linical Infectious Diseases

Emergence and Recovery of Ceftazidime-avibactam Resistance in blaKPC-33-Harboring Klebsiella pneumoniae Sequence Type 11 Isolates in China
Qingyu Shi,1,2,a Dandan Yin,1,2,a Renru Han,1,2 Yan Guo,1,2 Yonggui Zheng,1,2 Shi Wu,1,2 Yang Yang,1,2 Shirong Li,3 Rong Zhang,4 and Fupin Hu1,2

1Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China, 2Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, Shanghai, China, 3Laboratory Medicine, Huashan Hospital (West campus), Fudan University, Shanghai, China, and 4Department of Clinical Laboratory Medicine, Second Affiliated Hospital of Zhejiang University, Hangzhou, China

This is the first report of ceftazidime–avibactam resistance caused by the blaKPC-33 mutation through the D179Y variant during the treat- ment of blaKPC-2-positive Klebsiella pneumoniae-related infections in China. The blaKPC-33-containing K. pneumoniae was susceptible to meropenem–vaborbactam, cefepime–zidebactam, tigecycline, and polymyxin B. The blaKPC-33 gene was located on a 77 551-bp transform- able plasmid harboring qnrS1 and blaLAP-2. Detecting blaKPC-33-positive K. pneumoniae clinical strains is important for infection control.
Keywords. Carbapenem-resistant K. pneumoniae; Ceftazidime-avibactam; Meropenem-vaborbactam; blaKPC-2; blaKPC-33.

Carbapenemase-producing Enterobacterales (CPE)-related infections have been a major public healthcare problem, causing global concerns. Ceftazidime–avibactam has been ap- proved as an effective alternative antibiotic for clinical treat- ment against blaKPC- and blaOXA-48-producing isolates in recentyears.

However, resistance has gradually emerged along with its
wide usage. Herein, we present the first case of the emergence of ceftazidime–avibactam-resistant KPC-33 carbapenemase- producing K. pneumoniae in China, and described its molecular and genetic characteristics in detail.

METHODS
Three multidrug-resistant Klebsiella pneumoniae strains, H1, H2, and H3, were isolated from sputum specimens of a hospi- talized patient at Huashan Hospital in Shanghai, China. They were identified using MALDI-TOF MS (bioMérieux, Marcy-
l’Étoile, France). Escherichia coli ATCC 25922 and bla -
Quality control and interpretation of the results were also per- formed according to 2020 CLSI breakpoints [1] for all agents except tigecycline and polymyxin B, which were interpreted using the interpretative criteria according to the Food and Drug Administration and the European Committee on Antimicrobial Susceptibility Testing (EUCAST), respectively. The efflux pump inhibitor phenyl-arginine-β-naphthylamide (25 mg/L) was added into the broth to observe ceftazidime-avibactam MIC variation; no less than a quadruple MIC decrease was con- sidered to be significant.
Conjugation experiments were performed to explore the transferability of the ceftazidime–avibactam resistance using rifampicin-resistant E. coli EC600 as a recipient strain. The transconjugants were selected on Luria-Bertani agar plates containing ceftazidime–avibactam (8 mg/L) and rifampicin (200 mg/L), and were verified by PCR for the presence of blaKPC.

Bacterial DNA was digested with Xba I and S1-nuclease sub
containing K. pneumoniaeKPC-2ATCC 1705 were used as controls forjected to PFGE and S1-PFGE, respectively. PFGE was per-formed using a CHEF Mapper system (Bio-Rad Laboratories,

Downloaded from https://academic.oup.com/cid/article/71/Supplement_4/S436/6046209 by University of New England user on 01 June 2021
screening carbapenemase genes and testing antimicrobial sus- ceptibility. Escherichia coli EC600 and Salmonella braenderup H9812 were used as recipients for conjugation experiments and reference markers for pulsed-field gel electrophoresis (PFGE), respectively. The minimal inhibition concentration (MIC) was determined using the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method [1].

aQ. S. and D. Y. contributed equally to this work.
Correspondence: F. Hu, Institute of Antibiotics, Huashan Hospital, Fudan University 12
M. Wulumuqi Road, Shanghai 200040, China ([email protected]).
Clinical Infectious Diseases® 2020;71(S4):S436–9
© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: [email protected].
DOI: 10.1093/cid/ciaa1521
Hercules, CA, USA). PFGE patterns were interpreted in accord- ance with the criteria of Tenover et al. [2] using BioNumerics software version 6.5. Genomic DNAs of K. pneumoniae strains were subjected to whole-genome sequencing using Illumina (Illumina, San Diego, CA, USA) short-read sequencing (150- bp paired-end reads). Sequences were trimmed with sickle (GitHub) and de novo assembled using SPAdes 3.12.0. To eval- uate and compare the assembly results, Pilon 1.18 was used for base correction. Multilocus sequence typing (MLST) and anti- microbial resistance genes analysis were performed using MLST

2.0 (https://cge.cbs.dtu.dk/services/MLST/) and ResFinder 3.2 (https://cge.cbs.dtu.dk/services/ResFinder-3.2/), respectively.
Clinical characteristics including age, gender, disease rec- ognition, specimen origin and separation time, antibiotic exposure and duration time, in-hospital time, and disease prognosiswere systematically extracted from the electronic medical records.
Ethics committee approval was obtained from the institu- tional review board of Huashan Hospital for these isolates, and verbal informed consent from patient’s parents was also ac- cepted and approved.

RESULTS
phenyl-arginine-β-naphthylamide did not change the MIC of ceftazidime–avibactam. The plasmid harboring blaKPC-33 from strain H2 was successfully transferred into the EC600
E. coli recipient strain, making the transconjugants resistant to ceftazidime–avibactam, ertapenem, quinolone, and some ceph- alosporins (Table 1).
PFGE showed that the H1, H2, and H3 strains were highly homologous, showing a consistent fingerprint spectrum. Per the whole-genome sequencing analysis, H2 belonged to theThe first carbapenem-resistant K. pneumoniae (H1, bla
pos-ST11 type, harboring the aadA2b, rmtB, qnrS1, blaLAP-2, blaSHV-12,
KPC-2
bla , bla , bla , dfrA14, fosA, sul2, and tet(A) re-itive) was isolated from the sputum of a 42-year-old man, who
KPC-33
CTX-M-65
TEM-1B underwent an endoscopic-assisted transsphenoidal approach resection for prolactin-producing pituitary macroadenoma
3 days after hospitalization. Subsequently, ceftazidime– avibactam and tigecycline were used to replace the empir- ical meropenem regimen for anti-infection. However, after using ceftazidime–avibactam for 16 days, the patient’s body temperature rose again, concurrent with the symptoms of se- vere pulmonary infection. The second carbapenem-resistant
K. pneumoniae strain (H2, blaKPC-33 positive) was isolated on day 45 from the sputum. Considering the side effects and patient’s economic condition, imipenem, amoxicillin–clavulanic, and
gradually reduced doses of polymyxin B were used instead. Unfortunately, recurrent pneumonia emerged on day 63, and
the third carbapenem-resistant K. pneumoniae (H3, blaKPC-2
sistance genes. blaKPC-33 gene emergence was due to a single base mutation at G532T, causing the D179Y amino acid muta- tion of the blaKPC-2 gene. The plasmid harboring blaKPC-33 was a
77 551-bp conjugative plasmid (plasmid pKPC-H2; average GC
content, 53.79%).
In this plasmid, qnrS1, blaLAP-2, and blaKPC-33 were located at the same gene island. Mobile elements such as Tn3-family transposons, TnAs1, IS26, ISKpn27, ISKpn6, ISKpn19, and IS3 were also distributed densely around the re- sistant genes; these can cluster and combine with resistance genes to transfer multiple resistance of plasmids. pKPC-H2 was 100% covered and showed 99.66% identity when mapped to the plasmid (GenBank accession no. CP050279.1) with an 18 962- bp fragment deletion in the downstream region, containing the
repeat resistance genes qnrS1 and blaLAP-2. Additionally, a single-positive) was isolated from the sputum. Clinical and microbi- ologic details, timelines, and antibiotic therapies used are sum- marized in Figure 1.
H1, H2, and H3 were blaKPC-positive isolates, and were highly resistant to β-lactams, aminoglycosides, quinolones, and sulfanilamides (Table 1). H1 and H3 were highly resistant
to carbapenems and susceptible to ceftazidime–avibactam; while H2 (blaKPC-33 positive) was resistant to ceftazidime– avibactam but susceptible to imipenem with MIC = 0.25 mg/L.

Further, cefepime–zidebactam and meropenem–vaborbactam, as well as tigecycline and polymyxin B, showed good in vitro activities against all 3 isolates. The addition of
nucleotide deletion at position 86 of the OmpK35-encoding gene was found, producing a frameshift mutation and a prema- ture stop codon in the coding sequence.

DISCUSSION
As one of the most effective antibiotics against blaKPC-2-positive strains, ceftazidime–avibactam has been widely used fol- lowing its approval in China on May 21, 2019. However, with its ever-growing clinical application, the rate of ceftazidime– avibactam resistance in carbapenem-resistant K. pneumoniae- infected patients has reached approximately 10% [3]. The basic resistance mechanisms are as follows: (1) KPC or OXA-48
Downloaded from https://academic.oup.com/cid/article/71/Supplement_4/S436/6046209 by University of New England user on 01 June 2021
Time courses of infection and treatment of the patient with multidrug-resistant K. pneumoniae infection. The blocks from dark to light indicate high, mediate, low-grade fever, and normal temperatures.

Table 1. Minimal Inhibitory Concentrations (MICs) of K. pneumoniae Strains H1, H2, H3, and the blaKPC-33-Positive E. coli Transconjugant of H2

MIC (μg/mL) for:

Antimicrobial agents K. pneumoniae H1 (blaKPC-2) K. pneumoniae H2 (blaKPC-33) K. pneumoniae H3 (blaKPC-2) Transconjugant E. coli H2-EC600 (blaKPC-33) E. coli
EC600
Amikacin >128 >128 >128 ≤1 ≤1
Cefazolin >32 >32 >32 8 4
Cefuroxime >32 >32 >32 >32 16
Ceftriaxone >32 >32 >32 8 ≤0.5
Ceftazidime >32 >32 >32 >32 0.5
Cefepime >128 >128 >128 4 0.125
Aztreonam >128 >128 >128 2 ≤1
Imipenem 32 0.25 64 0.125 0.125
Meropenem >64 4 >64 0.03 ≤0.03
Ertapenem >64 16 >64 2 ≤0.03
Ceftolozane-tazobactam >128 >128 >128 16 1
Cefoperazone-sulbactam >128 >128 >128 ≤1 ≤1
Piperacillin-tazobactam >256 >256 >256 ≤2 ≤2
Ceftazidime-avibactam 2 >64 4 16 0.25
Meropenem-vaborbactam 2 1 2 0.06 0.03
Cefepime-tazobactam >64 64 >64 0.125 0.125
Cefepime-zidebactam 2 1 2 0.125 0.125
Ciprofloxacin >8 >8 >8 4 0.25
Levofloxacin >16 >16 >16 4 0.25
Trimethoprim-sulfamethoxazole >32 >32 >32 ≤0.25 ≤0.25
Tigecycline 2 2 4 0.25 0.125
Polymyxin B 0.25 0.25 1 0.25 0.25

Downloaded from https://academic.oup.com/cid/article/71/Supplement_4/S436/6046209 by University of New England user on 01 June 2021
carbapenemases co-producing metallo-β-lactamases such as NDM, VIM, or IMP [4]; (2) single amino acid substitutions of blaKPC, particularly at positions 164, 167, 169, and 179 within the Ω-loop of class A β-lactamases [5]; and (3) transposition of KPC with porin deficiency [6].
Herein, we report the first case of ceftazidime–avibactam re- sistance development during the treatment of CPE infections due to the change from KPC-2 to KPC-33 carbapenemase via the D179Y variant in China. Such mutations of plasmid- borne blaKPC-2 can reduce the MICs of carbapenems (often re-
storing susceptibility to imipenem and low-level resistance to
meropenem) [7, 8], because the mutations within the blaKPC Ω-loop (positions 165–179) enhance ceftazidime affinity and restrict avibactam binding [5]. Mutations such as R164S, L169P, D179Y, A177E, V240G, and T243M, and deletions in positions 167 and 168 are constantly emerging worldwide with the use of ceftazidime–avibactam, with D179Y as the most common amino acid substitution, especially in KPC [9–12]. Additionally, Ying Zhang et al. [13] reported that deficient OmpK35 and/
or OmpK36 expression could be found in blaKPC-2 positive
K. pneumoniae (ST11 type) at Huashan Hospital. The frame- shift mutation of the OmpK35-encoding gene found in our case indicates that OmpK35 porin may also be a minor contributor towards the drug resistance [13].
In the present case, ceftazidime–avibactam (2.5 g; admin- istered intravenously every 8 h) seems not to work, though it
has been previously confirmed to be effective against blaKPC-2- positive K. pneumoniae, since the patient experienced persistent fever during the whole exposure period. The blaKPC-33-positive strain emerged following the treatment of ceftazidime– avibactam for 16 days; this has been typically reported to occur after 10 to 19 days [7], and sometimes, after 33 days [14].
Subsequently, the blaKPC-2-positive strain dominated again after the imipenem substitution therapy for ceftazidime–avibactam resistance. Thus, we speculated that blaKPC-33 mutation via the D179Y variant of blaKPC-2 was caused mainly by the selective pressure of ceftazidime–avibactam usage.
Therefore, clinicians should be vigilant to ceftazidime–avibactam resistance when it is used for treating infections caused by blaKPC-2-positive iso- lates; reintroduction of carbapenems is worthy of consideration on occasion.
Another potentially suitable antibiotic was meropenem– vaborbactam, which showed excellent in vitro antimicro- bial activity and has been proven to be effective clinically [8]. Additionally, unlike ceftazidime–avibactam, no blaKPC mu-
tations have been found to confer resistance to meropenem–
vaborbactam [7].

Moreover, blaKPC-positive K. pneumoniae with the ST258 clonal background are considered to represent the majority of ceftazidime–avibactam-resistant isolates [14]; however, the strain isolated from our patient was ST11-type K. pneumoniae. In China, 96.2% of ST11-type isolates produce KPC-2
Downloaded from https://academic.oup.com/cid/article/71/Supplement_4/S436/6046209 by University of New England user on 01 June 2021
carbapenemase, one of the most common carbapenemase types in K. pneumoniae (76.5%) [15]. More importantly, blaKPC-2 pos- itive K. pneumoniae ST11 strains can easily evolve pan-drug resistance through chromosomal mutations [16]. We speculate that mutant blaKPC-33-positive K. pneumoniae ST11 strains may represent another main type of ceftazidime–avibactam resistant strains in China in the near future; thus, more attention may be required to monitor its prevalence.

Though detecting blaKPC-33-positive strains is important for the choice of medication, it is easily mis-detected due to the in- conspicuous characteristic of carbapenem resistance. NG-Test CARBA 5 and the RESIST-5, two popular direct rapid detection
methods for blaKPC-2-positive strains, show negative results for strains harboring blaKPC-33. Routine molecular screening for blaKPC has also been advocated to facilitate its rapid detection [17].
Thus, ceftazidime–avibactam is one of the few antibiotics ac- tive against CPE. During its use, it is important to define optimal dosing strategies, insist on rigorous infection control practices, develop quick and efficient assays for detecting resistance, and further analyze resistance mechanisms. Once relevant cases are found, actions such as contact isolation and environmental cleaning may also be performed to avoid nosocomial outbreaks.

Notes
Author contributions. F. H. and R. Z. conceptualized and designed the overall study. Q. S., D. Y., R. H. and S. L. collected and analyzed the data. Y. G., Y. Z., S. W. and Y. Y. directed and managed the planning and execution of the project. F. H., Q. S. and D. Y. wrote and revised the paper. All authors reviewed and approved the final version of the manuscript.
Financial support. This work was supported by the National Natural Science Foundation of China (grant numbers 81871690, 81902101, and 81861138051), the National Mega-project for Innovative Drugs (grant number 2019ZX09721001-006-004) and China Antimicrobial Surveillance Network (grant number 2020QD049).

Supplement sponsorship. This supplement was sponsored by MSD.
Potential conflicts of interest. All authors declare no conflict of interest.

References
1. Clinical and Laboratory Standards Institute. Performance standards for anti- microbial susceptibility testing, 30th ed. CLSI supplement M100. Clinical and Laboratory Standards Institute, Wayne, PA. 2020.
2. Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA re- striction patterns produced by pulsed-field gel electrophoresis: criteria for bacte- rial strain typing. J Clin Microbiol 1995; 33:2233–9.
3. Shields RK, Nguyen MH, Press EG, Chen L, Kreiswirth BN, Clancy CJ. Emergence of ceftazidime-avibactam resistance and restoration of carbapenem susceptibility in Klebsiella pneumoniae carbapenemase- producing K pneumoniae: A Case Report and Review of Literature. Open Forum Infect Dis, 2017; 4:ofx101.
4. van Duin D, Bonomo RA. Ceftazidime/avibactam and ceftolozane/tazobactam: second-generation beta-lactam/beta-lactamase inhibitor combinations. Clin Infect Dis, 2016; 63:234–241.
5. Winkler ML, Papp-Wallace KM, Bonomo RA. Activity of ceftazidime/avibactam against isogenic strains of Escherichia coli containing KPC and SHV beta- lactamases with single amino acid substitutions in the Omega-loop. J Antimicrob Chemother 2015; 70:2279–86.
6. Nelson K, Hemarajata P, Sun D, et al. Resistance to ceftazidime-avibactam is due to transposition of KPC in a porin-deficient strain of Klebsiella pneumoniae with increased efflux cctivity. Antimicrob Agents Chemother 2017; 61:e00989–17.
7. Shields RK, Chen L, Cheng S, et al. Emergence of ceftazidime-avibactam resist- ance due to plasmid-borne blaKPC-3 mutations during treatment of carbapenem- resistant Klebsiella pneumoniae infections. Antimicrob Agents Chemother 2017; 61:e02097–16.
8. Athans V, Neuner EA, Hassouna H, et al. Meropenem-vaborbactam as salvage therapy for ceftazidime-avibactam-resistant Klebsiella pneumoniae bacteremia and abscess in a liver transplant recipient. Antimicrob Agents Chemother 2019; 63:e01551–18.
9. Hemarajata P, Humphries RM. Ceftazidime/avibactam resistance associated with L169P mutation in the omega loop of KPC-2. J Antimicrob Chemother, 2019; 74:1241–1243.
10. Shields RK, Nguyen MH, Press EG, Chen L, Kreiswirth BN, Clancy CJ. In vitro selection of meropenem resistance among ceftazidime-avibactam- resistant, meropenem-susceptible Klebsiella pneumoniae isolates with variant KPC-3 carbapenemases. Antimicrob Agents Chemother 2017; 61:e00079–17.
11. Levitt PS, Papp-Wallace KM, Taracila MA, et al. Exploring the role of a conserved class A residue in the omega-loop of KPC-2 beta-lactamase: a mechanism for ceftazidime hydrolysis. J Biol Chem 2012; 287:31783–93.
12. Antinori E, Unali I, Bertoncelli A, Mazzariol A. Klebsiella pneumoniae carbapenemase (KPC) producer resistant to ceftazidime-avibactam due to a dele- tion in the blaKPC3 gene. Clin Microbiol Infect 2020; 26:946.e1–946.e3.
13. Zhang Y, Jiang X, Wang Y, et al. Contribution of beta-lactamases and porin pro- teins OmpK35 and OmpK36 to carbapenem resistance in clinical isolates of KPC-2-producing Klebsiella pneumoniae. Antimicrob Agents Chemother, 2014; 58:1214–17.
14. Giddins MJ, Macesic N, Annavajhala MK, et al. Successive emergence of ceftazidime-avibactam resistance through distinct genomic adaptations in blaKPC-2-harboring Klebsiella pneumoniae sequence type 307 isolates. Antimicrob Agents Chemother 2018; 62:e02101–17.
15. Wang Q, Wang X, Wang J, et al. Phenotypic and genotypic characterization of carbapenem-resistant enterobacteriaceae: data from a longitudinal large-scale cre study in China (2012–2016). Clin Infect Dis 2018; 67:S196–205.
16. Xu J, Zhao Z, Ge Y, He F. Rapid emergence of a pandrug-resistant Klebsiella pneumoniae ST11 isolate in an inpatient in a teaching hospital in China after treatment with multiple broad-spectrum antibiotics. Infect Drug Resist 2020; 13:799–804.
17. Humphries RM, McKinnell JA. Continuing challenges for the clinical laboratory for detection of Tigecycline carbapenem-resistant enterobacteriaceae. J Clin Microbiol 2015; 53:3712–14.