Motos et al. Critical Care (2023) 27:60 https://doi.org/10.1186/s13054-023-04331-x RESEARCH Open Access Inhaled amikacin for pneumonia treatment and dissemination prevention: an experimental model of severe monolateral Pseudomonas aeruginosa pneumonia Ana Motos1,2,3,4†, Hua Yang1,2,3†, Gianluigi Li Bassi2,14, Minlan Yang1,2,3, Andrea Meli1,5, Denise Battaglini1,3,6, Roberto Cabrera1,2,4, Joaquim Bobi1,2, Francesco Pagliara1,6, Gerard Frigola7, Marta Camprubí‑Rimblas4,8, Laia Fernández‑Barat1,2,3,4, Montserrat Rigol1,2, Antoni Ferrer‑Segarra1,9, Kasra Kiarostami1,2,3, Daniel Martinez7, David P. Nicolau10, Antonio Artigas4,8, Paolo Pelosi6,11, Jordi Vila12,13,15 and Antoni Torres1,2,3,4* Abstract Background Pseudomonas aeruginosa pneumonia is commonly treated with systemic antibiotics to ensure adequate treatment of multidrug resistant (MDR) bacteria. However, intravenous (IV) antibiotics often achieve suboptimal pulmonary concentrations. We therefore aimed to evaluate the effect of inhaled amikacin (AMK) plus IV meropenem (MEM) on bactericidal efficacy in a swine model of monolateral MDR P. aeruginosa pneumonia. Methods We ventilated 18 pigs with monolateral MDR P. aeruginosa pneumonia for up to 102 h. At 24 h after the bacterial challenge, the animals were randomized to receive 72 h of treatment with either inhaled saline (control), IV MEM only, or IV-MEM plus inhaled AMK (MEM + AMK). We dosed IV MEM at 25 mg/kg every 8 h and inhaled AMK at 400 mg every 12 h. The primary outcomes were the P. aeruginosa burden and histopathological injury in lung tissue. Secondary outcomes included the P. aeruginosa burden in tracheal secretions and bronchoalveolar lavage fluid, the development of antibiotic resistance, the antibiotic distribution, and the levels of inflammatory markers. Results The median (25–75th percentile) P. aeruginosa lung burden for animals in the control, MEM only, and MEM + AMK groups was 2.91 (1.75–5.69), 0.72 (0.12–3.35), and 0.90 (0–4.55) l­og10 CFU/g (p = 0.009). Inhaled therapy had no effect on preventing dissemination compared to systemic monotherapy, but it did have significantly higher bactericidal efficacy in tracheal secretions only. Remarkably, the minimum inhibitory concentration of MEM increased to > 32 mg/L after 72-h exposure to monotherapy in 83% of animals, while the addition of AMK prevented this increase (p = 0.037). Adjunctive therapy also slightly affected interleukin-1β downregulation. Despite finding high AMK concentrations in pulmonary samples, we found no paired differences in the epithelial lining fluid concentration between infected and non-infected lungs. Finally, a non-significant trend was observed for higher amikacin penetra‑ tion in low-affected lung areas. † Ana Motos and Hua Yang are equally contributed *Correspondence: Antoni Torres atorres@clinic.cat Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Motos et al. Critical Care (2023) 27:60 Page 2 of 11 Conclusions In a swine model of monolateral MDR P. aeruginosa pneumonia, resistant to the inhaled AMK and sus‑ ceptible to the IV antibiotic, the use of AMK as an adjuvant treatment offered no benefits for either the colonization of pulmonary tissue or the prevention of pathogen dissemination. However, inhaled AMK improved bacterial eradication in the proximal airways and hindered antibiotic resistance. Keywords Inhaled amikacin, Severe pneumonia, Pseudomonas aeruginosa, Animal model, Multidrug resistance, Monolateral pneumonia Background caused by P. aeruginosa, according to the ARRIVE Current guidelines for the management of hospital- guidelines [23]. This study was conducted according to acquired pneumonia (HAP) and ventilator-associated the European guidelines for the Care of Animal Experi- pneumonia (VAP) stress the need for preventive strate- mentation at the Division of Animal Experimentation, gies and accurate etiologic diagnosis [1, 2]. Unfortunately, Department of Pulmonary and Critical Care Medicine, routinely employed antibiotic agents are often ineffective Hospital Clínic, Barcelona, Spain. Documented approval against gram-negative pathogens, with multidrug resist- from the appropriate Institutional Review Board was ance (MDR) a common problem, even in combination obtained prior to the study started (number approval therapy [3]. The treatment of severe gram-negative pneu- 9772). monia therefore remains a major challenge [3, 4]. Pseudomonas aeruginosa pneumonia is usually treated The animal model with a combination of intravenous antibiotics to ensure Eighteen female Large White-Landrace pigs underwent the adequate treatment of MDR isolates [1, 2]. However, 96 h of mechanical ventilation. Animal preparation, insufficient lung distribution and adverse side effects including anesthesia induction and maintenance, airway limit this approach [5], leading to inhaled antibiotics management, and hemodynamic invasive monitoring were gaining increasing attention as a site-specific treatment performed as previously described [24]. Immediately after [6]. Inhaled has have improved bactericidal properties by preparation and stabilization, each animal was challenged ensuring high concentrations in tracheal secretions and with 15 mL of 1 ­ 07 CFU/mL log-phase culture of AMK- epithelial lining fluid (ELF) [7, 8], while lowering systemic resistant and MEM-susceptible P. aeruginosa at minimum concentrations to curtail its nephrotoxic and ototoxic inhibitory concentrations (MICs) of > 256 and 0.25 mg/L, effects [9, 10]. Experimental and clinical studies have respectively. We did select P. aeruginosa strain resistant to elicited conflicting results when assessing inhaled AMK AMK to consider the worst scenario for AMK efficacy, but in VAP [7, 11–19]. Unfortunately, the latest two rand- susceptibility to IV antibiotic was still ensured. omized clinical trials of inhaled AMK have failed to dem- The challenge was instilled bronchoscopically into the onstrate any benefit in primary outcomes (i.e., change in right upper, middle, and lower lobes, with the animals clinical pulmonary infection score and survival at days kept in a lateral-right slight Trendelenburg position to 28–32, respectively) for mechanically ventilated patients ensure a right predominance of infection. This was main- with pneumonia [14, 19]. The most recent management tained for 24 h, before the lateral position was changed guidelines for HAP/VAP advise against the routine use from one side to the other every 6 h. The pneumonia of adjunctive inhaled therapy, recommending it only diagnosis was confirmed 24 h after the bacterial chal- for susceptible MDR bacteria [2, 20]. Given that study lenge based on a decrease in the arterial partial pressure design, dosing, and nebulization technique may have of oxygen (­PaO2)/fraction of oxygen in the inhaled gas obscured the impact of inhaled AMK on VAP dissemi- ­(FiO2), plus at least one of the following signs of infec- nation and the emergence of resistance [21, 22] further tion: temperature ≥ 38.5 °C, leukocytosis ≥ 20.109 cells/L, research is warranted. or purulent secretions. In this study, we aimed to analyze the effects of inhaled Protocol. AMK of monolateral pneumonia caused by P. aeruginosa The 18 animals were randomized into one of three in pigs. groups after creating the model scenario: a control group (CONTROL, n = 6), a meropenem group (MEM, n = 6), and an amikacin plus meropenem group (MEM + AMK, Materials and methods n = 6). The CONTROL group received a 4-mL inhaled Study design solution of 0.9% NaCl every 12 h and no intravenous We analyzed the effects of inhaled AMK in a translational antibiotics. The MEM group received 25 mg/kg of MEM large animal (porcine) model of monolateral pneumonia Motos et al. Critical Care (2023) 27:60 Page 3 of 11 intravenously every 8 h, plus the inhaled saline. The version 8.0 (GraphPad Software, San Diego, CA, USA) MEM + AMK group received the inhaled saline and IV for the statistical analyses. MEM, plus a 3.2 mL dose of BAY 41–6551 inhalation solution (125 mg/mL of AMK, 400 mg total) every 12 h. Results The aerosolized antibiotic was delivered with the recently Study populations patented NEKTAR Pulmonary Drug Delivery System Eighteen Large White-Landrace female pigs (Novartis Pharmaceuticals, San Carlos, CA, USA), which (32.4 ± 1.8 kg) were included, of which 17 completed the is a high-efficiency vibrating mesh synchronized nebu- study. One pig in the control group was euthanized at lizer for the intrapulmonary delivery of inhaled AMK 76 h due to severe respiratory and hemodynamic insta- [17]. The nebulizer unit was connected to the ventilator bility and received only four saline doses by nebulizer. circuit between the Y-piece and endotracheal tube. Two cables connected the control module to both the nebu- AMK nebulization lizer and the air pressure-feedback unit (for breath syn- After meeting the diagnostic criteria for pneumo- chronization) in the inspiratory limb of the ventilator nia (Additional file 1: Table S1), each animal received 7 circuit [17]. nebulized doses of either saline (CONTROL and MEM During the procedure, animals were kept prone to groups) or AMK. Table 1 shows the ventilatory settings favor the bilateral distribution of the aerosol. We main- during nebulization and the adverse events. The CON- tained the ventilatory settings from before to after nebu- TROL group had the highest positive end-expiratory lization, including positive end-expiratory pressure, tidal pressure, while inspiratory flow and respiratory rate did volume, respiratory rate, F ­ iO2, and humidification (this not differ significantly among the study groups. Of note, system is not significantly affected by active humidifica- adverse events were comparable between pigs receiving tion [17]). Respiratory, hemodynamic, clinical, micro- saline and AMK. biological, and inflammatory assessments were recorded at scheduled time points throughout the experiment, as Primary outcome shown in Additional file 1: Figure S1. Drug distribution Lung P. aeruginosa burden was analyzed in the MEM and MEM + AMK groups at Figure 1 shows the differences in right lung tissue P. aer- the first aerosolization or the first meropenem adminis- uginosa concentration among study groups (p = 0.025), tration, as previously described [15]. with no statistically significant post hoc differences found The animals were killed 96 h after the bacterial chal- between the MEM and MEM + AMK groups (p > 0. 99). lenge, and the lungs were exposed, excised, and weighed. Overall, 88.9%, 61.1%, and 66.7% of the right lung lobes We obtained three samples from the most affected region were colonized by P. aeruginosa in the CONTROL, of each of the three right and two left lobes for histologi- MEM, and MEM + AMK groups, respectively (p = 0.16). cal, pharmacological, and microbiological assessments. Likewise, the left lung (Fig. 1) differed significantly Lung biopsies were processed and analyzed according to among study groups (p = 0.033), without significant vari- previously established protocols [24]. Further details are ations between antimicrobial-treated animals: 50% in the reported in the Online Data Supplement. CONTROL group were colonized by P. aeruginosa com- pared with 8.3% each in the remaining groups (p = 0.027). Overall, the median P. aeruginosa tissue concentration Statistical analysis across all pulmonary lobes was 0.72 (0.12–3.35) in the Data are reported as means ± standard deviation or MEM group, 0.90 (0–4.55) in the MEM + AMK group, medians (interquartile range) for normally and non- and 2.91 (1.75–5.69) l­og10 CFU/g in the CONTROL normally distributed continuous variables, respectively. group (p = 0.009). Categorical values are presented as percentages. Differ- An increase in the MIC of meropenem was found in ences among study groups and/or assessment times for three isolates, with two cases from the MEM group and continuous variables were analyzed by restricted maxi- one from the MEM + AMK group. Interestingly, left lung mum likelihood analysis, based on a repeated-measures colonization was driven by MEM-resistant isolates in approach (including study treatment and times of assess- both treated groups. ments or lobes). All two-sided comparisons among Figure 1 also shows the results of the histopathologi- groups were performed with Bonferroni correction. Dif- cal analysis of the 90 lung tissue samples. Significant ferences between categorical variables were analyzed by differences in histological features were found among Fisher’s exact test. A two-sided p-value ≤ 0.05 was con- the therapeutic groups (p = 0.038). Confluent pneu- sidered statistically significant. We used SAS 9.4 (SAS monia with bacteria and inflammatory cells between Institute Inc., Cary, NC, USA) and GraphPad Prism interlobular septa was observed in the CONTROL and Motos et al. Critical Care (2023) 27:60 Page 4 of 11 Table 1 Ventilator settings and adverse effects during nebulization Control MEM MEM + AMK p-value Group size, n 39 42 42 Ventilator setting RR (breaths/min) 24.1 ± 4.4 26.6 ± 9.7 25.6 ± 6.2 0.28 Inspiratory flow (L/min) 30.3 ± 5.6 33.7 ± 11.8 31.1 ± 7.0 0.18 PEEP ­(cmH2O) 9.9 ± 1.1 8.7 ± 1.4* 9.1 ± 0.9† < 0.001 Adverse effects, n (%) Coughing 2 (5.1) 1 (2.4) 1 (2.4) 0.73 Bronchoconstriction 0.0 0.0 0.0 1.00 Oxygen desaturation 0.0 0.0 2 (4.8) 0.14 Increased mucus production 26 (66.7) 32 (76.2) 31 (73.8) 0.61 Data are reported as means and standard deviations, medians (interquartile range), or (for adverse effects) the incidence among all nebulizations per group AMK, inhaled amikacin; MEM, IV meropenem; PEEP, positive end-expiratory pressure; RR, respiratory rate Post hoc analysis with Bonferroni correction: *p < 0.05 control versus MEM † p < 0.05 control versus MEM + AMK Fig. 1 Lung tissue P. aeruginosa burden. A–C Graphs depict overall, right, and left lung tissue P. aeruginosa colonization, respectively. The red dashed line represents the diagnostic clinical cut-off value for infection. In each group, the median value is indicated by the center horizontal line, and the 95% confidence intervals are indicated by the lower and upper horizontal lines. Red dots represent new isolates resistant to meropenem. D Histopathological analysis of the 90 tissue samples. E Representative photomicrographs of pulmonary tissue retrieved at autopsy. The lung injury score is reported per study group: the CONTROL and MEM + AMK groups show a pattern of confluent pneumonia with bacteria and inflammatory cells between interlobular septa; by contrast, the MEM group shows A more predominant histopathology pattern of pneumonia with inflammatory cells within the alveolar regions, but not confluent through the interlobular septal region. Hematoxylin & eosin staining. Magnification: Upper section =  × 40; Lower section =  × 100. Abbreviations: CONTROL, control; MEM, IV meropenem group; MEM + AMK, meropenem and inhaled amikacin group Motos et al. Critical Care (2023) 27:60 Page 5 of 11 MEM + AMK groups, whereas the MEM group showed significantly in the MEM group (p = 0.037). Specifically, a more predominant histopathology pattern of pneumo- the MIC in isolates from animals in the MEM group nia with inflammatory cells within the alveolar regions reached 64 (18–64) mg/L at the end of the study. and not confluent through the interlobular septal region. The lung appearance, lung/body weight ratio, and signs of pneumonia at gross examination during autopsy are Antibiotic pharmacokinetics detailed in Additional file 1: Figure S2. Signs of pneumo- Figure 3 depicts the concentrations of AMK and MEM nia were less frequent in the MEM + AMK group. in plasma, ELF, and tracheal secretions. Of note, we used a P. aeruginosa strain with an MIC of 0.25 mg/L Secondary outcomes for MEM and administered MEM at a dose of 25 mg/ Microbiological studies Kg every 8 h, aiming to achieve a drug concentration Figure 2 shows the colonization of tracheal aspirates and greater than the MIC for at least 6 h [15]. The plasma bronchoalveolar lavage (BAL) fluids by P. aeruginosa MEM concentration was 2.67 ± 2.11 µg/mL 4 h after colonization throughout the study. P. aeruginosa coloni- administration, while AMK was not detected at any zation within tracheal secretions differed among study time. The ELF concentrations of AMK and MEM groups (p < 0.001). Specifically, MEM + AMK therapy were 41.2 ± 37.8 and 6.47 ± 4.96 µg/mL, respectively had bactericidal effects on tracheal secretions, contrast- (p = 0.002). No paired differences in BAL concentration ing with CONTROL (p < 0.001) and MEM (p = 0.002) were found between infected and non-infected lungs throughout. The P. aeruginosa concentration in BAL (p = 0.63). MEM was not detected in 4 of 6 tracheal fluids varied among the groups (p = 0.012). Compared aspirates 4 h after dose, while two samples had low with the CONTROL group, MEM alone (p = 0.027) and MEM levels. By contrast, the median (IQR; min–max) combined with AMK (p = 0.026) had antipseudomonal AMK concentrations in tracheal aspirates were 4310 effects, though without differences between treated (12.5–26,900; 12.5–33,800) mg/L. animals. Unexpectedly, P. aeruginosa was detected Finally, MEM was detected in none of the pulmo- in only two animals in the MEM group and one in the nary tissue samples of any lung or in the most or least MEM + AMK group (p = 0.77). affected areas, while AMK was detected in some tissue The MIC of MEM for P. aeruginosa isolates remained samples (Fig. 3). Although not significant (p = 0.088), unchanged from the inoculated strain in the CON- more preserved areas showed higher concentrations of TROL and MEM + AMK groups, but increased AMK than damaged regions. Fig. 2 P. aeruginosa concentrations in tracheal secretions and bronchoalveolar lavage fluids. P. aeruginosa concentrations (log CFU/mL) and MICs are plotted as line graphs with means and standard errors of the mean. A P. aeruginosa concentrations in tracheal secretions differed among the study groups (p < 0.001) and over time (p < 0.001). Statistical significance against the CONTROL group for the MEM and MEM + AMK groups are shown by an asterisk and dagger, respectively. The double dagger shows a significant reduction of P. aeruginosa burden in the MEM + AMK group compared with the MEM group at 48 h (p < 0.001) and 96 h (p = 0.001). B Equally, P. aeruginosa concentrations in BAL fluids varied among treatments (p = 0.012), being significantly decreased in the MEM and MEM + AMK groups compared with the CONTROL group at 96 h. C The MIC of MEM for P. aeruginosa isolates did not change from that for the inoculated strain in the CONTROL and MEM + AMK groups, but increased to 11.5 ± 10.5 mg/L at 72 h and 46.8 ± 11.26 mg/L at 96 h in the MEM group. *p < 0.05 CONTROL versus MEM; †p < 0.05 CONTROL versus MEM + AMK; ‡ p < 0.05 MEM versus MEM + AMK. Abbreviations: AMK, inhaled amikacin; BAL, bronchoalveolar lavage; CFU, colony-forming unit; CONTROL, control; MEM, IV meropenem; MIC, minimum inhibitory concentration Motos et al. Critical Care (2023) 27:60 Page 6 of 11 Fig. 3 Antimicrobial pharmacokinetics. A, B AMK and MEM concentrations among study groups by assessment time and matrix. Of note, the AMK concentration only exceeded its MIC for P. aeruginosa in tracheal secretions. C ELF drug concentrations in the right medium (infected) and left upper (non-infected) lobes did not differ when analyzing paired concentrations (p = 0.63). D Non-significantly higher AMK tissue concentrations were observed in more preserved zones. Abbreviations: AMK, inhaled amikacin; ELF, epithelial lining fluid; MEM, IV meropenem; MIC, minimum inhibitory concentration Table 2 Clinical parameters Control MEM MEM + AMK p-value Temperature (°C) 38.3 ± 0.7 38.3 ± 0.6 38.1 ± 0.6 0.06 Tracheal secretions Quantity 2 (1–3) 1 (0–1)* 1 (0–1)† < 0.001 Purulent (%) 100 98.4 94.6 0.11 WBC ­(109/L) 13.3 (8.6–30.2) 16.0 (10.4–24.0) 13.6 (7.5–23.7) 0.91 Hemoglobin (g/L) 9.2 ± 0.9 9.4 ± 1.1 7.9 ± 1.4†, ‡ < 0.001 9 Platelets ­(10 /L) 313.3 ± 125.6 272.0 ± 104.0 249.4 ± 142.2 0.07 Creatinine (mg/dL) 1.00 (0.90–1.09) 1.07 (0.94–1.25)* 1.07 (1.01–1.22)† 0.017 PT (sec) 11.4 (10.5–12.0) 11.8 (10.5–13.8) 11.3 (10.5–12.4) 0.28 Data are reported as means ± standard deviations or as medians (interquartile range) for normally and non-normally distributed parameters, respectively. The PT reference range is 9–12 s in pigs AMK, inhaled amikacin; MEM, IV meropenem; PT, prothrombin time; PTT, thromboplastin time Post hoc analysis with Bonferroni correction: *p < 0.05 control versus MEM † p < 0.05 control versus MEM + AMK ‡ p < 0.05 MEM versus MEM + AMK Motos et al. Critical Care (2023) 27:60 Page 7 of 11 Clinical variables improved in the MEM and MEM + AMK groups, but Table 2 summarizes the clinical parameter data. Study without inhaled AMK offering additional benefit. We interventions reduced only the quantity of tracheal secre- observed a comparable increase in the pulmonary shunt tions (p < 0.001), while anemia was more pronounced in for the CONTROL group (p = 0.007). As shown in Addi- the MEM + AMK group (p < 0.001). Finally, creatinine tional file 1: Table S2, hemodynamic parameters did not levels were higher in the MEM and MEM + AMK groups improve in the MERO + AMK group, especially in com- than in the CONTROL group (p = 0.015), but without parison with the MEM group. significant differences between the treatment groups. Inflammatory markers Pulmonary mechanics and hemodynamics Figure 4 shows the serum concentrations of systemic Additional file 1: Figure S3 shows the data for pulmonary inflammatory markers. The P. aeruginosa challenge function and mechanics. This highlights that oxygenation caused a significant increase in all assessed cytokines, Fig. 4 Inflammatory markers. Bars show the fold-change from baseline (log2) and pneumonia diagnosis among study groups at the diagnosis of pneumonia and treatment with antimicrobials timepoints. A IL-1β varied significantly among study groups (p = 0.025) and over time (p = 0.043). Post hoc comparisons confirmed that IL-1β was downregulated by MEM + AMK treatment at 48, 72, and 96 h compared with the CONTROL and MEM groups. B–D IL-6, IL-8, and IL-10 did not vary among study groups, but did show a downward trend over time. Post hoc analysis with Bonferroni correction: *p < 0.05 CONTROL versus MEM; †p < 0.05 CONTROL versus MEM + AMK; ‡p < 0.05 MEM versus MEM + AMK. Abbreviations: AMK, inhaled amikacin; CONTROL, control; IL, interleukin; MEM, IV meropenem Motos et al. Critical Care (2023) 27:60 Page 8 of 11 except interleukin (IL) 8. However, antibiotic treat- in which P. aeruginosa colonized the left non-infected ments decreased serum levels of IL-1β, IL-6, and IL-10. lung, suggesting a role for acquired resistance. Of note, systemic IL-1β was significantly downregulated As expected, tracheal secretions revealed high AMK by inhaled AMK compared with the control and MEM concentrations and MEM concentrations below the groups (p = 0.025). Similarly, systemic IL-6 and Il-10 were limit of detection (0.10 mg/L). AMK was not detected upregulated at the diagnosis of pneumonia and showed in plasma though out the experiment, suggesting poor downward trends throughout treatment, but without AMK translocation from the lungs into the bloodstream, showing significant differences among study groups. reinforcing the idea that using such drugs can prevent systemic toxicity [18]. In contrast, high AMK concentra- tions in tracheal secretions correlate with rapidly steri- Discussion lized bronchial secretions. These results should not be This study describes the development of a porcine model neglected as they suggest a favorable prophylactic effect of monolateral severe P. aeruginosa pneumonia resist- on the progression from ventilator-associated tracheo- ant to AMK and susceptible to MEM, which we used bronchitis (VAT). Indeed, Palmer et al. showed a faster to explore the inhaled amikacin therapy. Our findings resolution of signs of infection when assessing the effects demonstrate that MEM treatment drove a reduction in of adjunctive nebulized antibiotic therapy in patients lung tissue concentration of P. aeruginosa, and that add- with VAT [28]. ing inhaled AMK as adjunctive therapy only reduced the AMK concentrations in the ELF were significantly higher bacterial burden of tracheal secretions. Unfortunately, than MEM concentrations, but we still below the MIC. we found no effect of the inhaled therapy on preventing The large difference between the MICs of MEM and AMK dissemination compared with systemic monotherapy, (i.e., 0.5 vs 256 mg/L, respectively) means that although although histological analysis revealed significantly fewer the AMK concentration in the ELF reached higher figures, signs of pneumonia than in the CONTROL group. the maximum concentration (at least 10 times the MIC Our results are consistent with the latest data from of the infecting pathogen) was not achieved. By contrast, randomized controlled trials [14, 19]. Indeed, in the the MEM free fraction concentration remained above the IASIS trial [14] found only a marginal effect with inhaled MIC achieved 100% of the time in the ELF. adjunctive amikacin/fosfomycin and only in tracheal We also measured the AMK and MEM levels in both aspirate samples, of which significantly fewer were posi- the infected and non-infected lungs, and found similar tive on days 3 and 7 compared to placebo. In the INHALE concentrations in the ELF bilaterally. However, when we trial [19], which used the same dosage of AMK (i.e., measured the AMK concentrations in tissue samples, 400 mg every 12 h), did show more frequent eradication we found a non-significant trend of higher antimicrobial among patients infected with P. aeruginosa and treated concentration in the more preserved areas. This indicated with inhaled AMK; however, this did not translate to that inhaled AMK did not efficiently reach poorly aerated improved survival. The reason why inhaled antibiotics do lung parenchyma. The deposition of inhaled drugs in the not prove benefits may be related to the susceptibility of lungs and in areas of pneumonia with loss of aeration is pathogens [25]. All enrolled patients in both trials were often questionable and may constitute a major limitation infected by pathogens susceptible to the intravenous when using this approach to treat ventilated patients [25]. antibiotics, as P. aeruginosa was susceptible to merope- Indeed, previous studies have shown that inhaled antibi- nem in our model. Furthermore, P. aeruginosa was resist- otics may not reach consolidated lung segments [12, 15], ant to AMK. Therefore, inhaled adjunctive therapy, even with research by Elman et al. in infected piglets revealing if effective, was unlikely to have a measurable effect. In that the pulmonary concentration of inhaled AMK was a recent meta-analysis of six randomized controlled tri- reduced in cases with extensive parenchymal infection als, inhaled adjunctive therapy achieved higher clinical [12]. In our setting, probably the infected lung has dif- resolution (odds ratio, 1.96; 95% CI 1.30–2.96) in patients ferent lung characteristics with a lower compliance and with pneumonia due to MDR pathogens, albeit not in higher airway resistance reducing drug distribution [29]. those with susceptible bacteria [26]. Notably, we did not change the ventilator settings dur- Even with 72 h of IV MEM therapy, inhaled AMK sup- ing nebulization, including the humidification and venti- pressed the emergence of the MEM-resistant subpopula- lator circuit, according to the manufacturer’s instructions. tion compared with systemic MEM therapy alone. The The European Investigators Network for Nebulized Anti- most recent clinical studies indicated that inhaled treat- biotics in Ventilator-associated Pneumonia agreed on spe- ment may hinder the development of resistance [27]. In cific recommendations for ventilator settings [30], but we our study, only one animal in the AMK group developed did not follow all of these in our protocol. Specifically, the MEM resistance, and of note, this was in the only animal recommendations specify that the mesh nebulizer should Motos et al. Critical Care (2023) 27:60 Page 9 of 11 be placed 10–15 cm before the Y-piece on the inspiratory anatomical differences of the tracheobronchial tree of limb, whereas we placed it only a few centimeters from piglets from human anatomy are critical factors that may the Y-piece [31]. Also, turning off active humidification affect lung deposition of inhaled particles [36]. is recommended during nebulization to avoid hygro- scopic growth and a rainout effect in the circuits and the endotracheal tube [32]. Although the penetration of AMK Conclusions may be modified by using our ventilator configuration, the In a swine model of monolateral MDR P. aeruginosa impact of this was not investigated. pneumonia resistant to the inhaled AMK and suscepti- As for how the study treatments affected other clinical, ble to the IV antibiotic, the use of AMK as an adjuvant pulmonary mechanics, and hemodynamics variables, we treatment offered no benefits in terms of pulmonary tis- found that even the most efficacious treatment had mini- sue colonization or the prevention of pathogen dissemi- mal impact. Only oxygenation was improved in treated nation. However, it improved bacterial eradication in animals, but without any additional benefit associated the proximal airways and hindered antibiotic resistance with the use of inhaled AMK, though it did have a slight and showed a non-significant trend to producing higher effect on the systemic IL-1β downregulation. AMK concentrations in more preserved lung areas. Finally, no adverse effects were reported. Creatinine levels were higher in both the MEM and MEM + AMK Abbreviations groups than in the CONTROL group, but without sig- ALAT Asociacion Latinoamericana del Torax nificant differences between the treatment groups, or AMK Amikacin BAL Bronchoalveolar lavage indeed, evidence of nephrotoxicity [17, 18]. CFU Colony-forming units Our findings help to delineate where inhaled antibiot- CRE Carbapenem-Resistant Enterobacterales ics might have utility in the management of VAP or ven- ELF Epithelial lining fluid ERS European Respiratory Society tilated HAP, whereas they may be limited to patients with ESCMID European Society of Clinical Microbiology and Infectious Diseases difficult-to-treat pathogens. Although their impact on ESICM European Society of Intensive Care Medicine preventing dissemination seems to be trifling, the high FiO2 Fraction of oxygen in the inhaled gas HAP Hospital-acquired pneumonia efficacy on tracheal secretions may provide a therapeu- IL Interleukin tic opportunity for VAT. Second, as observed over the IQR Interquartile range 100 h of this study, the use of a nebulized antibiotic may IV Intravenous MDR Multidrug resistant impede resistance to IV antibiotics in selected high-risk MEM Meropenem patients or in intensive care units with high MDR rates MIC Minimum inhibitory concentration [33]. Third, future studies should explore the optimal PaO2 Arterial partial pressure of oxygen PEEP Positive end-expiratory pressure method for measuring inhaled antibiotic concentrations RR Respiratory rate to ensure adequate dosage regimens to reach distal por- SEM Standard errors of the mean tions of highly infected pulmonary regions [34]. VAP Ventilator-associated pneumonia VAT Ventilator-associated tracheobronchitis This study presents some limitations that deserve fur- ther discussion. First, we used a 72-h course of therapy, which is unlikely in the most probable clinical scenario. Supplementary Information The online version contains supplementary material available at https://​doi.​ Second, the susceptibility of P. aeruginosa to MEM org/​10.​1186/​s13054-​023-​04331-x. makes it difficult to show the window of efficacy for inhaled AMK. Furthermore, only one strain was tested. Additional file 1. Supplementary methods and results Third, pharmacokinetic analyses were performed for only 4 h after dose administration, limiting the picture Acknowledgements of antimicrobial distribution to a brief period. Moreover, We thank Christina Sutherland of the Center of Anti-Infective Research & the differences between ELF and tissue samples may sug- Development (Hartford, CT, USA) and Pure Honey Technologies (Billerica, MA, USA) for her technical assistance with the MS-HPLC assays. We are in debt to gest that the former are not the best surrogate of pulmo- Laura Muñoz for her support in the MALDI-TOF analysis. We would also thank nary concentrations, particularly for inhaled drugs with Cristina Miralles for inclusion and processing all tissue samples. a high risk of bronchial tree contamination and where Author contributions their deposition is important [34, 35]. The AMK dose HY and AM participated in protocol development, study design, study man‑ may also be questioned because it is a concentration- agement, data collection, statistical analysis, data interpretation, and writing dependent antibiotic, and twice-daily administration the first draft of the report. MY, AMe, DB, JB, RC, FP, GF, MC, AFS, LFB, GLB and KK participated in data collection, data interpretation, and critical review similar to the IASIS trial may have produced variable ELF of the first draft of the report. LFB, MR, DM, DP, AA, PP, and JV participated concentrations [14, 19]. Finally, we studied young animals in data interpretation and revision and review of the report. GLB and AT with no comorbidities under deep sedation. Moreover, participated in protocol development, study design, study management, data Motos et al. Critical Care (2023) 27:60 Page 10 of 11 interpretation, and revision and review of the report. All authors read and pneumonia (HAP)/ventilator-associated pneumonia (VAP) of the Euro‑ approved the final manuscript. pean respiratory society (ERS), European society of intensive care medi‑ cine (ESICM), European society of clinical microbiology and infectious Funding diseases (ESCMID) and Asociacion Latinoamericana del Torax (ALAT). Eur Financial support was provided by the Institut d’Investigacions Biomèdiques Respir J. 2017;50(3):1700582. August Pi I Sunyer (IDIBAPS); Ministerio de Ciencia e Innovación; Centro 2. Kalil AC, Metersky ML, Klompas M, Muscedere J, Sweeney DA, Palmer de Investigación Biomédica en Red- Enfermedades Respiratorias (CIBERES, LB, et al. Management of adults with hospital-acquired and ventilator- CB06/06/0028); Quality Research Group from Generalitat de Catalunya associated pneumonia: 2016 clinical practice guidelines by the infectious (SGR/944); University of Barcelona; “ICREA Academia 2019” award to Prof. diseases society of America and the American thoracic society. Clin Infect Antoni Torres from the University of Barcelona; La Marató de TV3 (201831-10); Dis. 2016;63(5):e61–111. “La Caixa” Foundation (LCF/PR/HR19/52160019). 3. WHO Regional Office for Europe/European Centre for Disease Prevention and Control. Antimicrobial resistance surveillance in Europe 2022–2020 Availability of data and materials data. WHO Regional Office for Europe. 2022. The datasets used and/or analyzed during the current study are available from 4. Tamma PD, Aitken SL, Bonomo RA, Mathers AJ, van Duin D, Clancy CJ. the corresponding author on reasonable request. Infectious diseases society of America guidance on the treatment of extended-spectrum beta-lactamase producing enterobacterales (ESBL-E), carbapenem-resistant enterobacterales (CRE), and pseudomonas aerugi‑ Declarations nosa with difficult-to-treat resistance (DTR-P. aeruginosa). Clin Infect Dis. 2021;72(7):1109–16. Ethics approval and consent to participate 5. Roberts JA, Lipman J. Pharmacokinetic issues for antibiotics in the criti‑ This study was conducted according to the European guidelines for the Care cally ill patient. Crit Care Med. 2009;37(3):840–51. of Animal Experimentation at the Division of Animal Experimentation, Depart‑ 6. Palmer LB. Aerosolized antibiotics in the intensive care unit. Clin Chest ment of Pulmonary and Critical Care Medicine, Hospital Clínic, Barcelona, Med. 2011;32(3):559–74. Spain. Documented approval from University of Barcelona Institutional Review 7. Goldstein I, Wallet F, Nicolas-Robin A, Ferrari F, Marquette CH, Rouby JJ. Board was obtained prior to the study started (number approval 9772). Lung deposition and efficiency of nebulized amikacin during Escheri‑ chia coli pneumonia in ventilated piglets. Am J Respir Crit Care Med. Consent for publication 2002;166(10):1375–81. Not applicable. 8. Niederman MS, Chastre J, Corkery K, Fink JB, Luyt CE, Garcia MS. BAY41- 6551 achieves bactericidal tracheal aspirate amikacin concentrations Competing interest in mechanically ventilated patients with Gram-negative pneumonia. A. Torres and G. Li Bassi received an unrestrictive grant from Bayer AG (Berlin, Intensive Care Med. 2012;38(2):263–71. Germany) through their affiliated institution. Bayer AG, the manufacturer 9. Pien FD, Ho PW. Antimicrobial spectrum, pharmacology, adverse of inhaled amikacin, provided funds to support this research. Bayer AG had effects, and therapeutic use of amikacin sulfate. Am J Hosp Pharm. no role in the data collection, data analysis, or data interpretation. Bayer AG 1981;38(7):981–9. reviewed the final manuscript. 10. Bodey GP, Valdivieso M, Feld R, Rodriguez V. Pharmacology of amikacin in humans. Antimicrob Agents Chemother. 1974;5(5):508–12. Author details 11. Ehrmann S, Chastre J, Diot P, Lu Q. Nebulized antibiotics in mechanically 1 Servei de Pneumologia i Al•lèrgia Respiratòria, Pneumology Depart‑ ventilated patients: a challenge for translational research from technol‑ ment, Hospital Clínic, Thorax Institute, Calle Villarroel 170, Esc 6/8 Planta 2, ogy to clinical care. Ann Intensive Care. 2017;7(1):78. 08036 Barcelona, Spain. 2 Institut d’Investigacions Biomèdiques August Pi i 12. Elman M, Goldstein I, Marquette CH, Wallet F, Lenaour G, Rouby JJ, et al. Sunyer (IDIBAPS), Barcelona, Spain. 3 University of Barcelona, Barcelona, Spain. Influence of lung aeration on pulmonary concentrations of nebulized 4 Centro de Investigación Biomedica En Red- Enfermedades Respiratorias and intravenous amikacin in ventilated piglets with severe bronchopneu‑ (CIBERES), Barcelona, Spain. 5 Fondazione IRCCS Cà Granda Ospedale Maggiore monia. Anesthesiology. 2002;97(1):199–206. Policlinico Internal Medicine Department, Respiratory Unit and Adult Cystic 13. Goldstein I, Wallet F, Robert J, Becquemin MH, Marquette CH, Rouby Fibrosis Center, and Department of Pathophysiology and Transplantation, JJ. Lung tissue concentrations of nebulized amikacin during mechani‑ Università degli Studi di Milano, Milan, Italy. 6 Anesthesia and Critical Care, San cal ventilation in piglets with healthy lungs. Am J Respir Crit Care Med. Martino Policlinico Hospital, IRCCS for Oncology and Neurosciences, Genoa, 2002;165(2):171–5. Italy. 7 Department of Pathology, Hospital Clinic, Barcelona, Spain. 8 Critical Care 14. 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