Activity of Aztreonam/Avibactam and Recently Approved β-Lactamase Inhibitor Combinations against Enterobacterales and Pseudomonas aeruginosa from Intensive Care Unit and Non-Intensive Care Unit Patients

We evaluated the activities of aztreonam/avibactam and recently approved β-lactamase inhibitor combinations (BLICs) to compare the antimicrobial susceptibility patterns of Enterobacterales and Pseudomonas aeruginosa isolated from intensive care unit (ICU) and non-ICU patients. Clinical isolates (1/patient) were consecutively collected from 72 United States medical centres in 2020–2022 and susceptibility tested by broth microdilution. The results for 5421 isolates from ICU patients were analysed and compared to those for 20,649 isolates from non-ICU patients. Isolates from ventilator-associated pneumonia patients were analysed separately. Aztreonam/avibactam inhibited 100.0%/>99.9% Enterobacterales and 100.0%/98.3% of carbapenem-resistant Enterobacterales (CRE) from ICU/non-ICU patients at ≤8 mg/L, respectively. The CRE susceptibility rates were 88.5%/82.9% for ceftazidime/avibactam, 82.1%/81.2% for meropenem/vaborbactam, and 78.2%/72.6% for imipenem/relebactam among ICU/non-ICU isolates. Among the P. aeruginosa isolates from ICU/non-ICU patients, the susceptibility rates were 96.3%/97.6% for ceftazidime/avibactam, 97.2/98.4% for ceftolozane/tazobactam, 97.1%/98.0% for imipenem/relebactam, 77.8%/84.6% for piperacillin/tazobactam, and 76.9%/85.8% for meropenem; aztreonam/avibactam inhibited 78.0%/81.9% of P. aeruginosa at ≤8 mg/L. In summary, lower susceptibility rates were observed among ICU than non-ICU isolates. Aztreonam/avibactam exhibited potent in vitro activity and broad-spectrum activity against Enterobacterales from ICU and non-ICU patients, including CRE and isolates non-susceptible to newer BLICs. Against P. aeruginosa, aztreonam/avibactam showed a spectrum of activity comparable to that of piperacillin/tazobactam, meropenem, and ceftazidime.


Introduction
Healthcare-associated infections represent an important challenge when treating critically ill patients.Mortality rates and the length of hospital stays are markedly higher among intensive care unit (ICU) patients with an infection compared to non-infected patients [1].An important factor that contributes to poor patient outcomes is a delay in the introduction of effective antimicrobial therapy, which is more likely among patients infected with antimicrobial-resistant organisms than among patients infected with susceptible organisms [2][3][4].
Various factors contribute to increasing antimicrobial resistance in ICU patients, including the use of indwelling devices, the high occurrence of invasive procedures, the presence of comorbidities, prolonged hospital stays, the excessive use of antimicrobial agents, and the spread of resistant organisms [1,5].Gram-negative organisms, mainly Enterobacterales and Pseudomonas aeruginosa, are responsible for most infections in United States (US) ICUs, and antimicrobial resistance is remarkably problematic among these organisms [6].Moreover, inappropriate empiric therapy is clearly more frequent in Gramnegative than Gram-positive infections [7].
Multidrug-resistant (MDR) Gram-negative bacteria are the leading causes of nosocomial pneumonia, including ventilator-associated pneumonia (VAP) and bloodstream infection in ICU patients.Notably, carbapenem resistance has increased in the last decade, and it is commonly caused by carbapenemases that are able to hydrolyse most β-lactams currently available for clinical use in the US and Europe [8,9].
Various antimicrobial agents targeting MDR Gram-negative organisms have been licensed in recent years, including ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam, and cefiderocol [10].These agents represent an important improvement in the treatment of infections caused by MDR Gram-negative organisms, especially carbapenem-resistant Enterobacterales (CRE); however, resistance to these agents appears to be increasing in some US hospitals [11].
Aztreonam/avibactam is under development to treat patients with Gram-negative infections, especially those caused by CRE and Stenotrophomonas maltophilia, and it has recently (April 2024) been granted marketing authorization by the European Medicines Agency (EMA) in the European Union (https://www.ema.europa.eu/en/news/new-antibioticfight-infections-caused-multidrug-resistant-bacteria;accessed on 6 May 2024) [12,13].Aztreonam is stable to hydrolysis by metallo-β-lactamases (MBLs), and avibactam protects aztreonam from hydrolysis by serine β-lactamases, such as chromosomal derepressed AmpC, extended-spectrum β-lactamases, and Klebsiella pneumoniae carbapenemases (KPCs).In this investigation, we evaluated the in vitro activity of aztreonam/avibactam and recently approved β-lactamase inhibitor combinations (BLICs) and compared the antimicrobial susceptibility patterns of Enterobacterales and P. aeruginosa isolated from ICU and non-ICU patients.

Discussion
Infection is a common occurrence among patients in the ICU, and ICU-acquired infections are considered an independent risk factor for hospital mortality, even after adjustment for APACHE II or SOFA scores [16].In a 24 h point prevalence study conducted at 1150 centres in 88 countries on 13 September 2017, 54% of patients had suspected or proven infection, and 70% of all patients were receiving prophylactic or therapeutic antimicrobial agents.Moreover, the mortality rate was 32% among patients with infection, and infection due to CRE was independently associated with an increased risk of death [17].
The main objective of the present study was to evaluate the antimicrobial susceptibility of Enterobacterales and P. aeruginosa from ICU patients in comparison to those from non-ICU patients.Although some of the recently approved β-lactam agents remain equally and highly active against ICU and non-ICU isolates, our results indicate a clear tendency of higher resistance rates among ICU than non-ICU isolates.Among Enterobacterales, the susceptibility rates were markedly lower among the ICU compared to the non-ICU isolates for ceftolozane/tazobactam (90.1% vs. 95.5%),piperacillin/tazobactam (82.4% vs. 90.5%),ceftazidime (81.7% vs. 88.1%), and ceftriaxone (77.2% vs. 84.6%).Although the susceptibility rates were similar or slightly higher among the ICU compared to the non-ICU isolates for many antimicrobial agents, the MDR, XDR, and CRE rates were clearly higher among the ICU than among the non-ICU isolates (Table 1 and Figure 3).Among P. aeruginosa, the susceptibility rates were lower among the ICU compared to the non-ICU isolates for most antimicrobial agents, with the highest differences being observed with meropenem (76.9% vs. 85.8%),imipenem (77.4% vs. 84.3%),

Discussion
Infection is a common occurrence among patients in the ICU, and ICU-acquired infections are considered an independent risk factor for hospital mortality, even after adjustment for APACHE II or SOFA scores [16].In a 24 h point prevalence study conducted at 1150 centres in 88 countries on 13 September 2017, 54% of patients had suspected or proven infection, and 70% of all patients were receiving prophylactic or therapeutic antimicrobial agents.Moreover, the mortality rate was 32% among patients with infection, and infection due to CRE was independently associated with an increased risk of death [17].
The main objective of the present study was to evaluate the antimicrobial susceptibility of Enterobacterales and P. aeruginosa from ICU patients in comparison to those from non-ICU patients.Although some of the recently approved β-lactam agents remain equally and highly active against ICU and non-ICU isolates, our results indicate a clear tendency of higher resistance rates among ICU than non-ICU isolates.Among Enterobacterales, the susceptibility rates were markedly lower among the ICU compared to the non-ICU isolates for ceftolozane/tazobactam (90.1% vs. 95.5%),piperacillin/tazobactam (82.4% vs. 90.5%),ceftazidime (81.7% vs. 88.1%), and ceftriaxone (77.2% vs. 84.6%).Although the susceptibility rates were similar or slightly higher among the ICU compared to the non-ICU isolates for many antimicrobial agents, the MDR, XDR, and CRE rates were clearly higher among the ICU than among the non-ICU isolates (Table 1 and Figure 3).Among P. aeruginosa, the susceptibility rates were lower among the ICU compared to the non-ICU isolates for most antimicrobial agents, with the highest differences being observed with meropenem (76.9% vs. 85.8%),imipenem (77.4% vs. 84.3%),piperacillin/tazobactam (77.8% vs. 84.6%),and ceftazidime (81.4% vs. 87.8%).Moreover, the P. aeruginosa isolates from the VAP patients exhibited susceptibility rates lower than the ICU isolates, especially for meropenem (70.8% vs. 76.9%),imipenem (73.7% vs. 77.4%),and piperacillin tazobactam (75.6% vs. 77.8%;Table 1).
Antimicrobial resistance rates are likely to be higher among bacteria recovered from ICU patients than non-ICU patients since various risk factors related to antimicrobial resistance are more commonly observed in ICU patients, such as the use of indwelling devices, invasive procedures, prolonged hospital stays, and a high use of antimicrobial agents; however, studies comparing the antimicrobial susceptibility rates of ICU and non-ICU isolates are scarce [1,18,19].The results of the present study clearly show higher resistance rates among Enterobacterales and P. aeruginosa isolates from ICU patients and VAP patients compared to isolates from non-ICU patients.
Another important finding of this study was the reduced activity of cefiderocol and the newer β-lactamase inhibitor combinations ceftazidime/avibactam, meropenem/vaborbactam, and imipenem/relebactam against CRE and MDR Enterobacterales.Although these compounds have shown almost complete activity against US CRE and MDR Enterobacterales in previous investigations, resistance appears to be increasing recently in the US [11,20].The main reason for the increasing resistance to these antimicrobial agents appears to be the growing prevalence of MBL-producing Enterobacterales in US medical centres, since ceftazidime/avibactam, meropenem/vaborabactam, and imipenem/relebactam are not active against MBL-producing isolates, and cefiderocol has demonstrated limited activity against NDM-producing Enterobacterales [11,21,22].
The limitations of this study should be contemplated when interpreting the results.The INFORM Program was not designed to assess the susceptibility profile of bacterial isolates from ICU patients in comparison to non-ICU patients; patient-unique isolates were consecutively collected unrelated to the hospital unit.After the isolates were collected and tested for susceptibility, we analysed the results of the ICU and non-ICU isolates.Variability in the criteria used to label a bacterial isolate as "clinically significant" could also be considered a study limitation, since this term was not defined in the study protocol.Instead, the criteria were determined by local algorithms, which may vary among participant medical centres.Regardless of these limitations, these results provide valuable information on the susceptibility profile of Enterobacterales and P. aeruginosa causing infections in the ICUs of US medical centres.

Organism Collection
Isolates were collected from 72 US medical centres in 2020-2022 as part of the International Network For Optimal Resistance Monitoring (INFORM) program [23].Each participating centre was asked to collect a certain number of consecutive patient unique isolates from designated infection types during a certain period of the year, independent of the hospital unit where the patient was located.In this investigation, we assessed the susceptibility results for Enterobacterales and P. aeruginosa collected from ICU patients and compared them to those for isolates from patients hospitalized in other units (non-ICU).A total of 21,996 Enterobacterales isolates, comprising 4117 isolates from ICU patients and 17,879 isolates from non-ICU patients, and 4074 P. aeruginosa isolates, comprising 1304 isolates from ICU patients and 2770 isolates from non-ICU patients, were collected and analysed during the investigation period.The isolates were considered clinically significant by algorithms established by participant centres.Moreover, ICU isolates recovered from patients with ventilator-associated pneumonia (VAP), including 630 Enterobacterales and 308 P. aeruginosa isolates, were analysed separately.

Susceptibility Testing
The isolates were susceptibility tested using the reference broth microdilution method, as described by CLSI [24].Aztreonam/avibactam, ceftazidime/avibactam, ceftolozane/ tazobactam, imipenem/relebactam, and piperacillin/tazobactam were tested with a βlactamase inhibitor at a fixed concentration of 4 mg/L; meropenem/vaborbactam was tested with vaborbactam at a fixed concentration of 8 mg/L [14,24].Cefiderocol was tested only against CRE.MIC values were interpreted according to CLSI and/or US FDA breakpoint criteria unless otherwise noted.When aztreonam/avibactam was tested against Enterobacterales, both the pharmacokinetic/pharmacodynamic (PK/PD) and EUCAST susceptible breakpoints (≤8 mg/L and ≤4 mg/L, respectively) were applied for comparison.EUCAST criteria [25] were applied for meropenem/vaborbactam against P. aeruginosa because this compound is not approved for the treatment of P. aeruginosa infections in the US, and neither CLSI nor the US FDA have published breakpoint criteria for this organismdrug combination.CLSI does not currently publish a colistin-susceptible breakpoint and categorizes isolates with an MIC ≤2 mg/L as intermediate and ≥4 mg/L as resistant; thus, the percentages of intermediate resistance are shown in Table 1.The isolates were categorized as MDR or XDR according to the criteria defined in 2012 by the joint European and US Centers for Disease Control [26].These criteria define MDR as non-susceptible to ≥1 agent in ≥3 antimicrobial classes and XDR as susceptible to ≤2 classes.Carbapenem-resistant Enterobacterales (CRE) isolates were defined as displaying imipenem or meropenem MIC values at ≥4 mg/L.Imipenem was not applied to Proteus mirabilis or indole-positive Proteeae due to their intrinsically elevated MIC values.Categorical interpretations followed CLSI and/or US FDA criteria unless otherwise noted [14,15].

Screening for β-Lactamases
CRE isolates were tested for β-lactamase-encoding genes using Next-Generation Sequencing (NGS).Total genomic DNA was prepared using a KingFisher Cell and Tissue DNA kit (ThermoFisher Scientific, Waltham, MA, USA) or a MagMax DNA Multi-Sample Ultra 2.0 extraction kit (ThermoFisher) on a KingFisher Flex Magnetic Particle Processor (ThermoFisher).DNA libraries were constructed using either the Nextera XT library construction protocol and index kit or Illumina DNA prep (Illumina, San Diego, CA, USA), with sequencing performed on either a MiSeq Sequencer with a MiSeq Reagent Kit v3 (600 cycles) or a NextSeq 1000 Sequencer using NextSeq1000/2000 P2 Reagents (300 cycles).The generated FASTQ files were assembled using SPAdes Assembler and subjected to proprietary software version 3.15.3(Element Iowa City [JMI Laboratories]) for the screening of β-lactamase genes [27].

Figure 3 .
Figure 3. Bar graph showing the frequencies of key resistance phenotypes among Enterobacterales isolated from intensive care unit (ICU) patients, ventilator-associated pneumonia (VAP) patients, and non-ICU patients (non-ICU).

Figure 3 .
Figure 3. Bar graph showing the frequencies of key resistance phenotypes among Enterobacterales isolated from intensive care unit (ICU) patients, ventilator-associated pneumonia (VAP) patients, and non-ICU patients (non-ICU).

Susceptible (No. of Isolates Tested) a Antimicrobial Agent ICU VAP Non-ICU
cAll Enterobacterales species were included in the analysis, but CLSI excludes Morganella, Proteus, and Providencia species.d % Intermediate or not resistant.e % inhibited at ≤8 mg/L.f This compound is not approved for treatment of P. aeruginosa infections in the US.EUCAST criteria were applied since neither CLSI nor US FDA have published breakpoint criteria.

Table 2 .
Antimicrobial susceptibility of most common Enterobacterales species isolated from ICU and non-ICU patients from US hospitals (2018-2020).