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

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

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

Efficacy of cefepime and imipenem in experimental murine pneumonia caused by porin-deficient Klebsiella pneumoniae producing CMY-2 β-lactamase

Previous studies have shown decreased in vitro activity of zwitterionic cephalosporins and carbapenems against porin-deficient Klebsiella pneumoniae expressing a plasmid-mediated AmpC-type β-lactamase (PACBL). The in vitro and in vivo activities of cefepime and imipenem were evaluated against the porin-deficient strain K. pneumoniae C2 and its CMY-2-producing derivative [K. pneumoniae C2(pMG248)]. The MICs (in micrograms/milliliter) of cefepime and imipenem against K. pneumoniae C2 were 0.125 and 0.25, respectively, while the corresponding values against K. pneumoniae C2(pMG248) were 8 and 16. Cefepime showed a greater inoculum effect than imipenem against both strains. Imipenem showed a significant post-antibiotic effect (> 2 h) against K. pneumoniae C2(pMG248) at 1x, 2x, 4x, 6x, and 8x MIC. The maximum concentrations of drug in serum of cefepime and imipenem in a pneumonia model using mice were 124.1 and 16.9 μg/ml, respectively. ΔT/MIC for K. pneumoniae C2 and C2(pMG248) were 1.29 h and 0.34 h for imipenem and 2.96 h and 1.27 h for cefepime. Both imipenem (30 mg/kg of body weight every 3 h) and cefepime (60 mg/kg every 4 h), administered for 72 h, increased the survival rate (86.6% and 100%) compared with untreated control animals (26.6%, P < 0.003) infected with K. pneumoniae C2. For the CMY-2-producing strain, imipenem, but not cefepime, increased the survival rate compared to the controls (86.6% and 40% versus 40%, P < 0.01). Bacterial concentration of the lungs was significantly decreased by both antimicrobials. In conclusion, imipenem was more active in terms of survival than cefepime for the treatment of murine pneumonia caused by a porin-deficient K. pneumoniae expressing PACBL CMY-2.Consejería de Salud, Junta de Andalucía 00/153Red Española para la Investigación en Patología Infecciosa REIPI-ISCIII-C03/1

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 Risk Factors for Multidrug-Resistant Organisms Colonization in Long-Term Care Facilities Around the World: A Review

Elderly people confined to chronic care facilities face an increased risk of acquiring infections by multidrug-resistant organisms (MDROs). This review presents the current knowledge of the prevalence and risk factors for colonization by MDROs in long-term care facilities (LTCF), thereby providing a useful reference to establish objectives for implementing successful antimicrobial stewardship programs (ASPs). We searched in PubMed and Scopus for studies examining the prevalence of MDROs and/or risk factors for the acquisition of MDROs in LTCF. One hundred and thirty-four studies published from 1987 to 2020 were included. The prevalence of MDROs in LTCF varies between the different continents, where Asia reported the highest prevalence of extended-spectrum ß-lactamase (ESBL) Enterobacterales (71.6%), carbapenem resistant (CR) Enterobacterales (6.9%) and methicillin-resistant Staphylococcus aureus (MRSA) (25.6%) and North America the highest prevalence to MDR Pseudomonas aeruginosa (5.4%), MDR Acinetobacter baumannii (15.0%), vancomycin-resistant Enterococcus spp. (VRE) (4.0%), and Clostridioides difficile (26.1%). Furthermore, MDRO prevalence has experienced changes over time, with increases in MDR P. aeruginosa and extended spectrum ß-lactamase producing Enterobacterales observed starting in 2015 and decreases of CR Enterobacterales, MDR A. baumannii, VRE, MRSA and C. difficile. Several risk factors have been found, such as male sex, chronic wounds, the use of medical devices, and previous antibiotic use. The last of these aspects represents one of the most important modifiable factors for reducing colonization with MDROs through implementing ASPs in LTCF.The study was funded by the Instituto de Salud Carlos III, the Spanish Ministry of Economy, Industry, and Competitiveness (grant number: PI17-02195) and was partially funded by the European Development Regional Fund “A way to achieve Europe”. A.R.V. and A.B.G.G. are supported by the Subprograma Río Hortega, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain. A.R.V. grant number: CM18/00122. A.B.G.G. grant number: CM19/00029. C.M.G. and J.C.C.R. are supported by the Instituto de Salud Carlos III (grant number: PI17-02195) and co-financed by European Development Regional Fund ‘A way to achieve Europe’ ERDF, Spanish Network for the Research in Infectious Diseases (REIPI RD16/0016/0009).G.P. is supported 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 (REIPI RD16/0016/0001)- co-financed by European Development Regional Fund “A way to achieve Europe”, Operative program Intelligent Growth 2014–2020. M.E.P.I. is a postdoctoral researcher belonging to the program “Nicolás Monardes” (C1-0038-2019), Servicio Andaluz de Salud, Junta de Andalucía, Spain. J.M.C. received funding for research from Plan Nacional de I+D+i 2013–2016 and Instituto de Salud Carlos III, Subdireccion General de Redes y Centros de InvestigacionCooperativa, Ministry of Economy, Industry and Competitiveness, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0001, RD16/0016/0009), co-financed by the European Development Regional Fund “A way to achieve Europe”.Ye

Statistical analysis plan. Study protocol for a randomized clinical trial to assess 7 versus 14-days of treatment for Pseudomonas aeruginosa bloodstream infections (SHORTEN-2 trial)

Efficacy and safety of 7 versus 14 days of antibiotic treatment for Pseudomonas aeruginosa bacteraemia: a multicentre, randomized clinical trial (SHORTEN-2) with a DOOR/RADAR analysis.Peer reviewe