OriPlex: Origami-enabled multiplexed detection of respiratory pathogens.

Origami biosensors leverage paper foldability to develop total analysis systems integrated in a single piece of paper. This capability can also be utilized to incorporate additional features that would be difficult to achieve with rigid substrates. In this article, we report a new design for 3D origami biosensors called OriPlex, which leverages the foldability of filter paper for the multiplexed detection of bacterial pathogens. OriPlex immunosensors detect pathogens by folding nanoparticle reservoirs containing different types of nanoprobes. This releases antibody-coated nanoparticles in a central channel where targets are captured through physical interactions. The OriPlex concept was demonstrated by detecting the respiratory pathogens Pseudomonas aeruginosa (PA) and Klebsiella pneumoniae (KP) with a limit of detection of 3.4·103 cfu mL-1 and 1.4·102 cfu mL-1, respectively, and with a turn-around time of 25 min. Remarkably, the OriPlex biosensors allowed the multiplexed detection of both pathogens spiked into real bronchial aspirate (BAS) samples at a concentration of 105 cfu mL-1 (clinical infection threshold), thus demonstrating their suitability for diagnosing lower tract respiratory infections. The results shown here pave the way for implementing OriPlex biosensors as a screening test for detecting superbugs requiring personalized antibiotics in suspected cases of nosocomial pneumonia.

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.

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).

Pseudomonasaeruginosa antimicrobial susceptibility profiles, resistance mechanisms and international clonal lineages: update from ESGARS-ESCMID/ISARPAE Group.

SCOPE: Pseudomonas aeruginosa, a ubiquitous opportunistic pathogen considered one of the paradigms of antimicrobial resistance, is among the main causes of hospital-acquired and chronic infections associated with significant morbidity and mortality. This growing threat results from the extraordinary capacity of P. aeruginosa to develop antimicrobial resistance through chromosomal mutations, the increasing prevalence of transferable resistance determinants (such as the carbapenemases and the extended-spectrum ß-lactamases), and the global expansion of epidemic lineages. The general objective of this initiative is to provide a comprehensive update of P. aeruginosa resistance mechanisms, especially for the extensively drug-resistant (XDR)/difficult-to-treat resistance (DTR) international high-risk epidemic lineages, and how the recently approved ß-lactams and ß-lactam/ß-lactamase inhibitor combinations may affect resistance mechanisms and the definition of susceptibility profiles. METHODS: To address this challenge, the European Study Group for Antimicrobial Resistance Surveillance (ESGARS) from the European Society of Clinical Microbiology and Infectious Diseases launched the 'Improving Surveillance of Antibiotic-Resistant Pseudomonas aeruginosa in Europe (ISARPAE)' initiative in 2022, supported by the Joint programming initiative on antimicrobial resistance network call and included a panel of over 40 researchers from 18 European Countries. Thus, a ESGARS-ISARPAE position paper was designed and the final version agreed after four rounds of revision and discussion by all panel members. QUESTIONS ADDRESSED IN THE POSITION PAPER: To provide an update on (a) the emerging resistance mechanisms to classical and novel anti-pseudomonal agents, with a particular focus on ß-lactams, (b) the susceptibility profiles associated with the most relevant ß-lactam resistance mechanisms, (c) the impact of the novel agents and resistance mechanisms on the definitions of resistance profiles, and (d) the globally expanding XDR/DTR high-risk lineages and their association with transferable resistance mechanisms. IMPLICATION: The evidence presented herein can be used for coordinated epidemiological surveillance and decision making at the European and global level.

The imperative for quality control programs in Monkeypox virus DNA testing by PCR: CIBERINFEC quality control.

To evaluate molecular assays for Mpox diagnosis available in various clinical microbiology services in Spain through a quality control (QC) approach. A total of 14 centers from across Spain participated in the study. The Reference Laboratory dispatched eight serum samples and eight nucleic acid extracts to each participating center. Some samples were spiked with Mpox or Vaccinia virus to mimic positive samples for Mpox or other orthopox viruses. Participating centers provided information on the results obtained, as well as the laboratory methods used. Among the 14 participating centers seven different commercial assays were employed, with the most commonly used kit being LightMix Modular Orthopox/Monkeypox (Mpox) Virus (Roche®). Of the 12 centers conducting Mpox determinations, concordance ranged from 62.5% (n = 1) to 100% (n = 11) for eluates and from 75.0% (n = 1) to 100% (n = 10) for serum. Among the 10 centers performing Orthopoxvirus determinations, a 100% concordance was observed for eluates, while for serum, concordance ranged from 87.5% (n = 6) to 100% (n = 4). Repeatedly, 6 different centers reported a false negative in serum samples for Orthopoxvirus diagnosis, particularly in a sample with borderline Ct = 39. Conversely, one center, using the TaqMan™ Mpox Virus Microbe Detection Assay (Thermo Fisher), reported false positives in Mpox diagnosis for samples spiked with vaccinia virus due to cross-reactions. We observed a positive correlation of various diagnostic assays for Mpox used by the participating centers with the reference values. Our results highlight the significance of standardization, validation, and ongoing QC in the microbiological diagnosis of infectious diseases, which might be particularly relevant for emerging viruses.

A paper biosensor for overcoming matrix effects interfering with the detection of sputum pyocyanin with competitive immunoassays.

Detecting sputum pyocyanin (PYO) with a competitive immunoassay is a promising approach for diagnosing Pseudomonas aeruginosa respiratory infections. However, it is not possible to perform a negative control to evaluate matrix-effects in competitive immunoassays, and the highly complex sputum matrix often interferes with target detection. Here, we show that these issues are alleviated by performing competitive immunoassays with a paper biosensor. The biosensing platform consists of a paper reservoir, which contains antibody-coated gold nanoparticles, and a substrate containing a competing recognition element, which is a piece of paper modified with an albumin-antigen conjugate. Detection of PYO with a limit of detection of 4.7·10-3 µM and a dynamic range between 4.7·10-1 µM and 47.6 µM is accomplished by adding the sample to the substrate with the competing element and pressing the reservoir against it for 5 min. When tested with patient samples, the biosensor was able to qualitatively differentiate spiked from non-spiked samples, whereas ELISA did not show a clear cut-off between them. Furthermore, the relative standard deviation was lower when determining sputum with the paper-based biosensor. These features, along with a mild liquefaction step that circumvents the use of harsh chemicals or instruments, make our biosensor a good candidate for diagnosing Pseudomonas infections at the bedside through the detection of sputum PYO.

Molecular characterization of a suspected IMP-type carbapenemase-producing Pseudomonas aeruginosa outbreak reveals two simultaneous outbreaks in a tertiary-care hospital.

OBJECTIVE: To describe IMP-type carbapenemase-producing Pseudomonas aeruginosa outbreaks at Galdakao University Hospital between March 2021 to December 2021. DESIGN: Outbreak report. SETTING: Galdakao University Hospital is a tertiary-care hospital in the Basque Country (northern Spain). PATIENTS: All patients with a positive IMP-type carbapenemase producing Pseudomonas aeruginosa (IMP-PA) culture were included in this study, both colonization and infection cases. METHODS: An outbreak investigation was conducted, in which molecular epidemiology analysis [pulsed-field gel electrophoresis and whole-genome sequencing (WGS)] and environmental screenings were performed. RESULTS: Between March and December 2021, 21 cases of IMP-PA were detected in Galdakao University Hospital: 18 infection cases and 3 colonization cases. In total, 4 different pulsotypes were detected belonging to 4 clones according to WGS: ST175 (n = 14), ST633 (n = 3), ST179 (n = 3), and ST348 (n = 1). IMP-13 was detected in most isolates belonging to the ST175 clone and in all ST179 and ST348 clones, whereas IMP-29 was detected in isolates belonging to the ST633 clone. Clinical isolates belonging to the ST175 clone were isolated mainly from patients admitted to the respiratory ward, and isolates belonging to the ST633 clone from patients admitted to the ICU. Two environmental isolates belonging to the ST175 clone were detected in the respiratory ward. CONCLUSIONS: Molecular and genomic epidemiology revealed that there had been 2 independent IMP-PA outbreaks, one of long duration in the respiratory ward and the other more limited in the ICU.

Improved cytometric analysis of untouched lung leukocytes by enzymatic liquefaction of sputum samples.

BACKGROUND: Phenotyping sputum-resident leukocytes and evaluating their functional status are essential analyses for exploring the cellular basis of pathological processes in the lungs, and flow cytometry is widely recognized as the gold-standard technique to address them. However, sputum-resident leukocytes are found in respiratory samples which need to be liquefied prior to cytometric analysis. Traditional liquefying procedures involve the use of a reducing agent such as dithiothreitol (DTT) in temperature-controlled conditions, which does not homogenize respiratory samples efficiently and impairs cell viability and functionality. METHODS: Here we propose an enzymatic method that rapidly liquefies samples by means of generating O2 bubbles with endogenous catalase. Sputum specimens from patients with suspected pulmonary infection were treated with DTT, the enzymatic method or PBS. We used turbidimetry to compare the liquefaction degree and cell counts were determined using a hemocytometer. Finally, we conducted a comparative flow cytometry study for evaluating frequencies of sputum-resident neutrophils, eosinophils and lymphocytes and their activation status after liquefaction. RESULTS: Enzymatically treated samples were better liquefied than those treated with DTT or PBS, which resulted in a more accurate cytometric analysis. Frequencies of all cell subsets analyzed within liquefied samples were comparable between liquefaction methods. However, the gentle cell handling rendered by the enzymatic method improves cell viability and retains in vivo functional characteristics of sputum-resident leukocytes (with regard to HLA-DR, CD63 and CD11b expression). CONCLUSION: In conclusion, the proposed enzymatic liquefaction method improves the cytometric analysis of respiratory samples and leaves the cells widely untouched for properly addressing functional analysis of lung leukocytes.

Prevalence of the fimbrial operon mrkABCD, mrkA expression, biofilm formation and effect of biocides on biofilm formation in carbapenemase-producing Klebsiella pneumoniae isolates belonging or not belonging to high-risk clones.

BACKGROUND: The role of mrkA adhesin expression, biofilm production, biofilm viability and biocides in the biofilm of carbapenemase-producing Klebsiella pneumoniae isolates was investigated. METHODS: Seventeen isolates representing different sequence types and carbapenemases were investigated. mrkA expression was determined by real-time reverse transcription polymerase chain reaction. Biofilm production (25°C and 37°C, with and without humidity) was determined by the crystal violet assay. The effect of isopropanol, povidone-iodine, sodium hypochlorite, chlorhexidine digluconate, benzalkonium chloride, ethanol and triclosan on biofilm was determined. The effect of povidone-iodine on biofilm biomass and thickness was also determined by confocal laser scanning microscopy. RESULTS: mrkA expression ranged from 28.2 to 1.3 [high or intermediate level; 64% of high-risk (HR) clones] and from 21.5 to 1.3 (50% of non-HR clones). At 25°C, biofilm formation was observed in 41% of isolates (absence of humidity) and 35% of isolates (presence of humidity), whereas at 37°C, biofilm formation was observed in 76% of isolates with and without humidity. At 25°C, biofilm producers were more frequently observed in HR clones (45% with humidity and 55% without humidity) than non-HR clones (17% with and without humidity). Biofilm viability from day 21 was higher at 25°C than 37°C. The greatest decrease in biofilm formation was observed with povidone-iodine (29% decrease), which also decreased biofilm thickness. CONCLUSIONS: Biofilm formation in carbapenemase-producing K. pneumoniae is related to mrkA expression. Biofilm formation is affected by temperature (37°C>25°C), whereas humidity has little effect. Biofilm viability is affected by temperature (25°C>37°C). At 25°C, HR clones are more frequently biofilm producers than non-HR clones. Povidone-iodine can decrease biofilm production and biofilm thickness.

Rapid Identification and Classification of Pathogens That Produce Carbapenemases and Cephalosporinases with a Colorimetric Paper-Based Multisensor.

Infections caused by bacteria that produce ß-lactamases (BLs) are a major problem in hospital settings. The phenotypic detection of these bacterial strains requires culturing samples prior to analysis. This procedure may take up to 72 h, and therefore it cannot be used to guide the administration of the first antibiotic regimen. Here, we propose a multisensor for identifying pathogens bearing different types of ß-lactamases above the infectious dose threshold within 90 min that does not require culturing samples. Instead, bacterial cells are preconcentrated in the cellulose scaffold of a paper-based multisensor. Then, 12 assays are performed in parallel to identify whether the pathogens produce carbapenemases and/or cephalosporinases, including metallo-ß-lactamases, extended-spectrum ß-lactamases (ESBLs), and AmpC enzymes. The multisensor generates an array of colored spots that can be quantified with image processing software and whose interpretation leads to the detection of the different enzymes depending on their specificity toward the hydrolysis of certain antibiotics, and/or their pattern of inhibition or cofactor activation. The test was validated for the diagnosis of urinary tract infections. The inexpensive paper platform along with the uncomplicated colorimetric readout makes the proposed prototypes promising for developing fully automated platforms for streamlined clinical diagnosis.

Optimized detection of lung IL-6 via enzymatic liquefaction of low respiratory tract samples: application for managing ventilated patients.

Lung IL-6 is a promising biomarker for predicting respiratory failure during pulmonary infections. This biomarker is found in respiratory samples which need to be liquefied prior to analysis. Traditional liquefying methods use reducing agents such as dithiothreitol (DTT). However, DTT impairs immunodetection and does not liquefy highly viscous samples. We propose an enzymatic method that liquefies samples by means of generating O2 bubbles with endogenous catalase. Low respiratory tract specimens from 48 mechanically ventilated patients (38 with SARS-CoV-2 infection) were treated with DTT or with the enzymatic method. We used turbidimetry to compare the liquefaction degree and IL-6 was quantified with ELISA. Finally, we used AUC-ROC, time-to-event and principal component analysis to evaluate the association between respiratory compromise or local inflammation and IL-6 determined with both methods. Enzymatically treated samples were better liquefied than those reduced by DTT, which resulted in higher ELISA signals. Lung IL-6 levels obtained with the enzymatic procedure were negatively correlated with the oxygenation index (PaO2/FiO2) and the time of mechanical ventilation. The proposed enzymatic liquefaction method improves the sensitivity for lung IL-6 detection in respiratory samples, which increases its predictive power as a biomarker for evaluating respiratory compliance.

Immunodetection of Lung IgG and IgM Antibodies against SARS-CoV-2 via Enzymatic Liquefaction of Respiratory Samples from COVID-19 Patients.

Lung-secreted IgG and IgM antibodies are valuable biomarkers for monitoring the local immune response against respiratory infections. These biomarkers are found in lower airway secretions that need to be liquefied prior to analysis. Traditional methods for sample liquefaction rely on reducing disulfide bonds, which may damage the structure of the biomarkers and hamper their immunodetection. Here, we propose an alternative enzymatic method that uses O2 bubbles generated by endogenous catalase enzymes in order to liquefy respiratory samples. The proposed method is more efficient for liquefying medium- and high-viscosity samples and does not fragment the antibodies. This prevents damage to antigen recognition domains and recognition sites for secondary antibodies that can decrease the signal of immunodetection techniques. The suitability of the enzymatic method for detecting antibodies in respiratory samples is demonstrated by detecting anti-SARS-CoV-2 IgG and IgM to viral N-protein with gold standard ELISA in bronchial aspirate specimens from a multicenter cohort of 44 COVID-19 patients. The enzymatic detection sharply increases the sensitivity toward IgG and IgM detection compared to the traditional approach based on liquefying samples with dithiothreitol. This improved performance could reveal new mechanisms of the early local immune response against respiratory infections that may have gone unnoticed with current sample treatment methods.

Mobile origami immunosensors for the rapid detection of urinary tract infections.

Urinary tract infections (UTI) have a high prevalence and can yield poor patient outcomes if they progress to urosepsis. Here we introduce mobile origami biosensors that detect UTIs caused by E. coli at the bedside in less than 7 minutes. The origami biosensors are made of a single piece of paper that contains antibody-decorated nanoparticles. When the urine sample contains E. coli, the biosensors generate colored spots on the paper strip. These are then quantified with a mobile app that calculates the pixel intensity in real time. The tests are highly specific and do not cross-react with other common uropathogens. Furthermore, the biosensors only yielded one false negative result when queried with a panel containing 57 urine samples from patients, which demonstrates that they have excellent sensitivity and specificity. This, along with the rapid assay time and smartphone-based detection, makes them useful for aiding in the diagnosis of UTIs at the point of care.

Rapid Detection of Pseudomonas aeruginosa Biofilms via Enzymatic Liquefaction of Respiratory Samples.

Respiratory infections caused by multi-drug-resistant Pseudomonas aeruginosa often yield poor outcomes if not detected right away. However, detecting this pathogen in respiratory samples with a rapid diagnostic test is challenging because the protective biofilms created by the pathogen are themselves surrounded by a high-viscosity sputum matrix. Here, we introduce a method for liquefying respiratory samples and disrupting bacterial biofilms on the spot within a minute. It relies on the generation of oxygen bubbles by bacterial catalase through the addition of hydrogen peroxide. When coupled with a mobile biosensor made of paper, the resulting diagnostic kit was able to detect P. aeruginosa infections in sputa from patients with excellent sensitivity and specificity within 8 min. The quick turnaround time along with few infrastructure requirements make this method ideal for the rapid screening of P. aeruginosa infections at the point of care.

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.

Social Behavior of Antibiotic Resistant Mutants Within Pseudomonas aeruginosa Biofilm Communities.

The complex spatial structure and the heterogeneity within biofilms lead to the emergence of specific social behaviors. However, the impact of resistant mutants within bacterial communities is still mostly unknown. Thus, we determined whether antibiotic resistant mutants display selfish or altruistic behaviors in mixed Pseudomonas aeruginosa biofilms exposed to antibiotics. ECFP-tagged P. aeruginosa strain PAO1 and its EYFP-tagged derivatives hyperproducing the ß-lactamase AmpC or the efflux pump MexAB-OprM were used to develop single or mixed biofilms. Mature biofilms were challenged with different concentrations of ß-lactams to monitor biofilm structural dynamics, using confocal laser scanning microscopy (CLSM), and population dynamics, through enumeration of viable cells. While exposure of single wild-type PAO1 biofilms to ß-lactams lead to a major reduction in bacterial load, it had little effect on biofilms formed by the resistant mutants. However, the most reveling finding was that bacterial load of wild-type PAO1 was significantly increased when growing in mixed biofilms compared to single biofilms. In agreement with CFU enumeration data, CLSM images revealed the amplification of the resistant mutants and their protection of susceptible populations. These findings show that mutants expressing diverse resistance mechanisms, including ß-lactamases, but also, as evidenced for the first time, efflux pumps, protect the whole biofilm community, preserving susceptible populations from the effect of antibiotics. Thus, these results are a step forward to understanding antibiotic resistance dynamics in biofilms, as well as the population biology of bacterial pathogens in chronic infections, where the coexistence of susceptible and resistant variants is a hallmark.

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.

Antibiotic treatment of biofilm infections.

Bacterial biofilms are associated with a wide range of infections, from those related to exogenous devices, such as catheters or prosthetic joints, to chronic tissue infections such as those occurring in the lungs of cystic fibrosis patients. Biofilms are recalcitrant to antibiotic treatment due to multiple tolerance mechanisms (phenotypic resistance). This causes persistence of biofilm infections in spite of antibiotic exposure which predisposes to antibiotic resistance development (genetic resistance). Understanding the interplay between phenotypic and genetic resistance mechanisms acting on biofilms, as well as appreciating the diversity of environmental conditions of biofilm infections which influence the effect of antibiotics are required in order to optimize the antibiotic treatment of biofilm infections. Here, we review the current knowledge on phenotypic and genetic resistance in biofilms and describe the potential strategies for the antibiotic treatment of biofilm infections. Of note is the optimization of PK/PD parameters in biofilms, high-dose topical treatments, combined and sequential/alternate therapies or the use antibiotic adjuvants.