Dynamic evolution of ceftazidime-avibactam resistance from a single patient through the IncX3_NDM-5 plasmid transfer and blaKPC mutation.

The rapid dissemination of carbapenem-resistant Enterobacterales (CRE) especially carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a great threat to global public health. Ceftazidime-avibactam, a novel ß-lactam/ß-lactamase inhibitor combination, has been widely used due to its excellent antibacterial activity against KPC-producing K. pneumoniae. However, several resistance mechanisms have been reported since its use. Here, we conducted a series of in vitro experiments to reveal and demonstrate the dynamic evolution of ceftazidime-avibactam resistance including interspecies IncX3_NDM-5 plasmid transfer between Enterobacter cloacae and K. pneumoniae and blaKPC mutation from blaKPC-2 to blaKPC-33. Through the analysis of conjugation frequency and fitness cost, the IncX3_NDM-5 plasmid in this study showed strong transmissibility and stability in E. coli EC600 and clinical strain K. pneumoniae 5298 as recipient strain. With increasing ceftazidime-avibactam concentration, the conjugation frequency remained at 10-3-10-5, while the mutation frequency of K. pneumoniae 5298 was 10-6-10-8 at the same concentration. Further plasmid analysis (the IncX3_NDM plasmid from this study and other 658 plasmids from the NCBI database) revealed the diverse origin and genetic structure of blaNDM-5 carrying plasmids. E. coli (42.9%), China (43.9%), IncX3 (66.6%) are the most common strains, regions, and Inc types respectively. By analysing of genetic environment detected in IncX3 plasmids, the dominant structures (168/258, 65.1%) were identified: ISKox3-IS26-blaNDM-5-IS5-ISAba125-Tn3000-Tn3. In additon, several structural variations were found in the core gene structure. In conclusion, the high fitness and transmissibility of the IncX3_NDM-5 plasmids were noteworthy. More importantly, the diverse ceftazidime-avibactam resistance mechanisms including blaNDM-5 tranfer and blaKPC-2 mutation highlighted the importance of the continuous monitoring of antimicrobial susceptibility and carbapenemases subtype during ceftazidime-avibactam treatment.

Emergence of ceftazidime-avibactam resistance in blaKPC-33-harbouring ST11 Klebsiella pneumoniae in a paediatric patient.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses immense threats to the health of infected patients worldwide, especially children. This study reports the infection caused by CRKP in a paediatric intensive care unit (PICU) child and its drug-resistant mutation during the treatment. Twelve Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains were isolated from the child. Broth microdilution method, plasmid transformation assay, and whole genome sequencing (WGS) were performed to investigate the antimicrobial susceptibility, resistance mechanisms, and genetic structural features of CRKPs. The results showed that 12 strains were highly resistant to most available antimicrobial agents. Among them, K. pneumoniae FD11 and K. pneumoniae FD12 were resistant to ceftazidime-avibactam (CZA, MIC >64 mg/L) and restored the carbapenem susceptibility (Imipenem, MIC =0.25 mg/L; Meropenem, MIC =2 mg/L). The patient improved after treatment with CZA in combination with aztreonam. Plasmid transformation assay demonstrated that the blaKPC-33-positive transformant increased MICs of CZA by at least 33-fold and 8-fold compared with the recipient Escherichia coli DH5α and blaKPC-2-positive transformants. WGS analysis revealed that all strains belonged to the ST11-KL64 type and showed highly homologous (3-26 single nucleotide polymorphisms [SNPs]). A single base mutation (G532T) of blaKPC-2 resulted in a tyrosine to aspartic acid substitution at Ambler amino acid position 179 (D179Y), which conferred CZA resistance in K. pneumoniae. This is the first report of a drug-resistant mutation evolving into blaKPC-33 during the treatment of blaKPC-2-positive CRKP in paediatric-infected patients. It advises clinicians that routine sequential antimicrobial susceptibility testing and KPC genotyping are critical during CZA therapy in children infected with CRKP.

The mechanism of ceftazidime and cefiderocol hydrolysis by D179Y variants of KPC carbapenemases is similar and involves the formation of a long-lived covalent intermediate.

Klebsiella pneumoniae carbapenemase (KPC) variants have been described that confer resistance to both ceftazidime-avibactam and cefiderocol. Of these, KPC-33 and KPC-31 are D179Y-containing variants derived from KPC-2 and KPC-3, respectively. To better understand this atypical phenotype, the catalytic mechanism of ceftazidime and cefiderocol hydrolysis by KPC-33 and KPC-31 as well as the ancestral KPC-2 and KPC-3 enzymes was studied. Steady-state kinetics showed that the D179Y substitution in either KPC-2 or KPC-3 is associated with a large decrease in both kcat and KM such that kcat/KM values were largely unchanged for both ceftazidime and cefiderocol substrates. A decrease in both kcat and KM is consistent with a decreased and rate-limiting deacylation step. We explored this hypothesis by performing pre-steady-state kinetics and showed that the acylation step is rate-limiting for KPC-2 and KPC-3 for both ceftazidime and cefiderocol hydrolysis. In contrast, we observed a burst of acyl-enzyme formation followed by a slow steady-state rate for the D179Y variants of KPC-2 and KPC-3 with either ceftazidime or cefiderocol, indicating that deacylation of the covalent intermediate is the rate-limiting step for catalysis. Finally, we show that the low KM value for ceftazidime or cefiderocol hydrolysis of the D179Y variants is not an indication of tight binding affinity for the substrates but rather is a reflection of the deacylation reaction becoming rate-limiting. Thus, the hydrolysis mechanism of ceftazidime and cefiderocol by the D179Y variants is very similar and involves the formation of a long-lived covalent intermediate that is associated with resistance to the drugs.

Characteristics of a ceftadine/avibatam resistance KPC-33-producing Klebsiella Pneumoniae strain with capsular serotype K19 belonging to ST15.

OBJECTIVES: The aim of this study was to characterize the blaKPC-33 in a ST15-K19 ceftazidime-avibactam (CAZ-AVI)-resistant Klebsiella pneumoniae strain after the antibiotic CAZ-AVI was approved for use in Wuxi No. 2 People's Hospital, China. METHODS: Antimicrobial susceptibility testing was performed by the microdilution broth method. Whole genome sequencing (WGS) was performed using PacBio II and MiSeq sequencers. High-quality reads were assembled using the SOAPdenovo and GapCloser v1.12, and genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Genomic characteristics were analysed by using bioinformatics methods. RESULTS: K. pneumoniae strain KPHRJ showed resistance to CAZ-AVI. WGS analysis showed that strain KPHRJ had one 5 536 506 bp chromosome (57.25% G+C content) and one plasmid (133 451 bp, G+C 54.29%). KPHRJ was classified as ST15 and K19 serotype. Resistome analysis showed that KPHRJ carries seven antimicrobial resistance genes (ARGs). WGS analysis and conjugation experiments demonstrated that the blaKPC-33 gene was carried by plasmid pKPHRJ, flanked by two copies of IS26 mobile elements (IS26-ISKpn27-blaKPC-33-ISKpn6-korC-TnAs1-tetR-tetA-Tn3-IS26). Besides these acquired resistance genes, mutations in porin protein-coding genes, such as OmpK36 and OmpK37, which may reduce susceptibility to the CAZ-AVI, were also identified from the genome. CONCLUSION: Here, we present the WGS of a CAZ-AVI resistant K. pneumoniae isolate, strain KPHRJ, with capsular serotype K19 and belonging to ST15. CAZ-AVI resistance is likely conferred by a KPC-2 variant, blaKPC-33 and mutations in porin-coding genes. We speculate that the approval of the CAZ-AVI in hospital could contribute to the emergence of these genomic features by providing a selective pressure leading to the emergence of CAZ-AVI resistant bacteria.

Ceftazidime-Avibactam in Combination with Imipenem as Salvage Therapy for ST11 KPC-33-Producing Klebsiella pneumoniae.

A 22-year-old man, after a hematopoietic stem cell transplant, suffered long-term pneumonia caused by blaKPC-2-positive K. pneumoniae and blaKPC-33-positive K. pneumoniae alternately and finally achieved pathogenic clearance and improvement of clinical infectious conditions after using ceftazidime-avibactam in combination with imipenem as salvage therapy. This case provides a reference for treating infection caused by K. pneumoniae with a KPC variant in countries lacking new antimicrobial agents.

In vivo Selection of Imipenem Resistance Among Ceftazidime-Avibactam-Resistant, Imipenem-Susceptible Klebsiella pneumoniae Isolate With KPC-33 Carbapenemase.

We describe in vivo evolution of carbapenem and ceftazidime-avibactam resistance by analyzing four longitudinal Klebsiella pneumoniae clinical isolates from a patient with pneumonia following antimicrobial treatment. The patient had fever, cough associated with expectoration, and new infiltration was found on the chest CT. Antimicrobial susceptibility was determined, and whole genome sequencing (WGS) was performed to investigate its dynamic change of resistance phenotype. Population analysis profile was performed to investigate the population of Klebsiella pneumoniae. The infection started with a KPC-2-producing K. pneumoniae (ZRKP01, ceftazidime-avibactam-S/carbapenem-R). Then, after ceftazidime-avibactam treatment, the strain switched to D179Y mutant that is KPC-33 (ZRKP02, ceftazidime-avibactam-R/carbapenem-S), which restored carbapenem susceptibility. However, the restored carbapenem susceptibility in vivo was not stable and the subsequent use of imipenem against KPC-33-producing K. pneumoniae infection resulted in a reversion of KPC-2 producers (ZRKP03 and ZRKP04, ceftazidime-avibactam-S/carbapenem-R). Genetic analysis demonstrated that all four K. pneumoniae isolates belonged to sequence type 11and had identical capsular polysaccharide (KL47), identical porin genes, and same plasmid replicon types. Phylogenetic analysis indicated that four K. pneumoniae isolates showed a high degree of relatedness. Single nucleotide polymorphisms analysis indicated that the number of mutations observed in the KPC-33 isolate was more than in the wild-type KPC-2 isolates and the four KPC-Kp isolates evolved from a longitudinal evolution of K. pneumoniae harboring bla KPC-2 gene. This is the first report to observe the in vivo evolution of wild-type KPC-2 to KPC-33 and then the reversion to its original wild-type KPC-2. Through WGS, we demonstrated the role of selective pressure of antibiotic in the mutation and reversion of bla KPC genes, which leading to the dynamic change of KPC enzymes and the dynamic emergence of resistance to ceftazidime-avibactam and carbapenems. Statement: Recently, studies reported the emergence of ceftazidime-avibactam-resistant strains. The KPC mutations mediating ceftazidime-avibactam resistance are generally associated with the restoration of carbapenem susceptibility. However, clinical significance of this observation is unclear. In this manuscript, we demonstrate the role of selective pressure of antibiotic in the mutation and reversion of bla KPC genes, which leading to the dynamic change of KPC enzymes and the dynamic emergence of resistance to ceftazidime-avibactam and carbapenems. To the best of our knowledge, this is the first report to observe the in vivo evolution of wild-type KPC-2 to KPC-33 and then the reversion to its original wild-type KPC-2. It should be noted that understanding the clinical significance of this observation is of critical importance, and reversion to carbapenem susceptibility would not imply a potential role for carbapenems monotherapy. We hope our study will draw attention to clinicians, so that this agent can be used most effectively for the longest period of time.

Emergence of ceftazidime-avibactam resistance through distinct genomic adaptations in KPC-2-producing Klebsiella pneumoniae of sequence type 39 during treatment.

Three ceftazidime-avibactam-resistant KPC-2-producing Klebsiella pneumoniae strains of ST39 were isolated in Greece, from rectal swabs of three patients after 10-15 days of treatment. The patients were treated with ceftazidime-avibactam as monotherapy or in combination with colistin. Two of these strains harbored a D179Y or a D179V substitution in the Ω loop of KPC-2, corresponding to KPC-33, or to the novel KPC-57, respectively. The third strain had a 15 amino acid insertion after position 259 in the KPC-2, corresponding to KPC-44.

Emergence and Recovery of Ceftazidime-avibactam Resistance in blaKPC-33-Harboring Klebsiella pneumoniae Sequence Type 11 Isolates in China.

This is the first report of ceftazidime-avibactam resistance caused by the blaKPC-33 mutation through the D179Y variant during the treatment 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 transformable plasmid harboring qnrS1 and blaLAP-2. Detecting blaKPC-33-positive K. pneumoniae clinical strains is important for infection control.