Mechanisms of ceftolozane/tazobactam resistance in Pseudomonas aeruginosa isolates from South Korea:
A diagnostic study
Article information
Abstract
Purpose
To identify ceftolozane/tazobactam (C/T)-resistant Pseudomonas aeruginosa isolates in Korea and to investigate the resistance mechanism.
Methods
We performed antibiotic susceptibility testing for 42 carbapenem-resistant, non-carbapenemase-producing P. aeruginosa isolates from Korea. Multilocus sequence typing was performed for all isolates. Whole-genome sequencing was performed for four carbapenem-resistant P. aeruginosa isolates, including two C/T-resistant isolates and two C/T-susceptible isolates.
Results
We identified two C/T-resistant P. aeruginosa isolates (P70 and P112) among 42 carbapenem-resistant, non-carbapenemase-producing P. aeruginosa isolates. We found multiple amino acid substitutions in AmpC, AmpR, efflux pump systems, and a porin protein in C/T-resistant P. aeruginosa isolates. These were not reported previously in C/T-resistant P. aeruginosa isolates.
Conclusion
C/T-resistant P. aeruginosa isolates with multiple amino acid alterations were identified, implying that resistance to this novel antibiotics should be constantly monitored.
INTRODUCTION
Ceftolozane/tazobactam (C/T) is a novel antibiotic with broad-spectrum activity against gram-negative bacteria, including multidrug-resistant (MDR) Pseudomonas aeruginosa. Although many reports show that C/T can successfully treat infections caused by MDR P. aeruginosa, the emergence of C/T resistance has also been reported [1,2]. The resistance is primarily due to high induction of AmpC and/or repression of the AmpR transcriptional regulator, which is associated with their amino acid mutations [1,3]. In addition, several extended-spectrum β-lactamases, including GES, PER, and BEL have been known to be responsible for C/T resistance [4].
METHODS
In this study, we identified two C/T-resistant P. aeruginosa isolates (P70 and P112) among 42 carbapenem-resistant, non-carbapenemase-producing isolates (28 isolates from urine and 14 isolates from blood), were collected from November 2006 to August 2007 from 10 South Korean hospitals [5], and investigated their resistance mechanisms. Multilocus sequence typing analysis showed that the 42 isolates belonged to 26 different seguence types (STs): ST235 (seven isolates), ST641 (six isolates), ST233 (two isolates), ST357 (two isolates), ST654 (two isolates), ST1082 (two isolates), and the other STs represented a single isolate. While P70 was identified from a patient with urinary tract infection in Chonnam National University Hospital (Gwangju, Korea), P112 were from a patient with bacteremia in Gyeongsang National University Hospital (Jinju, Korea).
Whole-genome sequencing was performed on four carbapenem-resistant P. aeruginosa isolates, including two C/T-resistant isolates (P70 and P112) and two C/T-susceptible isolates (P17 and P171), using the Illumina HiSeq 2000 system preliminary performance parameters with paired-end 101 bp reads. The C/T-susceptible P. aeruginosa isolates, P17 and P171, with C/T minimum inhibitory concentrations (MICs) of 4/4 and 2/4 mg/L, respectively, were randomly selected from 42 carbapenem-resistant non-carbapenemase-producing isolates. P17 and P171 belong to ST27 and ST1085, respectively. The complete nucleotide sequences of P17, P70, P112, and P171 were submitted to GenBank under the accession numbers SRR11793832–SRR11793835.
RESULTS
The C/T-resistant isolates, P70 and P112, belonged to ST235 and ST983, respectively (Table 1). ST235 is the most commonly identified carbapenem-resistant clone found in South Korea. The MIC of C/T for both isolates was 16/4 mg/L using the broth microdilution method described by the Clinical and Laboratory Standards Institute. The two C/T-resistant P. aeruginosa isolates were also resistant to ceftazidime/avibactam, another novel drug combination, each with a MIC of 16/4 mg/L. In addition, the isolates were resistant to most antibiotics, including piperacillin/tazobactam, cefepime, ceftriaxone, ceftazidime, tetracycline, ciprofloxacin, and amikacin. They were only susceptible to polymyxins such as polymyxin B and colistin.
Ceftolozane/tazobactam-resistant Pseudomonas aeruginosa isolates identified in this study and their amino acid substitutions
We compared the sequences of the two C/T-resistant isolates with those of the two C/T-susceptible isolates and a reference P. aeruginosa strain, PAO1 (GenBank accession number NC_002516.2). The genomes of the four isolates were 6,264,404 bp in length, with G+C ratios of 66.14% to 66.69%. Alignment read depths from Illumina sequencing ranged from 322 to 439. We mapped reads from the four P. aeruginosa isolates to the PAO1 reference genome with 95.5% to 98.9% identity. Because they belonged to different clones, they showed great sequence variation at the amino acid level when compared with PAO1; their variant numbers were 29,331 (P17) to 58,451 (P70), corresponding to 0.47% to 0.93% of total genome. Thus, we compared the sequence variations in genes known to be associated with C/T resistance, ampC and its transcriptional regulator ampR, several efflux pump genes such as mexAB-oprM, mexGHI-opmD, mexCD-oprJ, and mexEF-oprN, and the porin gene oprD.
The amino acids that were identified in the two C/T-resistant P. aeruginosa isolates but were missing in the two C/ T-susceptible isolates and the reference strain, PAO1, are shown in Table 1. Three amino acid substitutions, G27D, A97V, and V205L, were identified in the AmpC of P70. AmpC mutations have not been described previously [1-4]. Each of the five amino acid alterations in P70 and P112 was found in ampR gene. Of these, three (H10P, P15L, and T212A) were shared by both C/T-resistant isolates. Amino acid substitutions in efflux pump proteins were more frequent in P70 than in P112. At P70, three substitutions were found in MexB of MexAB-OprM, five in MexGHI-OpmD (one in MexH, one in MexI, and three in OpmD), five in MexC of MexCD-OprJ, and four in MexEF-OprN (three in MexE and one in OprN). However, only four amino acid variations were found in P112: MexI, OpmM, OprJ, and OprN (Table 1). Similar to the efflux pumps, more amino acid alterations were identified in OprD of P70, with eight in P70 and three in P112. All three OprD amino acid substitutions at P112 were also observed at P70. Although it was not confirmed that all of the amino acid alterations were associated with C/T resistance, it should be noted that many mutations occurred in AmpC-AmpR, efflux pump systems, and a porin protein (OprD) of C/T-resistant P. aeruginosa isolates. It is more likely that the amino acid alterations found in both C/T-resistant isolates, three in AmpR, two in MexGHI-OpmD, and three in OprD, were responsible for C/T resistance, although additional experiments should be performed to confirm the association with C/T resistance. This has not been previously reported in C/T-resistant P. aeruginosa isolates.
DISCUSSION
In this study, we identified two C/T-resistant P. aeruginosa isolates among carbapenem-resistant, non-carbapenemase-producing isolates from South Korea. We found multiple amino acid substitutions in AmpC, AmpR, efflux pump systems, and a porin protein, although only two C/T-resistant isolates were identified.
Notes
No potential conflict of interest relevant to this article was reported.
AUTHOR CONTRIBUTIONS
Conception or design: YMW, SYK, KSK.
Acquisition, analysis, or interpretation of data: YMW, SYK, KSK.
Drafting the work or revising: YMW, SYK, KSK.
Final approval of the manuscript: YMW, SYK, KSK.
Acknowledgements
Some Escherichia coli isolates used in this study were obtained from the Asian Bacterial Bank (ABB) of the Asia Pacific Foundation for Infectious Diseases (APFID) (Seoul, South Korea).
This research was funded by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (grant no. NRF-2019R1A2C2004879 and 2022R1A2B5B02001716).