Siderophores promote cooperative interspecies and intraspecies cross-protection against antibiotics in vitro.

The antibiotic cefiderocol hijacks iron transporters to facilitate its uptake and resists ß-lactamase degradation. While effective, resistance has been detected clinically with unknown mechanisms. Here, using experimental evolution, we identified cefiderocol resistance mutations in Pseudomonas aeruginosa. Resistance was multifactorial in host-mimicking growth media, led to multidrug resistance and paid fitness costs in cefiderocol-free environments. However, kin selection drove some resistant populations to cross-protect susceptible individuals from killing by increasing pyoverdine secretion via a two-component sensor mutation. While pyochelin sensitized P. aeruginosa to cefiderocol killing, pyoverdine and the enterobacteria siderophore enterobactin displaced iron from cefiderocol, preventing uptake by susceptible cells. Among 113 P. aeruginosa intensive care unit clinical isolates, pyoverdine production directly correlated with cefiderocol tolerance, and high pyoverdine producing isolates cross-protected susceptible P. aeruginosa and other Gram-negative bacteria. These in vitro data show that antibiotic cross-protection can occur via degradation-independent mechanisms and siderophores can serve unexpected protective cooperative roles in polymicrobial communities.

Evolved bacterial siderophore-mediated antibiotic cross-protection.

Antibiotic cross-protection enables resistant bacteria to protect other bacteria that would be otherwise susceptible to the drug. Cefiderocol is the first siderophore cephalosporin antibiotic approved for treating Gram-negative bacterial infections, including carbapenem-resistant Pseudomonas aeruginosa strains. While highly effective, CFDC resistance has been detected clinically, and mechanisms of resistance and cross-protection are not completely understood. In this study, we used experimental evolution and whole genome sequencing to identify cefiderocol resistance mechanisms and evaluated the trade-offs of evolving resistance. We found some cefiderocol-resistant populations evolved cross-protective social behavior, preventing cefiderocol killing of susceptible siblings. Notably, cross-protection was driven by increased secretion of bacterial iron-binding siderophores, which is unique from previously described antibiotic degradation mediated cross-protection. While concerning, we also showed that resistance can be selected against in drug-free environments. Deciphering the costs associated with antibiotic resistance might aid the development of evolution-informed therapeutic approaches to delay the evolution of antibiotic resistance.

Antimicrobial action of 1,10-phenanthroline-based compounds on carbapenemase-producing Acinetobacter baumannii clinical strains: efficacy against planktonic- and biofilm-growing cells.

Therapeutic options are limited for patients infected with Acinetobacter baumannii due to its multidrug-resistance profile. So, the search for new antimicrobials against this gram-negative bacterial pathogen has become a worldwide priority. The present study aimed to evaluate the effects of 1,10-phenanthroline (phen), 1,10-phenanthroline-5,6-dione (phendione), [Ag(phendione)2]ClO4 (Ag-phendione) and [Cu(phendione)3](ClO4)2·4H2O (Cu-phendione) on 26 carbapenemase-producing A. baumannii strains. The susceptibility to carbapenems was performed by detecting the metallo-beta-lactamase (MBL) genes by PCR and by determining the MIC. Also, disk diffusion method was applied to evaluate the susceptibility to other antimicrobial classes. The test compounds were evaluated on both planktonic- and biofilm-growing bacterial cells. The results revealed that all A. baumannii strains had the intrinsic blaoxa-51 gene, and at least one of the blaoxa-23 or blaoxa-24 genes. The geometric mean MIC and minimum bactericidal concentration (MBC) values, respectively, were as follows: Cu-phendione (1.56 and 2.30 µM), Ag-phendione (2.48 and 3.63 µM), phendione (9.44 and 9.70 µM), and phen (70.46 and 184.28 µM). The test compounds (at 0.5 × MIC) affected the biofilm formation and disrupted the mature biofilm, in a typically dose-dependent manner, reducing biomass and viability parameters. Collectively, silver and copper-phendione derivatives presented potent antimicrobial action against planktonic- and biofilm-forming cells of carbapenemase-producing A. baumannii.

Disarming Pseudomonas aeruginosa Virulence by the Inhibitory Action of 1,10-Phenanthroline-5,6-Dione-Based Compounds: Elastase B (LasB) as a Chemotherapeutic Target.

Elastase B (lasB) is a multifunctional metalloenzyme secreted by the gram-negative pathogen Pseudomonas aeruginosa, and this enzyme orchestrates several physiopathological events during bacteria-host interplays. LasB is considered to be a potential target for the development of an innovative chemotherapeutic approach, especially against multidrug-resistant strains. Recently, our group showed that 1,10-phenanthroline-5,6-dione (phendione), [Ag(phendione)2]ClO4 (Ag-phendione) and [Cu(phendione)3](ClO4)2.4H2O (Cu-phendione) had anti-P. aeruginosa action against both planktonic- and biofilm-growing cells. In the present work, we have evaluated the effects of these compounds on the (i) interaction with the lasB active site using in silico approaches, (ii) lasB proteolytic activity by using a specific fluorogenic peptide substrate, (iii) lasB gene expression by real time-polymerase chain reaction, (iv) lasB protein secretion by immunoblotting, (v) ability to block the damages induced by lasB on a monolayer of lung epithelial cells, and (vi) survivability of Galleria mellonella larvae after being challenged with purified lasB and lasB-rich bacterial secretions. Molecular docking analyses revealed that phendione and its Ag+ and Cu2+ complexes were able to interact with the amino acids forming the active site of lasB, particularly Cu-phendione which exhibited the most favorable interaction energy parameters. Additionally, the test compounds were effective inhibitors of lasB activity, blocking the in vitro cleavage of the peptide substrate, aminobenzyl-Ala-Gly-Leu-Ala-p-nitrobenzylamide, with Cu-phendione having the best inhibitory action (K i = 90 nM). Treating living bacteria with a sub-inhibitory concentration (½ × MIC value) of the test compounds caused a significant reduction in the expression of the lasB gene as well as its mature protein production/secretion. Further, Ag-phendione and Cu-phendione offered protective action for lung epithelial cells, reducing the A549 monolayer damage by approximately 32 and 42%, respectively. Interestingly, Cu-phendione mitigated the toxic effect of both purified lasB molecules and lasB-containing bacterial secretions in the in vivo model, increasing the survival time of G. mellonella larvae. Collectively, these data reinforce the concept of lasB being a veritable therapeutic target and phendione-based compounds (mainly Cu-phendione) being prospective anti-virulence drugs against P. aeruginosa.

Anti-Virulence Strategy against the Multidrug-Resistant Bacterial Pathogen Pseudomonas aeruginosa: Pseudolysin (Elastase B) as a Potential Druggable Target.

Pseudomonas aeruginosa is a non-fermentative, gram-negative bacterium that is one of the most common pathogens responsible for hospital-acquired infections worldwide. The management of the infections caused by P. aeruginosa represents a huge challenge in the healthcare settings due to the increased emergence of resistant isolates, some of them resistant to all the currently available antimicrobials, which results in elevated morbimortality rates. Consequently, the development of new therapeutic strategies against multidrug-resistant P. aeruginosa is urgent and needful. P. aeruginosa is wellrecognized for its extreme genetic versatility and its ability to produce a lush variety of virulence factors. In this context, pseudolysin (or elastase B) outstands as a pivotal virulence attribute during the infectious process, playing multifunctional roles in different aspects of the pathogen-host interaction. This protein is a 33-kDa neutral zinc-dependent metallopeptidase that is the most abundant peptidase found in pseudomonal secretions, which contributes to the invasiveness of P. aeruginosa due to its ability to cleave several extracellular matrix proteins and to disrupt the basolateral intercellular junctions present in the host tissues. Moreover, pseudolysin makes P. aeruginosa able to overcome host defenses by the hydrolysis of many immunologically relevant molecules, including antibodies and complement components. The attenuation of this striking peptidase therefore emerges as an alternative and promising antivirulence strategy to combat antibiotic-refractory infections caused by P. aeruginosa. The anti-virulence approach aims to disarm the P. aeruginosa infective arsenal by inhibiting the expression/activity of bacterial virulence factors in order to reduce the invasiveness of P. aeruginosa, avoiding the emergence of resistance since the proliferation is not affected. This review summarizes the most relevant features of pseudolysin and highlights this enzyme as a promising target for the development of new anti-virulence compounds.

Heterogeneous production of proteases from Brazilian clinical isolates of Pseudomonas aeruginosa / La producción heterogénea de las proteasas a partir de aislamientos clínicos brasileños de Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa is an important human pathogen that causes severe infections in a wide range of immunosuppressed patients. Herein, we evaluated the proteolytic profiles of 96 Brazilian clinical isolates of P. aeruginosa recovered from diverse anatomical sites. Methods: Cell-associated and extracellular proteases were evidenced by gelatin-SDS-PAGE and by the cleavage of soluble gelatin. Elastase was measured by using the peptide substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide. The prevalence of elastase genes (lasA and lasB) was evaluated by PCR. Results: Bacterial extracts were initially applied on gelatin-SDS-PAGE and the results revealed four distinct zymographic profiles as follows: profile I (composed by bands of 145, 118 and 50kDa), profile II (118 and 50kDa), profile III (145kDa) and profile IV (118kDa). All the proteolytic enzymes were inhibited by EDTA, identifying them as metalloproteases. The profile I was the most detected in both cellular (79.2%) and extracellular (84.4%) extracts. Overall, gelatinase and elastase activities measured in the spent culture media were significantly higher (around 2-fold) compared to the cellular extracts and the production level varied according to the site of bacterial isolation. For instance, tracheal secretion isolates produced elevated amount of gelatinase and elastase measured in both cellular and extracellular extracts. The prevalence of elastase genes revealed that 100% isolates were lasB-positive and 85.42% lasA-positive. Some positive/negative correlations were showed concerning the production of gelatinase, elastase, isolation site and antimicrobial susceptibility. Conclusion: The protease production was highly heterogeneous in Brazilian clinical isolates of P. aeruginosa, which corroborates the genomic/metabolic versatility of this pathogen (AU)

Antecedentes: Pseudomonas aeruginosa (P. aeruginosa) es un importante patógeno humano que causa graves infecciones en diversos tipos de pacientes inmunodeprimidos. En este trabajo evaluamos los perfiles proteolíticos de 96 aislamientos clínicos brasileños de P. aeruginosaaislados de diferentes localizaciones anatómicas. Métodos: Las proteasas extracelulares y de extractos celulares fueron analizadas por SDS-PAGE copolimerizada con gelatina y a través de clivaje de gelatina en solución. La elastasa fue medida usando el substrato peptídico N-succinil-Ala-Ala-Ala-p-nitroanilida. La prevalencia de genes codificantes para elastasa (lasA y lasB) fue evaluada por PCR. Resultados: En primer lugar, los extractos de las bacterias fueron aplicados en geles de SDS-PAGE-gelatina, los cuales, después de revelados, revelaron 4 perfiles enzimográficos, así: perfil I(compuesto por bandas de 145, 118 y 50kDa), perfil II (118 y 50kDa), perfil III (145kDa) y perfil IV (118kDa). Todas las enzimas proteolíticas fueron inhibidas por EDTA, siendo, por tanto, identificadas como metaloproteasas. El perfil I fue el más detectado tanto en los extractos celulares (79,2%) como en los extracelulares (84,4%). Las actividades de gelatinasa y elastasa medidas en el medio de cultivo fueron significativamente más elevadas (cerca de 2 veces) que en los extractos celulares y el nivel de producción varió de acuerdo al sitio del cual fue aislada la cepa. Por ejemplo, cepas aisladas de secreción traqueal produjeron cantidades elevadas de gelatinasa y elastasa medidas tanto en el extracto celular como en los extractos extracelulares. La prevalencia de los genes de elastasa reveló que el 100% de los aislamientos fueron lasB positivos y 85.42% lasA positivos. En algunos casos se observó una correlación positiva/negativa respecto a la producción de gelatinasa, elastasa, sitio de aislamiento y susceptibilidad antimicrobiana. Conclusión: La producción de proteasas fue altamente heterogénea en los aislamientos clínicos brasileños de P. aeruginosa, lo cual corroboran la versatilidad genómica/metabólica de este patógeno (AU)

Heterogeneous production of proteases from Brazilian clinical isolates of Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is an important human pathogen that causes severe infections in a wide range of immunosuppressed patients. Herein, we evaluated the proteolytic profiles of 96 Brazilian clinical isolates of P. aeruginosa recovered from diverse anatomical sites. METHODS: Cell-associated and extracellular proteases were evidenced by gelatin-SDS-PAGE and by the cleavage of soluble gelatin. Elastase was measured by using the peptide substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide. The prevalence of elastase genes (lasA and lasB) was evaluated by PCR. RESULTS: Bacterial extracts were initially applied on gelatin-SDS-PAGE and the results revealed four distinct zymographic profiles as follows: profile I (composed by bands of 145, 118 and 50kDa), profile II (118 and 50kDa), profile III (145kDa) and profile IV (118kDa). All the proteolytic enzymes were inhibited by EDTA, identifying them as metalloproteases. The profile I was the most detected in both cellular (79.2%) and extracellular (84.4%) extracts. Overall, gelatinase and elastase activities measured in the spent culture media were significantly higher (around 2-fold) compared to the cellular extracts and the production level varied according to the site of bacterial isolation. For instance, tracheal secretion isolates produced elevated amount of gelatinase and elastase measured in both cellular and extracellular extracts. The prevalence of elastase genes revealed that 100% isolates were lasB-positive and 85.42% lasA-positive. Some positive/negative correlations were showed concerning the production of gelatinase, elastase, isolation site and antimicrobial susceptibility. CONCLUSION: The protease production was highly heterogeneous in Brazilian clinical isolates of P. aeruginosa, which corroborates the genomic/metabolic versatility of this pathogen.

Anti-Pseudomonas aeruginosa activity of 1,10-phenanthroline-based drugs against both planktonic- and biofilm-growing cells.

OBJECTIVES: The beneficial antimicrobial properties of 1,10-phenanthroline (phen)-based drugs, together with the imperative need to develop new chemotherapeutic options for prevention/treatment of infections caused by MDR Gram-negative bacteria, led us to evaluate the effects of phen, 1,10-phenanthroline-5,6-dione (phendione), [Ag(phendione)2]ClO4 and [Cu(phendione)3](ClO4)2·4H2O on planktonic- and biofilm-growing Pseudomonas aeruginosa. METHODS: Thirty-two non-duplicated Brazilian clinical isolates of P. aeruginosa with distinct genetic backgrounds were used in all experiments. The effect of test compounds on planktonic bacterial proliferation was determined as recommended by CLSI protocol. The effect on biofilm formation was evaluated by crystal violet incorporation (biomass determination) and XTT (viability assay). Mature biofilm disorganization was evidenced by staining with crystal violet. RESULTS: Phen-based compounds presented anti-P. aeruginosa activity, but with different potencies concerning the geometric mean MIC: [Cu(phendione)3](2+) (7.76 µM) > [Ag(phendione)2](+) (14.05 µM) > phendione (31.15 µM) > phen (579.28 µM). MICs of each compound were similar irrespective of whether the P. aeruginosa isolates were susceptible or resistant to classical antimicrobials (ceftazidime, meropenem and imipenem). The pretreatment of bacteria with phen, phendione and phendione's metal derivatives at 0.5 × MIC value inhibited biofilm formation, particularly the use of [Cu(phendione)3](2+) and [Ag(phendione)2](+), which significantly reduced both biomass (48% and 44%, respectively) and viability (78% and 77%, respectively). The compounds studied also disrupted mature biofilm in a dose-dependent manner, especially [Ag(phendione)2](+) and [Cu(phendione)3](2+) (IC50, 9.39 and 10.16 µM, respectively). CONCLUSIONS: Coordination of phendione to Ag(+) and Cu(2+) represents a new promising group of anti-infective agents, which revealed a potent anti-P. aeruginosa action against both planktonic- and biofilm-growing cells.

Candida parapsilosis (sensu lato) isolated from hospitals located in the Southeast of Brazil: Species distribution, antifungal susceptibility and virulence attributes.

Candida parapsilosis (sensu lato), which represents a fungal complex composed of three genetically related species - Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis, has emerged as an important yeast causing fungemia worldwide. The goal of the present work was to assess the prevalence, antifungal susceptibility and production of virulence traits in 53 clinical isolates previously identified as C. parapsilosis (sensu lato) obtained from hospitals located in the Southeast of Brazil. Species forming this fungal complex are physiologically/morphologically indistinguishable; however, polymerase chain reaction followed by restriction fragment length polymorphism of FKS1 gene has solved the identification inaccuracy, revealing that 43 (81.1%) isolates were identified as C. parapsilosis sensu stricto and 10 (18.9%) as C. orthopsilosis. No C. metapsilosis was found. The geographic distribution of these Candida species was uniform among the studied Brazilian States (São Paulo, Rio de Janeiro and Espírito Santo). All C. orthopsilosis and almost all C. parapsilosis sensu stricto (95.3%) isolates were susceptible to amphotericin B, fluconazole, itraconazole, voriconazole and caspofungin. Nevertheless, one C. parapsilosis sensu stricto isolate was resistant to fluconazole and another one was resistant to caspofungin. C. parapsilosis sensu stricto isolates exhibited higher MIC mean values to amphotericin B, fluconazole and caspofungin than those of C. orthopsilosis, while C. orthopsilosis isolates displayed higher MIC mean to itraconazole compared to C. parapsilosis sensu stricto. Identical MIC mean values to voriconazole were measured for these Candida species. All the isolates of both species were able to form biofilm on polystyrene surface. Impressively, biofilm-growing cells of C. parapsilosis sensu stricto and C. orthopsilosis exhibited a considerable resistance to all antifungal agents tested. Pseudohyphae were observed in 67.4% and 80% of C. parapsilosis sensu stricto and C. orthopsilosis isolates, respectively. The secretion of phytase (93% versus 100%), aspartic protease (88.4% versus 90%), esterase (20.9% versus 50%) and hemolytic factors (25.6% versus 40%) was detected in C. parapsilosis sensu stricto and C. orthopsilosis isolates, respectively; however, no phospholipase activity was identified. An interesting fact was observed concerning the caseinolytic activity, for which all the producers (53.5%) belonged to C. parapsilosis sensu stricto. Collectively, our results add new data on the epidemiology, antifungal susceptibility and production of potential virulence attributes in clinical isolates of C. parapsilosis complex.

Virulence attributes in Brazilian clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen responsible for causing a huge variety of acute and chronic infections with significant levels of morbidity and mortality. Its success as a pathogen comes from its genetic/metabolic plasticity, intrinsic/acquired antimicrobial resistance, capacity to form biofilm and expression of numerous virulence factors. Herein, we have analyzed the genetic variability, antimicrobial susceptibility as well as the production of metallo-ß-lactamases (MBLs) and virulence attributes (elastase, pyocyanin and biofilm) in 96 strains of P. aeruginosa isolated from different anatomical sites of patients attended at Brazilian hospitals. Our results revealed a great genetic variability, in which 86 distinct RAPD types (89.6% of polymorphisms) were detected. Regarding the susceptibility profile, 48 strains (50%) were resistant to the antimicrobials, as follows: 22.92% to the three tested antibiotics, 12.5% to both imipenem and meropenem, 11.46% to ceftazidime only, 2.08% to imipenem only and 1.04% to both ceftazidime and meropenem. Out of the 34 clinical strains of P. aeruginosa resistant to both imipenem and meropenem, 25 (73.53%) were MBL producers by phenotypic method while 12 (35.29%) were PCR positive for the MBL gene SPM-1. All P. aeruginosa strains produced pyocyanin, elastase and biofilm, although in different levels. Some associations were demonstrated among the susceptibility and/or production of these virulence traits with the anatomical site of strain isolation. For instance, almost all strains isolated from urine (85.71%) were resistant to the three antibiotics, while the vast majority of strains isolated from rectum (95%) and mouth (66.67%) were susceptible to all tested antibiotics. Urine isolates produced the highest pyocyanin concentration (20.15±5.65 µg/ml), while strains isolated from pleural secretion and mouth produced elevated elastase activity (1441.43±303.08 FAU) and biofilm formation (OD590 0.676±0.32), respectively. Also, MBL-positive strains produced robust biofilm compared to MBL-negative strains. Collectively, the production of site-dependent virulence factors can be highlighted as potential therapeutic targets for the treatment of infections caused by heterogeneous and resistant strains of P. aeruginosa.