Emergence of meropenem/vaborbactam resistance in KPC-producing Klebsiella pneumoniae causing bloodstream infections in northern Italy, 2018

We aimed to investigate the incidence of meropenem/vaborbactam-resistance among KPC-producing Klebsiella pneumoniae (KPC-Kp) bloodstream infection in a large Italian hospital. Meropenem/vaborbactam-resistance was found in 8% (n = 5) KPC-Kp, while 5% (n = 3) strains exhibited cross-resistance to ceftazidime/avibactam. Genomic analysis revealed that meropenem/vaborbactam-resistance was associated to non-functional OmpK35 and OmpK36 porins and no specic mutation was associated to cross-resistance. No specic antimicrobial treatment was related to favorable clinical outcome, while cross-resistance was not associated to higher clinical and/or microbiological failures. Our study indicated that resistance to meropenem/vaborbactam was due to porins deciency and is associated to reduced susceptibility both to ceftazidime/avibactam and carbapenems. was due to OmpK35 and OmpK36 porins depletion in our KPC-Kp strains and no specic mutations were observed in bla KPC gene. At the same time, our results showed that no specic mutations were observed in KPC-Kp isolates resistant both to meropenem/vaborbactam and ceftazidime/avibactam in comparison to strains resistant to meropenem/vaborbactam alone. These results are in accordance with previous ndings that showed as cross-resistance to meropenem/vaborbactam and ceftazidime/avibactam was due to loss-of-function mutations in the OmpK35 and OmpK36 porins which are also associated to elevated MICs for ß-lactams [4,5]. We observed that patients with BSI due to KPC-Kp resistant both to meropenem/vaborbactam and ceftazidime/avibactam had short colonization time, thus suggesting that cross-resistance did not result from selection pressure due to prolonged antimicrobial therapy while can be plausible that patients acquired cross-resistant strains during hospitalization.


Introduction
Management and treatment of patients with infections due to Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) is a daily challenge in clinical practice. In the last decade, the treatment of serious KPC-kp infections was frequently based on the combination of last-resort agents associated with nephrotoxicity, sub therapeutic concentrations, and/or intermediate in vitro activity due to limited options [1].
In vitro studies demonstrated excellent activity of MEM/VAB against KPC-producers with a low ability to acquire resistance [3]. Recent clinical study showed that MEM/VAB had a superior rate of clinical cure against CRE infections in patients with bacteremia, compared to other available therapies [2]. However, limited information is still available on the e cacy of MEM/VAB in clinical practice and the in vivo evolution of emerging resistant strains [3]. Recently, in vitro studies demonstrated that mutations causing loss-of-function of OmpK35 and OmpK36 porins or reduced production can generate resistance to MEM/VAB and/or ceftazidime/avibactam (CAZ/AVI) [4,5].
Aim of this study was to investigate the epidemiology of meropenem/vaborbactam resistance among KPC-Kp strains isolated from patients with bloodstream infection (BSI) and characterize genomically MEM/VAB-resistant strains.

Materials And Methods
Between 1st January to 31st December 2018, we collected all KPC-producing K. pneumoniae isolated from patients with BSIs hospitalized in the large tertiarycare university hospital in Bologna. The study was conducted in accordance with the Declaration of Helsinki. Analysis was performed with anonymized database. Bacteria was identi ed by MALDI-TOF (Bruker Daltonics, Germay) and antimicrobial susceptibility testing was performed by MicroScan Walkaway system and con rmed by MIC test strip (Lio lchem, Italy). The MIC value for colistin was determined by reference broth microdilution method [4]. MIC results were interpreted according to EUCAST clinical breakpoints v11.0 (http://www.eucast.org/clinical_breakpoints/). Carbapenemase production was determined by MALDI-TOF and/or NG-Test CARBA 5 (NG Biotech, France), as previously described [6]. Molecular assay was performed to identify carbapenemase gene (Xpert Carba-R, Cepheid).
Whole-genome sequencing of KPC-producing K. pneumoniae (KPC-Kp) strains was performed to identify the molecular mechanism at the basis of meropenem/vaborbactam-resistance as previous described [7]. Brie y, libraries were prepared by the Nextera DNA ex sample preparation kit and sequenced using the Illumina Iseq100 platform (Illumina, San Diego, USA) with a 2x150 paired end run. All read sets were evaluated by FastQC software and then assembled with SPAdes v.3.10 with careful settings. Assembled genomes were screened for Sequence type, known antimicrobial resistance, Sequence type (ST) by CGE server (https://cge.cbs.dtu.dk/services/). Porin genes and Tn4401 isoform were manually investigated by BLAST analysis. A core genome single nucleotide polymorphism (SNP) phylogeny was generated using Parsnp software [8] using draft genomes of KPC-producing K. pneumoniae CC258 strains isolated in Italy, as previously described [8]. Single nucleotide polymorphisms (SNPs) and insertion-deletions (Indels) between MEM/VAB-resistant KPC-Kp genomes were investigated using Breseq [9].
Clinical outcome was de ned as all-cause mortality at day 30 after BSI onset (index BC collection day). Microbiologic failure was de ned as isolation of KPC-Kp strain with similar antimicrobial susceptibility pattern following 30 days of target antimicrobial treatment.

Results
During the study period, in vitro activity of MEM/VAB has been evaluated against 62 KPC-Kp strains isolated from patients with BSIs. The KPC-Kp distribution revealed that 45.2% of bacteremic patients were hospitalized in intensive care units (ICUs), 34% in medicine, 9.7% in haemato-oncology, 6.4% in surgical and 4.7% in transplantation wards.
Genomic analysis showed that meropenem/vaborbactam-resistant strains belonged to Clonal Complex 258 (ST1519, ST258 and ST512) and shared similar genetic resistance determinants responsible for resistance to ß-lactams, aminoglycoside, uoroquinolone and fosfomycin (Table 1). In particular, analysis of ß-lactam resistance genes showed that all isolates shared wild-type bla KPC-3 gene, while 5 out of 8 carried bla SHV-182 variant. In addition, environment of Retrospective clinical analysis showed that patients with BSI due to KPC-Kp resistant both to ceftazidime/avibactam and meropenem/vaborbactam had short time of CPE colonization (median 4 days, IQR 0-7). Clinical characteristics of patients with meropenem/vaborbactam-resistant KPC-Kp are shown in Table 2.
Clinical success was achieved in 50% (4/8) of patients. None of patients were treated with meropenem/vaborbactam-based treatment. Of note, clinical failure occurred for different antimicrobial treatments and in 33% (1/3) of meropenem/vaborbactam and ceftazidime/avibactam -resistant KPC-Kp strains. In particular, no relapsing infection was observed for all antimicrobial combination treatment. At the same time, microbiological failure was achieved in one patient treated with colistin-meropenem therapy due to persistent bacteremia.

Discussion
In this study, we evaluated the activities of meropenem/vaborbactam against 62 KPC-Kp clinical strains collected from bacteremic patients during 2018. Our ndings showed that resistance to meropenem/vaborbactam emerged in 13% of KPC-Kp strains isolated from bloodstream infection. Of note, meropenem/vaborbactam resistance was associated to high level of MICs for ceftazidime/avibactam. In particular, cross-resistance to ceftazidime/avibactam and meropenem/vaborbactam was observed in three KPC-Kp strains which exhibited high levels of MICs for both ceftazidime/avibactam, meropenem/vaborbactam and carbapenems.
Recent study showed that the selection of KPC-Kp mutants with reduced susceptibility to meropenem-vaborbactam was due to porin mutations associated to increase in the blaKPC gene copy number, whereas was not asccoiated to mutations in the coding region of blaKPC gene [10]. Our results are in agreement with these ndings as we found that meropenem/vaborbactam-resistance was due to OmpK35 and OmpK36 porins depletion in our KPC-Kp strains and no speci c mutations were observed in bla KPC gene. At the same time, our results showed that no speci c mutations were observed in KPC-Kp isolates resistant both to meropenem/vaborbactam and ceftazidime/avibactam in comparison to strains resistant to meropenem/vaborbactam alone. These results are in accordance with previous ndings that showed as cross-resistance to meropenem/vaborbactam and ceftazidime/avibactam was due to loss-of-function mutations in the OmpK35 and OmpK36 porins which are also associated to elevated MICs for ß-lactams [4,5].
We observed that patients with BSI due to KPC-Kp resistant both to meropenem/vaborbactam and ceftazidime/avibactam had short colonization time, thus suggesting that cross-resistance did not result from selection pressure due to prolonged antimicrobial therapy while can be plausible that patients acquired cross-resistant strains during hospitalization.
Overall clinical and microbiological failure was observed in two out of three patients with pneumonia, while no speci c treatment was associated to clinical success. At the same time, microbiological failure was observed only in a colistin-meropenem combination treatment. Of note, cross-resistance to meropenem/vaborbactam and ceftazidime/avibactam was not associated to clinical or microbiological failure.
The study is limited by the small number of patients with BSI due to meropenem/vaborbactam-resistant strains treated with different combination treatments.
Further study is mandatory to evaluate the clinical outcome of different antimicrobial therapies against BSI due meropenem/vaborbactam and/or ceftazidime/avibactam -resistant strains.
In conclusion, here we described the emergence of meropenem/vaborbactam resistance among KPC-Kp strains isolated from bacteremic patients. Further studies are planned to characterize speci c-mutations emerging in vivo under different antimicrobial therapy and evaluate the in vitro activity of novel antimicrobials (i.e. ce derocol, imipenem/cilastatin/relebactam) against meropenem/vaborbactam and/or ceftazidime/avibactam-resistant strains.
The authors declare that they have no con ict of interest.
Availability of data and material The data are available for scientists having interest.

Code availability
Not applicable

Ethics approval
The study was conducted in the context of normal clinical routine. The study was conducted in accordance with the Declaration of Helsinki. Samples were coded and analysis was performed with anonymized database.

Supplementary Files
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