Nineteen ECC isolates co-resistant to carbapenem and colistin were collected from the First Affiliated Hospital of Wenzhou Medical University in China, from 2011 to 2018. All isolates were identified by MALDI-TOF MSusing a VITEK® mass spectrometer (BioMerieux, Lyons, France).
Antimicrobial susceptibility testing
The MICs of carbapenems (meropenem, imipenem, and ertapenem) and colistin to the 19 ECC strains were performed by the agar dilution method according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) guidelines (32). And the breakpoint of carbapenem and colistin for ECC was interpreted according to the guidelines of CLSI (32) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) (36) respectively. Escherichia coli ATCC 25922 was used as a control strain in this study.
To screen for suspected carbapenemase production in the 19 ECC strains, the mCIM was performed. As recommended by CLSI(32), emulsify a 1 μL loopful of bacteria from an overnight blood agar plate in 2 mL trypsin soy broth (TSB). Add a 10-μg meropenem disk in each suspension using sterile forceps and incubate at 35℃ ± 2℃ in ambient air for 4 h ± 15 min. Remove the meropenem disk from each TSB-meropenem disk suspension and place it on the Mueller-Hinton agar (MHA) plate previously inoculated with the meropenem-susceptible E.coli ATCC 25922 indicator strain. Invert and incubate the MHA plates at 35℃ ± 2℃ in ambient air for 18-24 h. Carbapenemase positive when the antibacterial zone diameter of 6-15 mm or presence of pinpoint colonies within a 16-18 mm zone; Carbapenemase negative when the antibacterial zone diameter of ≥19 mm (clear zone).
Polymerase chain reaction (PCR) detection for resistance genes
The genomic DNA of the 19 ECC strains were extracted using the Biospin Bacterial Genomic DNA Extraction kit (Bioflux, Tokyo, Japan). Using these DNA templates we detected the carbapenemase gene (blaKPC, blaNDM, blaIMP, blaVIM, blaIMI, blaSPM, blaOXA-23, blaOXA-24, blaOXA-48, blaOXA-58, blaNmc-A, blaFRI-1, blaBIC, blaGIM, blaSME, blaAIM, blaDIM, blaSIM and blaGES), extended-spectrum β-lactamase gene (blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaCTX-M-14), AmpC cephalosporinase gene ampC, colistin resistance-related genes (mcr-1, mcr-2, mcr-3, mcr-4, mcr-5 and ecr). The primers used for PCR amplification were showed in table S1. The positive PCR products were sequenced by Shanghai Genomics Institute Technology Co. Ltd (Shanghai, China). And the sequences of the product were analyzed by BLAST searches against the NCBI database (www.ncbi.nl m.nih.gov/BLAST).
Efflux inhibitors assay
In order to detect whether the efflux pump has an effect on drug resistance, MICs of ertapenem and colistin were determined in the presence or absence of efflux pump inhibitor: CCCP (8 μg/L, 1/4 MIC). CCCP stock solution uses dimethyl sulfoxide (DMSO) as the solvent. When the efflux pump inhibitors were present, a reduction of at least four-fold in the MICs were considered indicative of efflux(37).
Quantitative real-time PCR for outer membrane protein and efflux pump
Total RNAs of 19 ECC strains were extracted using the Bacterial RNA Miniprep Kit (Biomiga, Shanghai, China) according to the manufacturer's instructions. Then, 500 ng of RNA were mixed with the reverse transcription system of the PrimeScript™ RT reagent Kit (Takara, Japan) to obtain 10 μL of cDNA. The expression levels of outer membrane gene (ompC, ompF) and efflux pump gene (acrA, acrB) were performed by quantitative real-time PCR. The rpoB gene was selected as the internal gene. And the relative expression levels of four genes in 19 ECC strains were compared with the expression levels in co-susceptible Enterobacter cloacae ATCC 700323 and clinical strains CG37.
qPCR was performed using a CFX-96 touchTM Real-Time PCR system (Bio-Rad, California, USA) and TB Green Premix Ex Taq II (Tli RNaseH Plus) (2×) (Takara, Japan) with the specific primers (refer to Table S1). Then, 100 ng of cDNA was added to each sample as the template. Cycling conditions were as follows: 95℃ for 30 s followed by 40 cycles of 9℃ for 5 s and 60℃ for 20 s. A melting curve was performed after each run (raising 0.5℃ per second, from 65℃ to 95℃). Each sample was run in triplicate and the means of Ct values were used for analysis. Quantification of the target genes was analyzed using the comparative threshold cycle 2-ΔΔCt method. All experiments were repeated in triplicate independently.
Lipid A characterization by MALDI-TOF MS
Lipid A was isolated by using an optimized large-scale protocol based on mild acid hydrolysis (38). Overnight cultures (200 ml at 37°C) in LB broth were harvested by centrifugation at 3,220 × g for 30 min. Bacterial pellets were washed with single-phase Bligh-Dyer mixture (chloroform/methanol/water, 1:2:0.8 [vol/vol]) and centrifuged at 3,220 × g for 15 min. The LPS pellets were suspended in sodium acetate buffer (50 mM [pH 4.5]) and incubated at 100°C for 30 to 45 min. Reactions were moved into a two-phase Bligh-Dyer mixture (chloroform/methanol/water, 1:1:0.9 [vol/vol]) and centrifuged at 3,220 × g for 15 min. The lower phases were removed to clean tubes and dried using rotary evaporation. The dried samples contained whole-cell extracts of lipid A.
Dried lipid A samples were resuspended in 100 μl chloroform/methanol (1:1 [vol/vol]), and 3 μl 2,5-dihydroxybenzoic acid (DHB) matrix (20 mg/ml in TA30 solvent) was mixed with 3 μl lipid A. Aliquots of the mixture were spotted directly onto the well of the MALDI-TOF MS plate (ground steel). Mass spectra were recorded for optimal ion signals in negative-ion mode using a Bruker autoflex MALDI-TOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA). Data were acquired and processed by flexControl and flexAnalysis 3.4 (Bruker Daltonics Inc.).