Emergence of the New KPC-49 Variant Conferring an ESBL Phenotype with Resistance to Ceftazidime-Avibactam in the ST131-H30R1 Escherichia coli High-Risk Clone

We report the emergence of an isolate belonging to the sequence type (ST)131-Escherichia coli high-risk clone with ceftazidime-avibactam resistance recovered from a patient with bacteremia in 2019. Antimicrobial susceptibility was determined and whole genome sequencing (Illumina-NovaSeq6000) and cloning experiments were performed to investigate its resistance phenotype. A KPC-3-producing E. coli isolate susceptible to ceftazidime-avibactam (MIC = 0.5/4 mg/L) and with non-wild type MIC of meropenem (8 mg/L) was detected in a blood culture performed at hospital admission. Following 10-days of standard ceftazidime-avibactam dose treatment, a second KPC-producing E. coli isolate with a phenotype resembling an extended-spectrum β-lactamase (ESBL) producer (meropenem 0.5 mg/L, piperacillin-tazobactam 16/8 mg/L) but resistant to ceftazidime-avibactam (16/4 mg/L) was recovered. Both E. coli isolates belonged to ST131, serotype O25:H4 and sublineage H30R1. Genomics analysis showed a core genome of 5,203,887 base pair with an evolutionary distance of 6 single nucleotide polymorphisms. A high content of resistance and virulence genes was detected in both isolates. The novel KPC-49 variant, an Arg-163-Ser mutant of blaKPC-3, was detected in the isolate with resistance to ceftazidime-avibactam. Cloning experiments revealed that blaKPC-49 gene increases ceftazidime-avibactam MIC and decreases carbapenem MICs when using a porin deficient Klebsiella pneumoniae strain as a host. Both blaKPC-3 and blaKPC-49 genes were located on the transposon Tn4401a as a part of an IncF [F1:A2:B20] plasmid. The emergence of novel blaKPC genes conferring decreased susceptibility to ceftazidime-avibactam and resembling ESBL production in the epidemic ST131-H30R1-E. coli high-risk clone presents a new challenge in clinical practice.


Introduction
Global expansion of Escherichia coli sequence type 131 (ST131) among multidrugresistant (MDR) Enterobacterales strains is a cause of great concern in Public Health. The ST131-E. coli clonal group associated with extended-spectrum-β-lactamase (ESBLs) production is the most frequent lineage among extraintestinal pathogenic E. coli (ExPEC) isolates [1,2]. The ST131 clone is part of the phylogenetic group B2 and predominantly corresponds to the O25b:H4 serotype [3]. The rapid and successful dissemination of the ST131 high-risk clone has been mainly attributed to the sublineage H30, defined by the presence of the specific fimbrial adhesin allele, fimH30 [4]. Within the H30 sublineage, two fluoroquinolone resistant clades have been widely identified causing human infections: clade 1 or H30R1 and clade 2 or H30Rx [4,5]. The ST131-H30R1-E. coli subclone has been additionally related to an extensive virulence profile [1,2] and with the production of ESBL enzymes, particularly CTX-M-27 (sublineage C1-M27) [6].
In Spain, KPC (Klebsiella pneumoniae carbapenemase) production among ST131-E. coli isolates has been scarcely reported [7]. KPC enzymes hydrolyze efficiently almost all βlactam antibiotics, and older β-lactamase inhibitors such as clavulanic acid are ineffective to prevent β-lactam degradation. Mobilization and diffusion of bla KPC genes have been mostly linked to a conserved 10-kb Tn3-based transposon (Tn4401) and a wide variety of conjugative plasmids that frequently harbor resistance genes against other antimicrobial groups [8]. Infections caused by these MDR KPC-producing Enterobacterales isolates are usually difficult to treat and are frequently associated with high mortality and morbidity rates [9,10]. In February 2015, the U.S. Food and Drug Administration (FDA) approved the ceftazidime-avibactam combination as an alternative to carbapenems in patients with complicated intra-abdominal and urinary tract infections caused by MDR Gram-negative bacteria isolates [11]. Since then, several studies have demonstrated the in vivo efficacy of ceftazidime-avibactam in the treatment of infections caused by KPC-producers [12][13][14]. Moreover, a recent study has supported the presumptive use of ceftazidime-avibactam against carbapenem resistance E. coli isolates, including members of the ST131 lineage [15]. Nevertheless, the recent description of K. pneumoniae high-risk clones producing new KPC variants conferring resistance to ceftazidime-avibactam is a cause of great concern in the clinical setting [16][17][18].
The aim of this work was to describe the emergence of the novel KPC-49 variant conferring ceftazidime-avibactam resistance in an ST131-H30R1-E. coli isolate, with an ESBL resembled phenotype, recovered from an infected patient during the ceftazidimeavibactam treatment.

Case Report
In April 2019, a 40-50 range age woman with a biliary cholangiocarcinoma was admitted at the Ramón y Cajal University Hospital (Madrid, Spain) with sepsis symptoms. Timeline of events during the hospital admission and antibiotic treatments received are represented in Figure 1. An E. coli isolate with an MDR phenotype consistent with carbapenemase production (Ec-1) was recovered at admission from a blood culture. Ec-1 showed non wild-type susceptibility to imipenem (MIC = 4 mg/L) and meropenem (MIC = 8 mg/L) and co-resistance to other antimicrobial groups, except ceftazidime-avibactam (MIC = 0.5/4 mg/L), tigecycline (MIC ≤ 0.5 mg/L), amikacin (MIC ≤ 8 mg/L), colistin (MIC ≤ 1 mg/L), and fosfomycin (MIC ≤ 32 mg/L) ( Table 1). The eazyplex ® SuperBug CRE system demonstrated the presence of a bla KPC gene, and following the regional guidelines [19], the patient was placed under contact precautions in a single room.
At admission, the patient was treated with ceftriaxone (2 g/12 h iv) and trimethoprim sulfamethoxazole (400/800 mg/12 h po) during 3 and 4 days, respectively. Piperacillintazobactam (4/0.5 g/8 h iv) was also added between days 3 and 4. Treatment with ceftazidime-avibactam (2/0.5 g/8 h iv) was started on day 4 and was discontinued at discharge (after 30 days). On day 4, following the protocol of contact precautions, the patient's skin was washed with a 4% soapy chlorhexidine antiseptic gel. On day 7, a sterile blood culture was recovered, but due to the recurrent fever, the antibiotic regimen was modified adding metronidazole (500 mg/8 h iv) and linezolid (600 mg/12 h iv) over ceftazidimeavibactam from day 10 to 27 (17 days) and to day 34 (24 days), respectively. On hospital day 12, a K. pneumoniae isolate with a phenotype compatible with KPC carbapenemase production was detected in a rectal culture. Unfortunately, this isolate was not preserved for subsequent analysis. On day 14 post-admission, 10 days after the ceftazidime-avibactam treatment initiation, a second KPC-producing E. coli isolate (Ec-2) was recovered in a blood sample. This isolate (Ec-2) exhibited a phenotype compatible with an ESBL producer and showed an MIC reduction of 1-fold dilution to imipenem (MIC = 2 mg/L) and 4-fold dilutions to meropenem (MIC = 0.5 mg/L) with piperacillin-tazobactam in the susceptible, increased exposure category (I) (MIC = 16/4 mg/L). On the contrary, an increase of 5-fold dilutions to ceftazidime-avibactam MIC (16/4 mg/L) was observed compared to Ec-1 isolate. Moreover, Ec-2 remained susceptible to tigecycline (MIC ≤ 0.5 mg/L), amikacin (MIC ≤ 8 mg/L), colistin (MIC ≤ 1 mg/L) and fosfomycin (MIC ≤ 32 mg/L) ( Table 1). The eazyplex ® SuperBug CRE system also confirmed the presence of a bla KPC in this isolate. On day 20, amikacin (500 mg/24 h iv) was administered until day 27 and blood cultures collected between days 23 and 33 were negative. Patient's fever remitted and patient was discharged 34 days post-admission ( Figure 1).

Genome Characteristics and E. coli Typing
Assembly of both Ec-1 and Ec-2 strains revealed an approximate genome size of 5.2 Mb with a G+C content of 50.7%. Information about genomes characteristics is summarized in Table S1. The functional classification of both annotated genomes using the KEGG2 database showed a majority of genes linked to metabolism (44%), followed by genetic information processing (28%) and signaling and cellular processes (25%). Phylogenetic analysis of Ec-1 and Ec-2 isolates showed a core genome of 5,203,887 base pair (bp) with six single nucleotide polymorphisms (SNPs) (evolutionary distance of 1.15 SNPs/Mb) and one of them was the R163S mutation in bla KPC-3 (Table S2) designated as bla KPC-49 (accession number MN619655). Ec-1 and Ec-2 isolates were identified as ST131 by the Achtman scheme. Both ST131 isolates belonged to the serotype O25:H4 and were assigned to phylogroup B2. Moreover, both strains carried the fimH30 allele and were identified as subclone H30R1 (clade 1). Table 1. Antimicrobial susceptibility results by broth microdilution and gradient strips for KPC-producing E. coli isolates (Ec-1 and Ec-2), KPC-producing E. coli transformants (pKPC-3-TM and pKPC-49-TM), the isogenic E. coli DH5α strain, KPC-producing K. pneumoniae transformants (pKPC-3-TM and pKPC-49-TM), and the SHV-5-producing K. pneumoniae CSUB10R strain (∆ompK35; ∆ompK36; ∆ompK37).

bla KPC Genetic Environment
KPC-3 and the novel KPC-49 variant were located on an IncFII plasmid also harboring FIA/FIB replicons. In silico pMLST typing confirmed that this IncF-FIA-FIB plasmid belonged to the F1:A2:B20 sequence type. Both bla KPC-3 and bla KPC-49 genes were found as part of the composite transposon Tn4401 (Tn3) designated as "isoform a" variant.

Discussion
To the best of our knowledge, we report for the first time an ST131-H30R1-E. coli strain producing a new KPC variant, the R163S mutant of bla KPC-3 , conferring decreased susceptibility to ceftazidime-avibactam and designated as KPC-49. This ST131-E. coli strain was detected in a blood sample recovered from a patient during the antibiotic treatment with ceftazidime-avibactam (day 10). Typing characterization by WGS identified this ST131 strain as the H30R1 subclone, serotype O25:H4. The ST131-H30R1-E. coli subclone has been recently described in Europe as an emerging MDR pathogen involved in rectal colonization and associated with CTX-M-27 production (C1-M27 subclade) [6].
Association of ST131 strains with the production of KPC enzymes has been scarcely described and the few reports are in countries with a high endemicity level of KPCproducing K. pneumoniae [20,21]. For instance, a high incidence of the high-risk clone ST307-KPC-3-producing K. pneumoniae has been recently reported in both colonized and infected patients in our institution during the last two years (unpublished data). The persistence of certain K. pneumoniae clones, such as ST307, in the patient microbiome plays a crucial role in the cross-species transfer of carbapenemase genes [22,23]. Interestingly, a KPC-producing-K. pneumoniae isolate was also detected in a rectal sample from our patient during the admission. So that, we cannot rule out that the presence of this isolate in the patient microbiome could have facilitated the horizontal acquisition of bla KPC-3 gene by the ST131-E. coli.
Overall, the dissemination of bla KPC among different Enterobacterales species has been mainly related to the transposable element Tn4401a which is often carried on different conjugative plasmids [24]. In fact, cross-species transfer of IncF bla KPC-3 -encoding plasmids from K. pneumoniae to E. coli have been previously described in infected patients [25]. On the other hand, IncFII plasmids with FIA/FIB replicons have also been detected among ST131-E. coli isolates, mostly linked to the bla ESBLs genes dissemination. IncF (F1:A2:B20) plasmids have been usually associated with the H30R1 clade and IncF (F2:A1:B-) plasmids with the H30Rx clade [26,27]. Coincidentally, we found the ST131-H30R1 subclone harboring an IncF (F2:A1:B20) plasmid but encoding bla KPC-3 and the novel bla KPC-49 variant, not an ESBL-encoding gene. Furthermore, in contrast with previous studies [28], we found in both ST131-E. coli isolates a complete Tn4401a transposon, identical to that described in K. pneumoniae strains during the last decade. In fact, Tn4401a has been previously identified in our hospital as the genetic platform involved in the mobilization and diffusion of the bla KPC-3 gene in K. pneumoniae [24]. On the other hand, although bla CTX-M genes has been largely found inserted in both IncF-type plasmids and chromosomal locations in ST131-E. coli isolates [28], CTX-M-encoding genes were not detected in our isolates.
Coinciding with the reported literature, a high number of virulence and resistance genes was identified in both KPC-3-and KPC-49-ST131-H30R1-E. coli isolates [1,5]. The ST131-E. coli isolate recovered at admission showed a multidrug resistance profile, leaving few therapeutics options, as ceftazidime-avibactam, to treat the blood infection.
Although the in vivo clinical efficacy of ceftazidime-avibactam has been widely demonstrated [12,13], several studies have recently reported in K. pneumoniae epidemic clones the emergence of new ceftazidime-avibactam resistant KPC enzymes derived from point mutations in bla KPC-2 and bla KPC-3 genes, frequently after the antibiotic exposure [16][17][18]. In all cases, in vitro meropenem susceptibility was fully or partially restored due to these bla KPC mutations resulting in KPC-producing K. pneumoniae isolates with a phenotype resembling that of ESBL producers [16][17][18]. According to these data, the impact of ceftazidime-avibactam resistance could be minimized due to the lower carbapenem MICs. However, some studies have shown that after the discontinuation of ceftazidimeavibactam treatment, carbapenem resistant phenotype can be restored in K. pneumoniae isolates that still display ceftazidime-avibactam resistance [18]. In our study, a higher susceptibility to meropenem, imipenem, and piperacillin-tazobactam was observed in the KPC-49-ST131 strain coinciding with the increased ceftazidime-avibactam MIC. In concordance with previous reports, this resistance phenotype resembled that of an ESBL producer [16][17][18]. Importantly, our region is considered an endemic area of ESBL-producing E. coli isolates [29] and the emergence of the ST131-E. coli producing KPC carbapenemases but with an ESBL resistance profile could lead to under reporting of infections by KPC producers with potential carbapenemase activity.
According to our results, this resistance phenotype could be consequence of the R163S mutation in bla KPC-3 . It is noteworthy that the bla KPC-49 was more clearly validated as a ceftazidime-avibactam resistance determinant in the porin-deficient strain K. pneumoniae CSUB10R. Previous studies have also demonstrated that mutated or non-functional OmpK35 and Ompk36 porins slightly contribute to increase ceftazidime-avibactam MICs in KPC-3-producing K. pneumoniae isolates [30,31]. It should also be noted that the K. pneumoniae CSUB10R strain shares a similar genetic background to the MDR-K. pneumoniae clinical strains that usually circulate in the hospital setting causing severe and difficult-to-treat infections. We believe that the potential contribution of the ST131-E. coli clone in the dissemination of novel bla KPC genes conferring ceftazidime-avibactam resistance, such as bla KPC-49 , in epidemic K. pneumoniae clinical strains should be a cause of great concern for public health.

Sequence Data
The bla KPC-49 gene and the complete genomes of both ST131-H30R1-E. coli isolates were deposited at DDBJ/ENA/GenBank under accession numbers MN619655, WIRF00000000 and WIRG00000000, respectively.

Conclusions
In the present work we report the in vivo emergence of a novel KPC variant (KPC-49) conferring a phenotype resembling that of ESBL producers in the ST131-E. coli high-risk clone during the treatment with ceftazidime-avibactam. The global expansion of the ST131-H30-E. coli high-risk clone in both community and hospital settings together with the successful acquisition of IncF-type plasmids harboring bla KPC genes that function as ESBL genes but also conferring resistance to new β-lactam and β-lactamase inhibitor combinations such as ceftazidime-avibactam, poses a new challenge to the patient management and the containment programs design to avoid the spread of MDR pathogens.