Efficacy of Tamoxifen Metabolites in Combination with Colistin and Tigecycline in Experimental Murine Models of Escherichia coli and Acinetobacter baumannii.

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.

Ultrasensitive and rapid identification of ESRI developer- and piperacillin/tazobactam-resistant Escherichia coli by the MALDIpiptaz test.

BACKGROUND: The excessive use of piperacillin/tazobactam (P/T) has promoted the emergence of P/T-resistant Enterobacterales. We reported that in Escherichia coli, P/T contributes to the development of extended-spectrum resistance to ß-lactam/ß-lactamase inhibitor (BL/BLI) (ESRI) in isolates that are P/T susceptible but have low-level resistance to BL/BLI. Currently, the detection of P/T resistance relying on conventional methods is time-consuming. To overcome this issue, we developed a cost-effective test based on MALDI-MS technology, called MALDIpiptaz, which aims to detect P/T resistance and ESRI developers in E. coli. METHODS: We used automated Clover MS Data Analysis software to analyse the protein profile spectra obtained by MALDI-MS from a collection of 248 E. coli isolates (91 P/T-resistant, 81 ESRI developers and 76 P/T-susceptible). This software allowed to preprocess all the spectra to build different peak matrices that were analysed by machine learning algorithms. RESULTS: We demonstrated that MALDIpiptaz can efficiently and rapidly (15 min) discriminate between P/T-resistant, ESRI developer and P/T-susceptible isolates and allowed the correct classification between ESRI developers from their isogenic resistance to P/T. CONCLUSION: The combination of excellent performance and cost-effectiveness are all desirable attributes, allowing the MALDIpiptaz test to be a useful tool for the rapid determination of P/T resistance in clinically relevant E. coli isolates.

Repurposing of the Tamoxifen Metabolites to Treat Methicillin-Resistant Staphylococcus epidermidis and Vancomycin-Resistant Enterococcus faecalis Infections.

Repurposing drugs provides a new approach to the fight against multidrug-resistant (MDR) bacteria. We have reported that three major tamoxifen metabolites, N-desmethyltamoxifen (DTAM), 4-hydroxytamoxifen (HTAM), and endoxifen (ENDX), presented bactericidal activity against Acinetobacter baumannii and Escherichia coli. Here, we aimed to analyze the activity of a mixture of the three tamoxifen metabolites against methicillin-resistant Staphylococcus epidermidis (MRSE) and Enterococcus species. MRSE (n = 17) and Enterococcus species (Enterococcus faecalis n = 8 and Enterococcus faecium n = 10) strains were used. MIC of the mixture of DTAM, HTAM, and ENDX and that of vancomycin were determined by microdilution assay. The bactericidal activity of the three metabolites together and of vancomycin against MRSE (SE385 and SE742) and vancomycin-resistant E. faecalis (EVR1 and EVR2) strains was determined by time-kill curve assays. Finally, changes in membrane permeability of SE742 and EVR1 strains were analyzed using fluorescence assays. MIC90 of tamoxifen metabolites was 1 mg/liter for MRSE strains and 2 mg/liter for E. faecalis and E. faecium strains. In the time-killing assays, tamoxifen metabolites mixture showed bactericidal activity at 4× MIC for MRSE (SE385 and SE742) and at 2× MIC and 4× MIC for E. faecalis (EVR1 and EVR2) strains, respectively. SE385 and EVR2 strains treated with the tamoxifen metabolites mixture presented higher membrane permeabilization. Altogether, these results showed that tamoxifen metabolites presented antibacterial activity against MRSE and vancomycin-resistant E. faecalis, suggesting that tamoxifen metabolites might increase the arsenal of drug treatments against these bacterial pathogens. IMPORTANCE The development of new antimicrobial therapeutic strategies requires immediate attention to avoid the tens of millions of deaths predicted to occur by 2050 as a result of MDR bacterial infections. In this study, we assessed the antibacterial activity of three major tamoxifen metabolites, N-desmethyltamoxifen (DTAM), 4-hydroxytamoxifen (HTAM), and endoxifen (ENDX), against methicillin-resistant Staphylococcus epidermidis (MRSE) and Enterococcus spp. (E. faecalis and E. faecium). We found that the tamoxifen metabolites have antibacterial activity against MRSE, E. faecalis, and E. faecium strains by presenting MIC90 between 1 and 2 mg/liter and bactericidal activity over 24 h. In addition, this antibacterial activity is paralleled by an increased membrane permeability of these strains. Our results showed that tamoxifen metabolites might be potentially used as a therapeutic alternative when treating MRSE and E. faecalis strains in an animal model of infection.

Repurposing of the Tamoxifen Metabolites to Combat Infections by Multidrug-Resistant Gram-Negative Bacilli.

The development of new strategic antimicrobial therapeutic approaches, such as drug repurposing, has become an urgent need. Previously, we reported that tamoxifen presents therapeutic efficacy against multidrug-resistant (MDR) Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli in experimental infection models by modulating innate immune system cell traffic. The main objective of this study was to analyze the activity of N-desmethyltamoxifen, 4-hydroxytamoxifen, and endoxifen, three major metabolites of tamoxifen, against these pathogens. We showed that immunosuppressed mice infected with A. baumannii, P. aeruginosa, or E. coli in peritoneal sepsis models and treated with tamoxifen at 80 mg/kg/d for three days still reduced the bacterial load in tissues and blood. Moreover, it increased mice survival to 66.7% (for A. baumannii and E. coli) and 16.7% (for P. aeruginosa) when compared with immunocompetent mice. Further, susceptibility and time-kill assays showed that N-desmethyltamoxifen, 4-hydroxytamoxifen, and endoxifen exhibited minimum inhibitory concentration of the 90% of the isolates (MIC90) values of 16 mg/L, and were bactericidal against clinical isolates of A. baumannii and E. coli. This antimicrobial activity of tamoxifen metabolites paralleled an increased membrane permeability of A. baumannii and E. coli without affecting their outer membrane proteins profiles. Together, these data showed that tamoxifen metabolites presented antibacterial activity against MDR A. baumannii and E. coli, and may be a potential alternative for the treatment of infections caused by these two pathogens.

Antibacterial Activity of Colloidal Silver against Gram-Negative and Gram-Positive Bacteria.

Due to the emergence of antimicrobial resistance, new alternative therapies are needed. Silver was used to treat bacterial infections since antiquity due to its known antimicrobial properties. Here, we aimed to evaluate the in vitro activity of colloidal silver (CS) against multidrug-resistant (MDR) Gram-negative and Gram-positive bacteria. A total of 270 strains (Acinetobacter baumannii (n = 45), Pseudomonas aeruginosa (n = 25), Escherichia coli (n = 79), Klebsiella pneumoniae (n = 58)], Staphylococcus aureus (n = 34), Staphylococcus epidermidis (n = 14), and Enterococcus species (n = 15)) were used. The minimal inhibitory concentration (MIC) of CS was determined for all strains by using microdilution assay, and time-kill curve assays of representative reference and MDR strains of these bacteria were performed. Membrane permeation and bacterial reactive oxygen species (ROS) production were determined in presence of CS. CS MIC90 was 4-8 mg/L for all strains. CS was bactericidal, during 24 h, at 1× and 2× MIC against Gram-negative bacteria, and at 2× MIC against Gram-positive bacteria, and it did not affect their membrane permeabilization. Furthermore, we found that CS significantly increased the ROS production in Gram-negative with respect to Gram-positive bacteria at 24 h of incubation. Altogether, these results suggest that CS could be an effective treatment for infections caused by MDR Gram-negative and Gram-positive bacteria.