Tania Cebrero-Cangueiro

Background/Objectives: Infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa are steadily increasing, thus the discovery and development of new and effective agents are needed. Antimicrobial peptides (AMPs) are a heterogeneous group of innate defense system peptides with broad antimicrobial activity. In this study, 17 AMPs were tested, identifying CAP-18, a cathelicidin-based compound, as the most active. CAP-18 was optimized by synthesizing structural derivatives, which were selected for further studies based on their activity against a collection of MDR and colistin-resistant P. aeruginosa strains. Methods: AMPs collection was initially tested against different P. aeruginosa strains, identifying CAP-18 as the most active. CAP-18 derivatives were synthetized and assessed by the Minimum Inhibitory Concentration (MIC), time-kill kinetics, cytotoxicity against human cell lines, hemolytic activity, and therapeutic index (IC₅₀/MIC₉₀). The mechanism of action was assessed by Transmission Electron Microscopy (TEM), and in vivo efficacy was determined through a murine skin infection model. Results: CAP-18 and D-CAP-18 had a MIC₉₀ of 4 and 2 μg/mL, respectively, whereas CAP-18₃₁ and D-CAP-18₃₁ presented MIC₉₀ values of 16 mg/L. The shorter derivatives of CAP-18 showed a lower activity. Time-kill curves revealed a fast bactericidal effect. These derivatives showed low toxicity against different human cell lines and low hemolysis, resulting in a wide therapeutic index (IC₅₀/MIC₉₀), with D-CAP-18 having the best therapeutic index (137.4). TEM provided insight into the mechanism of action, revealing bacterial membrane damage. In vivo studies of both CAP-18 and D-CAP-18 showed good activity with a 3 log decrease compared to the infected control group. Conclusions: Among the investigated four peptides, D-CAP-18 is the most promising candidate to treat skin infections caused by MDR P. aeruginosa since it shows potent activity both in vitro and in vivo, and a high therapeutic index.

This study aimed to evaluate the potential of tamoxifen and N-desmethyltamoxifen metabolites as therapeutic agents against multidrug-resistant Escherichia coli and Acinetobacter baumannii, using a repurposing approach to shorten the time required to obtain a new effective treatment against multidrug-resistant bacterial infections. Characterisation and virulence studies were conducted on E. coli (colistin-susceptible C1-7-LE and colistin-resistant MCR-1+) and A. baumannii (tigecycline-susceptible Ab#9 and tigecycline-resistant Ab#186) strains. The efficacy of the metabolite mix (33.3% each) and N-desmethyltamoxifen in combination with colistimethate sodium (CMS) or tigecycline was evaluated in experimental models in mice. In the pneumonia model, N-desmethyltamoxifen exhibited significant efficacy against Ab#9 and both E. coli strains, especially E. coli MCR-1+ (−2.86 log₁₀ CFU/g lungs, −5.88 log₁₀ CFU/mL blood, and −50% mortality), and against the Ab#186 strain when combined with CMS (−2.27 log₁₀ CFU/g lungs, −2.73 log₁₀ CFU/mL blood, and −40% mortality) or tigecycline (−3.27 log₁₀ CFU/g lungs, −4.95 log₁₀ CFU/mL blood, and −50% mortality). Moreover, the metabolite mix in combination with both antibiotics decreased the bacterial concentrations in the lungs and blood for both A. baumannii strains. In the sepsis model, the significant efficacy of the metabolite mix was restricted to the colistin-susceptible E. coli C1-7-LE strain (−3.32 log₁₀ CFU/g lung, −6.06 log₁₀ CFU/mL blood, and −79% mortality). N-desmethyltamoxifen could be a new therapeutic option in combination with CMS or tigecycline for combating multidrug-resistant GNB, specifically A. baumannii.

In the context of difficult-to-treat carbapenem-resistant Pseudomonas aeruginosa infections, we evaluated imipenem, meropenem, and doripenem combinations against eleven carbapenemase-producing P. aeruginosa isolates. According to the widespread global distribution of high-risk clones and carbapenemases, four representative isolates were selected: ST175 (OXA-2/VIM-20), ST175 (VIM-2), ST235 (GES-5), and ST111 (IMP-33), for efficacy studies using a sepsis murine model. Minimum inhibitory concentration (mg/L) ranges were 64–256 for imipenem and 16–128 for meropenem and doripenem. In vitro, imipenem plus meropenem was synergistic against 72% of isolates and doripenem plus meropenem or imipenem against 55% and 45%, respectively. All combinations were synergistic against the ST175, ST235, and ST155 clones. In vivo, meropenem diminished the spleen and blood bacterial concentrations of four and three isolates, respectively, with better efficacy than imipenem or doripenem. The combinations did not show efficacy compared with the more active monotherapies, except for imipenem plus meropenem, which reduced the ST235 bacterial spleen concentration. Mortality decreased with imipenem plus meropenem or doripenem for the ST175 isolate. Results suggest that carbapenem combinations are not an alternative for severe infections by carbapenemase-producing P. aeruginosa. Meropenem monotherapy showed in vivo efficacy despite its high MIC, probably because its dosage allowed a sufficient antimicrobial exposure at the infection sites.

Multidrug-resistant (MDR) Pseudomonas aeruginosa is a public health problem causing both community and hospital-acquired infections, and thus the development of new therapies for these infections is critical. The objective of this study was to analyze in vitro the activity of pentamidine as adjuvant in combinations to antibiotics against seven clinical P. aeruginosa strains. The Minimum Inhibitory Concentration (MIC) was determined following standard protocols, and the results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints; however, the gentamicin activity was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. The bactericidal in vitro activity was studied at 1×MIC concentrations by time–kill curves, and also performed in three selected strains at 1/2×MIC of pentamidine. All studies were performed in triplicate. The pentamidine MIC range was 400–1600 μg/mL. Four of the strains were MDR, and the other three were resistant to two antibiotic families. The combinations of pentamidine at 1×MIC showed synergistic activity against all the tested strains, except for pentamidine plus colistin. Pentamidine plus imipenem and meropenem were the combinations that showed synergistic activity against the most strains. At 1/2×MIC, pentamidine plus antibiotics were synergistic with all three analyzed strains. In summary, pentamidine in combination with antibiotics showed in vitro synergy against multidrug-resistant P. aeruginosa clinical strains, which suggests its possible use as adjuvant to antibiotics for the therapy of infections from MDR P. aeruginosa.

S. pneumoniae is the leading cause of community-acquired pneumonia in the solid organ transplant recipient (SOTR); nevertheless, the prevalence of colonization and of the colonizing/infecting serotypes has not been studied in this population. In this context, the aim of the present study was to describe the rate, characteristics, and clinical impact of S. pneumoniae nasopharyngeal carriage. A prospective observational cohort of Solid Organ Transplant recipients (SOTR) was held at the University Hospital Virgen del Rocío, Seville, Spain with the aim to evaluate the S. pneumoniae colonization and the serotype prevalence in SOTR. Two different pharyngeal swabs samples from 500 patients were included in two different seasonal periods winter and spring/summer. Optochin and bile solubility tests were performed for the isolation of thew strains. Antimicrobial susceptibility studies (MICs, mg/l) of levofloxacin, trimethoprim-sulfamethoxazole, penicillin, amoxicillin, cefotaxime, ceftriaxone, erythromycin, azithromycin and vancomycin for each isolate were determined by E-test strips. Capsular typing was done by sequential multiplex PCR reactions. A multivariate logistic regression analysis of factors potentially associated with pneumococcal nasopharyngeal carriage and disease was performed. Twenty-six (5.6%) and fifteen (3.2%) patients were colonized in winter and spring/summer periods, respectively. Colonized SOT recipients compared to non-colonized patients were more frequently men (79.5% vs. 63.1%, P < 0.05) and cohabitated regularly with children (59% vs. 32.2%, P < 0.001). The most prevalent serotype in both studied periods was 35B. Forty-five percent of total isolates were included in the pneumococcal vaccine PPV23. Trimethoprim-sulfamethoxazole and macrolides were the less active antibiotics. Three patients had non-bacteremic pneumococcal pneumonia, and two of them died. Pneumococcal colonization in SOTR is low with the most colonizing serotypes not included in the pneumococcal vaccines.