In vitro activity of human defensins HNP-1 and hBD-3 against multidrug-resistant ESKAPE Gram-negatives of clinical origin and selected peptidoglycan recycling-defective mutants.

The use of immune compounds as antimicrobial adjuvants is a classic idea recovering timeliness in the current antibiotic resistance scenario. However, the activity of certain antimicrobial peptides against ESKAPE Gram-negatives has not been sufficiently investigated. The objective of this study was to determine the activities of human defensins HNP-1 and hBD-3 alone or combined with permeabilizing/peptidoglycan-targeting agents against clinical ESKAPE Gram-negatives [Acinetobacter baumannii (AB), Enterobacter cloacae (EC), Klebsiella pneumoniae (KP), and acute/chronic Pseudomonas aeruginosa (PA)]. Lethal concentrations (LCs) of HNP-1 and hBD-3 were determined in four collections of multidrug resistant EC, AB, KP, and PA clinical strains (10-36 isolates depending on the collection). These defensins act through membrane permeabilization plus peptidoglycan building blockade, enabling that alterations in peptidoglycan recycling may increase their activity, which is why different recycling-defective mutants were also included. Combinations with physiological lysozyme and subinhibitory colistin for bactericidal activities determination, and with meropenem for minimum inhibitory concentrations (MICs), were also assessed. HNP-1 showed undetectable activity (LC > 32 mg/L for all strains). hBD-3 showed appreciable activities: LC ranges 2-16, 8-8, 8->32, and 8->32 mg/L for AB, EC, KP, and PA, being PA strains from cystic fibrosis significantly more resistant than acute origin ones. None of the peptidoglycan recycling-defective mutants showed greater susceptibility to HNP-1/hBD-3. Combination with colistin or lysozyme did not change their bactericidal power, and virtually neither did meropenem + hBD-3 compared to meropenem MICs. This is the first study comparatively analyzing the HNP-1/hBD-3 activities against the ESKAPE Gram-negatives, and demonstrates interesting bactericidal capacities of hBD-3 mostly against AB and EC. IMPORTANCE: In the current scenario of critical need for new antimicrobials against multidrug-resistant bacteria, all options must be considered, including classic ideas such as the use of purified immune compounds. However, information regarding the activity of certain human defensins against ESKAPE Gram-negatives was incomplete. This is the first study comparatively assessing the in vitro activity of two membrane-permeabilizing/peptidoglycan construction-blocking defensins (HNP-1 and hBD-3) against relevant clinical collections of ESKAPE Gram-negatives, alone or in combination with permeabilizers, additional peptidoglycan-targeting attacks, or the blockade of its recycling. Our data suggest that hBD-3 has a notable bactericidal activity against multidrug-resistant Acinetobacter baumannii and Enterobacter cloacae strains that should be considered as potential adjuvant option. Our results suggest for the first time an increased resistance of Pseudomonas aeruginosa strains from chronic infection compared to acute origin ones, and provide new clues about the predominant mode of action of hBD-3 against Gram-negatives (permeabilization rather than peptidoglycan-targeting).

Transferable AmpCs in Klebsiella pneumoniae: interplay with peptidoglycan recycling, mechanisms of hyperproduction, and virulence implications.

Chromosomal and transferable AmpC ß-lactamases represent top resistance mechanisms in different gram-negatives, but knowledge regarding the latter, mostly concerning regulation and virulence-related implications, is far from being complete. To fill this gap, we used Klebsiella pneumoniae (KP) and two different plasmid-encoded AmpCs [DHA-1 (AmpR regulator linked, inducible) and CMY-2 (constitutive)] as models to perform a study in which we show that blockade of peptidoglycan recycling through AmpG permease inactivation abolished DHA-1 inducibility but did not affect CMY-2 production and neither did it alter KP pathogenic behavior. Moreover, whereas regular production of both AmpC-type enzymes did not attenuate KP virulence, when blaDHA-1 was expressed in an ampG-defective mutant, Galleria mellonella killing was significantly (but not drastically) attenuated. Spontaneous DHA-1 hyperproducer mutants were readily obtained in vitro, showing slight or insignificant virulence attenuations together with high-level resistance to ß-lactams only mildly affected by basal production (e.g., ceftazidime, ceftolozane/tazobactam). By analyzing diverse DHA-1-harboring clinical KP strains, we demonstrate that the natural selection of these hyperproducers is not exceptional (>10% of the collection), whereas mutational inactivation of the typical AmpC hyperproduction-related gene mpl was the most frequent underlying mechanism. The potential silent dissemination of this kind of strains, for which an important fitness cost-related contention barrier does not seem to exist, is envisaged as a neglected threat for most ß-lactams effectiveness, including recently introduced combinations. Analyzing whether this phenomenon is applicable to other transferable ß-lactamases and species as well as determining the levels of conferred resistance poses an essential topic to be addressed.IMPORTANCEAlthough there is solid knowledge about the regulation of transferable and especially chromosomal AmpC ß-lactamases in Enterobacterales, there are still gaps to fill, mainly related to regulatory mechanisms and virulence interplays of the former. This work addresses them using Klebsiella pneumoniae as model, delving into a barely explored conception: the acquisition of a plasmid-encoded inducible AmpC-type enzyme whose production can be increased through selection of chromosomal mutations, entailing dramatically increased resistance compared to basal expression but minor associated virulence costs. Accordingly, we demonstrate that clinical K. pneumoniae DHA-1 hyperproducer strains are not exceptional. Through this study, we warn for the first time that this phenomenon may be a neglected new threat for ß-lactams effectiveness (including some recently introduced ones) silently spreading in the clinical context, not only in K. pneumoniae but potentially also in other pathogens. These facts must be carefully considered in order to design future resistance-preventive strategies.

Filling knowledge gaps related to AmpC-dependent ß-lactam resistance in Enterobacter cloacae.

Enterobacter cloacae starred different pioneer studies that enabled the development of a widely accepted model for the peptidoglycan metabolism-linked regulation of intrinsic class C cephalosporinases, highly conserved in different Gram-negatives. However, some mechanistic and fitness/virulence-related aspects of E. cloacae choromosomal AmpC-dependent resistance are not completely understood. The present study including knockout mutants, ß-lactamase cloning, gene expression analysis, characterization of resistance phenotypes, and the Galleria mellonella infection model fills these gaps demonstrating that: (i) AmpC enzyme does not show any collateral activity impacting fitness/virulence; (ii) AmpC hyperproduction mediated by ampD inactivation does not entail any biological cost; (iii) alteration of peptidoglycan recycling alone or combined with AmpC hyperproduction causes no attenuation of E. cloacae virulence in contrast to other species; (iv) derepression of E. cloacae AmpC does not follow a stepwise dynamics linked to the sequential inactivation of AmpD amidase homologues as happens in Pseudomonas aeruginosa; (v) the enigmatic additional putative AmpC-type ß-lactamase generally present in E. cloacae does not contribute to the classical cephalosporinase hyperproduction-based resistance, having a negligible impact on phenotypes even when hyperproduced from multicopy vector. This study reveals interesting particularities in the chromosomal AmpC-related behavior of E. cloacae that complete the knowledge on this top resistance mechanism.

The balance between antibiotic resistance and fitness/virulence in Pseudomonas aeruginosa: an update on basic knowledge and fundamental research.

The interplay between antibiotic resistance and bacterial fitness/virulence has attracted the interest of researchers for decades because of its therapeutic implications, since it is classically assumed that resistance usually entails certain biological costs. Reviews on this topic revise the published data from a general point of view, including studies based on clinical strains or in vitro-evolved mutants in which the resistance phenotype is seen as a final outcome, i.e., a combination of mechanisms. However, a review analyzing the resistance/fitness balance from the basic research perspective, compiling studies in which the different resistance pathways and respective biological costs are individually approached, was missing. Here we cover this gap, specifically focusing on Pseudomonas aeruginosa, a pathogen that stands out because of its extraordinary capacity for resistance development and for which a considerable number of recent and particular data on the interplay with fitness/virulence have been released. The revised information, split into horizontally-acquired vs. mutation-driven resistance, suggests a great complexity and even controversy in the resistance-fitness/virulence balance in the acute infection context, with results ranging from high costs linked to certain pathways to others that are seemingly cost-free or even cases of resistance mechanisms contributing to increased pathogenic capacities. The elusive mechanistic basis for some enigmatic data, knowledge gaps, and possibilities for therapeutic exploitation are discussed. The information gathered suggests that resistance-fitness/virulence interplay may be a source of potential antipseudomonal targets and thus, this review poses the elementary first step for the future development of these strategies harnessing certain resistance-associated biological burdens.

Bacterial virulence regulation through soluble peptidoglycan fragments sensing and response: knowledge gaps and therapeutic potential.

Given the growing clinical-epidemiological threat posed by the phenomenon of antibiotic resistance, new therapeutic options are urgently needed, especially against top nosocomial pathogens such as those within the ESKAPE group. In this scenario, research is pushed to explore therapeutic alternatives and, among these, those oriented toward reducing bacterial pathogenic power could pose encouraging options. However, the first step in developing these antivirulence weapons is to find weak points in the bacterial biology to be attacked with the goal of dampening pathogenesis. In this regard, during the last decades some studies have directly/indirectly suggested that certain soluble peptidoglycan-derived fragments display virulence-regulatory capacities, likely through similar mechanisms to those followed to regulate the production of several ß-lactamases: binding to specific transcriptional regulators and/or sensing/activation of two-component systems. These data suggest the existence of intra- and also intercellular peptidoglycan-derived signaling capable of impacting bacterial behavior, and hence likely exploitable from the therapeutic perspective. Using the well-known phenomenon of peptidoglycan metabolism-linked ß-lactamase regulation as a starting point, we gather and integrate the studies connecting soluble peptidoglycan sensing with fitness/virulence regulation in Gram-negatives, dissecting the gaps in current knowledge that need filling to enable potential therapeutic strategy development, a topic which is also finally discussed.

Role of Enzymatic Activity in the Biological Cost Associated with the Production of AmpC ß-Lactamases in Pseudomonas aeruginosa.

In the current scenario of growing antibiotic resistance, understanding the interplay between resistance mechanisms and biological costs is crucial for designing therapeutic strategies. In this regard, intrinsic AmpC ß-lactamase hyperproduction is probably the most important resistance mechanism of Pseudomonas aeruginosa, proven to entail important biological burdens that attenuate virulence mostly under peptidoglycan recycling alterations. P. aeruginosa can acquire resistance to new ß-lactam-ß-lactamase inhibitor combinations (ceftazidime-avibactam and ceftolozane-tazobactam) through mutations affecting ampC and its regulatory genes, but the impact of these mutations on the associated biological cost and the role that ß-lactamase activity plays per se in contributing to the above-mentioned virulence attenuation are unknown. The same questions remain unsolved for plasmid-encoded AmpC-type ß-lactamases such as FOX enzymes, some of which also provide resistance to new ß-lactam-ß-lactamase inhibitor combinations. Here, we assessed from different perspectives the effects of changes in the active center and, thus, in the hydrolytic spectrum resistance to inhibitors of AmpC-type ß-lactamases on the fitness and virulence of P. aeruginosa, using site-directed mutagenesis; the previously described AmpC variants T96I, G183D, and ΔG229-E247; and, finally, blaFOX-4 versus blaFOX-8. Our results indicate the essential role of AmpC activity per se in causing the reported full virulence attenuation (in terms of growth, motility, cytotoxicity, and Galleria mellonella larvae killing), although the biological cost of the above-mentioned AmpC-type variants was similar to that of the wild-type enzymes. This suggests that there is not an important biological burden that may limit the selection/spread of these variants, which could progressively compromise the future effectiveness of the above-mentioned drug combinations. IMPORTANCE The growing antibiotic resistance of the top nosocomial pathogen Pseudomonas aeruginosa pushes research to explore new therapeutic strategies, for which the resistance-versus-virulence balance is a promising source of targets. While resistance often entails significant biological costs, little is known about the bases of the virulence attenuations associated with a resistance mechanism as extraordinarily relevant as ß-lactamase production. We demonstrate that besides potential energy and cell wall alterations, the enzymatic activity of the P. aeruginosa cephalosporinase AmpC is essential for causing the full attenuation associated with its hyperproduction by affecting different features related to pathogenesis, a fact exploitable from the antivirulence perspective. Less encouraging, we also show that the production of different chromosomal/plasmid-encoded AmpC derivatives conferring resistance to some of the newest antibiotic combinations causes no significantly increased biological burdens, which suggests a free way for the selection/spread of these types of variants, potentially compromising the future effectiveness of these antipseudomonal therapies.

Effects of Inhaled Corticosteroids on the Innate Immunological Response to Pseudomonas aeruginosa Infection in Patients with COPD.

Inhaled corticosteroids (ICS) use is associated with an increased risk of Pseudomonas aeruginosa (PA) infection in patients with COPD. We aimed to evaluate the effects of ICS on alveolar macrophages in response to PA in COPD patients with and without baseline ICS treatment (COPD and COPD + ICS, respectively) as well as smoker and nonsmoker controls. To do so, cells were infected with PA and cotreated with budesonide (BUD) or fluticasone propionate (FLU). The analysis of NF-κB and c-jun activity revealed a significant increase in both factors in response to PA cotreated with BUD/FLU in smokers but not in COPD or COPD + ICS patients when compared with PA infection alone. The expression of Toll-like receptor 2 (TLR2) and the transcription factor c-jun were induced upon PA infection in nonsmokers only. Moreover, in the smoker and COPD groups, there was a significant increase in TLR2 and a decrease in c-jun expression when treated with BUD/FLU after PA infection, which were not observed in COPD + ICS patients. Therefore, the chronic use of ICS seemingly makes the macrophages tolerant to BUD/FLU stimulation compared with those from patients not treated with ICS, promoting an impaired recognition of PA and activity of alveolar macrophages in terms of altered expression of TLR2 and cytokine production, which could explain the increased risk of PA infection in COPD patients under ICS treatment.

Impact of Peptidoglycan Recycling Blockade and Expression of Horizontally Acquired ß-Lactamases on Pseudomonas aeruginosa Virulence.

In the current scenario of antibiotic resistance magnification, new weapons against top nosocomial pathogens like Pseudomonas aeruginosa are urgently needed. The interplay between ß-lactam resistance and virulence is considered a promising source of targets to be attacked by antivirulence therapies, and in this regard, we previously showed that a peptidoglycan recycling blockade dramatically attenuated the pathogenic power of P. aeruginosa strains hyperproducing the chromosomal ß-lactamase AmpC. Here, we sought to ascertain whether this observation could be applicable to other ß-lactamases. To do so, P. aeruginosa wild-type or peptidoglycan recycling-defective strains (ΔampG and ΔnagZ) harboring different cloned ß-lactamases (transferable GES, VIM, and OXA types) were used to assess their virulence in Galleria mellonella larvae by determining 50% lethal doses (LD50s). A mild yet significant LD50 increase was observed after peptidoglycan recycling disruption per se, whereas the expression of class A and B enzymes did not impact virulence. While the production of the narrow-spectrum class D OXA-2 entailed a slight attenuation, its extended-spectrum derivatives OXA-226 (W159R [bearing a change of W to R at position 159]), OXA-161 (N148D), and principally, OXA-539 (D149 duplication) were associated with outstanding virulence impairments, especially in recycling-defective backgrounds (with some LD50s being >1,000-fold that of the wild type). Although their exact molecular bases remain to be deciphered, these results suggest that mutations affecting the catalytic center and, therefore, the hydrolytic spectrum of OXA-2-derived enzymes also drastically impact the pathogenic power of P. aeruginosa. This work provides new and relevant knowledge to the complex topic of the interplay between the production of ß-lactamases and virulence that could be useful to build future therapeutic strategies against P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is one of the leading nosocomial pathogens whose growing resistance makes the development of therapeutic options extremely urgent. The resistance-virulence interplay has classically aroused researchers' interest as a source of therapeutic targets. In this regard, we describe a wide array of virulence attenuations associated with different transferable ß-lactamases, among which the production of OXA-2-derived extended-spectrum ß-lactamases stood out as a dramatic handicap for pathogenesis, likely as a side effect of mutations causing the expansion of their hydrolytic spectrums. Moreover, our results confirm the validity of disturbing peptidoglycan recycling as a weapon to attenuate P. aeruginosa virulence in class C and D ß-lactamase production backgrounds. In the current scenario of dissemination of horizontally acquired ß-lactamases, this work brings out new data on the complex interplay between the production of specific enzymes and virulence attenuation that, if complemented with the characterization of the underlying mechanisms, will likely be exploitable to develop future virulence-targeting antipseudomonal strategies.

Mammals' humoral immune proteins and peptides targeting the bacterial envelope: from natural protection to therapeutic applications against multidrug-resistant Gram-negatives.

Mammalian innate immunity employs several humoral 'weapons' that target the bacterial envelope. The threats posed by the multidrug-resistant 'ESKAPE' Gram-negative pathogens (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are forcing researchers to explore new therapeutic options, including the use of these immune elements. Here we review bacterial envelope-targeting (peptidoglycan and/or membrane-targeting) proteins/peptides of the mammalian immune system that are most likely to have therapeutic applications. Firstly we discuss their general features and protective activity against ESKAPE Gram-negatives in the host. We then gather, integrate, and discuss recent research on experimental therapeutics harnessing their bactericidal power, based on their exogenous administration and also on the discovery of bacterial and/or host targets that improve the performance of this endogenous immunity, as a novel therapeutic concept. We identify weak points and knowledge gaps in current research in this field and suggest areas for future work to obtain successful envelope-targeting therapeutic options to tackle the challenge of antimicrobial resistance.

Activity of mammalian peptidoglycan-targeting immunity against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most important opportunistic pathogens, whose clinical relevance is not only due to the high morbidity/mortality of the infections caused, but also to its striking capacity for antibiotic resistance development. In the current scenario of a shortage of effective antipseudomonal drugs, it is essential to have thorough knowledge of the pathogen's biology from all sides, so as to find weak points for drug development. Obviously, one of these points could be the peptidoglycan, given its essential role for cell viability. Meanwhile, immune weapons targeting this structure could constitute an excellent model to be taken advantage of in order to design new therapeutic strategies. In this context, this review gathers all the information regarding the activity of mammalian peptidoglycan-targeting innate immunity (namely lysozyme and peptidoglycan recognition proteins), specifically against P. aeruginosa. All the published studies were considered, from both in vitro and in vivo fields, including works that envisage these weapons as options not only to potentiate their innate effects within the host or for use as exogenously administered treatments, but also harnessing their inflammatory and immune regulatory capacity to finally reduce damage in the patient. Altogether, this review has the objective of anticipating and discussing whether these innate immune resources, in combination or not with other drugs attacking certain P. aeruginosa targets leading to its increased sensitization, could be valid therapeutic antipseudomonal allies.

Publisher Correction: Profiling the susceptibility of Pseudomonas aeruginosa strains from acute and chronic infections to cell-wall-targeting immune proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Comparative Analysis of Peptidoglycans From Pseudomonas aeruginosa Isolates Recovered From Chronic and Acute Infections.

Pseudomonas aeruginosa is one of the first causes of acute nosocomial and chronic infections in patients with underlying respiratory pathologies such as cystic fibrosis (CF). It has been proposed that P. aeruginosa accumulates mutations driving to peptidoglycan modifications throughout the development of the CF-associated infection, as a strategy to lower the immune detection hence ameliorating the chronic persistence. As well, some studies dealing with peptidoglycan modifications driving to a better survival within the host have been published in other gram-negatives. According to these facts, the gram-negative peptidoglycan could be considered as a pathogen-associated molecular pattern with very important implications regarding the host's detection-response, worthy to dissect in detail. For this reason, in this work we characterized for the first time the peptidoglycans of three large collections [early CF, late CF and acute infection (bloodstream) P. aeruginosa strains] from qualitative (HPLC), quantitative and inflammatory capacity-related perspectives. The final goal was to identify composition trends potentially supporting the cited strategy of evasion/resistance to the immune system and providing information regarding the differential intrinsic adaptation depending on the type of infection. Although we found several punctual strain-specific particularities, our results indicated a high degree of inter-collection uniformity in the peptidoglycan-related features and the absence of trends amongst the strains studied here. These results suggest that the peptidoglycan of P. aeruginosa is a notably conserved structure in natural isolates regardless of transitory changes that some external conditions could force. Finally, the inverse correlation between the relative amount of stem pentapeptides within the murein sacculus and the resistance to immune lytic attacks against the peptidoglycan was also suggested by our results. Altogether, this work is a major step ahead to understand the biology of peptidoglycan from P. aeruginosa natural strains, hopefully useful in future for therapeutic alternatives design.

Profiling the susceptibility of Pseudomonas aeruginosa strains from acute and chronic infections to cell-wall-targeting immune proteins.

In the current scenario of high antibiotic resistance, the search for therapeutic options against Pseudomonas aeruginosa must be approached from different perspectives: cell-wall biology as source of bacterial weak points and our immune system as source of weapons. Our recent study suggests that once the permeability barrier has been overcome, the activity of our cell-wall-targeting immune proteins is notably enhanced, more in mutants with impaired peptidoglycan recycling. The present work aims at analyzing the activity of these proteins [lysozyme and Peptidoglycan-Recognition-Proteins (PGLYRPs)], alone or with a permeabilizer (subinhibitory colistin) in clinical strains, along with other features related to the cell-wall. We compared the most relevant and complementary scenarios: acute (bacteremia) and chronic infections [early/late isolates from lungs of cystic fibrosis (CF) patients]. Although a low activity of lysozyme/PGLYRPs per se (except punctual highly susceptible strains) was found, the colistin addition significantly increased their activity regardless of the strains' colistin resistance levels. Our results show increased susceptibility in late CF isolates, suggesting that CF adaptation renders P. aeruginosa more vulnerable to proteins targeting the cell-wall. Thus, our work suggests that attacking some P. aeruginosa cell-wall biology-related elements to increase the activity of our innate weapons could be a promising therapeutic strategy.