In vitro development of resistance against antipseudomonal agents: comparison of novel β-lactam/β-lactamase inhibitor combinations and other β-lactam agents

ABSTRACT We subjected seven P. aeruginosa isolates to a 10-day serial passaging against five antipseudomonal agents to evaluate resistance levels post-exposure and putative resistance mechanisms in terminal mutants were analyzed by whole-genome sequencing analysis. Meropenem (mean, 38-fold increase), cefepime (14.4-fold), and piperacillin-tazobactam (52.9-fold) terminal mutants displayed high minimum inhibitory concentration (MIC) values compared to those obtained after exposure to ceftolozane-tazobactam (11.4-fold) and ceftazidime-avibactam (5.7-fold). Fewer isolates developed elevated MIC values for other β-lactams and agents belonging to other classes when exposed to meropenem in comparison to other agents. Alterations in nalC and nalD, involved in the upregulation of the efflux pump system MexAB-OprM, were common and observed more frequently in isolates exposed to ceftazidime-avibactam and meropenem. These alterations, along with ones in mexR and amrR, provided resistance to most β-lactams and levofloxacin but not imipenem. The second most common gene altered was mpl, which is involved in the recycling of the cell wall peptidoglycan. These alterations were mainly noted in isolates exposed to ceftolozane-tazobactam and piperacillin-tazobactam but also in one cefepime-exposed isolate. Alterations in other genes known to be involved in β-lactam resistance (ftsI, oprD, phoP, pepA, and cplA) and multiple genes involved in lipopolysaccharide biosynthesis were also present. The data generated here suggest that there is a difference in the mechanisms selected for high-level resistance between newer β-lactam/β-lactamase inhibitor combinations and older agents. Nevertheless, the isolates exposed to all agents displayed elevated MIC values for other β-lactams (except imipenem) and quinolones tested mainly due to alterations in the MexAB-OprM regulators that extrude these agents.

region (3).The Centers for Disease Control and Prevention reports that MDR P. aeruginosa caused over 32,000 cases of infections in hospitalized patients in 2017 leading to the death of 2,700 patients (4).
Several studies describe antipseudomonal β-lactam resistance mechanisms, including among isolates resistant to these newer β-lactam/β-LICs (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19); however, fewer studies have addressed the effect of exposing P. aeruginosa clinical isolates to these agents, specifically their effect on promoting the development of resistance compared to older agents.In this study, we subjected seven clinical P. aeruginosa isolates (Table 1) with low antipseudomonal β-lactam MIC values to a 10-day serial passage with one of the following five agents: ceftazidime-avibactam, ceftolozane-tazobactam, meropenem, cefepime, and piperacillin-tazobactam.We then used whole-genome sequencing (WGS) to identify putative resistance mechanisms to better understand the potential effects of prolonged exposure to currently available antipseudomonal therapies.
To determine whether exposure to different β-lactam or β-lactam/β-LICs affected the susceptibility to other β-lactams or agents from other antimicrobial classes (e.g., aminoglycosides, quinolones), exposed isolates were susceptibility tested against a broad set of antimicrobial agents.Overall, regardless of selective antimicrobial during lineage evolution, more exposed isolates exhibited >2-fold increase in MIC relative to their respective parent strains for ceftazidime-avibactam (24 isolates; Table 2), cefepime (n=26), piperacillin-tazobactam (n=26), and meropenem (n=26) when compared to ceftolozane-tazobactam (14 isolates).Many exposed isolates also displayed elevated MIC values (>2 fold) against ceftazidime and aztreonam (23 and 25 isolates, respectively).Still, only a few isolates developed MIC results that would be categorized as resistant or intermediate to the β-lactam agents/combinations tested except for aztreonam (17 resistant and 8 intermediate).Against non-β-lactam agents, 9 and 11 exposed isolates displayed >2-or 2-fold MIC increases for levofloxacin, respectively, including 12 isolates that presented intermediate MIC values for this agent and 6 that were resistant.
Similar to ceftazidime-avibactam, most lineages exposed to cefepime produced daughters exhibiting elevated MIC values ≥2-fold for all β-lactams except imipenem; however, in this instance, all seven isolates displayed elevated ceftolozane-tazobactam MICs (Table 2).Two-fold increases in the MIC values for amikacin, levofloxacin, tigecy cline, and tobramycin were noted for 3-5 isolates exposed to cefepime.
Interestingly, most lineages evolved under ceftolozane-tazobactam selection produced terminal strains displaying ≥2-fold increases in MIC values for all other  β-lactams tested.This increase was noted for 6-7 of the isolates exposed to ceftolozanetazobactam when tested against different β-lactam agents/combinations, except for imipenem.Isolates exposed to piperacillin-tazobactam also displayed high MIC values for most other β-lactams, but to a lesser extent when compared to ceftolozane-tazobactam.Exposure to meropenem leads to the development of elevated MIC values (>2-fold) against imipenem in 5/7 isolates, and all 5 developed resistant MIC values against imipenem.Despite some increased levofloxacin MIC values, MIC increases against other classes were less common for ceftolozane-tazobactam, meropenem, and piperacillintazobactam when compared to ceftazidime-avibactam and cefepime.
Our experiments established a difference in the terminal susceptibility phenotypes in lineages of P. aeruginosa exposed to more recently developed therapeutic strategies (e.g., ceftolozane-tazobactam, ceftazidime-avibactam) versus older treatment options (e.g., meropenem, cefepime, piperacillin-tazobactam) in terms of cross-resistance to other β-lactam, β-lactam/β-lactamase inhibitor combinations, and other antimicrobial classes.We then sought to determine shared evolutionary trajectories among lineages  All exposed isolates (35) MIC increase >2-fold relative to the exposure agent and performed WGS followed by an analysis of the accumulated genetic changes, specifically single nucleotide polymorphisms (SNPs) and insertions/deletions (indels).We limited our analysis to strains displaying >2-fold increases in MIC against their respective exposure agent.Five isolates exposed to ceftazidime-avibactam exhibited MICs >2-fold their parent strain, and two of these isolates possessed nonsense or missense mutations in nalD, a regulator of the MexAB-OprM efflux system (Table 3).In addition, one isolate displayed an alteration in the intragenic region upstream of nalC, which is also involved in the repression of mexAB-oprM.These nalC/nalD mutants exhibited increases in MIC values of 2-fold to 8-fold for ceftazidime-avibactam, cefepime, and ceftazidime and 4-fold to 16-fold for meropenem, piperacillin-tazobactam, and aztreonam compared to their baseline MIC.Expectedly, these isolates displayed no significant increase in imipenem MIC results as this carbape nem is not a substrate of MexAB-OprM.All these isolates displayed elevated levofloxacin (≥2-fold), two had elevated tigecycline (4-fold to 8-fold) and one had gentamicin elevated MIC values (4-fold).The remaining isolates exposed to ceftazidime-avibactam displayed elevated MIC values for most other β-lactams agents/combinations tested and displayed alterations in a gene encoding a carboxy terminal-processing peptidase, algO, or a putative glycosyltransferase family 4 protein-like gene.
Of the six cefepime-exposed isolates selected for sequencing, one isolate displayed an alteration in the TetR family transcriptional regulator amrR (Table 3).This isolate only displayed a 4-fold increase in the cefepime and levofloxacin MIC values.Another isolate had a mutation in ampD along with a missense alteration in a glycosyltransferase gene.This isolate exhibited elevated MIC values for all β-lactams ranging from 8-fold to 128-fold and no increase in MIC results for other classes.One isolate displaying elevated MIC values for levofloxacin, gentamicin, tigecycline, ceftazidime, and piperacillin-tazo bactam in addition to cefepime had an alteration in wbpG, an N-acetyl sugar amidotrans ferase involved in LPS biosynthesis (20,21).An 18 bp deletion in mpl was detected in one isolate which was resistant to ceftazidime, piperacillin-and ceftolozane-tazobactam but not carbapenems or ceftazidime-avibactam.The mpl gene encodes a murein peptide ligase and is involved in the recycling of the cell wall peptidoglycan (22).The remaining isolate harbored alterations in mexB, upstream of nalC, mlaE, and cpxS.Alterations in CpxS have been observed in isolates exposed to different antimicrobial agents alone or in combination (23).This isolate exhibited increased (≥2-fold) MIC values for levofloxacin and tigecycline and most β-lactams but not imipenem.
Of five ceftolozane-tazobactam-exposed isolates sequenced, two strains acquired identical alterations in CpxS (Thr163Pro; Table 3).One of these isolates also possessed a missense mutation in ftsI, encoding penicillin-binding protein (PBP) 3, while the other isolate carried mutations in galU, involved in LPS biosynthesis (24), and a hypothetical protein.The three other isolates exposed to ceftolozane-tazobactam carried mutations in the secretion protein secY (one isolate; Leu365Pro) or mpl (two isolates; Val124Gly or Ala159Thr).Three of the five sequenced isolates exposed to ceftolozane-tazobactam were also noted for increased susceptibility to ≥2 aminoglcyoside agents and/or colistin; these isolates possessed alterations in mpl (n=2) and secY, ftsI, and cpxS.
Multiple isolates exposed to piperacillin-tazobactam acquired mutations in genes and intergenic regions comparable to those identified under different selection regimes (Table 3).Unique alterations among these isolates were identified in a putative MerR-family transcriptional regulator homologous to PA2737 from P. aeruginosa PAO1, the glucose-6-phosphate isomerase Pgi, the protease PepA, and the peptidoglycan recycling-associated MupP.Levofloxacin MICs were elevated ≥2-fold in most of these isolates.Similar to those terminal isolates produced under ceftolozane-tazobactam selection, piperacillin-tazobactam-selected isolates displayed increased susceptibility to colistin (4/6 isolates) and aminoglycosides (3/6 isolates; Table 3).
Out of the six meropenem mutants sequenced, one displayed alterations in OprD (Ser40Asn/Ser278Pro) and increased 8-fold and 4-fold increases in MIC values for imipenem and colistin, respectively (Table 3).Three isolates contained alterations within or upstream of nalC observed along with alterations in the PBP3 (Val471Gly) or the transcriptional regulator PhoP (Thr79Ser).The isolate harboring alterations in phoP and the upstream region of nalC had elevated MIC values for all β-lactams, except ceftolo zane-tazobactam and colistin (16-fold increase).The two isolates with nalC missense mutations displayed twofold or eightfold increases in levofloxacin MIC values.One isolate displayed an alteration in the intergenic region between mexR and mexA along with a mutation in rpoB.The last isolate displayed alterations in the two-component system sensor histidine kinase GacS and a putative UDP-glucose 6-dehydrogenase homologous to PA3559 from P. aeruginosa PAO1.

DISCUSSION
P. aeruginosa was the fourth most common cause of bloodstream infections among isolates submitted during the initial 20 years of the SENTRY Antimicrobial Surveillance Program, contributing 5.3% of the isolates analyzed (25).This organism is an impor tant cause of nosocomial pneumonia infections, and its prevalence is higher among immunocompromised and burn patients.P. aeruginosa isolates are intrinsically resistant to many antimicrobial genes and the accumulation of mutations modulating the activities of intrinsic resistance mechanisms is more frequently identified among P. aeruginosa clinical isolates compared to other species (1).
In this study, we exposed P. aeruginosa clinical isolates from bloodstream or pneumonia infections displaying low baseline MIC values to common antipseudomo nal agents to a 10-day serial passaging with one of five β-lactam agents, including new β-lactam/β-LICs, to identify mechanisms of resistance selected during exposure to these agents.We also evaluated the changes in susceptibility to various other antimicrobial agents of the same (β-lactam) and diverse classes (e.g., aminoglycosides, colistin).Overall, the mean MIC values displayed greater increases for meropenem, cefepime, and piperacillin-tazobactam when compared to ceftazidime-avibactam and ceftolozane-tazobactam.We also noted reduced susceptibility against β-lactams other than the agent of exposure as well as agents from other antimicrobial classes (Table 2).Generally, reduced susceptibility to non-passaging antibiotics occurred equally for all agents, although evolution under meropenem selection produced fewer terminal strains with reduced susceptibility to other agents.It is noteworthy that the increased MIC values displayed by terminal isolates generally did not rise to resistant levels, except for aztreonam (17/35 isolates; 48.6%).Furthermore, isolates passaged under selection with piperacillin-tazobactam or ceftolozane-tazobactam generated daughter strains with increased susceptibility to colistin and/or multiple aminoglycosides.We observed no pattern in the accumulation of mutations based on isolate, sequence type, or infection source (data not shown).
The most common genetic alterations detected in the terminal isolates were those in or upstream (+131, 133, 143, or 144 bp) of nalC and nalD, which are involved in the regulatory pathway of the mexAB-oprM efflux system (Table 3); alterations in the mexR-mexA intergenic region were noted in two isolates, as was a single isolate with substitutions in AmrR or MexB.Increased levofloxacin and unchanged impenem MIC results were noted in most isolates displaying nalC, nalD, or mexR alterations.Overex pression of mexAB-oprM confers resistance to all β-lactams, except for imipenem, and the quinolones (26).Furthermore, these MexAB-OprM regulatory alterations were noted in isolates exposed to all agents but ceftolozane-tazobactam and isolates harboring only alterations in these targets usually displayed <2-fold increase in the ceftolozane-tazobac tam MIC values.In studies evaluating the transcription of different genes in clinical isolates resistant to β-lactams, mexAB-oprM overexpression did not seem to affect the susceptibility of ceftolozane-tazobactam (27), corroborating our results.
Despite not being affected by alterations affecting mexAB-oprM expression, isolates exposed to ceftolozane-tazobactam developed elevated MIC values for other β-lactam agents via mechanisms common to the lineages exposed to other β-lactam or β-lac tam/β-LIC pressure.Mutations leading to amino acid substitutions and deletions in Mpl, identified in four isolates exposed to ceftolozane-tazobactam (n = 2) and cefepime or piperacillin-tazobactam (one isolate each), were the second most common alteration observed.Mutations in mpl have been frequently cited in in vitro studies as a common mutational site resulting in overexpression ampC (28) .Mutations in mpl were also associated with increased susceptibility to colistin and aminoglycosides (Table 3).
Overexpression of the PDC-encoding gene has been reported as a cause of resistance to newer β-lactam/β-LICs in clinical isolates (8,15) and isolates exposed in vivo to these agents (29); multiple isolates possessed alterations in AmpD, mutations in which increase PDC expression (30), following exposure to piperacillin-tazobactam and cefepime (one isolate each), but not for other agents.Interestingly, the piperacillin-tazobactam-exposed isolate displaying a single amino acid substitution in AmpD also exhibited alterations in GalU and the upstream region of mexR and it is possible the additive effect of these alterations underlies the development of resistance in this isolate (8-fold to 256-fold increases in MIC results for different β-lactams).
Finally, numerous isolates obtained during our passaging experiments against all selection agents acquired mutations in genes related to cell wall and outer membrane homeostasis.Multiple alterations were noted in genes encoding PBP3, OprD, PhoP, ClpA, Pgi, and CpxS, all of which are known to contribute to β-lactam resistance in P. aeruginosa isolates (1,31).In addition, multiple genes of unknown or presumed function were identified in our study, some of which were the only alterations present in the terminal isolate and may influence the reduction of susceptibility in those strains by as-yet undiscovered mechanisms.Future work will focus on defining the role of those proteins and if their role in the development of resistance is shared among other clinical P. aeruginosa isolates.
Overall, our results demonstrate following exposure to meropenem, cefepime, and piperacillin-tazobactam terminal isolates displayed greater fold increases in MIC values compared to the isolates obtained after exposure to ceftolozane-tazobactam and ceftazidime-avibactam.This data may indicate these newer agents could help prevent the emergence of high-level resistance.

Isolates, antimicrobial exposure, and susceptibility testing
Seven P. aeruginosa isolates including six clinical isolates from patients suffering bloodstream infection (n = 3) or pneumonia (n = 3) were selected as parental strains.The susceptibility reference strain ATCC 27853 was also included as a parental strain.Baseline MIC values were determined in triplicate by the reference broth microdilution method (32).All parental strains were exposed to ceftazidime-avibactam, ceftolozane-tazobac tam, meropenem, cefepime, and piperacillin-tazobactam.All β-lactamase inhibitors were tested at a 4 mg/L fixed concentration.A summary of the susceptibility profiles and associated genotypic information for all parental strains is shown in Table 1.Strains were generally maintained on tryptic soy agar with 5% sheep's blood (BAP, Remel; Lenexa, KS) and susceptibility testing was performed using cation-adjusted Mueller-Hinton broth.For serial passaging, lineages for each drug-parent combination were initiated in an identical fashion as was performed for the determination of baseline MICs against each agent; however, lineages were only grown in singlicate.Following overnight incubation, the entire contents of the well with the highest concentration of each antimicrobial displaying visible growth were used to prepare a new 0.5 McFarland standard.This inoculum was diluted to a final concentration of 5 × 10 5 CFU/mL in a new panel with the same antimicrobial agent.This process was repeated for nine additional days.A single colony was selected and passaged twice on drug-free BAP prior to further testing and storage.
Terminal isolates were susceptibility tested by reference broth microdilution method (32) against all agents used in exposure and comparator agents.

FIG 1
FIG1 Fold change in MIC results for isolates exposed to each agent when compared to its baseline isolate.

5 TABLE 3 ( 6 TABLE 3 7 TABLE 3 8 TABLE 3
inhibitory concentration.Full-Length Text Antimicrobial Agents and Chemotherapy May 2024 Volume 68 Issue 10.1128/aac.01363-23MIC fold change and genetic alterations in P. aeruginosa isolates exposed to β-lactam agents a Fold change in MIC compared to the parent isolate Continued on next page) Full-Length Text Antimicrobial Agents and Chemotherapy May 2024 Volume 68 Issue 5 10.1128/aac.01363-23MIC fold change and genetic alterations in P. aeruginosa isolates exposed to β-lactam agents a MIC fold change and genetic alterations in P. aeruginosa isolates exposed to β-lactam agents a MIC fold change and genetic alterations in P. aeruginosa isolates exposed to β-lactam agents a

TABLE 2
Changes in MIC values for P. aeruginosa isolates exposed to β-lactam agents a