First description of increased resistance in carbapenem-susceptible Klebsiella pneumoniae with imipenem treatment driven by outer membrane remodelling in China

The emergence of carbapenem-resistant Kelbsiella pneumoniae (CRKP) posed threats to human health. Although there are numerous studies regarding porin alteration in association with the production of ESBLs and/or AmpC β-lactamase, a systematic research about the treatment-emergence of porins alteration in antibiotic resistance does not exist yet. The aim of this study was to investigate the underlying mechanism and evolution of resistance of K. pneumoniae during carbapenem treatment. Here, we reported three strains (FK-2624, FK-2723 and FK-2820) isolated from one patient before and after imipenem treatment during hospitalization. Antibiotic susceptibility testing indicated that FK-2624 was susceptible to almost antimicrobials but fosfomycin; FK-2723 and FK-2820 were MDR. After imipenem therapy, FK-2820 was evolved to carbapenem-resistant. PCR and Whole-Genome sequencing ( WGS) indicated that resistance genes bla SHV , oqxA and fosA5 were detected in FK-2624, in addition, FK-2723 and FK-2820 harbored bla DHA , qnrB , aac (6’)-Ib . Virulence factors K57, ybtA, mrkD, entB and iroN were detected simultaneously in all of three strains. The results of pairwise comparisons , multi-locus sequencing typing (MLST) and pulsed-field gel electrophoresis (PFGE) revealed high homology among the isolates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that isolate FK-2820 lacked OmpK 36 as there was a premature stop codon of the outer membrane porin encoding gene ompk36 confirmed by sequencing. Real-time RT-PCR revealed that the expression of ompK36 in FK-2820 was 0.093 times the control isolate ATCC 13883. Our study highlighted that the alteration of outer membrane porins due to the 14-day use of imipenem clinically play a potential role in leading

to the carbapenems-resistance of FK-2820.

Introduction
Klebsiella pneumoniae is a serious hospital-acquired pathogen causing many infections including urinary tract infections, pneumonia, bloodstream infections [1,2]. The abuse of expanded-spectrum cephalosporins for the remedy of these organisms has contributed to the appearance of Extended-spectrum β-Lactamases (ESBLs)/AmpC-producing K. pneumoniae. From then on, carbapenems were considered as the last resort for the treatment of infections caused by multidrugresistant (MDR) isolates due to their broadest antibacterial spectrum compared to other β-lactams. However, carbapenem-resistant K. pneumoniae (CRKP) have increasingly emerged under the antibacterial drug selection pressure. The production of carbapenemases is the mainly mechanism of CRKP since the first isolation of CRKP in America in 1996 [3]. And it could also result from porin alteration in association with the production of ESBLs and/or AmpC β-lactamase [4].
OmpK36 is a nonspecific porin in K. pneumoniae, which belongs to the OmpC porin group with small channel size [5]. Many reports suggest that loss of Ompk36, coupled with ESBL and/or AmpC production, plays an important role in conferring carbapenem resistance in K. pneumoniae [6,7]. But the evolution of outer membrane porin still lacks of research. The current study focused on the evolution of carbapenem resistance determinants of K. pneumoniae isolated from one inpatient and emphasis on determining appropriate antimicrobial course of treatment.

Bacterial isolates
During October 25, 2015 to February 14, 2016, K. pneumoniae strains FK2624, FK-2723 and FK-2820 were isolated from the sputum samples of a patient in ICU of the First Affiliated Hospital in Wenzhou, China. FK-2624 was the first strain of K.
pneumoniae isolated from the patient. After that, seven strains were separated successively. FK-2624 was chosen to conduct this study due to the drug resistance profiles were the same as the other seven. FK-2723 was the carbapenemsusceptible strain isolated from the same patient before carbapenem treatment.
Subsequently, and after a 14-day treatment of imipenem, FK-2820 was recovered.

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

Determination of genetic relatedness of same-patient isolates
Pairwise comparisons method, pulsed-field gel electrophoresis (PFGE) and multilocus sequencing typing (MLST) were used to establish relatedness between samepatient 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 [27], accession number CP000647) was performed with the default settings of programs. Finally, the alignment percentage (supplementary material Table S1) 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 [28].Genomic DNA was extracted from the K. pneumoniae Seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) were amplified and sequenced according to Diancourt et al. [29]. 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℃. Outer membrane porins (OMPs) were recovered by centrifugation (4200 rpm for 15 min), washed with 10 mM Tris-HCl, 5 mM MgCl 2 (PH 7.3), and lysed by sonication as described [30]. The 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 the OMPs were suspended in 10 mM Tris-HCl, 5 mM MgCl 2 (PH 7.3).
Samples mixed with loading buffer (TaKaRa 9173, Japan) were first denatured by heating at 100℃ for 3 min, then they were separated by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) using 12% of separation gel and 5% of concentration gel. Bands were visualized by dyeing 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 OMPs 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

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.

Clinical characteristics of the patient
This case was a 62-year-old male patient admitted to the department of neurosurgery in the First Affiliated Hospital of Wenzhou Medical University on October 26, 2015 with headache. A diagnosis of cerebral aneurysm with subarachnoid hemorrhage was made, then, secondary pulmonary infection occurred after surgery. Treatment was started with 2 g of cefoperazone/sulbactam every 8 h (q8h), 0.5 g levofloxacin two times a day (bid.). On day 26, K. pneumoniae FK-2624, it was only resistant to fosfomycin, isolated from sputum sample ( Table 2). After that, due to pneumonia, he was switched to tigecycline via vein infusion (ivgtt.) loading dose of 100 mg. On Day 50, chest CT suggested atelectasis, which required a tracheotomy. Then, day 52, Pseudomonas aeruginosa was detected in purulent sputum, so he was switched to fosfomycin with an ivgtt. loading dose of 8 g q12h, tobramycin with an ivgtt. loading dose of 80 mg q12h. On Day 71 after his admission, isolate FK-2723 was identified from the patient's sputum sample on carbapenems-resistant. The MICs of imipenem and ertapenem for FK-2820 were 8 and 16 μg/mL, respectively. Although it was still susceptible to meropenem, the MIC of meropenem increased four times, range from 0.25 μg/mL to 1 μg/mL. Phenotypic method was used for the investigation of carbapenemase production. mCIM was negative for FK-2820. PCR and WGS was used to search for the presence of resistance genes, the bla SHV , bla DHA , qnrB, aac (6')-Ib, oqxA and fosA5 genes were identified in strains FK-2723 and FK-2820. FK-2624 harbored bla SHV , oqxA and fosA5 genes (Table 2). In addition, sequence analysis showed that bla DHA , qnrB, aac (6')-Ib were co-harbored in the same plasmid. The results of BLASTn analysis revealed that the plasmid with 100% query coverage displayed 100% identity to plasmid pR47-309 (Genbank accession CP040696.1).
Of the fourteen virulence factors found in this study, ybtA, mrkD, entB, iroN and capsular serotype K57 were found in the three isolates (Table 2). These genes were associated with high-affinity iron chelators or siderophores, iron uptake, biofilm formation and infection (such astissue-invasive, pneumonia), potentially contributing to the increased virulence in K. pneumoniae.

Homology Analysis and Molecular epidemiology
Genome homology comparison suggested that over 98% homology in tested strains (Table S1). PFGE analysis revealed that the three strains isolated from the same patient to be clonally identical. In addition, they all belonged to ST 660 (Figure 2).

OMP analysis
SDS-PAGE showed that isolate FK-2820 did not express a full complement of porins compared with the positive control K. pneumoniae ATCC13883 (Figure 3). Isolate FK-2820 lacked a band of ~35 kDa corresponding to the OmpK36 major porin.  Table 2).

Discussion
In last years, bacterial antimicrobial resistance has indeed emerged as one of the main concern of public health and constitutes a major challenge in the future [32].  [36,37]. Around the world, sever outbreaks due to outer membrane alteration in Enterobacteriaceae have been reported [38][39][40]. In the current study, resistance has changed in three strains isolated from one patient, the aim of our study was to investigate the mechanism and evolution of treatmentemergent carbapenem-resistance. A recently study revealed that in vitro carbapenem resistance induction could lead the loss of OmpK 36 in induced isolate after 13 days of serial passaging [47]. In this study, we reported a link between a 14-day imipenem treatment and the loss of OmpK 36 in vivo. This will give more inspiration to clinical anti-infective treatment.
The limitation in our study was that there was only one patient had been investigated. A large-scale study on the inclusion of additional patients under strict antibiotic regimen is needed in further research to provide more evidence on the clinical significance of these newly emerged strains.
Here, we analyzed the three strains from a patient, data manifested that the alteration of ompk36 might due to a 14-day imipenem treatment, which further confer a high level carbapenem resistance. To our knowledge, this is the first description of a connection between imipenem treatment and alteration of OmpK36

22.
Li R, Zhu H, Ruan J, Qian W, Fang X, Shi Z, Li Y, Li S, Shan G, Kristiansen K et Primer Sequence 5' → 3' DNA amplification and sequencing Note: F, forward (5') primer. R, reverse primer.  Timeline representing the days at which the isolates were obtained from patient and the resp

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download. Table S1.docx