Hypoxia effect on genetic regulation and virulence in Acinetobacter baumannii and Pseudomonas aeruginosa, in vitro and in vivo, and on innate immune response in infections caused by both pathogens.

The emergence of multi-drug resistant Gram-negative bacilli infections is a well-recognized global health challenge in urgent need of effective solutions. These pathogens cause infections that are very difficult to treat due to the high rate of resistance strains to a lot or even all antimicrobials used in the clinical practice. Furthermore, these kinds of infections are associated to important mortality rates. Unfortunately, the increasing problem of multidrug resistance is not followed by the development of novel antimicrobials. For this reason, there is an important need to develop new strategies to combat multi-drug, extensively-drug and pan-drug resistantGram-negative bacilli. A new alternative to combat this kind of infections might be blocking specific bacterial virulence factors that bacteria need to infect. It is known that bacteria modulate their gene expression in function of the environment. Therefore, expression of virulence factors can change during the course of infection according to every microenvironment in which bacteria are found. Several studies have reported that hypoxia occurs in a wide range of infection, so it would be interesting to determine the gene profile of these multi-drug resistant pathogens in order to find out new virulence factors that we could block. Moreover, it is also important to know how hypoxia affects to this kind of infection to know better what is happening in vitro and in vivo. Because A. baumannii and P. aeruginosa are two of the most common pathogens that cause healthcare-acquired infection, we have chosen them to study their virulence mechanism under hypoxia in more depth. We have selected the strains A. baumannii ATCC 17978 and P. aeruginosa PAO1 because they are well-known sequenced strains which allow us to analyze virulence factors in a better way. On the other hand, septic shock patients present tissue hypoxia that might influence the disease. Moreover, septic shock is the most severe complication of sepsis and most studies about this syndrome report a high mortality rate. Hence, it would be useful to study the relationship between tissue hypoxia, HIF-1α levels and immune response, and to determine new biomarkers to predict the outcome.Premio Extraordinario de Doctorado U

Synergistic Activity of Niclosamide in Combination With Colistin Against Colistin-Susceptible and Colistin-Resistant Acinetobacter baumannii and Klebsiella pneumoniae

Colistin is among the few antibiotics effective against multidrug-resistant Acinetobacter baumannii and Klebsiella pneumoniae clinical isolates. However, in the last few years, colistin-resistant A. baumannii and K. pneumoniae strains have emerged. Therefore, combination therapies, between colistin and other old drugs, restoring the activity of colistin are required. The main objective of this study was to analyse the activity of niclosamide, an anthelmintic drug, in combination with colistin against colistin-susceptible (Col-S) and colistin-resistant (Col-R) A. baumannii and K. pneumoniae. The MIC were determined by microdilution assay and the time-kill curves were performed. The zeta potential of Col-S and Col-R of A. baumannii and K. pneumoniae in presence of niclosamide was assessed. Niclosamide in combination with colistin showed improved activity against Col-S and Col-R A. baumannii and K. pneumoniae. Time-killing curves showed synergic activity between niclosamide and colistin against Col-S and Col-R A. baumannii and K. pneumoniae, especially when niclosamide or colistin was added for second time at 4 h of the 24 h killing curve. Col-R A. baumannii and K. pneumoniae in presence of niclosamide exhibited a greater negative charge (−34.95 ± 0.35 mV and −38.85 ± 0.92 mV; P < 0.05) than Col-R A. baumannii and K. pneumoniae in absence of niclosamide (−26.85 ± 3.65 mV and −35.27 ± 0.72 mV). These data suggest that niclosamide might be combined with colistin, being a potential alternative for treatment of Col-R Gram-negative bacilli infections.Instituto de Salud Carlos IIIProyectos de Investigacion en Salud PI16/01378Ministerio de Economía y Competitividad CP15/0135

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

iTRAQ-Based quantitative proteomic analysis of acinetobacter baumannii under hypoxia and normoxia reveals the role of ompW as a virulence factor

Acinetobacter baumannii needs to adapt to hypoxia during infection. Understanding its proteome regulation during infection would allow us to determine new targets to develop novel treatments. iTRAQ proteomic analysis of A549 cell infection by the ATCC 17978 strain was performed. A total of 175 proteins were differentially expressed under hypoxia versus normoxia. We selected the hypoxia-downregulated protein OmpW to analyze its role as a virulence factor. The loss of OmpW decreased the adherence and invasion of A. baumannii in these host cells, without affecting its bacterial growth. Moreover, A549 cell viability with ΔOmpW infection was higher than that with the wild-type strain. ΔOmpW presented less biofilm formation. Finally, the minimum lethal dose required by the ΔOmpW mutant was higher than that of the wild-type strain in a murine peritoneal sepsis model, with lower bacterial loads in tissues and fluids. Therefore, OmpW seems to be a virulence factor necessary for A. baumannii pathogenesis.This work was funded by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad (grant PIE13/0004), by Plan Nacional de I+D+i 2013 to 2016, and by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (grant RD16/0016/0009), cofinanced by the European Development Regional Fund (A way to achieve Europe, Operative Program Intelligent Growth 2014 to 2020). Y.S. also received support (grant CB21/13/00006) from the CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, cofinanced by the European Development Regional Fund. M.L.G.-M. received a Formación de Profesorado Universitario (FPU) grant (grant FPU13/04545) from the Ministerio de Educación, Cultura y Deporte, Spain, and Y.S. received a Miguel Servet Tipo II contract from the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spain (grant CPII20/00018)

Effect of Hypoxia on the Pathogenesis of Acinetobacter baumannii and Pseudomonas aeruginosa In Vitro and in Murine Experimental Models of Infection.

Hypoxia modulates bacterial virulence and the inflammation response through hypoxia-inducible factor 1α (HIF-1α). Here we study the influence of hypoxia on Acinetobacter baumannii and Pseudomonas aeruginosa infections. In vitro, hypoxia increases the bactericidal activities of epithelial cells against A. baumannii and P. aeruginosa, reducing extracellular bacterial concentrations to 50.5% ± 7.5% and 90.8% ± 13.9%, respectively, at 2 h postinfection. The same phenomenon occurs in macrophages (67.6% ± 18.2% for A. baumannii at 2 h and 50.3% ± 10.9% for P. aeruginosa at 24 h). Hypoxia decreases the adherence of A. baumannii to epithelial cells (42.87% ± 8.16% at 2 h) and macrophages (52.0% ± 18.7% at 24 h), as well as that of P. aeruginosa (24.9% ± 4.5% in epithelial cells and 65.7% ± 5.5% in macrophages at 2 h). Moreover, hypoxia decreases the invasion of epithelial cells (48.6% ± 3.8%) and macrophages (8.7% ± 6.9%) by A. baumannii at 24 h postinfection and by P. aeruginosa at 2 h postinfection (75.0% ± 16.3% and 63.4% ± 5.4%, respectively). In vivo, hypoxia diminishes bacterial loads in fluids and tissues in animal models of infection by both pathogens. In contrast, mouse survival time was shorter under hypoxia (23.92 versus 36.42 h) with A. baumannii infection. No differences in the production of cytokines or HIF-1α were found between hypoxia and normoxia in vitro or in vivo We conclude that hypoxia increases the bactericidal activities of host cells against both pathogens and reduces the interaction of pathogens with host cells. Moreover, hypoxia accelerates the rate at which animals die despite the lower bacterial concentrations in vivo

Peptidoglycan recycling contributes to intrinsic resistance to fosfomycin in Acinetobacter baumannii

[Background] Acinetobacter baumannii is intrinsically resistant to fosfomycin; however, the mechanisms underlying this resistance are poorly understood.[Objectives] To identify and characterize genes that contribute to intrinsic fosfomycin resistance in A. baumannii.[Methods] More than 9000 individual transposon mutants of the A. baumannii ATCC 17978 strain (fosfomycin MIC ≥1024 mg/L) were screened to identify mutations conferring increased susceptibility to fosfomycin. In-frame deletion mutants were constructed for the identified genes and their susceptibility to fosfomycin was characterized by MIC determination and growth in the presence of fosfomycin. The effects of these mutations on membrane permeability and peptidoglycan integrity were characterized. Susceptibilities to 21 antibiotics were determined for the mutant strains.[Results] Screening of the transposon library identified mutants in the ampD and anmK genes, both encoding enzymes of the peptidoglycan recycling pathway, that demonstrated increased susceptibility to fosfomycin. MIC values for in-frame deletion mutants were ≥42-fold (ampD) and ≥8-fold (anmK) lower than those for the parental strain, and growth of the mutant strains in the presence of 32 mg/L fosfomycin was significantly reduced. Neither mutation resulted in increased cell permeability; however, the ampD mutant demonstrated decreased peptidoglycan integrity. Susceptibility to 21 antibiotics was minimally affected by mutations in ampD and anmK.[Conclusions] This study demonstrates that AmpD and AnmK of the peptidoglycan recycling pathway contribute to intrinsic fosfomycin resistance in A. baumannii, indicating that inhibitors of these enzymes could be used in combination with fosfomycin as a novel treatment approach for MDR A. baumannii.This work was 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 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 Europ’e, Operative program Intelligent Growth 2014‐2020, and a Grant from the European Society of Clinical Microbiology and Infectious Diseases awarded to M. J. M. M. J. M. is supported by the Subprograma Miguel Servet (CPII16/00061), Instituto de Salud Carlos III, Ministerio de Economía, Industria y Competitividad. M. L. G.-M. is supported by the Formación de Profesorado Universitario Program (FPU13/04545), Ministerio de Educación, Cultura y Deporte, Spain.Peer reviewe

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

Synergistic Activity of Niclosamide in Combination With Colistin Against Colistin-Susceptible and Colistin-Resistant Acinetobacter baumannii and Klebsiella pneumoniae

Colistin is among the few antibiotics effective against multidrug-resistant Acinetobacter baumannii and Klebsiella pneumoniae clinical isolates. However, in the last few years, colistin-resistant A. baumannii and K. pneumoniae strains have emerged. Therefore, combination therapies, between colistin and other old drugs, restoring the activity of colistin are required. The main objective of this study was to analyse the activity of niclosamide, an anthelmintic drug, in combination with colistin against colistin-susceptible (Col-S) and colistin-resistant (Col-R) A. baumannii and K. pneumoniae. The MIC were determined by microdilution assay and the time-kill curves were performed. The zeta potential of Col-S and Col-R of A. baumannii and K. pneumoniae in presence of niclosamide was assessed. Niclosamide in combination with colistin showed improved activity against Col-S and Col-R A. baumannii and K. pneumoniae. Time-killing curves showed synergic activity between niclosamide and colistin against Col-S and Col-R A. baumannii and K. pneumoniae, especially when niclosamide or colistin was added for second time at 4 h of the 24 h killing curve. Col-R A. baumannii and K. pneumoniae in presence of niclosamide exhibited a greater negative charge (−34.95 ± 0.35 mV and −38.85 ± 0.92 mV; P < 0.05) than Col-R A. baumannii and K. pneumoniae in absence of niclosamide (−26.85 ± 3.65 mV and −35.27 ± 0.72 mV). These data suggest that niclosamide might be combined with colistin, being a potential alternative for treatment of Col-R Gram-negative bacilli infections.This study was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (grant PI16/01378). YS is supported by the Subprograma Miguel Servet Tipo I from the Ministerio de Economía y Competitividad of Spain (CP15/01358).Peer reviewe

Extended-spectrum resistance to β-lactams/β-lactamase inhibitors (ESRI) evolved from low-level resistant Escherichia coli

[Objectives] Escherichia coli is characterized by three resistance patterns to β-lactams/β-lactamase inhibitors (BLs/BLIs): (i) resistance to ampicillin/sulbactam and susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (RSS); (ii) resistance to ampicillin/sulbactam and amoxicillin/clavulanic acid, and susceptibility to piperacillin/tazobactam (RRS); and (iii) resistance to ampicillin/sulbactam, amoxicillin/clavulanic acid and piperacillin/tazobactam (RRR). These resistance patterns are acquired consecutively, indicating a potential risk of developing resistance to piperacillin/tazobactam, but the precise mechanism of this process is not completely understood.[Methods] Clinical isolates incrementally pressured by piperacillin/tazobactam selection in vitro and in vivo were used. We determined the MIC of piperacillin/tazobactam in the presence and absence of piperacillin/tazobactam pressure. We deciphered the role of the blaTEM genes in the new concept of extended-spectrum resistance to BLs/BLIs (ESRI) using genomic analysis. The activity of β-lactamase was quantified in these isolates.[Results] We show that piperacillin/tazobactam resistance is induced in E. coli carrying blaTEM genes. This resistance is due to the increase in copy numbers and transcription levels of the blaTEM gene, thus increasing β-lactamase activity and consequently increasing piperacillin/tazobactam MICs. Genome sequencing of two blaTEM-carrying representative isolates showed that piperacillin/tazobactam treatment produced two types of duplications of blaTEM (8 and 60 copies, respectively). In the clinical setting, piperacillin/tazobactam treatment of patients infected by E. coli carrying blaTEM is associated with a risk of therapeutic failure.[Conclusions]This study describes for the first time the ESRI in E. coli. This new concept is very important in the understanding of the mechanism involved in the acquisition of resistance to BLs/BLIs.This study was supported by the Miguel Servet Tipo I Project grant, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad (CP15/00132), and 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 (RD16/0016/0009), cofinanced by the European Development Regional Fund ‘A Way to Achieve Europe’, Operative Program Intelligent Growth 2014–2020. This study was supported by the Assistance Publique – Hôpitaux de Paris (AP-HP), the University Paris-Sud, the Laboratory of Excellence in Research on Medication and Innovative Therapeutics (LERMIT) supported by a grant from the French National Research Agency [ANR-10-LABX-33]. Y. S. is supported by the Subprograma Miguel Servet Tipo I, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spain (CP15/00132). A. R-V. is supported by the Programa de fomento de investigación en Residentes HUVR/IBIS, Fundación Pública Andaluza Para la Gestión de la Investigación en Salud de Sevilla, Junta de Andalucía, Spain (068/18-HUVR-I). M. L. G-M. is supported by the program FPU (Formación de Profesorado Universitario; FPU13/04545), Ministerio de Educación, Cultura y Deporte, Spain. Transparency declaration

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.The present work has been supported by Pfizer, 2014 ASPIRE Awards in Vaccine Research in Europe (Pfizer Reference # WI191483),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 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.Peer reviewe