Skip to main content

Inhaled amikacin for pneumonia treatment and dissemination prevention: an experimental model of severe monolateral Pseudomonas aeruginosa pneumonia



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.


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.


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 penetration in low-affected lung areas.


In a swine model of monolateral MDR P. aeruginosa pneumonia, resistant to the inhaled AMK and susceptible 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.


Current guidelines for the management of hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) stress the need for preventive strategies and accurate etiologic diagnosis [1, 2]. Unfortunately, routinely employed antibiotic agents are often ineffective against gram-negative pathogens, with multidrug resistance (MDR) a common problem, even in combination therapy [3]. The treatment of severe gram-negative pneumonia therefore remains a major challenge [3, 4].

Pseudomonas aeruginosa pneumonia is usually treated with a combination of intravenous antibiotics to ensure the adequate treatment of MDR isolates [1, 2]. However, insufficient lung distribution and adverse side effects limit this approach [5], leading to inhaled antibiotics gaining increasing attention as a site-specific treatment [6]. Inhaled has have improved bactericidal properties by ensuring high concentrations in tracheal secretions and epithelial lining fluid (ELF) [7, 8], while lowering systemic concentrations to curtail its nephrotoxic and ototoxic effects [9, 10]. Experimental and clinical studies have elicited conflicting results when assessing inhaled AMK in VAP [7, 11,12,13,14,15,16,17,18,19]. Unfortunately, the latest two randomized clinical trials of inhaled AMK have failed to demonstrate any benefit in primary outcomes (i.e., change in clinical pulmonary infection score and survival at days 28–32, respectively) for mechanically ventilated patients with pneumonia [14, 19]. The most recent management guidelines for HAP/VAP advise against the routine use of adjunctive inhaled therapy, recommending it only for susceptible MDR bacteria [2, 20]. Given that study design, dosing, and nebulization technique may have obscured the impact of inhaled AMK on VAP dissemination and the emergence of resistance [21, 22] further research is warranted.

In this study, we aimed to analyze the effects of inhaled AMK of monolateral pneumonia caused by P. aeruginosa in pigs.

Materials and methods

Study design

We analyzed the effects of inhaled AMK in a translational large animal (porcine) model of monolateral pneumonia caused by P. aeruginosa, according to the ARRIVE guidelines [23]. This study was conducted according to the European guidelines for the Care of Animal Experimentation at the Division of Animal Experimentation, Department of Pulmonary and Critical Care Medicine, Hospital Clínic, Barcelona, Spain. Documented approval from the appropriate Institutional Review Board was obtained prior to the study started (number approval 9772).

The animal model

Eighteen female Large White-Landrace pigs underwent 96 h of mechanical ventilation. Animal preparation, including anesthesia induction and maintenance, airway management, and hemodynamic invasive monitoring were performed as previously described [24]. Immediately after preparation and stabilization, each animal was challenged with 15 mL of 107 CFU/mL log-phase culture of AMK-resistant and MEM-susceptible P. aeruginosa at minimum inhibitory concentrations (MICs) of > 256 and 0.25 mg/L, respectively. We did select P. aeruginosa strain resistant to AMK to consider the worst scenario for AMK efficacy, but susceptibility to IV antibiotic was still ensured.

The challenge was instilled bronchoscopically into the right upper, middle, and lower lobes, with the animals kept in a lateral-right slight Trendelenburg position to ensure a right predominance of infection. This was maintained for 24 h, before the lateral position was changed from one side to the other every 6 h. The pneumonia diagnosis was confirmed 24 h after the bacterial challenge based on a decrease in the arterial partial pressure of oxygen (PaO2)/fraction of oxygen in the inhaled gas (FiO2), plus at least one of the following signs of infection: temperature ≥ 38.5 °C, leukocytosis ≥ 20.109 cells/L, or purulent secretions.


The 18 animals were randomized into one of three 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, n = 6). The CONTROL group received a 4-mL inhaled solution of 0.9% NaCl every 12 h and no intravenous antibiotics. The MEM group received 25 mg/kg of MEM intravenously every 8 h, plus the inhaled saline. The MEM + AMK group received the inhaled saline and IV MEM, plus a 3.2 mL dose of BAY 41–6551 inhalation solution (125 mg/mL of AMK, 400 mg total) every 12 h. The aerosolized antibiotic was delivered with the recently patented NEKTAR Pulmonary Drug Delivery System (Novartis Pharmaceuticals, San Carlos, CA, USA), which is a high-efficiency vibrating mesh synchronized nebulizer for the intrapulmonary delivery of inhaled AMK [17]. The nebulizer unit was connected to the ventilator circuit between the Y-piece and endotracheal tube. Two cables connected the control module to both the nebulizer and the air pressure-feedback unit (for breath synchronization) in the inspiratory limb of the ventilator circuit [17].

During the procedure, animals were kept prone to favor the bilateral distribution of the aerosol. We maintained the ventilatory settings from before to after nebulization, including positive end-expiratory pressure, tidal volume, respiratory rate, FiO2, and humidification (this system is not significantly affected by active humidification [17]). Respiratory, hemodynamic, clinical, microbiological, and inflammatory assessments were recorded at scheduled time points throughout the experiment, as shown in Additional file 1: Figure S1. Drug distribution was analyzed in the MEM and MEM + AMK groups at the first aerosolization or the first meropenem administration, as previously described [15].

The animals were killed 96 h after the bacterial challenge, and the lungs were exposed, excised, and weighed. We obtained three samples from the most affected region of each of the three right and two left lobes for histological, pharmacological, and microbiological assessments. Lung biopsies were processed and analyzed according to previously established protocols [24]. Further details are reported in the Online Data Supplement.

Statistical analysis

Data are reported as means ± standard deviation or medians (interquartile range) for normally and non-normally distributed continuous variables, respectively. Categorical values are presented as percentages. Differences among study groups and/or assessment times for continuous variables were analyzed by restricted maximum likelihood analysis, based on a repeated-measures approach (including study treatment and times of assessments or lobes). All two-sided comparisons among groups were performed with Bonferroni correction. Differences between categorical variables were analyzed by Fisher’s exact test. A two-sided p-value ≤ 0.05 was considered statistically significant. We used SAS 9.4 (SAS Institute Inc., Cary, NC, USA) and GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA) for the statistical analyses.


Study populations

Eighteen Large White-Landrace female pigs (32.4 ± 1.8 kg) were included, of which 17 completed the study. One pig in the control group was euthanized at 76 h due to severe respiratory and hemodynamic instability and received only four saline doses by nebulizer.

AMK nebulization

After meeting the diagnostic criteria for pneumonia (Additional file 1: Table S1), each animal received 7 nebulized doses of either saline (CONTROL and MEM groups) or AMK. Table 1 shows the ventilatory settings during nebulization and the adverse events. The CONTROL group had the highest positive end-expiratory pressure, while inspiratory flow and respiratory rate did not differ significantly among the study groups. Of note, adverse events were comparable between pigs receiving saline and AMK.

Table 1 Ventilator settings and adverse effects during nebulization

Primary outcome

Lung P. aeruginosa burden

Figure 1 shows the differences in right lung tissue P. aeruginosa concentration among study groups (p = 0.025), with no statistically significant post hoc differences found between the MEM and MEM + AMK groups (p > 0. 99). Overall, 88.9%, 61.1%, and 66.7% of the right lung lobes were colonized by P. aeruginosa in the CONTROL, MEM, and MEM + AMK groups, respectively (p = 0.16). Likewise, the left lung (Fig. 1) differed significantly among study groups (p = 0.033), without significant variations between antimicrobial-treated animals: 50% in the CONTROL group were colonized by P. aeruginosa compared with 8.3% each in the remaining groups (p = 0.027). Overall, the median P. aeruginosa tissue concentration across all pulmonary lobes was 0.72 (0.12–3.35) in the MEM group, 0.90 (0–4.55) in the MEM + AMK group, and 2.91 (1.75–5.69) log10 CFU/g in the CONTROL group (p = 0.009).

Fig. 1
figure 1

Lung tissue P. aeruginosa burden. AC 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

An increase in the MIC of meropenem was found in three isolates, with two cases from the MEM group and one from the MEM + AMK group. Interestingly, left lung colonization was driven by MEM-resistant isolates in both treated groups.

Figure 1 also shows the results of the histopathological analysis of the 90 lung tissue samples. Significant differences in histological features were found among the therapeutic groups (p = 0.038). Confluent pneumonia with bacteria and inflammatory cells between interlobular septa was observed in the CONTROL and MEM + AMK groups, whereas the MEM group showed a more predominant histopathology pattern of pneumonia with inflammatory cells within the alveolar regions 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 detailed in Additional file 1: Figure S2. Signs of pneumonia were less frequent in the MEM + AMK group.

Secondary outcomes

Microbiological studies

Figure 2 shows the colonization of tracheal aspirates and bronchoalveolar lavage (BAL) fluids by P. aeruginosa colonization throughout the study. P. aeruginosa colonization within tracheal secretions differed among study groups (p < 0.001). Specifically, MEM + AMK therapy had bactericidal effects on tracheal secretions, contrasting with CONTROL (p < 0.001) and MEM (p = 0.002) throughout. The P. aeruginosa concentration in BAL fluids varied among the groups (p = 0.012). Compared with the CONTROL group, MEM alone (p = 0.027) and combined with AMK (p = 0.026) had antipseudomonal effects, though without differences between treated animals. Unexpectedly, P. aeruginosa was detected in only two animals in the MEM group and one in the MEM + AMK group (p = 0.77).

Fig. 2
figure 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

The MIC of MEM for P. aeruginosa isolates remained unchanged from the inoculated strain in the CONTROL and MEM + AMK groups, but increased significantly in the MEM group (p = 0.037). Specifically, the MIC in isolates from animals in the MEM group reached 64 (18–64) mg/L at the end of the study.

Antibiotic pharmacokinetics

Figure 3 depicts the concentrations of AMK and MEM in plasma, ELF, and tracheal secretions. Of note, we used a P. aeruginosa strain with an MIC of 0.25 mg/L for MEM and administered MEM at a dose of 25 mg/Kg every 8 h, aiming to achieve a drug concentration greater than the MIC for at least 6 h [15]. The plasma MEM concentration was 2.67 ± 2.11 µg/mL 4 h after administration, while AMK was not detected at any time. The ELF concentrations of AMK and MEM were 41.2 ± 37.8 and 6.47 ± 4.96 µg/mL, respectively (p = 0.002). No paired differences in BAL concentration were found between infected and non-infected lungs (p = 0.63). MEM was not detected in 4 of 6 tracheal aspirates 4 h after dose, while two samples had low MEM levels. By contrast, the median (IQR; min–max) AMK concentrations in tracheal aspirates were 4310 (12.5–26,900; 12.5–33,800) mg/L.

Fig. 3
figure 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

Finally, MEM was detected in none of the pulmonary tissue samples of any lung or in the most or least affected areas, while AMK was detected in some tissue samples (Fig. 3). Although not significant (p = 0.088), more preserved areas showed higher concentrations of AMK than damaged regions.

Clinical variables

Table 2 summarizes the clinical parameter data. Study interventions reduced only the quantity of tracheal secretions (p < 0.001), while anemia was more pronounced in the MEM + AMK group (p < 0.001). Finally, creatinine levels were higher in the MEM and MEM + AMK groups than in the CONTROL group (p = 0.015), but without significant differences between the treatment groups.

Table 2 Clinical parameters

Pulmonary mechanics and hemodynamics

Additional file 1: Figure S3 shows the data for pulmonary function and mechanics. This highlights that oxygenation improved in the MEM and MEM + AMK groups, but without inhaled AMK offering additional benefit. We observed a comparable increase in the pulmonary shunt for the CONTROL group (p = 0.007). As shown in Additional file 1: Table S2, hemodynamic parameters did not improve in the MERO + AMK group, especially in comparison with the MEM group.

Inflammatory markers

Figure 4 shows the serum concentrations of systemic inflammatory markers. The P. aeruginosa challenge caused a significant increase in all assessed cytokines, except interleukin (IL) 8. However, antibiotic treatments decreased serum levels of IL-1β, IL-6, and IL-10. Of note, systemic IL-1β was significantly downregulated by inhaled AMK compared with the control and MEM groups (p = 0.025). Similarly, systemic IL-6 and Il-10 were upregulated at the diagnosis of pneumonia and showed downward trends throughout treatment, but without showing significant differences among study groups.

Fig. 4
figure 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. BD 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


This study describes the development of a porcine model of monolateral severe P. aeruginosa pneumonia resistant to AMK and susceptible to MEM, which we used to explore the inhaled amikacin therapy. Our findings demonstrate that MEM treatment drove a reduction in lung tissue concentration of P. aeruginosa, and that adding inhaled AMK as adjunctive therapy only reduced the bacterial burden of tracheal secretions. Unfortunately, we found no effect of the inhaled therapy on preventing dissemination compared with systemic monotherapy, although histological analysis revealed significantly fewer signs of pneumonia than in the CONTROL group.

Our results are consistent with the latest data from randomized controlled trials [14, 19]. Indeed, in the IASIS trial [14] found only a marginal effect with inhaled adjunctive amikacin/fosfomycin and only in tracheal aspirate samples, of which significantly fewer were positive on days 3 and 7 compared to placebo. In the INHALE trial [19], which used the same dosage of AMK (i.e., 400 mg every 12 h), did show more frequent eradication among patients infected with P. aeruginosa and treated with inhaled AMK; however, this did not translate to improved survival. The reason why inhaled antibiotics do not prove benefits may be related to the susceptibility of pathogens [25]. All enrolled patients in both trials were infected by pathogens susceptible to the intravenous antibiotics, as P. aeruginosa was susceptible to meropenem in our model. Furthermore, P. aeruginosa was resistant to AMK. Therefore, inhaled adjunctive therapy, even if effective, was unlikely to have a measurable effect. In a recent meta-analysis of six randomized controlled trials, inhaled adjunctive therapy achieved higher clinical resolution (odds ratio, 1.96; 95% CI 1.30–2.96) in patients with pneumonia due to MDR pathogens, albeit not in those with susceptible bacteria [26].

Even with 72 h of IV MEM therapy, inhaled AMK suppressed the emergence of the MEM-resistant subpopulation compared with systemic MEM therapy alone. The most recent clinical studies indicated that inhaled treatment may hinder the development of resistance [27]. In our study, only one animal in the AMK group developed MEM resistance, and of note, this was in the only animal in which P. aeruginosa colonized the left non-infected lung, suggesting a role for acquired resistance.

As expected, tracheal secretions revealed high AMK concentrations and MEM concentrations below the limit of detection (0.10 mg/L). AMK was not detected in plasma though out the experiment, suggesting poor AMK translocation from the lungs into the bloodstream, reinforcing the idea that using such drugs can prevent systemic toxicity [18]. In contrast, high AMK concentrations in tracheal secretions correlate with rapidly sterilized bronchial secretions. These results should not be neglected as they suggest a favorable prophylactic effect on the progression from ventilator-associated tracheobronchitis (VAT). Indeed, Palmer et al. showed a faster resolution of signs of infection when assessing the effects of adjunctive nebulized antibiotic therapy in patients with VAT [28].

AMK concentrations in the ELF were significantly higher than MEM concentrations, but we still below the MIC. The large difference between the MICs of MEM and AMK (i.e., 0.5 vs 256 mg/L, respectively) means that although the AMK concentration in the ELF reached higher figures, the maximum concentration (at least 10 times the MIC of the infecting pathogen) was not achieved. By contrast, the MEM free fraction concentration remained above the MIC achieved 100% of the time in the ELF.

We also measured the AMK and MEM levels in both the infected and non-infected lungs, and found similar concentrations in the ELF bilaterally. However, when we measured the AMK concentrations in tissue samples, we found a non-significant trend of higher antimicrobial concentration in the more preserved areas. This indicated that inhaled AMK did not efficiently reach poorly aerated lung parenchyma. The deposition of inhaled drugs in the lungs and in areas of pneumonia with loss of aeration is often questionable and may constitute a major limitation when using this approach to treat ventilated patients [25]. Indeed, previous studies have shown that inhaled antibiotics may not reach consolidated lung segments [12, 15], with research by Elman et al. in infected piglets revealing that the pulmonary concentration of inhaled AMK was reduced in cases with extensive parenchymal infection [12]. In our setting, probably the infected lung has different lung characteristics with a lower compliance and higher airway resistance reducing drug distribution [29].

Notably, we did not change the ventilator settings during nebulization, including the humidification and ventilator circuit, according to the manufacturer’s instructions. The European Investigators Network for Nebulized Antibiotics in Ventilator-associated Pneumonia agreed on specific recommendations for ventilator settings [30], but we did not follow all of these in our protocol. Specifically, the recommendations specify that the mesh nebulizer should be placed 10–15 cm before the Y-piece on the inspiratory limb, whereas we placed it only a few centimeters from the Y-piece [31]. Also, turning off active humidification is recommended during nebulization to avoid hygroscopic growth and a rainout effect in the circuits and the endotracheal tube [32]. Although the penetration of AMK may be modified by using our ventilator configuration, the impact of this was not investigated.

As for how the study treatments affected other clinical, pulmonary mechanics, and hemodynamics variables, we found that even the most efficacious treatment had minimal impact. Only oxygenation was improved in treated animals, but without any additional benefit associated with the use of inhaled AMK, though it did have a slight effect on the systemic IL-1β downregulation.

Finally, no adverse effects were reported. Creatinine levels were higher in both the MEM and MEM + AMK groups than in the CONTROL group, but without significant differences between the treatment groups, or indeed, evidence of nephrotoxicity [17, 18].

Our findings help to delineate where inhaled antibiotics might have utility in the management of VAP or ventilated HAP, whereas they may be limited to patients with difficult-to-treat pathogens. Although their impact on preventing dissemination seems to be trifling, the high efficacy on tracheal secretions may provide a therapeutic opportunity for VAT. Second, as observed over the 100 h of this study, the use of a nebulized antibiotic may impede resistance to IV antibiotics in selected high-risk patients or in intensive care units with high MDR rates [33]. Third, future studies should explore the optimal method for measuring inhaled antibiotic concentrations to ensure adequate dosage regimens to reach distal portions of highly infected pulmonary regions [34].

This study presents some limitations that deserve further discussion. First, we used a 72-h course of therapy, which is unlikely in the most probable clinical scenario. Second, the susceptibility of P. aeruginosa to MEM makes it difficult to show the window of efficacy for inhaled AMK. Furthermore, only one strain was tested. Third, pharmacokinetic analyses were performed for only 4 h after dose administration, limiting the picture of antimicrobial distribution to a brief period. Moreover, the differences between ELF and tissue samples may suggest that the former are not the best surrogate of pulmonary concentrations, particularly for inhaled drugs with a high risk of bronchial tree contamination and where their deposition is important [34, 35]. The AMK dose may also be questioned because it is a concentration-dependent antibiotic, and twice-daily administration similar to the IASIS trial may have produced variable ELF concentrations [14, 19]. Finally, we studied young animals with no comorbidities under deep sedation. Moreover, anatomical differences of the tracheobronchial tree of piglets from human anatomy are critical factors that may affect lung deposition of inhaled particles [36].


In a swine model of monolateral MDR P. aeruginosa pneumonia resistant to the inhaled AMK and susceptible to the IV antibiotic, the use of AMK as an adjuvant treatment offered no benefits in terms of pulmonary tissue colonization or the prevention of pathogen dissemination. However, it improved bacterial eradication in the proximal airways and hindered antibiotic resistance and showed a non-significant trend to producing higher AMK concentrations in more preserved lung areas.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.



Asociacion Latinoamericana del Torax




Bronchoalveolar lavage


Colony-forming units


Carbapenem-Resistant Enterobacterales


Epithelial lining fluid


European Respiratory Society


European Society of Clinical Microbiology and Infectious Diseases


European Society of Intensive Care Medicine

FiO2 :

Fraction of oxygen in the inhaled gas


Hospital-acquired pneumonia




Interquartile range




Multidrug resistant




Minimum inhibitory concentration

PaO2 :

Arterial partial pressure of oxygen


Positive end-expiratory pressure


Respiratory rate


Standard errors of the mean


Ventilator-associated pneumonia


Ventilator-associated tracheobronchitis


  1. Torres A, Niederman MS, Chastre J, Ewig S, Fernandez-Vandellos P, Hanberger H, et al. International ERS/ESICM/ESCMID/ALAT guidelines for the management of hospital-acquired pneumonia and ventilator-associated pneumonia: guidelines for the management of hospital-acquired pneumonia (HAP)/ventilator-associated pneumonia (VAP) of the European respiratory society (ERS), European society of intensive care medicine (ESICM), European society of clinical microbiology and infectious diseases (ESCMID) and Asociacion Latinoamericana del Torax (ALAT). Eur Respir J. 2017;50(3):1700582.

    Article  PubMed  Google Scholar 

  2. Kalil AC, Metersky ML, Klompas M, Muscedere J, Sweeney DA, Palmer LB, et al. Management of adults with hospital-acquired and ventilator-associated pneumonia: 2016 clinical practice guidelines by the infectious diseases society of America and the American thoracic society. Clin Infect Dis. 2016;63(5):e61–111.

    Article  PubMed  PubMed Central  Google Scholar 

  3. WHO Regional Office for Europe/European Centre for Disease Prevention and Control. Antimicrobial resistance surveillance in Europe 2022–2020 data. WHO Regional Office for Europe. 2022.

  4. Tamma PD, Aitken SL, Bonomo RA, Mathers AJ, van Duin D, Clancy CJ. Infectious diseases society of America guidance on the treatment of extended-spectrum beta-lactamase producing enterobacterales (ESBL-E), carbapenem-resistant enterobacterales (CRE), and pseudomonas aeruginosa with difficult-to-treat resistance (DTR-P. aeruginosa). Clin Infect Dis. 2021;72(7):1109–16.

    Article  PubMed  Google Scholar 

  5. Roberts JA, Lipman J. Pharmacokinetic issues for antibiotics in the critically ill patient. Crit Care Med. 2009;37(3):840–51.

    Article  CAS  PubMed  Google Scholar 

  6. Palmer LB. Aerosolized antibiotics in the intensive care unit. Clin Chest Med. 2011;32(3):559–74.

    Article  PubMed  Google Scholar 

  7. Goldstein I, Wallet F, Nicolas-Robin A, Ferrari F, Marquette CH, Rouby JJ. Lung deposition and efficiency of nebulized amikacin during Escherichia coli pneumonia in ventilated piglets. Am J Respir Crit Care Med. 2002;166(10):1375–81.

    Article  PubMed  Google Scholar 

  8. Niederman MS, Chastre J, Corkery K, Fink JB, Luyt CE, Garcia MS. BAY41-6551 achieves bactericidal tracheal aspirate amikacin concentrations in mechanically ventilated patients with Gram-negative pneumonia. Intensive Care Med. 2012;38(2):263–71.

    Article  CAS  PubMed  Google Scholar 

  9. Pien FD, Ho PW. Antimicrobial spectrum, pharmacology, adverse effects, and therapeutic use of amikacin sulfate. Am J Hosp Pharm. 1981;38(7):981–9.

    CAS  PubMed  Google Scholar 

  10. Bodey GP, Valdivieso M, Feld R, Rodriguez V. Pharmacology of amikacin in humans. Antimicrob Agents Chemother. 1974;5(5):508–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ehrmann S, Chastre J, Diot P, Lu Q. Nebulized antibiotics in mechanically ventilated patients: a challenge for translational research from technology to clinical care. Ann Intensive Care. 2017;7(1):78.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Elman M, Goldstein I, Marquette CH, Wallet F, Lenaour G, Rouby JJ, et al. Influence of lung aeration on pulmonary concentrations of nebulized and intravenous amikacin in ventilated piglets with severe bronchopneumonia. Anesthesiology. 2002;97(1):199–206.

    Article  CAS  PubMed  Google Scholar 

  13. Goldstein I, Wallet F, Robert J, Becquemin MH, Marquette CH, Rouby JJ. Lung tissue concentrations of nebulized amikacin during mechanical ventilation in piglets with healthy lungs. Am J Respir Crit Care Med. 2002;165(2):171–5.

    Article  PubMed  Google Scholar 

  14. Kollef MH, Ricard JD, Roux D, Francois B, Ischaki E, Rozgonyi Z, et al. A randomized trial of the amikacin fosfomycin inhalation system for the adjunctive therapy of gram-negative ventilator-associated pneumonia: IASIS trial. Chest. 2017;151(6):1239–46.

    Article  PubMed  Google Scholar 

  15. Li Bassi G, Motos A, Fernandez-Barat L, Aguilera Xiol E, Chiurazzi C, Senussi T, et al. Nebulized amikacin and fosfomycin for severe pseudomonas aeruginosa pneumonia: an experimental study. Crit Care Med. 2019;47(6):e470–7.

    Article  CAS  PubMed  Google Scholar 

  16. Lu Q, Yang J, Liu Z, Gutierrez C, Aymard G, Rouby JJ, et al. Nebulized ceftazidime and amikacin in ventilator-associated pneumonia caused by Pseudomonas aeruginosa. Am J Respir Crit Care Med. 2011;184(1):106–15.

    Article  CAS  PubMed  Google Scholar 

  17. Luyt CE, Clavel M, Guntupalli K, Johannigman J, Kennedy JI, Wood C, et al. Pharmacokinetics and lung delivery of PDDS-aerosolized amikacin (NKTR-061) in intubated and mechanically ventilated patients with nosocomial pneumonia. Crit Care. 2009;13(6):R200.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Luyt CE, Eldon MA, Stass H, Gribben D, Corkery K, Chastre J. Pharmacokinetics and tolerability of amikacin administered as BAY41-6551 aerosol in mechanically ventilated patients with gram-negative pneumonia and acute renal failure. J Aerosol Med Pulm Drug Deliv. 2011;24(4):183–90.

    Article  CAS  PubMed  Google Scholar 

  19. Niederman MS, Alder J, Bassetti M, Boateng F, Cao B, Corkery K, et al. Inhaled amikacin adjunctive to intravenous standard-of-care antibiotics in mechanically ventilated patients with Gram-negative pneumonia (INHALE): a double-blind, randomised, placebo-controlled, phase 3, superiority trial. Lancet Infect Dis. 2020;20(3):330–40.

    Article  CAS  PubMed  Google Scholar 

  20. Rello J, Sole-Lleonart C, Rouby JJ, Chastre J, Blot S, Poulakou G, et al. Use of nebulized antimicrobials for the treatment of respiratory infections in invasively mechanically ventilated adults: a position paper from the European society of clinical microbiology and infectious diseases. Clin Microbiol Infect. 2017;23(9):629–39.

    Article  CAS  PubMed  Google Scholar 

  21. Torres A, Motos A, Battaglini D, Li BG. Inhaled amikacin for severe gram-negative pulmonary infections in the intensive care unit: current status and future prospects. Crit Care. 2018;22(1):343.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Rouby JJ, Monsel A, Leone M, Mimoz O, Laterre PF, Pugin J. The IASIS, INHALE and VAPORISE trials. Reasons for a triple failure: study design, aminoglycosides dosing and technique of nebulisation. Anaesth Crit Care Pain Med. 2020;39(2):179–83.

    Article  PubMed  Google Scholar 

  23. Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. BMJ Open Sci. 2020;4(1):100115.

    Google Scholar 

  24. Luna CM, Baquero S, Gando S, Patron JR, Morato JG, Sibila O, et al. Experimental severe Pseudomonas aeruginosa pneumonia and antibiotic therapy in piglets receiving mechanical ventilation. Chest. 2007;132(2):523–31.

    Article  PubMed  Google Scholar 

  25. Rouby JJ, Monsel A, Ehrmann S, Bougle A, Laterre PF. The INHALE trial: multiple reasons for a negative result. Lancet Infect Dis. 2020;20(7):778–9.

    Article  PubMed  Google Scholar 

  26. Sole-Lleonart C, Rouby JJ, Blot S, Poulakou G, Chastre J, Palmer LB, et al. Nebulization of antiinfective agents in invasively mechanically ventilated adults: a systematic review and meta-analysis. Anesthesiology. 2017;126(5):890–908.

    Article  CAS  PubMed  Google Scholar 

  27. Palmer LB, Smaldone GC. Reduction of bacterial resistance with inhaled antibiotics in the intensive care unit. Am J Respir Crit Care Med. 2014;189(10):1225–33.

    Article  PubMed  Google Scholar 

  28. Palmer LB, Smaldone GC, Chen JJ, Baram D, Duan T, Monteforte M, et al. Aerosolized antibiotics and ventilator-associated tracheobronchitis in the intensive care unit. Crit Care Med. 2008;36(7):2008–13.

    Article  CAS  PubMed  Google Scholar 

  29. Zhang C, Berra L, Klompas M. Should aerosolized antibiotics be used to treat ventilator-associated pneumonia? Respir Care. 2016;61(6):737–48.

    Article  PubMed  Google Scholar 

  30. Rouby JJ, Sole-Lleonart C, Rello J. European investigators network for nebulized antibiotics in ventilator-associated P. ventilator-associated pneumonia caused by multidrug-resistant gram-negative bacteria: understanding nebulization of aminoglycosides and colistin. Intensive Care Med. 2020;46(4):766–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Rello J, Rouby JJ, Sole-Lleonart C, Chastre J, Blot S, Luyt CE, et al. Key considerations on nebulization of antimicrobial agents to mechanically ventilated patients. Clin Microbiol Infect. 2017;23(9):640–6.

    Article  CAS  PubMed  Google Scholar 

  32. Kollef MH, Hamilton CW, Montgomery AB. Aerosolized antibiotics: do they add to the treatment of pneumonia? Curr Opin Infect Dis. 2013;26(6):538–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zilberberg MD, Shorr AF, Micek ST, Vazquez-Guillamet C, Kollef MH. Multi-drug resistance, inappropriate initial antibiotic therapy and mortality in Gram-negative severe sepsis and septic shock: a retrospective cohort study. Crit Care. 2014;18(6):596.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Rouby JJ, Monsel A. Nebulized antibiotics: epithelial lining fluid concentrations overestimate lung tissue concentrations. Anesthesiology. 2019;131(2):229–32.

    Article  PubMed  Google Scholar 

  35. Dhanani JA, Diab S, Chaudhary J, Cohen J, Parker SL, Wallis SC, et al. Lung pharmacokinetics of tobramycin by intravenous and nebulized dosing in a mechanically ventilated healthy ovine model. Anesthesiology. 2019;131(2):344–55.

    Article  CAS  PubMed  Google Scholar 

  36. Rouby JJ, Bouhemad B, Monsel A, Brisson H, Arbelot C, Lu Q, et al. Aerosolized antibiotics for ventilator-associated pneumonia: lessons from experimental studies. Anesthesiology. 2012;117(6):1364–80.

    Article  CAS  PubMed  Google Scholar 

Download references


We thank Christina Sutherland of the Center of Anti-Infective Research & 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 Laura Muñoz for her support in the MALDI-TOF analysis. We would also thank Cristina Miralles for inclusion and processing all tissue samples.


Financial support was provided by the Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS); Ministerio de Ciencia e Innovación; Centro de Investigación Biomédica en Red- Enfermedades Respiratorias (CIBERES, CB06/06/0028); Quality Research Group from Generalitat de Catalunya (SGR/944); University of Barcelona; “ICREA Academia 2019” award to Prof. Antoni Torres from the University of Barcelona; La Marató de TV3 (201831-10); “La Caixa” Foundation (LCF/PR/HR19/52160019).

Author information

Authors and Affiliations



HY and AM participated in protocol development, study design, study management, data collection, statistical analysis, data interpretation, and writing 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 of the first draft of the report. LFB, MR, DM, DP, AA, PP, and JV participated in data interpretation and revision and review of the report. GLB and AT participated in protocol development, study design, study management, data interpretation, and revision and review of the report. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Antoni Torres.

Ethics declarations

Ethics approval and consent to participate

This study was conducted according to the European guidelines for the Care of Animal Experimentation at the Division of Animal Experimentation, Department of Pulmonary and Critical Care Medicine, Hospital Clínic, Barcelona, Spain. Documented approval from University of Barcelona Institutional Review Board was obtained prior to the study started (number approval 9772).

Consent for publication

Not applicable.

Competing interest

A. Torres and G. Li Bassi received an unrestrictive grant from Bayer AG (Berlin, Germany) through their affiliated institution. Bayer AG, the manufacturer of inhaled amikacin, provided funds to support this research. Bayer AG had no role in the data collection, data analysis, or data interpretation. Bayer AG reviewed the final manuscript.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1.

Supplementary methods and results

Rights and permissions

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 The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Motos, A., Yang, H., Li Bassi, G. et al. Inhaled amikacin for pneumonia treatment and dissemination prevention: an experimental model of severe monolateral Pseudomonas aeruginosa pneumonia. Crit Care 27, 60 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • Inhaled amikacin
  • Severe pneumonia
  • Pseudomonas aeruginosa
  • Animal model
  • Multidrug resistance
  • Monolateral pneumonia