In Vivo Pharmacodynamics of β-Lactams/Nacubactam against Carbapenem-Resistant and/or Carbapenemase-Producing Enterobacter cloacae and Klebsiella pneumoniae in Murine Pneumonia Model

Carbapenem-resistant Enterobacterales (CRE) and carbapenemase-producing Enterobacterales (CPE) have become global threats. CRE− and CPE− derived infections have been associated with high mortality due to limited treatment options. Nacubactam is a β-lactamase inhibitor and belongs to the new class of diazabicyclooctane. The agent has an in vitro antimicrobial activity against several classes of β-lactamase-producing Enterobacterales. This study evaluated antimicrobial activity of combination therapies including β-lactams (aztreonam, cefepime, and meropenem) and nacubactam against four Enterobacter cloacae and six Klebsiella pneumoniae isolates with murine pneumonia model. Based on changes in bacterial quantity, antimicrobial activities of some regimens were assessed. Combination therapies including β-lactams (aztreonam, cefepime, and meropenem) with nacubactam showed enhanced antimicrobial activity against CRE E. cloacae (−3.70 to −2.08 Δlog10 CFU/lungs) and K. pneumoniae (−4.24 to 1.47 Δlog10 CFU/lungs) with IMP-1, IMP-6, or KPC genes, compared with aztreonam, cefepime, meropenem, and nacubactam monotherapies. Most combination therapies showed bacteriostatic (−3.0 to 0 Δlog10 CFU/lungs) to bactericidal (<−3.0 Δlog10 CFU/lungs) activities against CRE isolates. This study revealed that combination regimens with β-lactams (aztreonam, cefepime, and meropenem) and nacubactam are preferable candidates to treat pneumonia due to CRE and CPE.


Introduction
Carbapenem-resistant Enterobacterales (CRE)-caused infections have become a global issue. As therapeutic options are limited, the infections showed high mortality [1,2]. Carbapenemase-producing Enterobacterales (CPE) have spread globally at an alarming rate [3,4]. These bacterial strains have potentials to be highly resistant to not only for carbapenems, but also other commonly used antimicrobials, through the propagation of plasmids encoding carbapenem-hydrolyzing enzymes [5,6].
Escherichia coli and Klebsiella pneumoniae occupy the majority among detected CPE strains from infected patients [7][8][9]. However, Enterobacter cloacae was the second-most detected pathogen with carbapenemase genes in sputum specimens, followed by K. pneumoniae [10]. Among Enterobacterales strains, the production of β-lactamases was the main reason for resistance to β-lactams. Then, owing to the hydrolysis of almost all β-lactams, infections due to carbapenemase-producing Enterobacterales strains are the one of the most serious issues to manage in treatment.
In our previous in vivo study with murine thigh infection model, combination therapies including β-lactams (aztreonam (ATM), cefepime (FEP), and meropenem (MEM)) and NAC showed enhanced antibacterial activities against CRE pathogens [14]. However, these combination therapies have not been evaluated the antimicrobial activities against CRE− and/or CPE− derived pneumonia. Therefore, the antimicrobial efficacy of these combination therapies against E. cloacae and K. pneumoniae were assessed with a murine pneumonia model ( Figure 1).  Table 1 shows MICs of ATM, FEP, MEM, and NAC against the 10 study isolates. Among them, seven isolates are resistance to MEM according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints [15]. Combination regimens of ATM, FEP, and MEM with NAC showed 2-to >128-fold lower MICs, compared with those of β-lactam monotherapies against the CRE+ isolates. Additionally, these combination regimens showed the same to >128-fold lower MICs than those of β-lactam monotherapies against   Table 1 shows MICs of ATM, FEP, MEM, and NAC against the 10 study isolates. Among them, seven isolates are resistance to MEM according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints [15]. Combination regimens of ATM, FEP, and MEM with NAC showed 2-to >128-fold lower MICs, compared with those of βlactam monotherapies against the CRE+ isolates. Additionally, these combination regimens showed the same to >128-fold lower MICs than those of β-lactam monotherapies against the CRE−/CPE+ isolates. Same MICs were observed between combinations and monotherapies against CRE−/CPE− E. cloacae (20-5694). ATM, FEP, MEM, and NAC PK profiles in ELF and plasma are depicted in Figure S1. In this study, single doses of NAC and each β-lactam monotherapies were used at 100 mg/kg (subcutaneous doses). Table 2 shows the calculated PK parameters of the antimicrobials in plasma and ELF. At the start of antimicrobial therapies (0 h), bacterial counts in lungs of control mice were between 6.79 to 7.00 log 10 colony forming units (cfu)/lungs for E. cloacae and 6.59 to 7.21 log 10 cfu/lungs for K. pneumoniae. Bacterial number differences of growth control were between 0.06 to 2.58 ∆log 10 CFU/lungs for E. cloacae and −0.77 to 2.74 ∆log 10 CFU/lungs for K. pneumoniae after 24 h from the start of antimicrobial therapies. Bacterial number differences of NAC monotherapy (320, 160, and 80 mg/kg q8h) were between −2.27 and 1.19 ∆log 10 CFU/lungs for four E. cloacae isolates and −1.34 to 2.66 ∆log 10 CFU/lungs for six K. pneumoniae isolates ( Figure S2). Among total 10 study isolates, six isolates showed higher bacterial numbers in lungs at 24 h than at 0 h, while maximum dosage of NAC was adopted (320 mg/kg q8h).

Antimicrobial Efficacies against CRE− Isolates
Bacterial number differences in combination therapies were between −3.56 and −2.81 ∆log 10 CFU/lungs for E. cloacae and −4.46 and −1.70 ∆log 10 CFU/lungs for K. pneumoniae ( Figure 4). Comparing with the corresponding β-lactam monotherapies, combination therapies showed enhanced antimicrobial activities, when combined with FEP and AZT with NAC against CRE−/CPE− (K. pneumoniae ATCC700603) and CRE−/CPE+ (K. pneumoniae 990645). However, NAC did not affect antimicrobial activity of MEM therapy against these isolates. Moreover, no enhanced antimicrobial activity was found in combination therapies against E. cloacae (20-5694).
Antibiotics 2021, 10, x FOR PEER REVIEW 4 of 11 bacterial numbers in lungs at 24 h than at 0 h, while maximum dosage of NAC was adopted (320 mg/kg q8h).

Discussion
NAC has a dual mechanism of action. The agent inhibits some classes of serine βlactamases and PBP2 in Enterobacterales [11,16,17]. In our previous study, the same combination therapies including NAC and β-lactams showed enhanced antimicrobial activities against Enterobacterales have some carbapenemase-producing genes with a murine thigh infection model [14]. However, these combination therapies have not been evaluated against CRE− and CPE−caused pneumonia. In this in vitro study, the combination regimens of β-lactams with NAC showed 1-to >128-fold lower MICs against CPE and CRE isolates, comparing with the corresponding β-lactam monotherapies ( Table 1). As previous in vitro study suggested, our results also suggested that NAC enhanced in vitro antimicrobial activities of β-lactams against several classes of β-lactamase-producing E. cloacae and K. pneumoniae isolates [11][12][13].
Moreover, in this pharmacodynamics evaluation, NAC monotherapy could not show a bactericidal effect against all study isolates ( Figure S2). In contrast, combination therapies showed enhanced antimicrobial activities against CRE+ E. cloacae and K. pneumoniae isolates (Figures 2 and 3). At least one regimen among combination therapies of ATM,

Discussion
NAC has a dual mechanism of action. The agent inhibits some classes of serine β-lactamases and PBP2 in Enterobacterales [11,16,17]. In our previous study, the same combination therapies including NAC and β-lactams showed enhanced antimicrobial activities against Enterobacterales have some carbapenemase-producing genes with a murine thigh infection model [14]. However, these combination therapies have not been evaluated against CRE− and CPE−caused pneumonia. In this in vitro study, the combination regimens of β-lactams with NAC showed 1-to >128-fold lower MICs against CPE and CRE isolates, comparing with the corresponding β-lactam monotherapies ( Table 1). As previous in vitro study suggested, our results also suggested that NAC enhanced in vitro antimicrobial activities of β-lactams against several classes of β-lactamase-producing E. cloacae and K. pneumoniae isolates [11][12][13].
Moreover, in this pharmacodynamics evaluation, NAC monotherapy could not show a bactericidal effect against all study isolates ( Figure S2). In contrast, combination therapies showed enhanced antimicrobial activities against CRE+ E. cloacae and K. pneumoniae isolates (Figures 2 and 3). At least one regimen among combination therapies of ATM, FEP, and MEM with NAC showed bactericidal effects against all CRE+ E. cloacae and K. pneumoniae isolates, excluding the K. pneumoniae isolate (594).
Similar to our previous study using a murine thigh infection model [14], combination therapy of MEM and NAC was not effective (>0∆log 10 CFU/lungs) against CRE+ K. pneumoniae (594) (Figure 2). The isolate has the IMP-6 gene and showed low susceptibility to MEM and NAC, whereas MEM + NAC resulted in relatively lower MIC (2 µg/mL) ( Table 1). In vitro studies of the other group conducted showed that the efficacy of NAC is restricted to bacteria with IMP-type carbapenemase-producing genes [11][12][13]. NAC only binds to PBP2. On the other hand, ATM and FEP bind to PBP3 rather than PBP2 [11]. Thus, the NAC-derived enhancer effect was expected when the agent was co-administrated with ATM and FEP than with MEM. Furthermore, we could speculate that NAC was not effective to work as an PBP2 inhibitor to CRE+ K. pneumoniae (594) as the isolate has low susceptibility to both MEM and NAC. Although further study to clarify the mechanism is needed, MEM + NAC combination therapy against IMP-type carbapenemase-producing Enterobacterales could be unsuitable, especially when causative bacteria have high MICs (low susceptibilities) of MEM and NAC.
In general, optimizing the antimicrobial dosage according to the PK/PD theory is one of the important components to improve infected patient outcomes. As the PK/PD breakpoint of NAC has not been fully evaluated, it was difficult to interpret our PD study results. However, the antimicrobial activities of β-lactams correlate with %f T > MIC well [18,19]. NAC co-administration had no impact on PK profiles of β-lactams (ATM, FEP, and MEM) in plasma [14]. Additionally, we did not observe significant differences in the PK data of plasma between the thigh infection model and pneumonia model [14, Figure S1]. Due to the fact that the reappearance of human PK profiles of antimicrobials was difficult for us, as mice have higher clearance ability than that of humans, we used the same dosage regimens as previous in vivo studies [14] to obtain similar areas under the free drug concentration-time curves (f AUC) when β-lactams (ATM, FEP, and MEM) were dosed at 1 g q8h, and NAC (2, 1, and 0.5 g q8h) [20][21][22][23][24][25][26]. Using calculated PK parameters, we conducted PK/PD analysis and found the good relationships between %f T > MIC of β-lactams (ATM, FEP, and MEM) in ELF and plasma and antimicrobial activities ( Figure 5). Our data will give important insights to explore the optimal dosage regimen for pneumonia patients.
The combination therapy of β-lactams (ATM, FEP, and MEM) and NAC was highly effective against CPE and CRE caused pneumonia. However, we evaluated limited classes of carbapenemases producing Enterobacteriales in this study. In Japan, IMP-1 and -6 type CPE isolates are the most frequently detected [7,8]. However, there are regional differences of the β-lactamases prevalence in Enterobacterales [7,9,27]. Therefore, further studies are needed. As the other limitation, we did not evaluate inoculum effects, while we evaluated antimicrobial activities with similar inoculum size to previous report [28][29][30].
Although our study has a few limitations, this is the first in vivo study to evaluate antimicrobial activities of combination therapies including β-lactams and NAC with pneumonia model. Our PD data suggested combination therapied including NAC is a potent candidate for CRE caused pneumonia. This fact can give new therapeutic choice to antimicrobial treatment for CRE caused pneumonia, while the prevalence and burden of CRE infection are rising. Additionally, we revealed the relationship between these antimicrobial activities and antimicrobial PK data (in plasma and ELF). Therefore, our data would be a preferable reference to explore optimal antimicrobial dosages in the future.

Microorganisms
Four E. cloacae and six K. pneumoniae isolates were used in this study (Table 1). Clinical isolates of E. cloacae and K. pneumoniae were provided from department of Microbial laboratory in Aichi Medical University Hospital. CRE isolates have MEM MIC ≥ 4 µg/mL. CPE isolates have any detectable carbapenemase genes. According to previous our study methods [14], direct sequencing was conducted to screen carbapenemase and other βlactamase-encoding genes.

Susceptibility Test
The antimicrobial susceptibilities of ATM, FEP, MEM, and NAC were determined with the broth microdilution method, following recommended methods by CLSI [15]. For the combination regimens, the broth microdilution method was conducted in fixed concentration ratios of each β-lactam and NAC (1:1 (w/w)), according to CLSI method (M100-ED31: Table 5A-2). The β-lactam concentration was used to show MICs in combination therapies. The MIC studies were conducted a minimum of three times, and the geometric MIC was reported.

Animals
Pathogen-free, ICR mice (4-week-old) were acquired from Charles River Laboratories Japan, Inc. (Yokohama, Japan). They were provided food and water ad libitum. They were housed and used following the National Research Council recommendations.

Pneumonia Model
Neutropenic murine pneumonia model was used to evaluate antimicrobial activities of various antimicrobial regimens against E. cloacae and K. pneumoniae in the lungs of mice [30]. Mice were rendered transiently neutropenic with cyclophosphamide of intraperitoneal doses at Day-4 (150 mg/kg) and Day-1 (100 mg/kg) before inoculation. Study isolates for inoculation were stored at −80 • C. A bacterial suspension was prepared at approximately 10 9 cfu/mL with study isolates after 24 h incubation of the second transfer. Under anesthesia, mice were orally instilled the bacterial suspension (0.075 mL), and the nares were blocked while being held vertically for 60 s to aspirate the suspension into the lungs. After inoculation, mice were randomly divided into control or each antimicrobial treatment group (n = 6, respectively).

PK Studies
For the PK study of ATM, FEP, MEM, and NAC in ELF and plasma, each antimicrobial was dosed subcutaneously (single dose) as monotherapies to the infected mice. The neutropenic pneumonia mice were infected with K. pneumoniae ATCC43816. The mice were injected with ATM, FEP, MEM, or NAC (at 100 mg/kg, respectively). Mice were sacrificed at each time point (0.25, 0.5, 1, 2, 3, 4, and 5 h). Then, blood samples from the axillary artery and bronchoalveolar lavage fluid (BALF) samples were collected (n = 3 in each group). After the detection of urea concentration in each BALF and plasma sample with a urea assay kit (Urea Assay Kit, BioChain Institute, Inc., Newark, CA, USA), the antimicrobial concentrations in ELF were calculated: ELF antimicrobial concentration = BALF antimicrobial concentration × (the ratio of urea concentrations in plasma/BALF). AUC in ELF and plasma from time 0 to ∞ (AUC0-∞) were calculated according to the trapezoidal method. The PK parameters in the ELF and plasma were calculated with Phoenix WinNonlin software (ver. 8.1; Certara, L.P.).

Instrumentation and Chromatographic Conditions
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze ATM, FEP, MEM, and NAC concentrations at Meiji Seika Pharma Co., Ltd. Detailed conditions and procedures are in the Supplementary document.

PD Study
One hour after inoculation of study isolates, mice received antimicrobials (0.2 mL) subcutaneously (n = 6 in each group). We used same dosage of β-lactams and NAC with our previous in vivo study [14] to be similar f AUC values of NAC (2.0, 1.0, and 0.5 g), and β-lactams (1.0 g) in blood with clinical PK study data. The following eight-hourly (q8h) doses were evaluated: 700 mg/kg for ATM, 260 mg/kg for FEP, and 140 mg/kg for MEM, 320, 160, and 80 mg/kg for NAC. Control mice were administered NS (0.2 mL).
Twenty-four hours after antimicrobial treatments started, lungs were harvested from mice. Then, mice were sacrificed by CO 2 exposure. Removed lungs from each mouse were homogenized individually, and the homogenates were plated on trypticase soy agar with 5% sheep blood after serial dilutions with NS to determine the bacterial counts in lungs (cfu/lungs). In addition, six infected mice were harvested just before the start of antimicrobial therapies to know bacterial number in lungs at 0 h. The antimicrobial efficacies were defined as follows: detected bacterial counts in the treated group at 24 h and bacterial counts in the control group at 0 h (∆log 10 CFU/lungs).

PK/PD Analysis
The %T > MIC in ELF and plasma for unbound ATM, FEP, MEM, and NAC was calculated with PK parameters (Table S1 and Table S2) in pneumonia mice, serum protein binding ratio [14,[20][21][22], and MIC values, with the R software (ver. 3.6.1). For combination therapies, MICs of combinations were used. An inhibitory effect sigmoid I max model was adapted to reveal the correlations between %f T > MIC of each drug and ∆log 10 CFU/lungs of each antimicrobial regimen with Phoenix WinNonlin software (ver. 8.1; Certara, L.P.). To estimate the percentage of variance in efficacy, the coefficient of determination (R 2 ) was used.

Statistical Analysis
Mann-Whitney U test was used to compare in vitro MIC values of each β-lactam monotherapy and corresponding combination therapies. To compare the in vivo antimicrobial efficacy between the regimens, one-way ANOVA with Bonferroni correction was used. Statistical analysis was performed with JMP, version 10.0 (SAS, Tokyo, Japan). Differences were considered statistically significant at p < 0.05.

Conclusions
Combination therapies including ATM, FEP, and MEM with NAC showed potent in vivo antimicrobial activities against pneumonia caused by CRE and CPE E. cloacae and K. pneumoniae. These translational data support the potential role of NAC in combination with ATM, FEP, and MEM as a therapy for CRE and CPE K. pneumoniae and E. cloacae caused human pneumonia.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antibiotics10101179/s1, Figure S1: In vivo pharmacokinetic data of nacubactam, meropenem, cefepime, and aztreonam in ELF and plasma, Figure S2: In vivo efficacy of nacubactam monotherapy, Table S1: Apparent pharmacokinetic parameters in ELF in murine pneumonia model for calculation of f T > MIC, Table S2: Pharmacokinetic parameters in plasma in murine pneumonia model for calculation of %f T > MIC.

Institutional Review Board Statement:
The ethics committee of Aichi medical university reviewed this study protocol and approved (2020-30). To conduct PK studies of β-lactams and NAC, the animal experiment management committee and pharmaceutical research laboratories at Meiji Seika Pharma Co., Ltd. reviewed this study protocol and approved (C2020-010 and C2020-013).

Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets analyzed during this study are available and can be obtained, at request, on reasonable enquiry.