Integration of omics and bioinformatics to identify new therapeutic targets for Acinetobacter baumannii. Virulence role of CarO in Acinetobacter baumannii infections

Acinetobacter baumannii remains a significant and difficult-to-treat pathogen that causes a range of interactions with the human host from asymptomatic colonization and carriage in the skin, intestinal tract, and respiratory tract to invasive infection, such as nosocomial pneumonia or bacteraemia. Especially, this pathogen affects critically-ill and immunocompromised patients admitted to Intensive Care Units (ICUs), causing them severe infections, which are associated with long hospital stay and high mortality rates. The success of this bacterium is due to a combination of several factors, highlighting its extraordinary ability to develop antimicrobial resistance that results in the rapid nosocomial spread of strains resistant to almost all known antimicrobials, including including the last reservoirs of our antimicrobial arsenal such as the carbapenems, worldwide. Thus, in the recent global priority list of antimicrobial-resistant bacteria of the World Health Organization, carbapenem-resistant A. baumannii (CRAB) is considered as a “critical priority” for the development of new antimicrobials, due to the lack of therapeutic options. This situation has promoted the search of new therapeutic strategies to deal with multidrug-resistant (MDR) A. baumannii strains displaying additional carbapenem-resistance, and non-antimicrobial approaches aimed at bacterial virulence factors may represent a promising alternative. Nevertheless, our knowledge on A. baumannii pathogenesis and virulence traits is still relatively scarce. In this Doctoral Thesis, and in order to stop the evolution of A. baumannii infections, we aimed to study more deeply the pathogenesis of CRAB infections using omics and bioinformatics to ultimately discover new therapeutic targets for anti-A. baumannii drugs. Firstly, we studied MDR/CRAB isolates from tracheobronchial aspirate samples of ICU adult patients who suffered A. baumannii bacteraemic ventilator-associated pneumonia (VAP) or remained exclusively colonised by this pathogen. We analysed the in vitro and in vivo virulence of these isolates, in order to know if the invasive isolates exhibited higher virulence than the colonising ones, but no differences were found. Moreover, when the whole-genome sequencing (WGS) data of these isolates were analysed following different approaches, again we did not find any difference between both phenotypes regarding their clonal relationship, antimicrobial resistance mechanisms, or known virulence determinants. However, the invasive A. baumannii isolates exhibited higher levels of expression of the outer membrane protein (OMP) OmpA than the colonising ones, suggesting that those phenotypes depend on the regulation of already-known or still unknown virulence factors, instead of on the genomic content. Secondly, six bacteraemic MDR/CRAB clinical isolates obtained from six clinically homogeneous ICU adult patients with bacteraemia secondary to VAP, who were subjected to optimal colistin treatment but with radically different clinical outcomes, were comparatively analyzed by WGS complemented with proteomic and immunoblot techniques. These analyses indicated that the carO gene, encoding for the second most abundant β-barrel protein of the A. baumannii outer membrane (OM), CarO, was interrupted by different disruptive events in the isolates from patients who recovered from infection, while it was intact in the isolates from patients who did not survive. When the virulence role of A. baumannii CarO was analyzed in model systems, an isogenic mutant lacking carO (ATCC 17978 ΔcarO) showed lower ability in vitro to adhere and invade cultured human lung epithelial cells, and exhibited a higher minimum lethal dose and a lower dissemination potential into essential organs and fluids in a murine model of peritoneal sepsis. All of the above deficiencies were reverted in the ATCC 17978 ΔcarO mutant transformed with a carO expression plasmid restoring OM CarO levels. Thus, the results presented here reveal a previously unnoticed virulence role for the A. baumannii OMP CarO, which may be responsible of the poor clinical outcome and therefore a potential target for the development of novel anti-A. baumannii drugs

In vitro Activity of Pentamidine Alone and in Combination With Aminoglycosides, Tigecycline, Rifampicin, and Doripenem Against Clinical Strains of Carbapenemase-Producing and/or Colistin-Resistant Enterobacteriaceae

Enterobacteriaceae cause different types of community- and hospital-acquired infections. Moreover, the spread of multidrug-resistant Enterobacteriaceae is a public health problem and the World Health Organization pointed them among the pathogens in which the search of new antibiotics is critical. The objective of this study was to analyze the in vitro activity of pentamidine alone and in combination with gentamicin, tobramycin, amikacin, tigecycline, rifampicin, or doripenem against eight clinical strains of carbapenemase-producing and/or colistin-resistant Enterobacteriaceae: five carbapenemase-producing Klebsiella pneumoniae, one carbapenemase-producing Escherichia coli, and two colistin-resistant Enterobacter cloacae. MIC and MBC were determined following standard protocols. MIC results were interpreted for all the antibiotics according to the EUCAST breakpoints but for rifampicin in which the French FSM breakpoint was used. Bactericidal and synergistic activity of pentamidine alone and in combination with antibiotics at concentrations of 1xMIC was measured by time-kill curves. For one selected strain, K. pneumoniae OXA-48/CTX-M-15 time-kill curves were performed also at 1/2xMIC of pentamidine. All studies were performed in triplicate. Pentamidine MIC range was 200-800 μg/mL. The 50, 12.5, 62.5, 87.5, and 62.5% of the strains were susceptible to gentamicin, tobramycin, amikacin, tigecycline, and doripenem, respectively. Only the two E. cloacae strains were susceptible to rifampicin. Pentamidine alone at 1xMIC showed bactericidal activity against all strains, except for the E. cloacae 32 strain. The bactericidal activity of pentamidine alone was also observed in combination. The combinations of pentamidine were synergistic against E. cloacae 32 with amikacin and tobramycin at 24 h and with tigecycline at 8 h. Pentamidine plus rifampicin was the combination that showed synergistic activity against more strains (five out of eight). Pentamidine plus doripenem did not show synergy against any strain. At 1/2xMIC, pentamidine was synergistic with all the studied combinations against the K. pneumoniae OXA-48/CTX-M-15 strain. In summary, pentamidine alone and in combination shows in vitro activity against carbapenemase-producing and/or colistin-resistant Enterobacteriaceae. Pentamidine appears to be a promising option to treat infections caused by these pathogens.Plan Nacional de I+D+I 2013-2016 REIPI RD16/0016/0009Instituto de Salud Carlos III. Subdirección General de Redes y Centros de Investigación Cooperativa REIPI RD16/0016/0009Ministerio de Economía, Industria y Competitividad REIPI RD16/0016/0009Spanish Network for Research in Infectious Diseases REIPI RD16/0016/0009European Development Regional Fun

In vitro activity of pentamidine alone and in combination with antibiotics against multidrug-resistant clinical Pseudomonas aeruginosa strains

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.Instituto de Salud Carlos III Proyectos de Investigación en Salud PI18-01842Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Red Española de Investigación en Enfermedades Infecciosas REIPI RD16 / 0016/0009Fondo Regional de Desarrollo Europeo Una forma de lograr Europa, Operativa programa Crecimiento inteligente 2014-2020. T.CUniversidad de Sevilla. Servicio Andaluz de Salud, Junta de Andalucía C1-0038-2019Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III, cofinanciado por la Unión Fondo Regional de Desarrollo RD16 / 0016/0009Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III, cofinanciado por European Development Regional Fund (A Way to Achieve Europe) y por la Red Española de Investigación en Enfermedades Infecciosas JR17 / 0002

In vitro activity of piperazine derivates against multidrug-resistance bacteria

Motivation: The increasing prevalence of multidrug-resistance represent a serious challenge for clinical management and public health. Multidrug-resistant (MDR) bacteria is a common cause of infections, especially in inmunocompromised patient. Nowadays, colistin has re-emerged as one of the last therapeutic option against these kinds of infections, but colistin resistant strains have been reported, leaving no alternative of treatment. The aim of this work is to study in vitro the activity of piperazine derivates against MDR and colistin resistant bacteria. Methods: Clinical and standard strains: MDR: Acinetobacter baumannii (Ab; n=1), Klebsiella pneumoniae carbapenemases producing (n=4), Pseudomonas aeruginosa (Pa; n=2), Escherichia coli ATCC 25922 (n=1), colistin resistant A. baumannii (n=13). Piperazine derivatives: four different families were tested: 1, 2, 3, and 4. The derivivates were synthesized in the Pharmacy Faculty of Seville. A) Inhibition screening: all strains at a concentration of 5x105 CFU/mL were tested at 50 µM of each derivative. B) Minimal Inhibitory Concentration (MIC): were calculated for the derivates that inhibit the bacterial growth. C) Time-kill curves: were performed for six derivates against two colistin resistant A. baumannii clinical strains.  Results: A) Inhibition was observed only in colistin resistant A. baumannii clinical strains. B) Family 1, inhibited the growth of 46 % (6/13) of the strains. Family 2, inhibited the growth of 30% (4/13) of the strains. Family 3, inhibited the growth of 30% (4/13) of the strains. Family 4, inhibited the growth of 38% (5/13) of the strains. C) Family 1: MIC range was 50-3.12 µM. Family 2: MIC range was 50-6.25 µM. Family 3: MIC range was 50-1.56 µM. Family 4: MIC range was 50-3.12 µM. D) One piperazine derivates presented bactericidal activity at 24 hours against one of the tested strains.  Conclusions: Piperazine derivatives showed in vitro activity against colistin resistant A. baumannii clinical strains. Further studies, in vitro and in vivo need to be performed in order to confirm the activity of the piperazine derivates against infections due to these kinds of infections

Efficacy of Colistin and Its Combination With Rifampin in Vitro and in Experimental Models of Infection Caused by Carbapenemase-Producing Clinical Isolates of Klebsiella pneumoniae.

Despite the relevance of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) infections there are a scarce number of studies to evaluate in vivo the efficacy of combinations therapies. The bactericidal activity of colistin, rifampin, and its combination was studied (time-kill curves) against four clonally unrelated clinical isolates of CP-Kp, producing VIM-1, VIM-1 plus DHA-1(acquired AmpC β-lactamase), OXA-48 plus CTX-M-15 (extended spectrum β-lactamase) and KPC-3, respectively, with colistin MICs of 0.5, 64, 0.5, and 32 mg/L, respectively. The efficacies of antimicrobials in monotherapy and in combination were tested in a murine peritoneal sepsis model, against all the CP-Kp. Their efficacies were tested in the pneumonia model against the OXA-48 plus CTX-M-15 producers. The development of colistin-resistance was analyzed for the colistin-susceptible strains in vitro and in vivo. In vitro, colistin plus rifampin was synergistic against all the strains at 24 h. In vivo, compared to the controls, rifampin alone reduced tissue bacterial concentrations against VIM-1 and OXA-48 plus CTX-M-15 strains; CMS plus rifampin reduced tissue bacterial concentrations of these two CP-Kp and of the KPC-3 strain. Rifampin and the combination increased the survival against the KPC-3 strain; in the pneumonia model, the combination also improved the survival. No resistant mutants appeared with the combination. In conclusion, CMS plus rifampin had a low and heterogeneous efficacy in the treatment of severe peritoneal sepsis model due to CP-Kp producing different carbapenemases, increasing survival only against the KPC-3 strain. The combination showed efficacy in the less severe pneumonia model. The combination prevented in vitro and in vivo the development of colistin resistant mutants.España, Consejería de Salud Junta de Andalucía PI-0622-201

Prevalence and clinical impact of Streptococcus pneumoniae nasopharyngeal carriage in solid organ transplant recipients

Background: 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. Methods: 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. Results: 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. Conclusions: Pneumococcal colonization in SOTR is low with the most colonizing serotypes not included in the pneumococcal vaccines.Pfizer, 2014 ASPIRE Awards in Vaccine Research in Europe (Pfizer Reference # WI191483)Plan Nacional de I + D + i 2013–2016 , Instituto de Salud Carlos III, Ministerio de Economía, Industria y Competitividad REIPI RD16/0016/0009 Fondo Regional de Desarrollo Europeo "Una forma de alcanzar Europa", Programa operativo Crecimiento inteligente 2014–2020

In Vitro Activity of Pentamidine Alone and in Combination with Antibiotics against Multidrug-Resistant Clinical Pseudomonas aeruginosa Strains

© 2020 by the authors.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.This study was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (PI18-01842), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009)—co-financed by European Development Regional Fund A way to achieve Europe, Operative program Intelligent Growth 2014–2020. T.C.-C. is supported by the V Plan Propio of the University of Seville with a postdoctoral contract as research personnel in training. M.E.P.-I. is a researcher belonging to the program “Nicolás Monardes” (C1-0038-2019), Servicio Andaluz de Salud, Junta de Andalucía, Spain. G.L.-H. has a grant from the Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III, cofinanced by the European Development Regional Fund (A Way to Achieve Europe) and by the Spanish Network for Research in Infectious Diseases (grant REIPI RD16/0016/0009). R.Á.-M. has a grant Juan Rodes grant from the Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III, cofinanced by the European Development Regional Fund (A Way to Achieve Europe) and by the Spanish Network for Research in Infectious Diseases (JR17/00025)

Pangenome of Acinetobacter baumannii uncovers two groups of genomes, one of them with genes involved in CRISPR/Cas defence systems associated with the absence of plasmids and exclusive genes for biofilm formation

Acinetobacter baumannii is an opportunistic bacterium that causes hospital-acquired infections with a high mortality and morbidity, since there are strains resistant to virtually any kind of antibiotic. The chase to find novel strategies to fight against this microbe can be favoured by knowledge of the complete catalogue of genes of the species, and their relationship with the specific characteristics of different isolates. In this work, we performed a genomics analysis of almost 2500 strains. Two different groups of genomes were found based on the number of shared genes. One of these groups rarely has plasmids, and bears clustered regularly interspaced short palindromic repeat (CRISPR) sequences, in addition to CRISPR-associated genes (cas genes) or restriction-modification system genes. This fact strongly supports the lack of plasmids. Furthermore, the scarce plasmids in this group also bear CRISPR sequences, and specifically contain genes involved in prokaryotic toxin–antitoxin systems that could either act as the still little known CRISPR type IV system or be the precursors of other novel CRISPR/Cas systems. In addition, a limited set of strains present a new cas9-like gene, which may complement the other cas genes in inhibiting the entrance of new plasmids into the bacteria. Finally, this group has exclusive genes involved in biofilm formation, which would connect CRISPR systems to the biogenesis of these bacterial resistance structures.The project has been supported by Plan Nacional de I+D+i 2013‐2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009), and co‐ financed by European Development Regional Fund ‘A way to achieve Europe’, Operative program Intelligent Growth 2014‐2020. E.L.M. is supported by ‘Programa de Empleo Joven’ (FEDER/Junta de Andalucía, Fase I, II convocatoria), R.A.-M. by a Juan Rodes grant (JR17/00025), and G.L.-H. by a i-PFIS grant (IFI15/00128) from Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain

Role of PstS in the Pathogenesis of Acinetobacter baumannii Under Microaerobiosis and Normoxia

Acinetobacter baumannii is a successful pathogen responsible for infections with high mortality rate. During the course of infection it can be found in microaerobic environments, which influences virulence factor expression. From a previous transcriptomic analysis of A. baumannii ATCC 17978 under microaerobiosis, we know the gene pstS is overexpressed under microaerobiosis. Here, we studied its role in A. baumannii virulence. pstS loss significantly decreased bacterial adherence and invasion into A549 cells and increased A549 cell viability. pstS loss also reduced motility and biofilm-forming ability of A. baumannii. In a peritoneal sepsis murine model, the minimum lethal dose required by A. baumannii ATCC 17978 ΔpstS was lower compared to the wild type (4.3 vs 3.2 log colony forming units/mL, respectively), and the bacterial burden in tissues and fluids was lower. Thus, the loss of the phosphate sensor PstS produced a decrease in A. baumannii pathogenesis, supporting its role as a virulence factor.This study was supported by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad (grant number PIE13/0004; program Formación de Profesorado Universitario grant number FPU13/04545 to M. L. G. M.; and Subprograma Miguel Servet Tipo I grant number CP15/00132 to Y. S.) and Plan Nacional de I + D + i 2013–2016, Spanish Network for Research in Infectious Diseases, cofinanced by the European Development Regional Fund A Way to Achieve Europe, operative program Intelligent Growth 2014–2020 (grant numbers RD12/0015/0001, RD12/0015/0012, and RD16/0016/0009). M. E. P. I. is supported by Program Nicolás Monardes, Andalusian Health Service, Ministry of Health and Families

IgM-enriched immunoglobulin improves colistin efficacy in a pneumonia model by Pseudomonas aeruginosa.

We evaluated the efficacy of ceftazidime or colistin in combination with polyclonal IgM-enriched immunoglobulin (IgM-IG), in an experimental pneumonia model (C57BL/6J male mice) using two multidrug-resistant Pseudomonas aeruginosa strains, both ceftazidime-susceptible and one colistin-resistant. Pharmacodynamically optimised antimicrobials were administered for 72 h, and intravenous IgM-IG was given as a single dose. Bacterial tissues count and the mortality were analysed. Ceftazidime was more effective than colistin for both strains. In mice infected with the colistin-susceptible strain, ceftazidime reduced the bacterial concentration in the lungs and blood (-2.42 and -3.87 log10 CFU/ml) compared with colistin (-0.55 and -1.23 log10 CFU/ml, respectively) and with the controls. Colistin plus IgM-IG reduced the bacterial lung concentrations of both colistin-susceptible and resistant strains (-2.91 and -1.73 log10 CFU/g, respectively) and the bacteraemia rate of the colistin-resistant strain (-44%). These results suggest that IgM-IG might be useful as an adjuvant to colistin in the treatment of pneumonia caused by multidrug-resistant P. aeruginosa