The biotechnology research for the unresolved and challenging health problems

The big health threats are detected by Public Health indicators through surveillance systems, worldwide and/or at the European and/or Spanish levels. Solid and hematologic neoplasia’s, cardiovascular and respiratory diseases, chronic and aging related diseases, infectious diseases, among others, are the main health problems; in the under-developed countries, problems arisen from lower socio-sanitary conditions may magnify the effects of severe and other moderate diseases.At the clinical setting, the health workers, physicians and nurses detect unresolved problems in the prevention, diagnosis, support, care and treatment of diseases, when attending different patients. Depending on the presence or the absence of underlying debilitating and chronic conditions, the clinical manifestations and the outcomes of the diseases, in terms of disability or death, are different and evolving in time. Currently, important technological improvements, including new drugs and devices, new types of surgery, or new diagnosis methods, as examples, have permitted better management of many diseases. However, new challenges continuously appeared, depending on new diseases or old non-resolved problems, despite the previous advances or, in the field of the infectious diseases, because the ability to recognize old pathogens, the emergence of new virus, and the worldwide emergence of the multidrug- and extensively-drug-resistance to antimicrobials in bacteria.Clinicians must be involved in the formulation of hypothesis to look for new solutions in the management of the old and emergent health problems and diseases, to improve the care and the cure of the patients and to prevent diseases. Clinical research is the classical approach to answer the questions derived from those hypotheses and to achieve new knowledges, which can be applied directly in the attention to the patients. However, many questions need a clinical/experimental approach to obtain useful results and, consequently, a multidisciplinary team, including biologists, biotechnologists, chemists, engineers and physicians, among others, is needed. The Sanitary Research Institutes were configured in 2004 through the cooperation between Hospitals and Universities, to have the framework where a very close multidisciplinary work can be developed. However, we must have an open mind to efficiently collaborate with any scientific, in any institution, in any company, in any country, in the context of an open scientific world.

"In vitro" activity of pentamidine alone and in combination against clinical isolates of carbapenemase-producing and colistin-resistant Enterobacteriaceae

Motivation: The increase in antimicrobial resistance rates and the difficulty to treat patients with infections caused by these kind of pathogens may urgent the search of alternative treatments. The objective of this project was to study the in vitro activity of pentamidine (PEN) alone and in combination against these kinds of pathogens. Methods: Fifteen different clinical isolates: 9 Enterobacter spp., 5 Klebsiella pneumoniae and 1 Escherichia coli. MICs were determined by microdilution in Mueller-Hinton broth at 24 and 48 hours. The assignment of clinical categories was made by the EUCAST criteria. MBCs were measured following the standard protocols. Bactericidal and synergistic activity of PEN alone and in combination was measured by time-kill curves at concentrations of 1xMIC in different time point. The in vitro prevention of the development of resistance was analyzed in time–kill studies.Results: PEN MIC range was from 200-800 mg/L. All Enterobacter strains were susceptible to doripenem (DOR), meropenem, amikacin (AMK) and rifampicin (RIF); also were susceptible to gentamicin (GEN) and tobramycin (TOB), except E. cloacae 32 and 297. All Enterobacter spp. were resistant to colistin (COL). Fifty percent of the strains were resistant to tigecycline (TIG). K. pneumoniae OXA-48/CTX-M-15 was resistant only to fosfomycin (FOS). K. pneumoniae KPC-3 was susceptible only to GEN. E. coli NDM-1 and K. pneumoniae NDM-1 were susceptible to COL and TIG. K. pneumoniae VIM-1 was resistant to GEN and TOB and K. pneumoniae VIM-1/DHA-1 was resistant to TOB, DOR and COL. All strains were resistant to FOS except E. cloacae 190. PEN alone and in combination with GEN, AMK, TIG, TOB, RIF and DOR showed bactericidal activity after four hours against K. pneumoniae NDM-1, K. pneumoniae NDM-1/DHA-1 and K. pneumoniae OXA-48/CTX-M-15. PEN together with the tested antimicrobials but TIG showed bactericidal and synergistic activity at 24 hours against E. cloacae 32. In addition, PEN prevented the appearance of mutants resistant to aminoglycosides and TIG in the carbapenemase-producing strains, and TOB-resistant mutants in the E. cloacae 32 strain.Conclusions: The association of pentamidine with the antimicrobials studied confirms a good in vitro activity of these combinations against carbapenemase-producing and colistin-resistant Enterobacteriaceae, suggesting that they could be a new alternative for the treatment of infections caused by these kinds of pathogens

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

Perspectives for clinical use of engineered human host defense antimicrobial peptides.

Infectious diseases caused by bacteria, viruses or fungi are among the leading causes of death worldwide. The emergence of drug-resistance mechanisms, especially among bacteria, threatens the efficacy of all current antimicrobial agents, some of them already ineffective. As a result, there is an urgent need for new antimicrobial drugs. Host defense antimicrobial peptides (HDPs) are natural occurring and well-conserved peptides of innate immunity, broadly active against Gram-negative and Gram-positive bacteria, viruses and fungi. They also are able to exert immunomodulatory and adjuvant functions by acting as chemotactic for immune cells, and inducing cytokines and chemokines secretion. Moreover, they show low propensity to elicit microbial adaptation, probably because of their non-specific mechanism of action, and are able to neutralize exotoxins and endotoxins. HDPs have the potential to be a great source of novel antimicrobial agents. The goal of this review is to provide an overview of the advances made in the development of human defensins as well as the cathelicidin LL-37 and their derivatives as antimicrobial agents against bacteria, viruses and fungi for clinical use

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 silico discovery of Acinetobacter baumannii genes involved in microaerobiosis resistance

Motivation: the infectious ability of Acinetobacter baumannii combined with its antibiotic resistant profile turn this bacteria into a objective with global priority, being currently highlighted by the World Health Organization as one of the most relevant resistant bacteria. Thanks to the development of Next-Generation Sequencing Methods, we can apply bioinformatics tools to analyse data that give us information about the behaviour of the bacteria under different conditions, which gives us the opportunity to discover new pharmacological targets that allow us to fight against infections by A. baumannii.Methods: We use data from RNA-Seq technique, obtained from A. baumannii ATCC 17978 growth in two different conditions: normoxia (21% O2) and microaerobiosis (0.1-0.3% O2). The first one is the regular situation, while the second one is the condition that the bacteria suffer when an infection occurs, especially across an injury, during the inflammatory phase. The results of this transcriptomic experiment were subjected to a bioinformatic workflow, starting with the quality analysis and trimming process, followed by the alignment of the reads and their quantification, until the differential expression analysis, whose results were filtered according to fold change value (R2>=1) and p-value (padj<0.05). Additionally, we want to improve the current annotation from A. baumannii ATCC 17978 genome. For that purpose, three sources of candidates were combined: GenBank available information, Prokka predictions and AnaBlast predictions. With this annotation, we can associate possible paths and processes in which the differential expression genes could be implicated, by a functional enrichment analysis using GO terms and KEGG, and the packages from R programming languages, such as Bioconductor and Clusterprofiler.Results: Resultados obtenidos. No tienen porqué aparecer todos los apartados (se puede prescindir de alguno de ellos, o todos). No más de 2500 caracteres el total del resumen. Incluir aparte (en el apartado de debajo) 1-3 referencias bibliográficas.Conclusions: The differentially expressed genes are enriched in X and Y, and these pathways have later been reviewed and completed by manual annotation using specific proteins databases. This could give us key information about the behaviour of the bacteria when it is found in hypoxia during the infection, and we could even find some factors involved in its virulence

In vitro effect of ceftazidime-avibactam pressure on ceftazidime-avibactam resistance in KPC-producing Klebsiella pneumoniae clinical isolates.

Motivation: Infections caused by KPC-producing Klebsiella pneumoniae represent a challenge due to the limited available treatement choices. In this context, ceftazidime-avibactam (CAZ-AVI) is postulated as an alternative treatment effective against class A beta-lactamases such as KPC [1]. But, recent data reported the failure of CAZ-AVI treatment of infections by KPC-producing K. pneumoniae due to the development of CAZ-AVI resistance [2]. However, little is known concernig the CAZ-AVI resistance developement by CAZ-AVI selective pressure. Here, we aimed to determinate in vitro whether the exposure of KPC-producing K. pneumoniae clinical isolates to CAZ-AVI subinhibitory concentrations could lead the selection of CAZ-AVI resistant isolates. Methods: Seventeen KPC-producing K. pneumoniae clinical isolates (7 KPC-2, 9 KPC-3 and 1 KPC-11) were analyzed. Minimum inhibitory concentrations (MICs) of CAZ-AVI were determined by broth microdilution using a fixed AVI cocentration of 4 mg/L [3]. Moreover, these isolates were further exposed to increasing concentrations of CAZ and fixed 4 mg/L of AVI, from a sub-MIC up to 256/4 mg/L of CAZ-AVI (or the concentration able to kill the bacterial isolate) at 37ºC with shaking during 24h. New MICs to CAZ-AVI were determined in each condition and after 15 days without CAZ-AVI pressure. Therefore, in order to demonstrated that blaKPC gene is responsible for acquisition of CAZ-AVI resistance in KPC-producing K. pneumoniae, blaKPC-2 and blaKPC-3 were cloned into a reference K. pneumoniae CECT 997 strain. Resistance or susceptibility were determined according to EUCAST criteria [3]. Results: All (17/17, 100%) KPC-producing K. pneumoniae isolates were able to grow at high concentrations of CAZ-AVI (≥64/4 mg/L), increasing their resistance to CAZ-AVI ≥8-fold. Likewise, fifteen of the 17 (88.2%) resistant isolates maintained the acquired CAZ-AVI resistance 15 days after without CAZ-AVI pressure. In addition, the CECT 997 mutants with blaKPC-2 or blaKPC-3 were able to grow up to 256/4 mg/L of CAZ-AVI, displaying and maintaining CAZ-AVI MIC shift from <0.01/4 mg/L (susceptible) to 512/4 mg/L (resistant). Conclusions: These data suggest that exposure of KPC-producing K. pneumoniae to subinhibitory CAZ-AVI concentrations could lead to the selection of CAZ-AVI resistance and this resistance is stable over the time

Sequence-activity relationship, and mechanism of action of mastoparan analogues against extended-drug resistant Acinetobacter baumannii

The treatment of some infectious diseases can currently be very challenging since the spread of multi-, extended- or pan-resistant bacteria has considerably increased over time. On the other hand, the number of new antibiotics approved by the FDA has decreased drastically over the last 30 years. The main objective of this study was to investigate the activity of wasp peptides, specifically mastoparan and some of its derivatives against extended-resistant Acinetobacter baumannii. We optimized the stability of mastoparan in human serum since the specie obtained after the action of the enzymes present in human serum is not active. Thus, 10 derivatives of mastoparan were synthetized. Mastoparan analogues (guanidilated at the N-terminal, enantiomeric version and mastoparan with an extra positive charge at the C-terminal) showed the same activity against Acinetobacter baumannii as the original peptide (2.7 muM) and maintained their stability to more than 24 h in the presence of human serum compared to the original compound. The mechanism of action of all the peptides was carried out using a leakage assay. It was shown that mastoparan and the abovementioned analogues were those that released more carboxyfluorescein. In addition, the effect of mastoparan and its enantiomer against A. baumannii was studied using transmission electron microscopy (TEM). These results suggested that several analogues of mastoparan could be good candidates in the battle against highly resistant A. baumannii infections since they showed good activity and high stability

Genomic Evolution of Two Acinetobacter baumannii Clinical Strains from ST-2 Clones Isolated in 2000 and 2010 (ST-2_clon_2000 and ST-2_clon_2010)

Acinetobacter baumannii is a successful nosocomial pathogen due to its ability to persist in hospital environments by acquiring mobile elements such as transposons, plasmids, and phages. In this study, we compared two genomes of A. baumannii clinical strains isolated in 2000 (ST-2_clon_2000) and 2010 (ST-2_clon_2010) from GenBank project PRJNA308422

Synergic effect of oxyclozanide in combination with colistin against colistin-resistant and colistin-susceptible clinical strains of Klebsiella pneumoniae

Motivation: Colistin is among the few antibiotics effective against Klebsiella pneumoniae clinical isolates. However, in the last few years, colistin-resistant K. pneumoniae have been isolated (1). Therefore, combination therapies between colistin and old drug effective against these isolates are required. The objective of this study is to study in vitro the activity of oxyclozanide, an anthelmintic drug (2), in combination with colistin against colistin-susceptible (Col-S) and colistin-resistant K. pneumoniae.Methods: Col-R (KPc21) and Col-S (CECT 997) K. pneumonia strains were used. Checkerboard assay with colistin and oxyclozanide to study the synergy between both drugs was performed. Time-kill assays using both strains at 6 log CFU/ml, colistin and oxyclozanide were tested alone and in combination with sub-minimal inhibitory concentration (MIC) of colistin (0.25 µg/ml for CECT 997 strain and 16 µg/ml for KPc21 strain) and oxyclozanide at 2 µg/ml. Analysis of KPc21 and CECT 997 strains cell walls in presence of 2 µg/ml oxyclozanide during 24 h by transmission electron microscopy (TEM) was performed. Permeability assays and outer membrane proteins (OMPs) profile analysis by SDS-PAGE of both strains were performed.Results: Checkerboard assay showed a synergic effect between colistin and oxyclozanide against the KPc21 strain (Fold change = 8), but not for CECT 997 strain (Fold change = 2). Time-kill assays showed a synergic effect between colistin and oxyclozanide against the KPc21 strain (decreasing the bacterial growth by 3.24 log CFU/mL) at 24 h, but not against the CECT 997 strain whose bacterial growth was reduced by 0.45 log CFU/mL. Incubation with oxyclozanide at 24 h did not cause change on the OMPs profile of both strains. Futhermore, the images from TEM showed that oxyclozanide disrupted the bacterial cell envelope affecting its permeability. The membrane permeabilization assay confirmed these data, in which the Col-R strain had higher membrane permeability.Conclusions: From these in vitro data, we concluded that oxyclozanide potentiates the bactericidal activity of colistin by disrupting the bacterial cell envelope. For this reason, oxyclozanide would be a good adjuvant for colistin to treating the infections caused by K. pneumoniae