 and September 2012. Patients requiring intensive care or ventilator support were admitted directly to ICU from the emergency department or operation theatre and included patients of acute poisoning, neurological or cardiovascular emergency, acute respiratory failure and patients undergoing emergency and elective surgery. Patients transferred to the ICU from other wards and/or hospitals were excluded. Two rectal swabs were collected from all enrolled patients (n = 100), one each on day 1 (D1) and day 4 (D4) of ICU admission and transported in nylon flocked system in Amies liquid transport medium. Patients, in whom D4 sample could not be collected, were excluded from the study. To study risk factors for colonization; demographic data, clinical history, current antibiotic therapy, including carbapenems, invasive procedures and co-morbid conditions were recorded through chart review in a predesigned proforma (Additional file 1). Controls included, stool samples from 122 non-hospitalized patients attending the outpatient department for routine health checkup. This study was approved by the Institute Ethics Committee, V.M.M.C & Safdarjung Hospital (S.NO. VMMC/SJH/Ethics/SEP-11/29).
Isolation and identification
Approximately 0.5gm of stool or rectal swab was emulsified in 0.5 ml of sterile 0.85 % saline. Screening of CRE was done on in-house prepared media by four different methods and included: (i) overnight selective enrichment in 5 ml tryptic soy broth with a 10 μg ertapenem disk followed by plating onto MacConkey agar (CDC protocol) [11], (ii) direct plating onto MacConkey agar supplemented with imipenem at 1 mg/L (MacI), (iii) direct plating onto MacConkey agar supplemented with cefotaxime at 1 mg/L (MacC ESBL), (iv) MacConkey agar with standard imipenem, meropenem and ertapenem Disk (10 μg Disk), applied at the 4-, 8-, and 12-o’clock positions (MacD). Evaluation of bacterial growth was made after 18 to 24 h of incubation at 37 °C in ambient air. All morphologically distinct colonies were sub cultured on standard MacConkey agar plate and isolates were identified by conventional biochemical tests [12]. The media used in the study had been previously validated for isolation of CRE harbouring carbapenemase genes (KPC, IMP, VIM, NDM-1, OXA-48 and OXA-181).
Susceptibility testing and phenotypic detection of β-lactamases
Antibiotic susceptibility was performed by disk diffusion as per clinical and laboratory standards institute (CLSI) 2012 guidelines [13] for ampicillin (10 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), piperacillin (100 μg), ceftazidime (30 μg), cefotaxime (30 μg) cefoxitin (30 μg), piperacillin-tazobactam (100/10 μg), cefoperazone-sulbactam (75/30 μg), netilmicin (30 μg), amikacin (30 μg), gentamycin (10 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), tetracycline (30 μg). All Enterobacteriaceae were screened for carbapenem resistance using ertapenem (10 μg), meropenem (10 μg), imipenem (10 μg) disk and results were interpreted as per CLSI 2012 guidelines. All isolates with ertapenem zone diameter <22 mm were subjected to minimum inhibitory concentration (MIC) for ertapenem, meropenem, imipenem, colistin and tigecycline using E test (bioMérieux, France). Isolates with ertapenem MIC > 0.5 mg/L were defined as CRE. Resistant and susceptible to tigecycline were defined as MIC˃2 mg/L and ≤1 mg/L respectably (European committee on antimicrobial susceptibility testing; EUCAST 2011).
All CRE were screened for β-lactamases by phenotypic test and PCR. For phenotypic test commercially available KPC, MBL and ESBL/AmpC Screen kit (Rosco Diagnostica, Denmark) containing meropenem and cefotaxime alone and with various β-lactamase inhibitors (boronic acid, dipicolinic acid, clavulanic acid, cloxacillin and clavulanic acid + cloxacillin) were used. Zone diameters were recorded. Tests were interpreted as per manufacture instruction.
PCR for β-lactamases among CRE
DNA was isolated by phenol chloroform method as per Sambrook et al. [14]. PCR was performed for ESBL (SHV, TEM and CTX-M), AmpC (MOX, FOX, DHA, CIT, ACC, EBC) and Class A (KPC1 and 2), Class B (NDM-1, IMP, VIM) and class D (OXA- 48 and OXA-181) carbapenemases. PCR product were electrophoresed on 1.5 % agarose gel along with a 100 bp DNA ladder (as a molecular wt marker) and visualized using a UV transilluminator. For TEM [5], SHV [5], CTX-M [15], AmpC [16], NDM-1 [5], IMP [5], VIM [5], KPC [5] and OXA-181 [17] previously designed primers were used. For OXA–48, primer (self-deigned) used was FP (5′-GCGTGTATTAGCCTTATC-3′) and RP (5′-CGCGGTTCGGTAGTGTGTTT-3′), which amplifies a 760 bp product. Cycling conditions were: 95 °C for 5 min; 30 cycles of 95 °C for 30s, 60 °C for 30s and 72 °C for 60s; and 72 °C for 5 min. Selected amplicons were sequenced. All primers were procured from Integrated DNA Technologies (IDT).
CRE growing on selective media and positive for any carbapenemase gene were defined as carbapenemase producing Enterobacteriaceae (CPE). Non-CPE were defined as all CRE isolates growing on selective media and negative for any carbapenemase gene.
Controls used for antibiotic susceptibility, phenotypic test and PCR included ESBL-positive Klebsiella pneumoniae ATCC 700603, ESBL-negative Escherichia coli ATCC 25922, KPC-positive Klebsiella pneumoniae ATCC BAA-1705, KPC-negative Klebsiella pneumoniae ATCC BAA-1706. For IMP, VIM, NDM-1, OXA-48 and OXA-181 in house strains confirmed by sequencing were used as controls.
Figure 1 shows the flowchart of steps to study colonization with CPE.
Statistical analysis
Antibiotic resistance data was analyzed using WHONET 5.6. χ
2 test was used for comparison of prevalence fecal carriage of CPE in ICU patients D1 vs D4 and ICU vs controls. Statistical significance was taken as p value <0.05.
Risk factors for colonization with CPE among ICU patients were compared using χ2 or Fisher exact test, as appropriate. All p values were two tailed. p value <0.05 was considered statistically significant. Adjusted odds ratios and 95 % confidence intervals (CIs) were computed for the significant factors. Variables that were present in more than 10 % of ICU patients with CPE colonization with p value <0.05 were entered into backward stepwise logistic regression models in multivariate analysis using SPSS version 20.0 (SPSS, Chicago, IL)