Emergence of KPC-113 and KPC-114 variants in ceftazidime-avibactam-resistant Klebsiella pneumoniae belonging to high-risk clones ST11 and ST16 in South America

ABSTRACT Two novel variants of Klebsiella pneumoniae carbapenemase (KPC) associated with resistance to ceftazidime-avibactam (CZA) and designated as KPC-113 and KPC-114 by NCBI were identified in 2020, in clinical isolates of Klebsiella pneumoniae in Brazil. While K. pneumoniae of ST16 harbored the bla KPC-113 variant on an IncFII-IncFIB plasmid, K. pneumoniae of ST11 carried the bla KPC-114 variant on an IncN plasmid. Both isolates displayed resistance to broad-spectrum cephalosporins, β-lactam inhibitors, and ertapenem and doripenem, whereas K. pneumoniae producing KPC-114 showed susceptibility to imipenem and meropenem. Whole-genome sequencing and in silico analysis revealed that KPC-113 presented a Gly insertion between Ambler positions 264 and 265 (R264_A265insG), whereas KPC-114 displayed two amino acid insertions (Ser-Ser) between Ambler positions 181 and 182 (S181_P182insSS) in KPC-2, responsible for CZA resistance profiles. Our results confirm the emergence of novel KPC variants associated with resistance to CZA in international clones of K. pneumoniae circulating in South America. IMPORTANCE KPC-2 carbapenemases are endemic in Latin America. In this regard, in 2018, ceftazidime-avibactam (CZA) was authorized for clinical use in Brazil due to its significant activity against KPC-2 producers. In recent years, reports of resistance to CZA have increased in this country, limiting its clinical application. In this study, we report the emergence of two novel KPC-2 variants, named KPC-113 and KPC-114, associated with CZA resistance in Klebsiella pneumoniae strains belonging to high-risk clones ST11 and ST16. Our finding suggests that novel mutations in KPC-2 are increasing in South America, which is a critical issue deserving active surveillance.

and is now associated with novel KPC variants (15)(16)(17)(18)(19).In this study, we report two novel KPC variants associated with resistance to CZA emerging in K. pneumoniae strains belonging to the high-risk clones ST11 and ST16, in Brazil, 2 years after CZA approval in this country (20).
In 2020, two CZA-resistant K. pneumoniae strains (330 and 331) were isolated from blood and rectal swab cultures from two different patients admitted to a teaching hospital.
Carbapenemase-positive K. pneumoniae strain 330 was isolated from a 61-year-old male patient admitted to the ICU ward with a medical history of alcoholic cirrhosis, hepatocellular carcinoma, hypertension, type 2 diabetes mellitus, and hepatic ence phalopathy.The patient underwent a liver transplant.A surveillance rectal swab was collected, being negative for carbapenemase producers.However, after transplant, the patient's respiratory status deteriorated, polymerase chain reaction COVID-19 test was negative, and based on chest X-ray abnormalities the patient was treated with polymyxin B, fluconazole, teicoplanin, meropenem, and sulfamethoxazole/trimethoprim.After a month of hospitalization, blood cultures tested positive for KPC-producing K. pneumo niae (strain 330), and pulmonary sepsis was the focus of the infection.The patient was treated with ceftazidime-avibactam, gentamicin, and sulfamethoxazole/trimethoprim.However, the patient's condition deteriorated rapidly, with multiple organ dysfunctions, metabolic acidosis, and positive blood cultures for Candida tropicalis.Then therapy with ceftazidime-avibactam, polymyxin B, teicoplanin, and micafungin was started.However, clinical condition worsened and the patient died within 24 h.
Carbapenemase-positive K. pneumoniae strain 331 was isolated from a 59-year-old female patient with a medical history of chronic kidney disease, hypertension, smok ing, and alcoholism.The patient was admitted to the nephrology ward for a kidney transplant.The patient did not experience any complications during hospitalization.A surveillance rectal swab was collected 1 week after transplant, and culture was negative for carbapenemase producers.However, a week later, a new surveillance rectal swab was collected being positive for carbapenemase-producing K. pneumoniae (strain 331).The patient was treated with meropenem for 14 days and a week later was discharged.
In order to evaluate the in vivo activity of meropenem against meropenem-suscep tible KPC-114-positive K. pneumoniae 331, healthy Galleria mellonella larvae weigh ing ~250 mg were selected and inoculated with 10 µL of 1.5 × 10 8 CFU/mL K. pneumoniae 331.After 1 h, larvae were treated with 10 µL of 0.5 mg/mL meropenem or 10 µL of 1.0 mg/mL meropenem, in order to achieve clinical human doses of 1 g and 2 g meropenem, respectively, as standardized by the EUCAST for intravenous regimens (22,23).The inoculum was delivered in the last right proleg, and the treatments were injected in the last left proleg by using a sterile insulin syringe.Five larvae were included in each tested group, and assays were performed in duplicate.While 60% of untreated G. mellonella larvae died at 48 h post-infection, 100% survival was observed in both G. mellonella groups treated with clinical doses of 1 g and 2 g meropenem (Fig. S2).Likewise, 100% survival was observed in the uninfected control group treated with 10 µL of sterile saline.Although this result suggests that meropenem could be an option for decolonization or treatment of meropenem-susceptible KPC-114-positive K. pneumoniae, additional investigation is necessary, in order to investigate the emergence of possible additional new mutations conferring resistance to CZA and meropenem.In this study, the colonized patient was treated with meropenem for 14 d and a week later was discharged, confirming favorable use of empiric therapy in KPC-positive K. pneumoniae colonized patients (24).
Carbapenem-resistant K. pneumoniae has been the most important species recov ered from surveillance rectal swabs of hospitalized patients and the most common cause of subsequent infections (25,26).In fact, colonization at multiple sites with carbapenem-resistant K. pneumoniae has been the strongest predictor of bloodstream infection development in previous large cohorts of carbapenem-resistant K. pneumo niae rectal carriers (27).In patients colonized by KPC-positive K. pneumoniae, utility of the Giannella Risk Score (https://www.pharmacyjoe.com/giannella-risk-score-calculator-for-infection-with-carbapenem-resistant-klebsiella-pneumoniae/) to predict infection risk has been previously confirmed (24,28).
Strikingly, KPC-114 was not detected by the NG-Test CARBA 5 lateral flow immu nochromatographic test (NG Biotech) (29).Previous studies have demonstrated that KPC-31, KPC-33, KPC-68, KPC-71, KPC-78, KPC-90, KPC-104, KPC-106, KPC-139, KPC-141, KPC142, and KPC-143 variants also have been not detected by NG-Test CARBA-5 (30,31).Therefore, most likely R264_A265insG and S181_P182insSS mutations confer protein conformations leading to inefficient binding of immobilized monoclonal antibodies targeted to recognize KPC-type enzymes inhibited by avibactam, consequently resulting in the failure of the detection method.On the other hand, CZA-resistant isolates displaying susceptibility to meropenem could not be identified as KPC producers (32), leading to a misleading detection by diagnostic laboratories.Thus, since CZA has become the first-line option for the treatment of infections due to KPC-2 producers, it is imperative to improve screening methods for the detection of KPC variants displaying resistance to CZA, in order to facilitate its rapid and accurate detection.
Although a limitation of this study is the lack of kinetic data, shortly after NCBI designation of the novel bla KPC allele identified in the CZA-resistant K. pneumoniae strain 330 as bla KPC-113 (GenBank accession number: OM728506.1),a report from China described the presence and kinetic properties of KPC-113, identified in Pseudomonas aeruginosa, confirming considerable hydrolyzing abilities to carbapenems and ceftazi dime and the significantly weakened inhibitory effect of avibactam (33).
In summary, we described two novel KPC variants, KPC-113 and KPC-114, associated with resistance to CZA in high-risk clones of K. pneumoniae belonging to ST11 and ST16.Our finding suggests that novel mutations in KPC-2 are increasing in South America, which is a critical issue deserving active surveillance.

FIG 1
FIG 1 Heatmap displaying the resistome and virulome of K. pneumoniae 330/ST16 and K. pneumoniae 331/ST11 harboring KPC-113 and KPC-114, respectively.The colored regions represent the presence of antibiotic resistance genes (ARGs) and virulence genes (VGs) and the location (plasmid or chromosome).Blank fragments represent their absence.

TABLE 1
Antimicrobial resistance profile of K. pneumoniae strains producing KPC-113 and KPC-114 variants to β-lactam antibiotics