Bacterial isolates
Between October 2015 to February 2016, K. pneumoniae strains FK2624, FK-2723 and FK-2820 were isolated from the sputum samples of a patient in the First Affiliated Hospital in Wenzhou, China. FK-2624 was the first strain of K. pneumoniae isolated from the patient. After that, multiple swabs for bacterial isolation were taken successively. FK-2624 was chosen to conduct this study as it represented the drug resistance profiles for five of the early isolates (Table S1). Thereafter, FK-2723 was the carbapenem-susceptible strain isolated from the patient immediately before carbapenem treatment was enacted. Subsequently, and after a 14-day treatment with the carbapenem, imipenem, FK-2820 was isolated. Species identification was performed using the Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDITOF MS, Bruker Daltonics, US). After collection, isolates were stored in 30% glycerol at –80°C. All of the investigation protocols in this study were approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (ethical number 2019-75). Informed consent was waived because this retrospective study focused on the bacterial isolates and had no impact on interventions to the patient.
Antimicrobial susceptibility testing, detection of resistance genes and virulence factors
The modified carbapenem inactivation method (mCIM) and EDTA synergy test was carried out according to CLSI recommendations for phenotypic screening of carbapenemase producers[22, 23]. Antimicrobial susceptibility test was determined by the agar dilution method and interpreted according to the recommendations in the latest CLSI guidelines, including ceftriaxone (0.015-64 μg/mL), ceftazidime (0.015-64 μg/mL), cefotaxime (0.015-64 μg/mL), imipenem (0.015-16 μg/mL), meropenem (0.015-16 μg/mL), ertapenem (0.0035-16 μg/mL), levofloxacin (0.0035-16 μg/mL), ciprofloxacin (0.0035-16 μg/mL), tobramycin (0.125-256 μg/mL), gentamicin (0.125-256 μg/mL), amikacin (0.25-128 μg/mL) and fosfomycin (0.25-1024 μg/mL). The minimum inhibitory concentrations (MICs) of colistin (0.125-16 μg/mL) and tigecycline (0.125-16 μg/mL) were determined by the broth microdilution method. The latest EUCAST breakpoints (available at http://www. eucast.org/clinical breakpoints/) were used for colistin and tigecycline. All of the antibiotics were purchased from Becton Dickinson (Sparks, MD). E. coli ATCC 25922 was used as the quality control strain.
A panel of various, relevant antimicrobial resistance genes were amplified by PCR, and the positive amplicons were further confirmed by DNA sequencing. These resistance genes included those encoding the extended spectrum β-lactamase genes (blaCTX-M, blaPER, blaSHV, blaTEM and blaVEB) [24], carbapenemase genes (blaGES, blaIMI/NMC-A, blaSME, blaKPC, blaVIM, blaIMP, blaNDM and blaOXA-48) [25, 26], AmpC β-lactamase genes (blaCMY, blaFOX, blaMOX, and blaDHA) [27], plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6’)-Ib-cr) [28], oqxAB multidrug efflux pump genes [29] and the aminoglycoside resistant genes (aac(6’)–Ib, aac(3’)–Ib, rmtB, armA, APH, ANT, rmtA, rmtB rmtC and rmtD) [30-32]. DNA sequences were characterized by sequence comparisons using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) [33].
To check for the presence of genes that associated with virulence in K. pneumoniae [34-36], virulence profiles were established by PCR, namely capsular serotype K1, K2, K5, K20, K54, K57, hypermucoviscosity phenotype (magA), allantoin metabolism (allS), regulator of mucoid phenotype A (rmpA), iron system capture (iroN), adhesion type 3 fimbriae (mrkD), iron transport and phosphotransferase function (kfu), siderophore (entB) and siderophore yersiniabactin (ybtA).
Whole-Genome Sequencing (WGS)
Genomic DNA of K. pneumoniae FK-2624, FK-2723 and FK-2820 were purified using the Bioflux DNA purification kit (Bioflux BSC12S1, Beijing) as recommended by the manufacturer. A total amount of 1μg DNA per sample was used as input material for the DNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB E7645S, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. At last, PCR products were purified (AMPure XP system A63880, Beckman, USA) and libraries were analyzed for size distribution by Agilent2100 Bioanalyzer and quantified using real-time PCR. The genomes were sequenced by Illumina NovaSeq PE150.
Sequence reads for each isolate were assembled individually. All good quality paired reads were assembled using the SOAP denovo [37] into a number of contigs. After assembly, the contigs were scaffolded using MeDuSa [38] and ordered of scaffolds was done using MAUVE version 02.25 [39] using as a reference a complete close genome detected using MagiBlast. Annotated was performed using the National Center for Biotechnology Information’s Prokaryotic Genome Annotation Pipeline [40]. To identify potential antibiotic resistance genes in the genomic sequence of the isolates, sequence alignment of the protein sequences of antibiotic resistance genes in the Center for Genomic Epidemiology (http://www. genomicepidemiology.org/). Virulence genes were identified using the virulence factor database (http://www.mgc.ac.cn/VFs/) and PathogenFinder (https://cge.cbs.dtu.dk/services/PathogenFinder/). Additionally, Kleborate (version 0.3.0) was used to identify resistant and virulent genes with their corresponding subtypes [41, 42]. The nucleotide sequence of the genome of FK-2624, FK-2723 and FK-2820 have been submitted to GenBank with accession no. VIGL00000000, VIGM00000000 and VIGK00000000, respectively. Alignments with the three assembled genomes were done using MAUVE version 02.25 [39]. Sequences that were different between isolates were tested for different GC content, using window-acgt form GLIMMER v3.02 [43]. Putative plasmids were assembled and the resulting plasmid assemblies were annotated with Prokka (v1.12) [44]. Graphical representation was generated using the R packages.
Roary (version 3.12.0) was used to align the assembled genomes and two additional ST660 MLST genomes found in NCBI (GCA_002853165 and GCA_002205995.1) and extract the core genome [45]. The core genome was used to generate a phylogenetical tree using RaxML (version 8.2.9), with a GTR+GAMMA model, and a bootstrapping of 1000 replicates [46].
MLST determination of genetic relatedness of isolates
Pairwise comparisons method, pulsed-field gel electrophoresis (PFGE) and multi-locus sequencing typing (MLST) were used to establish relatedness between same-patient isolates.
The sequences of the three complete genomes of K. pneumoniae isolates were independently used in sequence alignments. The sequence reads were mapped to the reference genomes using the Bowtie2 software, which is good for mapping short sequence reads to medium-sized and large genomes. The alignment of clean data of 3 isolates with reference K. pneumoniae MGH 78578 (MDR bacterium isolated from a patient [47], accession number CP000647) was performed with the default settings of programs. Finally, the alignment percentage (supplementary material Table S2) was showed by Bowtie2, revealed a high degree of genetic conservation was observed between the three K. pneumoniae strains.
PFGE was carried out on our strains according to the method described previously with minor modification [48]. Genomic DNA was extracted from the K. pneumoniae isolates, followed by restriction enzyme Xba I (Takara 1093A, Japan) digestion for 2 h. PFGE was performed using a CHEF-Mapper XA PFGE system (Bio-Rad, USA) for 18 h with a switch time 6–36 s. Then DNA fingerprints were revealed by GelRed staining. The banding patterns were visualized by GelDoc XR gel imaging system (Bio-Rad, USA) and cluster analysis of similarity values of the PFGE profiles were finally performed by Quantity One program (BioRad Laboratories, USA). The sequence types (STs) of FK-2624, FK-2723 and FK-2820 were determined by MLST. Seven housekeeping genes (gapA, infB, mdh, pgi,phoE, rpoB, and tonB) were amplified and sequenced according to Diancourt et al. [49]. Alleles and sequence types were assigned by the MLST database (http://www.pasteur.fr/mlst/Kpneumoniae.html).
Outer membrane protein isolation and SDS-PAGE
FK-2624, FK-2723 and FK-2820 were cultured in Muller Hinton broth with shaking overnight at 37℃. Cells were isolated by centrifugation (4200 rpm for 15 min), the cell pellets were washed with 10 mM Tris-HCl, 5 mM MgCl2 (pH 7.3), and the cells then lysed by sonication as described [50]. The cell lysate supernatants were treated with 2 % solution of sodium lauroylsarcosinate for 30 min at room temperature, centrifuged 30 min at 17000 rpm and the pellets containing outer membrane proteins (OMPs) were suspended in 10 mM Tris-HCl, 5 mM MgCl2 (pH 7.3).
Samples containing the OMPs were mixed with loading buffer (TaKaRa 9173, Japan) and denatured by heating at 100℃ for 3 min. Proteins were then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 12% polyacrylamide separation gel. Bands corresponding to the major protein species were visualized by staining the gels with 0.2% Coomassie brilliant blue (Solarbio C8430, China) in 10% acetic acid and 45% methanol. K. pneumoniae ATCC 13883 (an isolate with known expression of OmpK35 and OmpK36) served as a control strain for OMP profiling.
Analysis of ompK35 and ompk36 genes
The coding sequences of the ompK35 and ompK36 genes for the representative isolates were amplified and sequenced using primers listed in Table 1. Amplification was carried out with the following thermal cycling conditions: 5 min at 94℃ and 35 cycles of amplification consisting of 30 s at 94℃, 30 s at 58℃, and 1 min at 72℃, with 10 min at 72℃ for the final extension. DNA fragments were analyzed by electrophoresis in a 1% agarose gel at 120 V for 20 min, stained with GelRed (Biotium 41003, USA). DNA sequence analysis compared with those of K. pneumoniae NTUH-2044 (NCBI accession number AP 006725) was conducted.
Isolates were examined by real-time RT-PCR to measure the steady-state levels of the transcripts as an indication of the expression of ompK35 and ompK36. For RNA isolation, K. pneumoniae isolates were grown in fresh LB medium at 37 ◦C overnight. Total RNA was extracted from 3 mL of culture using a RNeasy Mini Kit (Qiagen 74524, Germany) according to the manufacturer’s instructions. The extracted RNA samples were stored at −80℃. Purified RNA was reverse transcribed into cDNA for RT-PCR analysis using a cDNA synthesis kit (TaKaRa 6210A, Japan) according to the manufacturer’s instructions. Gene expression levels were measured with RT-PCR using TB Green® Premix Ex Taq™ (TaKaRa RR420Q, Japan). Primers were designed on the basis of the nucleotide sequence in GenBank (Table 1). Expression of each gene was normalized to that of a housekeeping gene (rpoB), the primers used for RT-PCR were rpoB-F (5’-AAGGCGAATCCAGCTTGTTCAGC-3’) and rpoB-R (5’-TGACGTTGCATGTTCGCACCCATCA-3’). The relative expression of ompK35 and ompK36 was then calibrated against the corresponding expression by K. pneumoniae ATCC 13883. For quality-controlling strain, relative expression was equal to 1.0. All experiments were performed in triplicate and mean of the Ct values were used for analysis. Gene expression levels were calculated using 2-ΔΔCt method [51].
Statistical Analysis
The statistical correlation of the expression of ompk35 and ompk36 was calculated by Student’s t test. SPSS (version 17; IBM, USA, IL) was used for statistical analysis. A P value lower than 0.05 was considered statistically significant.