Successive Emergence of Ceftazidime-Avibactam Resistance through Distinct Genomic Adaptations in blaKPC-2-Harboring Klebsiella pneumoniae Sequence Type 307 Isolates.
ABSTRACT: Ceftazidime-avibactam (CAZ-AVI) is a promising novel treatment for infections caused by carbapenem-resistant Enterobacteriaceae (CRE). Despite improved treatment outcomes compared to those achieved with aminoglycoside- and colistin-based regimens, the rapid evolution of CAZ-AVI resistance during treatment has previously been reported in Klebsiella pneumoniae sequence type 258 (ST258) blaKPC-3-harboring isolates. Here, we report the stepwise evolution and isolation of two phenotypically distinct CAZ-AVI-resistant Klebsiella pneumoniae isolates from a patient with pancreatitis. All susceptible (n = 3) and resistant (n = 5) isolates were of the ST307 clonal background, a rapidly emerging clone. Taking advantage of short-read Illumina and long-read Oxford Nanopore sequencing and full-length assembly of the core chromosome and plasmids, we demonstrate that CAZ-AVI resistance first occurred through a 532G ? T blaKPC-2 point mutation in blaKPC-2 (D179Y protein substitution) following only 12 days of CAZ-AVI exposure. While subsequent isolates exhibited substantially decreased meropenem (MEM) MICs (?2 ?g/ml), later cultures demonstrated a second CAZ-AVI resistance phenotype with a lower CAZ-AVI MIC (12 ?g/ml) but also MEM resistance (MIC > 128 ?g/ml). These CAZ-AVI- and MEM-resistant isolates showed evidence of multiple genomic adaptations, mainly through insertions and deletions. This included amplification and transposition of wild-type blaKPC-2 into a novel plasmid, an IS1 insertion upstream of ompK36, and disruption of the rfb gene locus in these isolates. Our findings illustrate the potential of CAZ-AVI resistance to emerge in non-K. pneumoniae ST258 clonal backgrounds and alternative blaKPC variants. These results raise concerns about the strong selective pressures incurred by novel carbapenemase inhibitors, such as avibactam, on isolates previously considered invulnerable to CAZ-AVI resistance. There is an urgent need to further characterize non-KPC-mediated modes of carbapenem resistance and the intrinsic bacterial factors that facilitate the rapid emergence of resistance during treatment.
Project description:In December 2018, a ceftazidime-avibactam (CAZ-AVI)-resistant KPC-2-producing Klebsiella pneumoniae strain was isolated in Finland. CAZ-AVI resistance was observed 34 days after CAZ-AVI treatment in a trauma patient transferred from a hospital in Greece who had been colonised with blaKPC-2-producing K. pneumoniae ST39, and later developed a bloodstream infection. The CAZ-AVI-resistant strain contained a novel 15 amino acid insertion in the KPC-2 protein causing structural changes proximal to the KPC-2 active site.
Project description:Ceftazidime-avibactam (CAZ-AVI) and aztreonam-avibactam (AZT-AVI) are novel antibiotic combinations active against multidrug-resistant Gram-negative pathogens. This study aimed to evaluate their in vitro activities and inoculum effects in carbapenem-resistant <i>Enterobacterales</i> (CRE), including carbapenemase-producing (CP)-CRE and non-CP-CRE. A total of 81 independent clinical isolates of carbapenem-resistant <i>Escherichia coli</i> and <i>Klebsiella pneumoniae</i> were collected. CAZ-AVI and AZT-AVI minimal inhibitory concentrations (MICs) were evaluated by broth microdilution using standard and high inocula. The inoculum effect was defined as an ?8-fold increase in MIC with high inoculum. Phenotypic determination of ?-lactam resistance mechanism and PCR for carbapenemase genes were performed. Of the 81 CRE isolates, 35 (43%) were CP-CRE. Overall, 73% of the isolates were susceptible to CAZ-AVI, and 95% had low AZT-AVI MICs (?8 µg/mL). The MIC<sub>50/</sub>MIC<sub>90</sub>s of CAZ-AVI and AZT-AVI were 4/?512 µg/mL and 0.5/4 µg/mL, respectively. CAZ-AVI was more active against non-CP-CRE than against CP-CRE (susceptibility 80% vs. 63%, <i>p</i> = 0.08; MIC<sub>50</sub>/MIC<sub>90</sub>, 2/16 ?g/mL vs. 4/?512 ?g/mL), whereas AZT-AVI was more active against CP-CRE (MIC<sub>50</sub>/MIC<sub>90</sub>, 0.25/1 ?g/mL vs. 0.5/8 ?g/mL). All four isolates with high AZT-AVI MIC (?16 ?g/mL) were resistant to CAZ-AVI, but only 18% (4/22) of CAZ-AVI-resistant isolates had high AZT-AVI MIC. The rates of the inoculum effect for CAZ-AVI and AZT-AVI were 18% and 47%, respectively (<i>p</i> < 0.001). Interestingly, the frequency of the AZT-AVI inoculum effect was higher in <i>K. pneumoniae</i> than <i>E. coli</i> (64% vs. 8%, <i>p</i> < 0.001). AZT-AVI is more active against CRE than CAZ-AVI, even in CP-CRE and CAZ-AVI-resistant isolates. The presence of a substantial inoculum effect may contribute to clinical failure in high-inoculum infections treated with AZT-AVI.
Project description:BACKGROUND:Detection of ceftazidime/avibactam (CAZ/AVI) antibacterial activity is absolutely vital with the rapid growth of carbapenem resistant Enterobacteriaceae (CRE). But now, there is no available automated antimicrobial susceptibility testing card for CAZ/AVI, so Kirby-Bauer has become an economical and practical method for detecting CAZ/AVI antibacterial activity against Enterobacteriaceae. RESULT:In this study, antimicrobial susceptibility testing of CAZ/AVI against 386 Enterobacteriaceae (188 Klebsiella pneumoniae, 122 Escherichia coli, 76 Enterobacter cloacae) isolated from clinical patients was performed by broth microdilution. Of the 386 strains, 54 extended spectrum β lactamases negative (ESBL(-)), 104 extended spectrum β lactamases positive (ESBL(+)), 228 CRE. 287 isolates were susceptible to CAZ/AVI and 99 isolates were resistant to CAZ/AVI. At the same time, to obtain optimal content avibactam (AVI) disk containing ceftazidime (30 μg), inhibition zone diameter of four kinds of ceftazidime (30 μg) disk containing different AVI content (0 μg, 10 μg, 25 μg, 50 μg) were tested by Kirby-Bauer method. The microdilution broth method interpretation was used as the standard to estimate susceptible or resistance and then coherence analysis was carried out between Kirby-Bauer and broth microdilution. The result shows the inhibition zone diameter of 30 μg/50 μg disk, susceptible isolates: 20.5 mm-31.5 mm, resistance isolates: 8.25 mm-21.5 mm. The inhibition zone diameter of 30 μg/25 μg disk, susceptible isolates: 19.7 mm-31.3 mm, resistance isolates: 6.5 mm-19.2 mm. The inhibition zone diameter of 30 μg/10 μg disk, susceptible isolates: 19.5 mm-31 mm, resistance isolates: 6.5 mm-11 mm. The inhibition zone diameter of ceftazidime (30 μg), susceptible isolates: 6.5 mm-27.5 mm, resistance isolates 6.5 mm. CONCLUSION:Our results show that 30 μg/50 μg, 30 μg/25 μg, 30 μg/10 μg CAZ/AVI disk have significant statistical differences to determinate CAZ/AVI antibacterial activity, but for 30 μg/50 μg disk, there has a cross section between susceptible isolates (minimum 20.5 mm) and resistance isolates (maximum 21.5 mm). For 30 μg/25 μg disk, it is hard to distinguish the difference between susceptible isolates (minimum 19.7 mm) and resistance isolates (maximum 19.2 mm), so 30 μg/10 μg CAZ/AVI disk is more conducive to determinate antibacterial activity.
Project description:Background: For years, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae have represented a serious health problem in hospitals worldwide. Since its approval in 2015, ceftazidime-avibactam (CAZ-AVI) had been successfully used for treating complicated KPC-K. pneumoniae infections, until increasing reports of resistance began to emerge. Methods: Phenotypic tests and molecular analysis were performed in four multidrug-resistant K. pneumoniae isolates, collected from two patients following treatment with CAZ-AVI. Results: In this study, we report two cases of emergence of CAZ-AVI resistance in KPC-3-producing K. pneumoniae isolates, collected from two patients following treatment with CAZ-AVI. Molecular analysis highlighted the D179Y mutation in the bla KPC-3 gene, whose role in the loss of hydrolytic activity (resulting in decreased carpabenem minimum inhibitory concentrations and negative phenotypic tests) of the enzyme has already been shown. Conclusion: Most surveillance schemes aimed at detecting carbapenem-resistant Enterobacteriaceae (CRE) rely on confirmatory phenotypic tests for detecting carbapenemase production. As reports of these treatment-induced, altered CRE phenotypes are increasing, the initial susceptibility testing should be followed by a combination of phenotypic and molecular methods, to make sure that no potential carbapenemase-producing bacteria are missed.
Project description:The emergence and prevalence of carbapenem-resistant <i>Enterobacteriaceae</i> (CRE) have drawn worldwide attention. Ceftazidime/avibactam (CAZ/AVI) gives us a valuable alternative strategy to treat CRE infections. Unfortunately, CAZ/AVI resistance could occur during CAZ/AVI treatment. The CAZ/AVI-resistant Carbapenem-resistant <i>Klebsiella pneumoniae</i> (CR-KP) (KP137060) and earlier CAZ/AVI-susceptible isolate (KP135194) from the same hospitalized patient were collected at Fujian Medical University Union Hospital between October and November 2019. In this study, CAZ/AVI MICs of CAZ/AVI-susceptible and -resistant isolates (KP135194 and KP137060) were 4 mg/L and 128 mg/L, respectively; and the two isolates had the same antibiotic resistance pattern to other carbapenems. Two strains were then submitted for whole-genome sequencing and bioinformatic analysis. <i>ompK36</i> was not detected in two isolates. No mutation was observed in <i>bla</i> <sub>KPC-2</sub>, <i>ompK35</i> and <i>ompK37</i> in this study and there was no significant difference of the expression in <i>bla</i> <sub>KPC-2</sub>, <i>ompK35</i> and <i>ompK37</i> between the two isolates (<i>p</i>>0.05). Two isolates were sequence type 11 and harbored <i>bla</i> <sub>KPC-2</sub>, <i>bla</i> <sub>SHV-182</sub> and <i>bla</i> <sub>TEM-1B</sub>. Compared with KP135194, KP137060 harbored an additional <i>bla</i> <sub>NDM-5</sub> positive plasmid. <i>bla</i> <sub>NDM-5</sub> gene could be successfully transferred into <i>E. coli</i> J53 at a conjugation frequency of 1.14×10<sup>-4</sup>. Plasmid stability testing showed that <i>bla</i> <sub>KPC-2</sub>- and <i>bla</i> <sub>NDM-5</sub>-harboring plasmids were still stably maintained in the hosts. Growth assay and growth competition experiments showed there was no significant difference in fitness cost between two CR-KP isolates. Our study described the acquisition of a <i>bla</i> <sub>NDM-5</sub>-harboring plasmid leading to resistance to ceftazidime/avibactam in KPC-2-producing <i>Klebsiella pneumoniae</i> during treatment. This phenomenon deserves further exploration.
Project description:Ceftazidime-avibactam (CAZ-AVI) shows promising activity against carbapenem-resistant <i>Klebsiella pneumoniae</i> (CRKP), however, CAZ-AVI resistance have emerged recently. Mutations in KPCs, porins OmpK35 and/or OmpK36, and PBPs are known to contribute to the resistance to CAZ-AVI in CRKP. To identify novel CAZ-AVI resistance mechanism, we generated 10 CAZ-AVI-resistant strains from 14 CAZ-AVI susceptible KPC-producing <i>K. pneumoniae</i> (KPC-Kp) strains through <i>in vitro</i> multipassage resistance selection using low concentrations of CAZ-AVI. Comparative genomic analysis for the original and derived mutants identified CAZ-AVI resistance-associated mutations in KPCs, PBP3 (encoded by <i>ftsI</i>), and LamB, an outer membrane maltoporin. CAZ-AVI susceptible KPC-Kp strains became resistant when complemented with mutated <i>bla</i><sub>KPC</sub> genes. Complementation experiments also showed that a plasmid borne copy of wild-type <i>lamB</i> or <i>ftsI</i> gene reduced the MIC value of CAZ-AVI in the induced resistant strains. In addition, <i>bla</i><sub>KPC</sub> expression level increased in four of the six CAZ-AVI-resistant strains without KPC mutations, indicating a probable association between increased <i>bla</i><sub>KPC</sub> expression and increased resistance in these strains. In conclusion, we here identified a novel mechanism of CAZ-AVI resistance associated with mutations in porin LamB in KPC-Kp.
Project description:Infections due to carbapenemase-producing Gram-negative pathogens are associated with limited treatment options and consequently lead to increased mortality and morbidity. In response, combinations of existing ?-lactams and novel ?-lactamase inhibitors, such as ceftazidime-avibactam (CAZ-AVI), have been developed as alternative treatment options. To understand the development of resistance and evolutionary trajectories under CAZ-AVI exposure, we studied the effects of ceftazidime (CAZ) and CAZ-AVI on the carbapenemase OXA-48 and the epidemic OXA-48 plasmid in Escherichia coli Exposure of CAZ and CAZ-AVI resulted in single (P68A) and double (P68A,Y211S) amino acid substitutions in OXA-48, respectively. The antimicrobial susceptibility data and enzyme kinetics showed that the P68A substitution was responsible for an increased activity toward CAZ, whereas P68A,Y211S led to a decrease in the inhibitory activity of AVI. X-ray crystallography and molecular modeling of the mutants demonstrated increased flexibility within the active site, which could explain the elevated CAZ hydrolysis and reduced inhibitory activity of AVI. Interestingly, these substitutions resulted in collateral effects compromising the activity of OXA-48 toward carbapenems and penicillins. Moreover, exposure to CAZ-AVI selected for mutations within the OXA-48-encoding plasmid that severely reduced fitness in the absence of antimicrobial selection. These evolutionary trade-offs may contribute to limit the evolution of OXA-48-mediated CAZ and CAZ-AVI resistance, as well as potentially resensitize isolates toward other therapeutic alternatives.IMPORTANCE The recent introduction of novel ?-lactam/?-lactamase inhibitor combinations like ceftazidime-avibactam has increased our ability to treat infections caused by multidrug-resistant Gram-negative bacteria, including carbapenemase-producing Enterobacterales However, the increasing number of cases of reported resistance to ceftazidime-avibactam is a concern. OXA-48 is a carbapenemase that has no significant effect on ceftazidime, but is inhibited by avibactam. Since isolates with OXA-48 frequently harbor extended-spectrum ?-lactamases that are inhibited by avibactam, it is likely that ceftazidime-avibactam will be used to treat infections caused by OXA-48-producing Enterobacterales. Our data show that exposure to ceftazidime-avibactam can lead to changes in OXA-48, resulting in increased ability to hydrolyze ceftazidime and withstand the inhibitory effect of avibactam. Thus, resistance toward ceftazidime-avibactam among OXA-48-producing Enterobacterales should be monitored. Interestingly, the compromising effect of the amino acid substitutions in OXA-48 on other ?-lactams and the effect of ceftazidime-avibactam exposure on the epidemic OXA-48 plasmid indicate that the evolution of ceftazidime-avibactam resistance comes with collateral effects.
Project description:Two ceftazidime-avibactam (CAZ-AVI)-resistant Klebsiella pneumoniae carbapenemase (KPC)-positive K. pneumoniae strains, including one pandrug resistant, were isolated in 2019 from two Greek hospitals. The strains were sequence types (ST)s 258 and 147 and both harboured similar self-transmissible IncA/C2 plasmids encoding a novel Lys234Arg variant of the Vietnamese extended-spectrum ?-lactamase (VEB)-1, not inhibited by AVI (VEB-25). Conjugal transfer of VEB-25-encoding plasmids to Escherichia coli yielded CAZ-AVI-resistant clones, supporting that VEB-25 is directly linked to the derived phenotype.
Project description:Ceftazidime-avibactam is a novel ?-lactam/?-lactamase inhibitor with activity against carbapenem-resistant Enterobacteriaceae (CRE) that produce Klebsiella pneumoniae carbapenemase (KPC). We report the first cases of ceftazidime-avibactam resistance to develop during treatment of CRE infections and identify resistance mechanisms. Ceftazidime-avibactam-resistant K. pneumoniae emerged in three patients after ceftazidime-avibactam treatment for 10 to 19 days. Whole-genome sequencing (WGS) of longitudinal ceftazidime-avibactam-susceptible and -resistant K. pneumoniae isolates was used to identify potential resistance mechanisms. WGS identified mutations in plasmid-borne blaKPC-3, which were not present in baseline isolates. blaKPC-3 mutations emerged independently in isolates of a novel sequence type 258 sublineage and resulted in variant KPC-3 enzymes. The mutations were validated as resistance determinants by measuring MICs of ceftazidime-avibactam and other agents following targeted gene disruption in K. pneumoniae, plasmid transfer, and blaKPC cloning into competent Escherichia coli In rank order, the impact of KPC-3 variants on ceftazidime-avibactam MICs was as follows: D179Y/T243M double substitution > D179Y > V240G. Remarkably, mutations reduced meropenem MICs ?4-fold from baseline, restoring susceptibility in K. pneumoniae from two patients. Cefepime and ceftriaxone MICs were also reduced ?4-fold against D179Y/T243M and D179Y variant isolates, but susceptibility was not restored. Reverse transcription-PCR revealed that expression of blaKPC-3 encoding D179Y/T243M and D179Y variants was diminished compared to blaKPC-3 expression in baseline isolates. In conclusion, the development of resistance-conferring blaKPC-3 mutations in K. pneumoniae within 10 to 19 days of ceftazidime-avibactam exposure is troubling, but clinical impact may be ameliorated if carbapenem susceptibility is restored in certain isolates.
Project description:Klebsiella pneumoniae carbapenemase (KPC)-producing Pseudomonas aeruginosa (KPC-PA) has been reported sporadically. However, epidemiological and antimicrobial susceptibility data specific for KPC-PA are lacking. We collected 374 carbapenem-resistant P. aeruginosa (CRPA) isolates from seven hospitals in China from June 2016 to February 2019 and identified the <i>bla</i><sub>KPC-2</sub> gene in 40.4% (<i>n</i> = 151/374) of the isolates. Approximately one-half of all KPC-PA isolates (<i>n</i> = 76/151; 50.3%) were resistant to ceftazidime-avibactam (CAZ-AVI). Combining Kraken2 taxonomy identification and Nanopore sequencing, we identified eight plasmid types, five of which carried <i>bla</i><sub>KPC-2</sub>, and 13 combination patterns of these plasmid types. In addition, we identified IS<i>26</i>-ΔTn<i>6296</i> and Tn<i>1403</i>-like-ΔTn<i>6296</i> as the two mobile genetic elements that mediated <i>bla</i><sub>KPC-2</sub> transmission. <i>bla</i><sub>KPC-2</sub> plasmid curing in 28 strains restored CAZ-AVI susceptibility, suggesting that <i>bla</i><sub>KPC-2</sub> was the mediator of CAZ-AVI resistance. Furthermore, the <i>bla</i><sub>KPC-2</sub> copy number was found to correlate with KPC expression and, therefore, CAZ-AVI resistance. Taken together, our results suggest that KPC-PA is becoming a clinical threat and that using CAZ-AVI to treat this specific pathogen should be done with caution. <b>IMPORTANCE</b> Previous research has reported several cases of KPC-PA strains and three KPC-encoding P. aeruginosa plasmid types in China. However, the prevalence and clinical significance of KPC-PA are not available. In addition, the susceptibility of the strains to CAZ-AVI remains unknown. Samples in this study were collected from seven tertiary hospitals prior to CAZ-AVI clinical approval in China. Therefore, our results represent a retrospective study establishing the baseline efficacy of the novel β-lactam/β-lactamase combination agent for treating KPC-PA infections. The observed correlation between the <i>bla</i><sub>KPC</sub> copy number and CAZ-AVI resistance suggests that close monitoring of the susceptibility of the strain during treatment is required. It would also be beneficial to screen for the <i>bla</i><sub>KPC</sub> gene in CRPA strains for antimicrobial surveillance purposes.