Ethical statement
This study was approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences “IR.SBMU.MSP.REC.1397. 629”. In order to maintain patients confidentiality participants were anonymous and no personal information was collected or included in the study.
Bacterial isolates
K. pneumoniae and E. coli isolates were collected from hospitalized patients infected in Iran hospitals from September 2016 to August 2018. The isolates were cultured from different clinical specimens, including wound, urine, blood, and tracheal aspirates. Each isolate was identified at species level based on the biochemical reactions, including reaction on SH2/indole/motility (SIM) medium, triple tugar iron (TSI) agar, urease production on urea agar, growth on Simmon citrate agar medium, methyl red/Vogues-Proskauer (MR/VP), and ornithine decarboxylase (OD) test (13). All isolates were stored in tryptic soy broth (TSB) tube with 20% glycerol at −70°C.
Antimicrobial susceptibility testing
Antimicrobial susceptibility of all E. coli and K. pneumoniae isolates was determined by the Kirby-Bauer disk diffusion method on Mueller Hinton agar (Merck, Germany) and interpreted as recommended by the Clinical and Laboratory Standards Institute (2018 CLSI breakpoints) or Food and Drug Administration (FDA) breakpoints guidelines (for tigecycline) (14, 15). Antibiotic discs used were as follow: penicillins [piperacillin (PIP, 100 μg)] , β-lactam/β-lactamase inhibitor combinations [piperacillin/tazobactam (PTZ, 100/10 μg )] , cephems [ceftazidime (CAZ, 30 μg), cefotaxime (CTX, 30 μg), cefepime (FEP, 30 μg), cefpodoxime (CPD, 30 μg)] , monobactams [aztreonam (ATM, 30 μg)], carbapenems [imipenem (IPM, 10 μg), meropenem (MEM, 10 μg), ertapenem (ETP, 10 μg), doripenem (DOR, 10 μg)], aminoglicosides [gentamicin(GEN,10 μg)], Amikacin (AK, 30 μg)], Fluoroquinolones [ciprofloxacin (CIP, 5 μg)], inhibitors [trimethoprim-sulfamethoxazole (TS, 2.5 μg)], fosfomycins [fosfomycin/trometamol (FOT, 200 μg)], tigecycline (TGC, 15 μg), and nalidixic acid (NA, 30 μg), (Mast Group, Merseyside, UK). The minimum inhibitory concentrations (MICs) of seven antibiotics, including imipenem, meropenem, ceftazidime, cefotaxime, cefepime, ciprofloxacin, and colistin were determined by broth microdilution method, and the results were analyzed according to the CLSI guidelines (14). The 2016 EUCAST breakpoints were used (available at http://www.eucast.org/clinical_breakpoints/) for colistin. The antibiotic powders were purchased from Sigma-Aldrich (St. Louis, MO, USA). E. coli ATCC 25922 was used as a quality control strain for disk diffusion and MIC results.
Phenotypic detection of β-lactamases
Detection of ESBLs was tested for all the isolates by combination disc diffusion test (CDDT) containing ceftazidime (CAZ) and cefotaxime (CTX) with CAZ 30 μg + CA 10 μg and CTX 30 μg + CA 10 μg per disc (Mast Group, Merseyside, UK). K. pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as positive and negative controls for ESBL production, respectively (22).
Phenotypic detection of metallo β-lactamases
Combined disk diffusion test were used to detect metallo-β-Lactamase (MBL) activity (16). Pseudomonas aeruginosa ATCC 27853 and P.aeruginosa PA40 (Accession number: KM359725) were used as negative and positive controls for MBL production, respectively.
Screening for carbapenemase production
The Carba NP test was performed for the detection of carbapenemase activity in isolates as described previously (17, 18).
Biofilm formation assay
Assessment of biofilm formation was performed by the colorimetric microtiter plate assay in triplicates (20, 21). Overnight cultures of bacterial isolates were suspended in tryptic soy broth (TSB) (Merck- Germany) at 37 °C. Then, 200 μL bacterial suspension with turbidity of 0.5 McFarland standard were transferred into the sterile 96-well polystyrene microplates (JET Biofil, Guangzhou, China). TSB without bacteria was used as negative control. After 24 hours of incubation at 37°C, each well rinsed three times with phosphate buffered saline (PBS, pH 7.3) to remove any non-adherent cells. Fixation and staining the adherent cells were performed by methanol and 1% crystal violet (Merck, Germany). Then, plates were gently rinsed off with PBS and destained by 33% glacial acetic acid and finally OD of each well were measured at 492 nm. The criteria for categorization of isolates were including: strong biofilm producer (4 x ODc < OD), moderate biofilm producer (2 x ODc < OD < 4 xODc), weak biofilm producer (ODc < OD <2 x ODc) and no biofilm producer (OD < ODc) (19, 20).
Detection of resistance genes
DNA was extracted using the DNA extraction kit (High Pure PCR Template Preparation Kit-Roche, Germany, Lot. No. 10362400) according to the manufacturer's instruction. Detection of resistance genes, including ESBL-encoding genes (blaTEM, blaSHV, blaCTX-M, blaGES, blaPER, and blaVEB), carbapenemases genes (blaNDM, blaKPC, blaVIM, and blaIMP), and two fosfomycin resistance genes (fosA and fosC2), was performed by polymerase chain reaction (PCR) amplification using the specific primers (21-25) and confirmed by sequencing. PCR products were purified using a PCR purification Kit (Bioneer Co., Korea) and then, nucleotide sequencing of amplicons was performed by an ABI PRISM 3700 sequencer (Macrogen Co., Korea). Nucleotide sequences were analyzed using Chromas software version 1.45 (http://www.technelysium.com.au) and NCBI BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Real-time PCR assay
All isolates were subjected to real-time PCR for amplification of blaNDM and mcr genes. DNA sequencing was performed on amplified products of selected positive samples to confirm the results. The primers used were described by the Center of Disease Control and Prevention (26, 27).
Molecular analysis of colistin resistance
Analysis of plasmid-mediated colistin resistance was performed by PCR amplification of mcr-1, mcr-2, mcr-3, and mcr-4. All colistin-resistant K. pneumoniae isolates were also examined for the presence of mutations in the chromosomally-encoded modifications of the LPS, including mgrB, pmrA, pmrB, phoP, and phoQ genes (28, 29). Insertion sequences (ISs) were identified using the IS finder tool (https://www-is.biotoul.fr/index.php). Genomic DNA from two colistin-sensitive K. pneumoniae clinical isolates and K. pneumoniae ATCC 700603 were used as control.
Real-time quantitative reverse transcription PCR
Colistin-resistant isolates were assessed for expression of pmrC, pmrA, pmrB, pmrD, pmrE, and pmrK genes using specific primers (25, 29, 30). rpsL gene encoding a ribosomal protein was used as housekeeping gene to normalize the levels of transcripts tested. Total RNA was extracted from the cultures grown in the mid-log phase of growth in Luria-Bertani broth (Merck, Darmstadt, Germany) by the RNX-Plus Kit (Cat. No., RN7713C, Sinaclon, Iran) according to the manufacturer’s instruction. The contaminating DNA was removed by RNase-free DNase I (Fermentas, Thermo Fisher Scientific Inc., USA). The total RNA concentration was determined by Nanodrop (WPA Biowave II Nanospectrophotometer, USA). DNase-treated RNA was reverse-transcribed into cDNA using the Takara Kit (Japan). Real-time PCR assay was performed on synthesized cDNA using the Power SYBR Green PCR Master Mix (Bioneer, Korea) on a Corbett Rotor-Gene 6000 real-time rotary analyzer (Corbett Life Science, Australia). Each amplification protocol included a first denaturation step of 10 min at 94°C, followed by 40 cycles of 20 s at 94°C and 45 s at 59°C. All samples were run in triplicate. Control samples without reverse transcriptase confirmed the absence of contaminating cDNA. Data were compared to those obtained with the rpsL gene. The expression level of transcripts was calculated based on 2−ΔΔCT method (relative) against that for the susceptible isolate, K. pneumoniae ATCC 700603. Experiments were repeated three times. The parameter Ct was defined as the threshold cycle number at which the first detectable fluorescence generated by the binding of SYBR Green I dye to the minor groove of double-stranded DNA began to increase exponentially.
Plasmid manipulation and analysis
NDM positive strains were selected for plasmid analysis. Plasmid DNA of isolates, transconjugants, and transformants was extracted by using the Roche kit (Cat. No. 11 754 777 001) according to the manufacturer’s instructions. Electroporation was used to transform plasmids encoding blaNDM into E. coli TOPO10. The blaNDM transformants were selected on MH agar (Merck- Germany) supplemented with meropenem (0.5 mg/L) (Sigma–Aldrich). Conjugation experiments were carried out in LB broth with sodium-azide-resistant E. coli J53AzR as the recipient. Cultures of donor and recipient cells in logarithmic phase were added to 4 mL of fresh LB broth and were then incubated at 37°C overnight without shaking. The transconjugants were selected on MH agar (Merck- Germany) supplemented with meropenem (0.5 mg/L) or ceftazidime (1, 2 and 4 mg/L) with sodium azide (100 mg/L) (Sigma–Aldrich).
PCR-based replicon typing
All transconjugants and transformants were typed by a PCR method based on replicons of the major plasmid incompatibility groups among Enterobacteriaceae (31).
Multi-locus sequence type(MLST) analysis
Genotyping by MLST analysis was conducted to characterize diversity and epidemiology of blaNDM- carrying K. pneumoniae isolates (32). Briefly, PCR for seven housekeeping genes, including rpoB, gapA, mdh, phoE, pgi, infB, and tonB was carried out. Results were analyzed according to the Institute Pasteur Klebsiella MLST database (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html). Unique sequence (allele) number for each gene was assigned on the basis of the information in the K. pneumoniae MLST database to determine specific sequence types (ST). A combination of the allelic sequences of the seven genes yielded the allelic profile for each isolate.
Repetitive extragenic palindromic (rep)-PCR typing
Rep-PCR analyses were conducted with the single primer BoxA1R (5′-CTA CGG CAA GGC GAC GCT GAC G-3′) (33). To determine phylogenetic relationships, rep-PCR profiles were analyzed by GelCompar II software (Applied Maths, Belgium) using the Pearson’s correlation coefficient with unweighted paired group method using arithmetic averages (UPGMA) as well as at the 80% similarity level (33).
Statistical analysis
Chi-squared test was performed using SPSS software, 21.0 (SPSS Inc., Chicago, IL, USA) to check for any significant differences between datasets. A significant level of P ≤ 0.05 was considered statistically significant.