pubs.acs.org/journal/aidcbc Article Direct Quantitative Immunochemical Analysis of Autoinducer Peptide IV for Diagnosing and Stratifying Staphylococcus aureus Infections Enrique-J. Montagut, Gerardo Acosta, Fernando Albericio, Miriam Royo, Gerard Godoy-Tena, Alicia Lacoma, Cristina Prat, Juan-Pablo Salvador, and María-Pilar Marco* Cite This: ACS Infect. Dis. 2022, 8, 645−656 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: An immunochemical strategy to detect and quantify AIP-IV, the quorum sensing (QS) signaling molecule produced by Staphylococcus aureus agr type IV, is reported here for the first time. Theoretical calculations and molecular modeling studies have assisted on the design and synthesis of a suitable peptide hapten (AIPIVS), allowing to obtain high avidity and specific antibodies toward this peptide despite its low molecular weight. The ELISA developed achieves an IC50 value of 2.80 ± 0.17 and an LOD of 0.19 ± 0.06 nM in complex media such as 1/2 Tryptic Soy Broth. Recognition of other S. aureus AIPs (I−III) is negligible (cross-reactivity below 0.001%), regardless of the structural similarities. A pilot study with a set of clinical isolates from patients with airways infection or colonization demonstrates the potential of this ELISA to perform biomedical investigations related to the role of QS in pathogenesis and the association between dysfunctional agr or the agr type with unfavorable clinical outcomes. The AIP-IV levels could be quantified in the low nanomolar range in less than 1 h after inoculating agr IV-genotyped isolates in the culture broth, while those genotyped as I−III did not show any immunoreactivity after a 48 h growth, pointing to the possibility to use this technology for phenotyping S. aureus. The research strategy here reported can be extended to the rest of the AIP types of S. aureus, allowing the development of powerful multiplexed chips or point-of-care (PoC) diagnostic devices to unequivocally identify its presence and its agr type on samples from infected patients. KEYWORDS: quorum sensing, autoinducer peptides, Staphylococcus aureus, agr group, antibodies, diagnostic Staphylococcus aureus is a threatening pathogen and is the these infections due to its virulence and antibiotic resistance, leading cause of a broad spectrum of infective processes such leading to high morbidity and mortality rates.10−12 as endocarditis, pneumonia, skin and soft tissue infections, The current techniques used to assess the etiology of lower osteoarticular infections, and device-related infections. Up to respiratory tract infections often provide poor or inaccurate 60% of human population1 is colonized by this Gram-positive results aggravated, in the case of S. aureus, by the existence of bacterium2 that shows a particular ability to evade the primary small colony variants (SCVs), which are frequently overlooked innate immune response3,4 and therefore acts as a very efficient or misidentified in culture plates.13,14 In addition, culture infective agent. It belongs to the so-called ESKAPE group of diagnostic techniques may take up to 72 h to identify the pathogens (Enterococcus faecium, S. aureus, Klebsiella pneumo- microorganism causing an unaffordable delay to deliver results niae, Acinetobacter baumannii, Pseudomonas aeruginosa, and to the patient, which prompts the use of broad-spectrum Enterobacter spp.) which include six microorganisms causing antibiotics contributing to the generation of antimicrobial nosocomial infections and exhibit extreme virulence and resistance (AMR).15 There is a clear and urgent need of multidrug resistance behavior.5 Particularly, methicillin-resist- finding novel diagnostic approaches for fast and accurate ant S. aureus (MRSA) is estimated to account for 25% of the S. pathogen identification to complement the current diagnostic aureus strains with a prevalence of up to 50% in some areas, generating a social and economic burden by means of both community-acquired infections (CAIs) and healthcare-associ- Received: December 28, 2021 ated infections (HAIs).6−8 S. aureus is one of the earliest Published: February 17, 2022 pathogens isolated from the airways of cystic fibrosis patients, being positive for more than 70% of neonates and 45% of those becoming persistently colonized.9 S. aureus is also frequently involved in ventilator-associated pneumonia, complicating © 2022 The Authors. Published by American Chemical Society https://doi.org/10.1021/acsinfecdis.1c00670 645 ACS Infect. Dis. 2022, 8, 645−656 ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article methods for infectious diseases. Quantitative real-time PCR, (2002−2017), pointing at the higher prevalence of agr IV on focused on the identification of particular genome sequences, colonizing strains, agr II on HAIs, and agr I on CAIs, while agr and matrix-assisted laser desorption ionization-time-of-flight- II would be more prevalent in adults, and agr III would be mass spectrometry (MALDI-TOF-MS), based on the analysis associated with infections in children.39 On the other hand, it of the mass distribution of bacterial proteins, are able to more has also been reported that a large proportion of clinical rapidly unequivocally identify S. aureus16−18 and other isolates are consistently found to have a mutationally pathogens. However, these approaches often still require inactivated Agr system (Agr-negative), which have a survival prior culture enrichment steps, expensive equipment, highly advantage in the host. However, it has recently been reported trained personnel, and extensive validation for clinical that a minor fraction of these Agr-negative mutants can revert interpretation of the results on routine clinical analyses, their Agr activity upon phagocytosis.26 Therefore, agr typing which make their wide implementation difficult in all clinical could be of great interest to manage infections and distinguish settings or near the patient primary attention centers.16−18 An between colonization and infection.40−44 additional unmet challenge of the diagnosis of infectious Some authors have pointed at the possibility to use AIs as diseases is the difficulty to differentiate simple carriers from specific biomarkers of infection,45 but although AIP-I has been infected patients.19 Diagnostic approaches that fulfill the quantified in bacterial cultures using mass spectrometry with ASSURED (affordability, sensitivity, specificity, user-friend- limits of detection (LOD) in the low micromolar range,46,47 to liness, rapidity and robustness, no equipment needed, and our knowledge, direct quantification in clinical samples has not deliverable to end users)20 criteria are a recognized unmet yet been achieved. The possible reasons for this fact could be need for many diseases and particularly to diagnose infections. their potential low concentration levels in body fluids or the Implementation of point-of-care (PoC) devices and diagnostic lack of chemical stability of these AIPs in certain media. Hence, methods accomplishing such criteria would substantially the instability of thiolactone functionality and the possibility of improve and facilitate the management of infectious diseases. protease degradation of these peptides during the clean-up, A key aspect in achieving this goal is to select appropriate extraction, or preconcentration procedures have been reported specific biomarker targets whose detection clearly points to the to be between of the reasons because chromatography and pathogen causing the disease. certain bioanalytical techniques have failed to directly detect The quorum sensing (QS), a population-dependent bacteria AIPs in clinical samples.45 In this context, the potential of communication system that controls the genetic expression of immunochemical techniques to detect and quantify a whole virulence factors and crucial survival mechanisms during variety of molecules at low concentration levels is well known, pathogenesis, has attracted the attention of the scientific even in complex media without the need of previous extraction community.21 This communication process relies on the or clean-up steps, because of the extraordinary properties of release of low molecular weight signaling molecules to the the antibodies, which are able to bind antigens with high extracellular matrix, whose concentration will increase as a affinity and specificity, despite the presence of other function of population density.22 QS in Gram-negative bacteria substances. The final aim of this research line is to go deeper depends on characteristic molecules such as homoserine into the role of AIPs and agr types in pathogenesis providing lactones (HSLs) called autoinducers (AI), while in Gram- antibodies and immunochemical tools for their investigation. positive bacteria, these AIs are called peptides (AIP, auto- Moreover, immunochemical analytical tools would allow inducer peptide).23 S. aureus virulence is under the control of a quantifying AIPs in clinical samples and validating their value primary QS system called Agr (accessory gene regulator).24 It as biomarkers of S. aureus infection. As a starting point, we consists of two transcriptional units, RNAII (containing the have focused on AIP-IV based on the association of agr IV to genes agrA, agrB, agrC, and agrD, transcribed according to the colonizing strains found by Pérez-Montarelo et al. on the study following order agrBDCA), responsible of its own auto- mentioned above,39 which if confirmed, could allow discrim- induction, and RNAIII, which encodes a series of processes inating infection from colonization, preventing unnecessary related to virulence. Indeed, the agr operon regulates over 70 antibiotic treatment.39 genes, 23 of which control its pathogenicity and invasive infections.25 The activation of the agr loci switches the bacterium from being a sessile colonizer to a hostile, invasive ■ RESULTS AND DISCUSSION AIP-IV Immunizing Hapten Design. The production of pathogen.26 Moreover, S. aureus can be stratified into four antibodies against low molecular weight targets has several different groups (agr I, agr II, agr III, and agr IV) according to decisive steps, among which, the rational design of the the sequences of the agrC and agrD genes, which results in four immunizing hapten and its chemical synthesis should be different AIs, which are small (7−9 amino acids) cyclic highlighted. Knowledge of the chemical features of the target is thiodepsipeptides (the C-terminal carboxylic group forms a a key issue, and in this case, a main challenge was the reported thiolactone with the thiol of a Cys) produced in a strain- lack of stability associated with the thiolactone chemical specific manner.27 There have been reported clear associations function closing the peptide ring (see the AIP-IV structure in between the agr type and virulence, the ability to form biofilms, Figure 2).48 Hence, Park and co-workers49 attempted to and AMR profile.28−34 Hence, higher prevalence of generalized produce antibodies against AIP-IV by synthesizing a hapten exfoliative syndromes or osteoarticular infections has been with a lactone group instead of a thiolactone due to its better associated with agr group IV,35,36 TSS toxin1-producing stability. The antibodies produced against the lactone hapten isolates have been found to belong mainly to agr group III, were used for therapeutic purposes but were never assessed to and higher risk of endocarditis has been associated with detect and quantify AIPs in complex biological media or for infections from agr types I and II.37,38 Other interesting diagnostic purposes. However, it should be noticed that the associations have also been recently reported on a study lactone functionality used is not exempt from the risk of performed with 833 S. aureus strains (785 bacteremia and 48 hydrolysis. In fact, persecuting a similar goal, Debler and co- colonizing strains) collected in Spain over a period of 15 years workers50 used a lactam hapten to produce antibodies against 646 https://doi.org/10.1021/acsinfecdis.1c00670 ACS Infect. Dis. 2022, 8, 645−656 ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article Figure 1. Left: Graph showing the variations in the punctual charges of the amino acid residues affected by the thiolactone, lactam, or lactone functionalities of the proposed immunizing haptens. Right: Structural alignment for AIP-IVS with AIP-IVO and AIP-IVNH molecules where the RMSE values were obtained. Figure 2. Chemical structures of the native AIP-IV and the AIP-IV haptens designed. In bold, those that were finally used to raise antibodies against the S. aureus QS signaling molecule AIP-IV. HSLs, which are QS signaling molecules produced by Gram- the lactone molecules, respectively. It should be noticed that negative bacteria. In light of these precedents, the possibility of different conformations of the native AIP-IV at their minimum using a more stable AIP-IV lactam hapten instead of a lactone energy level showed RMSE values of just 0.094 ± 0.062 Å. On to raise antibodies was considered. However, before, we the other hand, certain chemical properties, such as its wanted to evaluate the impact that substituting the sulfur atom capability to establish hydrogen bonds with other molecules, by a nitrogen atom could have in the electronic and structural pointed at major differences for the lactam molecule, in respect properties of the molecule. to thiolactone or lactone. Despite these results, the risk of Theoretical calculations and molecular modeling studies are instability of lactone and the precedents of Debler and co- powerful instruments for designing and evaluating the workers50 with the HSLs prompted us to select the lactam as suitability of the hapten chemical structures to raise high- the stable alternative for the thiolactone functionality of the affinity antibodies. Using these tools, it is possible to ensure AIP-IV immunizing hapten (AIPIVNH hapten). Even though, that the electronic configuration and the geometrical owing to the conformational and chemical differences of the conformation of the hapten mimic as much as possible that lactam presented above, the synthesis of a hapten preserving of the analyte51−54 and also to assess conformational changes the thiolactone moiety (AIPIVS hapten) was also addressed. on hapten−carrier bioconjugates.55 Hence, the minimum The immunizing haptens designed incorporated a short energy conformers of the native AIP-IV structure (thiolactone) bifunctional oligo ethylene glycol (8-amino-3,6-dioxaoctanoic and of the corresponding lactam or lactone AIP-IV structures acid, O2Oc) spacer arm at the N-terminal Tyr residue of the were calculated using Molecular Mechanics MM+, followed by AIP-IV (see Figure 2). The amino end of O2Oc could be a semiempirical PM3 method. Regarding the electronic adequately modified for further protein bioconjugation. The distribution, aside from the “X” functionality, significant hapten maximized the exposure of the cyclic peptide structure differences were only encountered in the region close to the to the immune system, which is the most characteristic thiolactone/lactam functionalities (see the graph in Figure 1). molecule moiety. Moreover, the ethylene glycol units of the As it can be observed, the position Cβ, corresponding to the spacer arm ensured hydrophilicity while minimizing its Cys residue of the native AIP-IV that is directly coupled to S, antigenicity. NH, or O in the corresponding AIPIV, AIPIV(NH), and General Strategy for the Synthesis of Native AIPs (I− AIPIV(O) molecules, is the only one affected probably due to IV) and the AIP-IV Haptens. The synthesis of the AIP-IV the fact that while NH and O have donor electron-transfer hapten and the native AIP (I−IV) target analytes started with features, the sulfur atom has acceptor electron properties. The the common linear peptide precursor (see Figure S1), variation is particularly evident in the Cβ position of synthesized using a fluorenymethoxycarbonyl (Fmoc)/tert- AIPIV(O), changing the electronegativity of its punctual butyl (tBu) solid-phase peptide synthesis strategy on a 2- charge. In respect to the geometry, the impact of changing the chloro-trityl chloride (CTC) resin. All functional groups of the thiolactone functionality by a lactam or by a lactone was amino acid side chains were protected with tBu with the estimated calculating the RMSE (root-mean-square error) by exception of the thiol group of the internal Cys residue that overlapping the lactam or the lactone with the native will form thiolactone, which was protected with a 4- thiolactone peptide (see models in Figure 1). The RMSE methoxyltrityl group (Mmt). For the case of AIP (I−IV) values of 0.449 and 0.357 Å were recorded for the lactam and target analytes, a tert-butyloxycarbonyl (Boc) protecting group 647 https://doi.org/10.1021/acsinfecdis.1c00670 ACS Infect. Dis. 2022, 8, 645−656 ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article was used for the α-amino group of the N-terminal amino acid direct nucleophilic reaction of the amino groups of the Lys (Tyr) of each linear peptide precursor. The AIP (I−IV) linear residues with the succinimide ester moiety of the hapten peptidyl resins were treated with a low concentration of allowed us to obtain easily the corresponding AIPIVS-HCH trifluoroacetic acid (TFA) [TFA/H2O/DCM (2:4:96)] to and BSA bioconjugates. Bioconjugation of the AIPIVNH(SH) remove the Mmt from the Cys and to cleave the peptide from hapten was performed through a two-step procedure by first the CTC resin, maintaining intact Boc/tBu protecting groups. reacting the Lys residues of the proteins with succinimidyl Then, the linear precursor peptides were treated with iodoacetate (SIA), to introduce iodine as a living group, and benzotriazol-1-yloxytripyrrolidinophosphonium hexafluoro- subsequently linking the hapten through a selective reaction of phosphate (PyBOP)/N,N-diisopropylethylamine (DIEA) to the SH group of the terminal Cys residue. MALDI-TOF-MS form the thioester between the thiol of the internal Cys and analyses of the corresponding AIPIVS(NHS)- and AIPNH- the carboxylic acid of the C-terminal. Finally, Boc/tBu (SH)-BSA bioconjugates recorded hapten densities of 8 and 6, protecting groups were eliminated by standard acidolysis respectively (see Table S1). Antisera were raised against treatment (TFA/triisopropylsilane (TIS)/H2O, 95:2.5:2.5). AIPIVNH(SH)-HCH (As376, As377, and As378) and Synthesis of the AIPIVS(X) Haptens. For the first AIP-IV AIPIVS(NHS)-HCH (As379, As380, and As381) using the hapten proposed (AIPIVS(SH)), Boc-Cys(Trt)-OH was standard immunization protocol described in the experimental incorporated at the O2Oc end with the idea to perform an section. orthogonal bioconjugation with the proteins previously Development of AIP-IV (As380/AIPIVS(NHS)-BSA) derivatized with maleimide. The protected peptide was cleaved ELISA. The avidity of the antisera against homologous and from the solid support with TFA/H2O/DCM (2:4:96), heterologous BSA bioconjugates (in respect to the chemical without deprotecting the Trt of the Cys N-terminal, and structures of the haptens) was assessed by bidimensional cycled to form thiolactone in solution as mentioned above. titration experiments from which the most suitable concen- Unfortunately, the global deprotection with TFA/TIS/H2O trations for each As/bioconjugate competitor combinations (95:2.5:2.5), which removes the Trt of the Cys terminal and were selected. Although not all of them gave rise to usable the Boc/tBu groups, rendered two compounds, the expected competitive assays, in general, homologous combinations AIPIVS(SH) hapten and a secondary product resulting from performed better than the heterologous ones (Table S2). On the attack of the thiol group of the N-terminal Cys residue to the other hand, it is relevant mentioning that the antisera thiolactone, giving rise to a large thioester peptide cycle (see raised using the thiolactone-immunizing hapten (AIPIVS- Figure S2, top) according to NMR. To overcome this problem, (NHS)-HCH) provided immunochemical assays with sub- it was proposed using a less nucleophilic amino group stantially higher detectability than the antisera obtained (AIPIVS(NH2) hapten), instead of an SH, for bioconjugation immunizing with the lactam hapten (AIPIVN(SH)-HCH). to the proteins. However, also in this case, the formation of the As can be observed in Table S2, the ELISAs using As379 and undesired more stable large lactam cyclic peptide, resulting As380 (against AIPIVS) show lower IC50 values (79 and 6 nM, from the reaction of the amine group with thiolactone, was respectively) compared to the ELISAs using As376−378 (188, observed when the protecting groups were eliminated (see 360, and 294 nM of IC50, respectively). These results agree Figure S2, bottom). with the theoretical data discussed above, in which, addition- In light of these results, it was clear that the functional group ally to the electronic distribution, significant differences in the for bioconjugation did not have to have a nucleophilic geometry were predicted for the lactam in respect to characteristic, for which reason a third hapten with a carboxylic thiolactone (RMSE 0.449 Å). Moreover, as mentioned before, group in the form of NHS (N-hydroxysuccinimide) ester was there are clear differences in respect to the capabilities to proposed. Thus, the AIPIVS(NHS) hapten was prepared by establish hydrogen bond interactions of these two function- the reaction of AIP-IV with 3,6-dioxaoctandioic acid alities. bissuccinimidyl ester (dihydroxysuccimidyl-PEG (2) ester) From all the antisera raised against AIPIVS(NHS)-HCH, (see Figure S1), without further difficulties, and obtained with As380 was giving the best immunoassay features under 96% purity according to UV and HPLC−MS analyses. The homologous and standard immunoassay conditions [assay NHS ester could be directly used to form amide groups with buffer 10 mM PBST (0.05% Tween), 30 min competitive the Lys residues of the protein and, as it has an electrophilic step]. For this reason, As380/AIPIVS(NHS)-BSA was selected character, did not interfere reacting with the labile thiolactone. for further investigations with the aim of improving its Synthesis of the AIPIVNH(SH) Hapten. The AIP-IV-NH performance and getting knowledge of the behavior of the (SH) hapten lactam derivative (see Figure S3) was synthesized assay under different physicochemical conditions. With this using a Fmoc-Dap(Alloc)-OH, instead of a Fmoc-Cys(Mmt)- purpose, the effects of several factors such as pH, ionic OH, and the AIPIV linear precursor was elongated with a Boc- strength, concentration of Tween 20, competition, and Cys-(Trt) at the end. Prior to the cleavage of the linear preincubation time, as well as the concentration of organic precursor from the peptidyl resin, the Alloc group was solvents such as DMSO, were investigated. eliminated by treatment with tetrakis(triphenylphosphine)- The results of these studies are shown in Figure S10, where palladium(0) (Pd(Ph3P)4) and phenylsilane (PheSiH3). it can be observed that the As380/AIPIVS(NHS)-BSA Finally, the lactam ring was also formed using PyBOP/DIEA, immunochemical assay is quite robust in respect to variations and the side-chain protecting groups, including the Trt of the in the pH and concentration of Tween 20 in the assay buffer. Cys, were removed with a high TFA content solution to obtain Hence, the ELISA As380/AIPIVS(NHS)-BSA performed quite the desired hapten AIP-IV-NH(SH) with 98.4% purity well on a broad range of pH values, keeping the best features according to UV and HR-MS. between 4.5 and 8.5, and the concentration of Tween20 did Antibody Development. Bioconjugation of the hapten not affect substantially the assay if used below 0.05%, although AIPIVS(NHS) to HCH and BSA was carried out by just it should be noticed that higher concentrations had a negative adding the hapten to the solutions of the proteins in PBS. A effect on the detectability. At pH 9.5, the assay was still usable, 648 https://doi.org/10.1021/acsinfecdis.1c00670 ACS Infect. Dis. 2022, 8, 645−656 ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article although a slight decrease in detectability was observed (IC50 0.19 nM and an LOD of 0.22 ± 0.06 nM, a detectability which 7.98 nM at pH 7.5 vs 13.2 nM at pH 9.5). This fact could be was below the concentration values found for AIP-I in culture related to the instability of thiolactone at basic pH. Regarding media.46,47 The assay attains higher detectability than other conductivity, a slight improvement in the detectability was methods developed for the quantification of AIPs based on observed with the increase of the ionic strength, but a mass spectrometry.46,47,56−59 Even though AIPs have never proportional reduction of the maximum absorbance was also been directly quantified in clinical samples, the excellent produced. The addition of a small percentage of DMSO (2%) features and detectability of the developed assay make foresee to the competitive step slightly increased the detectability, its potential for further studies to gain knowledge about the although the slope was slightly affected (data not shown). The role of AIPs in the development of pathogenesis and to assess competition time was set at 30 min since although the same the convenience of using AIPs as biomarkers of disease. detectability could be reached at shorter incubation times, the The concentrations of the BSA conjugate and As dilution maximum signal of the assay decreased significantly. A slight used were 0.63 μg mL−1 and 1/4000, respectively. The improvement in assay detectability was observed if the analyte parameters for the ELISA in Tryptic Soy Broth (TSB) 1/2 and the antibody were preincubated overnight or just for 10 refer to the diluted sample. The concentrations are expressed min at room temperature (RT) (IC50 3.7 and 3.8 nM, in nM, and the data shown correspond to the average of the respectively, vs 4.9 nM if no preincubation) without affecting analytical parameters recorded from assays performed in three the maximum signal. All these results pointed at the robustness different days using at least two wells/replicates per of the assay since any analytical parameter was significantly concentration of the standard curve. affected but also at the possibility of a slight improvement, if Because of the reported evidence that associate the Agr type required. However, further studies were conducted without with different forms of infection or colonization, assessing the modifying the assay conditions (see Table S3 for a summary of specificity of the As380/AIPIVS(NHS)-BSA ELISA developed the assay conditions selected for the As380/AIPIVS(NHS)- in respect to the other S. aureus AIPs was of great BSA ELISA). importance.60 Thus, in addition to AIP-IV, AIPs I−III were Analytical Characterization of the AIP-IV ELISA. The also synthetized and used to build standard curves that were analytical features of the assay run under these conditions are enclosed in Table 1. The assay showed an IC50 value of 2.90 ± measured in the ELISA. Surprisingly, despite the great structural similarities (see Figure 3), the ELISA was highly specific for AIP-IV, and the cross-reactivity of other AIPs was Table 1. Analytical Features of the As380/AIPIVS (NHS)- less than 0.001% even for AIP-I, which differs from AIP-IV in BSA ELISA for the Detection of AIP-IV just one amino acid (see the red moieties of AIP-I and AIP-IV analytical in Figure 3), demonstrating that the tyrosine residue present in parameters PBST TSB diluted 1/2 the AIP-IV cycle is essential for the biorecognition. Amin 0.15 ± 0.04 0.09 ± 0.01 Implementation of the As380/AIPIVS(NHS)-BSA Amax 1.46 ± 0.01 1.60 ± 0.05 ELISA to the Analysis of AIP-IV in Culture Media. The slope −0.91 ± 0.03 −0.89 ± 0.03 possibility to use this ELISA to directly analyze the profile of IC50 2.89 ± 0.19 2.80 ± 0.17 release of AIP-IV by S. aureus in media culture was dynamic range 0.68 ± 0.14 to 0.55 ± 0.11 to investigated. With this purpose, on a first instance, the 14.26 ± 0.93 12.84 ± 6.20 LOD 0.22 ± 0.06 0.19 ± 0.06 potential non-specific interferences caused by the TSB were R2 0.996 ± 0.003 0.995 ± 0.003 evaluated by building AIP-IV calibration curves in this broth diluted several times with the assay buffer. Figure 4 shows the Figure 3. Left: Results from the specificity study in respect to the AIPs of the four S. aureus agr groups. AIPs I−IV were used to build standard curves and measure them with the As380/AIPIVS(NHS)-BSA ELISA. The cross-reactivity was less than 0.001% for AIP-I−III. Each calibration point was measured in triplicates on the same ELISA plate, and the results show the average and standard deviation of analysis made on three different days. Right: Chemical structures of the four AIPs. The structural differences between AIP-I and AIP-IV are highlighted in red. 649 https://doi.org/10.1021/acsinfecdis.1c00670 ACS Infect. Dis. 2022, 8, 645−656 ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article Figure 4. (A) AIP-IV calibration curves using the As380/AIPIVS(NHS)-BSA ELISA competitive indirect assay run in PBST buffer, TSB culture media, and TBS diluted with the buffer at different ratios. The results shown are the average and standard deviations of analysis made on two different days. Each calibration point was measured by duplicates on each day. (B) Graph showing the correlation between the AIP-IV-spiked concentration and the concentration measured with the As380/AIPIVS(NHS)-BSA ELISA. Accuracy experiments were run in TSB culture media diluted 1/2 using PBST. Each calibration point was measured in triplicates on the same ELISA plate, and the results show the average and standard deviation of analysis made on three different days. Table 2. Results from the Precision Studies of the ELISA for AIP-IV [AIP-IV] R1 R2 R3 mean SD % CV inter-day high 12.85 13.79 12.52 13.05 0.66 5.1 medium 2.64 2.30 2.35 2.43 0.18 7.4 low 0.43 0.21 0.42 0.35 0.12 34.8 inter-plate high 14.80 15.12 15.33 15.08 0.27 1.8 medium 3.12 3.57 3.01 3.23 0.30 9.2 low 0.83 0.96 0.65 0.81 0.15 19.0 intra-plate high 14.16 12.27 13.56 13.33 0.96 7.2 medium 3.33 2.87 2.79 3.00 0.29 9.7 low 0.77 0.63 0.65 0.68 0.08 11.5 calibration curves obtained under these conditions. As it is resolving power hybrid mass spectrometer using a combination possible to observe, undiluted TSB affects substantially the of a segmented quadrupole and an Orbitrap mass analyzer (the assay performance; however, a simple one-half dilution with Q-Exactive) coupled to an UPLC reaching an LOD and LOQ the assay buffer is sufficient to overcome these interferences. of 0.0035 and 0.10 μM, respectively,46 concentration values Moreover, further dilution of the matrix provided identical that still are higher than those of the ELISA reported in this curves to the one performed in buffer, which indicates that paper. further dilution does not affect the assay performance. The accuracy of the assay was evaluated by measuring a set Table 1 shows the analytical parameters of the calibration of TBS blind-spiked samples. As it is shown in Figure 4, a good curves run in buffer and in 1/2 diluted TSB. The 1/2 dilution correlation between the spiked concentrations and the ELISA provides an immunochemical assay with IC50 and LOD values results was observed with a slope of 0.94, very close to 1, and a of 5.60 ± 0.34 and 0.38 ± 0.12 nM (originally 2.80 ± 0.17 and regression coefficient of 0.97, pointing at the good accuracy of 0.19 ± 0.06 nM without taking into account the 1/2 dilution the assay to quantify AIP-IV in TSB culture broth samples. with the assay buffer), which do not compromise the Moreover, the intra-plate, inter-plate, and inter-day precision of detectability of the assay, considering the reported concen- the assay was also very good as it can be observed in Table 2, tration values of AIPs in the TSB culture broth (i.e., 2−15 μM where the coefficients of variation (CVs) found at three range47). Moreover, the detectability achieved by this ELISA is concentrations levels are shown. The results indicated an higher than that reported when using mass spectrometry (MS) excellent assay precision, although, as expected, the higher methods and does not require any samples pretreatment (see variability was found at the limit of quantitation of the assay Table S4 for a summary of the detectability of MS reported (IC80), particularly when the assay was performed on different approaches). Thus, AIP detection by MS has been unwieldy plates or different days (% CV intra-plate, 11.5%; inter-plate, requiring extensive sample clean-up.56,57 MALDI-TOF-MS has 19%; and inter-day, 34.8%). However, in all other situations, been used for rapid detection of AIPs from other Gram- the % CV remained lower, below 10%, demonstrating that the positive bacteria, on crude supernatant cultures, but only with assay can be used to provide reliable quantification data in qualitative purposes.58 Using a hybrid ion trap Orbitrap mass respect to the AIP-IV concentration levels found in complex spectrometer (the LTQ Orbitrap), Cech and co-workers were matrices. able to quantify AIP in MRSA cultures with a limit of TBS samples for precision studies were prepared by spiking quantification of 2.6 μM,59 and Junio and co-workers were able the TBS broth at three levels corresponding to the to achieve an LOD of 0.25 μM for AIP-I by directly injecting concentrations providing 80% (low), 50% (medium), and the culture broth, previously filtered through a 0.22 μm 20% (high) of the maximum signal of the assay in order to surfactant-free cellulose acetate, on a UHPLC coupled to the cover all the dynamic range. The CV was calculated following LTQ Orbitrap.47 More recently, Todd and co-workers were the equation CV (%) = σ/μ × 100. The ELISA was run in the also able to quantify AIP-I using a new generation of a high- TBS culture broth diluted 1/2 with the assay buffer. The TBS 650 https://doi.org/10.1021/acsinfecdis.1c00670 ACS Infect. Dis. 2022, 8, 645−656 ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article samples were measured in triplicate on the same ELISA plate recorded for two of the bacterial clinical isolates genotyped as (intra-plate) and on three different days (inter-day) or on three agr IV (#48 and #165). Measurable AIP-IV IR equiv could be different plates (inter-plate). The concentrations of the detected almost immediately after introducing the inoculum in replicates, mean, standard deviation, and ICs are expressed in the culture media (strain # 48 at time zero and strain #165 nM; R: replicate; σ: standard deviation; and μ: average. after 1 h) even if the OD600 values were below 0.5. Junio et Assessment of AIP-IV Production by Clinical Isolates. al.47 could only detect AIP-I 4 h after inoculating the bacteria As a pilot study, a set of clinical isolates from patients infected in the media culture but the concentration levels recorded or colonized with S. aureus, according to the current were in the micromolar range. The reason for this difference definitions, and showing distinct respiratory S. aureus may be diverse, including the agr type, the clinical status of the pathologies were selected. The samples were genotyped to patient from which the bacteria was isolated, or the lower obtain knowledge of their agr type (see Table 3 for data detectability of the methodology employed (LOD 0.25 μM vs 0.38 nM of the ELISA reported here). In contrast, the third Table 3. Description of the Clinical Isolates Used in This clinical isolate (#197), classified as doubtful genotype agr I/IV Pilot Study showed a significant lower and irregular production of AIP-IV. # clinical isolate Agr system infection type Tracing the origin of this sample, it was found that although this patient appeared to be infected by a S. aureus strain (197- 6_19850 I pneumonia 3_40448 II tracheobronchitis 63538-CC121) showing a regular morphology and behavior, 197_63535 (CC45, CC121)a I, I/IVa pneumonia the culture also showed small satellite colonies (197-63538- 165_36759 IV tracheobronchitis CC45), which further genotyping studies revealed to be agr I. 32_75664 III carrier Apparently, the inoculum used was taken from this last type of 48_86474 IV bronchia colonization colonies, which could explain the unexpected behavior of this Newman I sample. The fact that after 18 h of growth it was possible to USA300 I detect some AIP-IV IR equiv still has to be explained and may a The clinical isolate 197_63535 was found to be formed by two require further analyses; however, it could have been related to different clonal complexes. While CC45 was genotyped as agr I, a potential contamination with CC121. Despite the partic- CC121 was classified as doubtful I/IV according to the kit used. ularities of this last clinical isolate, these results point at the possibility of using this ELISA to identify the S. aureus and also regarding these samples) and inoculated on TSB. Bacterial to the agr strain type in a reliable manner and in a short time. growth was similar for all the strains, independent of the agr Further impact of the results reported in this paper could be system or type of infection, as evidenced by the measurement the use of the technology here presented to identify the absorbance at 600 nm and the OD600 at different time intervals dysfunctional agr strains by directly quantifying the QS for a 25 h period (see Figure S11 and Table S5 for OD600 signaling molecules in parallel to real-time PCR61 or other values and cfu mL−1 data of each point). Small sample aliquots indirect conventional methods such as CAMP or VLT (vesicle were taken at different time intervals (1 h, from 0 to 6 h of lysis test) assays, which asses agr functionality through the growth, 3 h, from 6 to 24 h growth, and after 48 h) and hemolysis effect of δ-lysine.62 Hence, several clinical studies measured with ELISA after a 1/2 dilution with the assay buffer, point to the higher probability of unfavorable clinical outcomes as described above. by invasive infection of dysfunctional agr S. aureus strains, Figure 5 shows the AIP-IV immunoreactivity equivalents (IR pointing to the possibility to use these AIPs as potential equiv) recorded for all the clinical strains. As expected, any biomarkers also to predict prognostic63,64 or to distinguish AIP-IV IR equiv were detected in the samples from the colonization from infection.62,65,66 Dysfunctional agr has been cultures where strains with agr systems I−III were grown. On associated with more abundant biofilm formation, deficient the other hand, AIP-IV IR equiv of up to 200 nM were bacteria autolysis, decrease in the antibiotic activity, persistent Figure 5. Graph showing the AIP-IV immunoreactivity equivalents (IRequiv) measured at different times from aliquots taken from the culture media where different S. aureus strains were grown. Culture broth samples were taken at the selected times, and the AIP-IV IRequiv were measured using the developed As380/AIPIVS(NHS)-BSA ELISA. IRequiv was only detected in the culture broth where clinical isolates genotyped as Agr IV (# 48 and #165) grew. Sample #197 was genotyped as agr I (CC-45); however, the clinical sample was found to contain two different strains, the second one (CC121) was classified as doubtful agr I/IV according to the kit used. Culture media of the rest of the clinical isolates genotyped as Agr I−III (# 6, 3, and 32, Newman and US300) did not show any immunoreactivity. See Table 3 for more information on the features of the samples measured. Each calibration point was measured in triplicates on the same ELISA plate, and the results show the average and standard deviation of analysis made on two different days. 651 https://doi.org/10.1021/acsinfecdis.1c00670 ACS Infect. Dis. 2022, 8, 645−656 ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article bacteremia, and higher mortality.64 In the same manner, some nanomolar range (0.22 ± 0.06 and 0.19 ± 0.06 nM in buffer authors also point to the implication of dysfunctional agr and in 1/2 TBS, respectively). Hence, a similar hapten in system on the formation of S. aureus SCV,67 often resistant to which thiolactone had been substituted by a more stable antibiotics such as gentamicin or aminoglycosides, being able lactam provided assays with LODs 2 orders of magnitude to bypass the effect of other antibiotics.68 Thus, S. aureus SCVs higher. The assay has shown to be very specific for AIP-IV as have been reported to be implicated in chronic infections, with demonstrated by cross-reactivity studies and by the measure- a high capacity for survival and adaptation to the host ment of the AIPs produced by S. aureus of agr types I−III. This immunological or inflammatory response.69 Although SCVs fact allows envisaging the possibility to use the technology here are not particularly virulent, some phenotypes represent reported for phenotyping the agr, if antibodies with similar transient states that under certain conditions can revert to features would be available for each of the AIPs released by the wild type or to a different phenotype (distinct from the SCV rest of the agr groups. AIP-IV could be detected in the low progenitor), leading to the development of an acute nanomolar range in the culture broth almost immediately (<1 infection.70,71 It is well known that during infectious processes, h) after inoculating samples from clinical isolates previously bacteria can switch from planktonic (acute infection) to sessile genotyped as agr IV. In contrast, any immunoreactivity was lifestyle (chronic infection). During this process, the detected in TBS samples from media were clinical isolates production of toxins, enzymes, and other survival mechanisms genotyped agr I−III had been growth under the same is adapted to the needs of the bacterial population, and all conditions. these processes may be regulated by the Agr system. Therefore, The antibodies against AIP-IV, reported here for the first the technology here reported when extended to other AIPs time, show great potential for basic research, biomedical could be extremely useful to perhaps identify SCVs and to investigations, or diagnostic purposes. As is known, there exist understand all these processes. a variety of immunochemical analytical configurations in which Further studies need to be performed in order to implement antibodies can be used, including lateral-flow immunoassays this technology to the direct analysis of clinical samples and to for PoC applications, microarray for multiplexed analysis, or a prove the value of the AIP levels in terms of the severity or the wide variety of electrochemical or optical biosensors suitable stage of the disease. The data presented in this paper are only for PoC or for benchtop laboratory analyzers. In light of the preliminary studies addressed to demonstrate the potential of results reported here, we envisage addressing our investigations the ELISA developed for diagnostic purposes, whose value may toward the development of specific antibodies against the go beyond just identifying the bacteria causing the disease. It different AIPs produced by the rest of agr types of S. aureus. A will be necessary to carry out a complete well-designed clinical multiplexed chip combining all these immunoreagents could study that takes into consideration all the clinical parameters of be a powerful diagnostic tool for identification and surveillance interest. However, we can ensure that the As380/AIPIVS- of S. aureus infection while stratifying the patients in respect of (NHS)-BSA ELISA is a highly sensitive, robust, and reliable the agr type. bioanalytical tool to specifically quantify AIP-IV. Complex In this paper, we report the development of a microplate- biological samples such as culture broth can be directly based ELISA which shows great potential for high-throughput analyzed without any previous sample treatment, other than sample analyses. More than 100 samples can be simultaneously diluting the sample two times with the assay buffer. The assay processed in about 1:5 h. Here, we describe the application of has high sample throughput capability, which has allowed this ELISA to monitor the profile of release of S. aureus strains obtaining information about the profile of release of eight in culture media. Further studies will be addressed to different bacterial strains grown in TBS media. The levels of implement the present technology to the direct analysis of AIP-IV released are in the high nanomolar range and can be clinical samples such as, for example, sputa, BAS, or BAL. ■ measured in less than 1 h of incubation in the culture broth. The clinical significance of QS molecules needs to be further explored due to the vast set of mechanisms that is under their METHODS control. Interesting studies are already addressing the clinical General Methods and Instruments. General methods significance and the role of QS molecules from Gram-negative related to chemical and immunochemical studies performed, as bacteria such as P. aeruginosa; for their QS signaling molecules, well as the main equipment used, can be found in the we have also reported the development of specific antibod- Supporting Information. ies.72−74 Similar studies could be addressed in the near future, Theoretical Calculations and Molecular Modeling. which may provide interesting information in respect to the Molecular modeling was performed using the Hyperchem 6.03 involvement of these QS molecules on pathogenesis, as well as software package (Hypercube Inc., Gainesville, FL). Theoreti- the possibility to develop powerful technologies for the cal geometries and electronic distributions were evaluated for diagnostic and surveillance of S. aureus infections. AIP-IV hapten derivatives (thiolactone or lactam ring) using ■ CONCLUSIONS A highly sensitive and specific immunochemical assay for direct semiempirical quantum mechanics MNDO and PM3 models. All calculations were performed using the standard computa- tional chemistry criteria. quantification of AIP-IV, the QS signaling molecule specifically Synthesis of AIPs I−IV and AIP-IV Immunizing released by agr IV-type S. aureus, has been developed. The Haptens. The experimental procedure for the synthesis of excellent analytical features have been accomplished thanks to AIPIVS, AIPIVNH, and AIPs I−IV is fully described in the the high-quality antibodies raised against a molecular Supporting Information, including the schematic synthetic modeling-assisted hapten design (AIPIVS), which preserves sequences for each immunizing hapten derivative (see Figures all the electronic, geometric, and chemical features of the S1−S3) and images of the HPLC-PADs and HR-MS spectra native AIP-IV. Preserving the thiolactone functionality has used to characterize the synthesized peptides (see Figures S4− found to be crucial to achieve a detectability in the low S9). 652 https://doi.org/10.1021/acsinfecdis.1c00670 ACS Infect. Dis. 2022, 8, 645−656 ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article Synthesis of the AIP-IV Bioconjugates. Lactam Hapten Implementation of the ELISA to the Analysis of Bioconjugates (AIPIVNH(SH)-BSA and AIPIVNH(SH)-HCH). A Clinical Isolates. Description of the Clinical Samples. Six solution of SIA (4.5 μmol) in DMF (0.1 mL) was added clinical S. aureus isolates were retrospectively selected from a dropwise to a solution of the protein (BSA or HCH, 5.6 mg collection of strains in the Hospital Universitari Germans Trias mL−1, 0.9 mL in borax/borate buffer), and the mixture was i Pujol. Strains were isolated from respiratory specimens stirred 4 h at RT. The mixture was purified by AKTA using a obtained from patients under mechanical ventilation admitted HiTrap desalting column and borax/borate as eluting buffer to at the intensive care unit.75 The isolates were stored at −80 °C isolate the protein−SIA intermediates. A fraction (20 μL) of in a maintenance freezing medium (Oxoid TP, 15731). The the BSA-SIA bioconjugate was kept for MALDI-TOF analysis. strains were identified as S. aureus by conventional assays Then, a solution of tris(2-carboxyethyl)phosphine (150 μL, 2.5 (Gram staining, selective culture media, coagulase test) and mg mL−1) was added dropwise to the AIPIVNH hapten (3.45 antibiotic susceptibility testing and were genotypically mg, 2.8 μmol) in 1:1 CH3CN/H2O (150 μL), and the mixture characterized by means of a DNA microarray (Clondiag). was stirred for 10 min at 40 °C and added (150 μL) to the According to array results, the strains were agr I (culture solutions of proteins (BSA or KLH, 2.8 mg mL−1, 1.8 mL in number 19850), agr II (culture number 40448), agr III borax/borate buffer). The mixtures were stirred overnight at 4 (culture number 75664), and agr IV (culture numbers 86474, °C, and on the next day, the bioconjugates were purified by 63535, and 36759). The clinical isolates and two S. aureus dialysis against 0.5 mM PBS (5 × 5 L) and Milli-Q water (1 × reference strains (USA300 and Newman, agr I) were cultured 5 L) and stored freeze-dried at −80 °C. A small fraction (20 overnight at 37 °C in TSB (5 mL). The next day, dilutions 1/ μL) of AIPIVNH(SH)BSA was kept for MALDI-TOF analysis, 50 in fresh TSB were prepared, and the optical density at 600 rendering a hapten density of six haptens per molecule of BSA nm (OD600) was measured. Then, the resulting solutions were (see Table S1). shaken at 37 °C until the selected time of growth was Thiolactone Hapten Bioconjugates [AIPIVS(NHS)-BSA and completed. When the time was finished, aliquots were AIPIVS(NHS)-HCH]. A solution of the AIPIVS(NHS) hapten extracted for colony forming units (cfus) counting and (2.9 mg, 2.3 μmol) in anhydrous DMF (0.1 mL) was added OD600 measurement. Afterward, each solution was centrifuged dropwise over the protein solutions (BSA or HCH, 2.5 mg 10 min at 3000 g, and aliquots were stored at −20 °C for AIP- mL−1, 2 mL in PBS 10 mM buffer), and the mixtures were left IV concentration measurement using the ELISA developed in to stir for 4 h at RT. Subsequently, the bioconjugates were this work. AIP-IV concentrations measured by ELISA are purified by dialysis against 0.5 mM PBS (5 × 5 L) and Milli-Q expressed as AIP-IV immunoreactivity equivalents (IRequiv). water (1 × 5 L) and stored freeze-dried at −80 °C. A small Matrix Effect Studies. To find out the most appropriate fraction (20 μL) of AIPIVS-BSA was kept for MALDI-TOF conditions to quantitate AIP-IV in culture samples, AIP-IV analysis, rendering a hapten density of eight haptens per standard calibration curves were prepared in the TSB culture molecule of BSA (see Table S1). broth diluted with PBST (1:2, 1:5, 1:10, and 1:20), and the ■ analytical parameters compared with the standard curve were prepared in PBST to select the dilution factor in which the ELISA nonspecific interferences caused from the matrix were As380/AIPIVS-BSA ELISA. Microtiter plates were coated minimized. with the AIPIVS(NHS)-BSA bioconjugate in coating buffer Accuracy Studies. Blind spiked samples were prepared in (0.31 μg mL−1, 100 μL/well) overnight at 4 °C and covered the TSB culture broth and measured using the above-described with adhesive plate sealers. The next day, the plates were ELISA. The samples were measured in triplicates, and the washed with PBST (4 × 300 μL/well), and solutions of the experiment was repeated on three different days. AIP-IV standard (2 μM to 0.13 nM in PBST, 50 μL/well) were added, followed by As380 (dil. 1/4000 in PBST, 50 μL/well), and left without agitation for 30 min at RT. After another ■ * ASSOCIATED CONTENT sı Supporting Information washing step, a solution of goat antirabbit IgG-HRP (1/6000 The Supporting Information is available free of charge at in PBST) was added (100 μL/well) and incubated for 30 min https://pubs.acs.org/doi/10.1021/acsinfecdis.1c00670. at RT. The plates were washed again, and the substrate General chemical and immunochemical methods and solution was added (100 μL) and left for 30 min at RT in the instruments; buffers; polyclonal antibody development; dark. The enzymatic reaction was stopped by adding of 4 N peptide synthesis [AIP-I−IV, AIPIVNH (SH) , and H2SO4 solution (50 μL/well), and the absorbance was read at AIPIVS(NHS)] and characterization; theoretical calcu- 450 nm. lations about immunogen’s structure; ELISA develop- Immunoassay Evaluation. The performance of the assays ment and characterization; and data related to the was evaluated through the modification of different phys- growth of the distinct S. aureus strains analyzed (PDF) icochemical parameters [competence time, incubation time, pH, ionic strength, the presence of a surfactant (% Tween 20), or solubility with the addition of organic solvents] in the competition step. ■ AUTHOR INFORMATION Corresponding Author Cross-Reactivity Studies. Standard solutions of synthetic María-Pilar Marco − Nanobiotechnology for Diagnostics AIPs (I−IV) were prepared (0.12 nM to 10 μM in PBST) and (Nb4D), Department of Surfactants and Nanobiotechnology, measured with the ELISA following the procedure described Institute for Advanced Chemistry of Catalonia (IQAC) of the above. The standard curves obtained were fitted to the four- Spanish Council for Scientific Research (CSIC), 08750 parameter equation, and the IC50 value was used to calculate Barcelona, Spain; CIBER de Bioingeniería, Biomateriales y the cross-reactivity according to the following equation: CR Nanomedicina (CIBER-BBN), 28029 Madrid, Spain; (%) = IC50 (cross-reactant)/IC50 (analyte) × 100. orcid.org/0000-0002-4064-1668; Phone: +34 93 653 https://doi.org/10.1021/acsinfecdis.1c00670 ACS Infect. Dis. 2022, 8, 645−656 ACS Infectious Diseases ■ pubs.acs.org/journal/aidcbc Article 4006184; Email: pilar.marco@cid.csic.es; Fax: +34 93 ACKNOWLEDGMENTS 2045904 This work has been funded (SAF2015-67476-R, RTI2018- Authors 096278-B-C21, both to M.-P. Marco) by MICIN/AEI/ 10.13039/501100011033 and by “ERDF A way of making Enrique-J. Montagut − Nanobiotechnology for Diagnostics Europe”, and to C. Prat through the Instituto de Salud Carlos (Nb4D), Department of Surfactants and Nanobiotechnology, III (PI17/01139). M.-P. Marco has also received funding from Institute for Advanced Chemistry of Catalonia (IQAC) of the the Fundació Marató de TV3 (TV32018-201825-30-31). Spanish Council for Scientific Research (CSIC), 08750 Enrique-J. Montagut wishes to thank the FPI fellowship Barcelona, Spain; CIBER de Bioingeniería, Biomateriales y (BES-2016-076496) from the MICIN/AEI/10.13039/ Nanomedicina (CIBER-BBN), 28029 Madrid, Spain 501100011033 and ESF investing in your future. We also Gerardo Acosta − CIBER de Bioingeniería, Biomateriales y thank the denomination of consolidated group from the Nanomedicina (CIBER-BBN), 28029 Madrid, Spain; Generalitat de Catalunya: 2017 SGR 1441 (M.-P. Marco) and Multivalent Systems for Nanomedicine (MS4N), Department 2017 SGR 1439 (M. Royo). CIBER Actions, CB06/01/0036 of Surfactants and Nanobiotechnology, Institute for Advanced (M.-P. Marco), CB06/06/0031 (C. Prats) and CB06/01/0074 Chemistry of Catalonia (IQAC) of the Spanish Council for (M. Royo) have been financed by the Instituto de Salud Carlos Scientific Research (CSIC), 08750 Barcelona, Spain III with assistance from the European Regional Development Fernando Albericio − CIBER de Bioingeniería, Biomateriales Fund (ERDF). The U2 (Custom Antibody Service (CAbS) y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain; and the U3 (Synthesis of Peptide Unit) of the ICTS Multivalent Systems for Nanomedicine (MS4N), Department “NANBIOSIS” at the IQAC-CSIC, are acknowledged for the of Surfactants and Nanobiotechnology, Institute for Advanced assistance and support related respectively to the development Chemistry of Catalonia (IQAC) of the Spanish Council for of the immunoreagents and the synthesis of the different Scientific Research (CSIC), 08750 Barcelona, Spain; peptide haptens used in this work. ■ Department of Organic Chemistry, Faculty of Chemistry, University of Barcelona, 08028 Barcelona, Spain; School of Chemistry and Physics, University of KwaZulu-Natal, 4000 REFERENCES Durban, South Africa (1) Hurley, M. N. Staphylococcus aureus in cystic fibrosis: problem Miriam Royo − CIBER de Bioingeniería, Biomateriales y bug or an innocent bystander? Breathe 2018, 14, 87−90. 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