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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.

The accessory gene regulator (agr) locus of Staphylococcus aureus is a quorum-sensing virulence regulator. Although there are many studies concerning the effect of dysfunctional agr on the outcomes of S. aureus infection, there is no systematic review to date. We systematically searched for clinical studies reporting outcomes of invasive S. aureus infections and the proportion of dysfunctional agr among their causative strains, and we performed a meta-analysis to obtain estimates of the odds of outcomes of invasive S. aureus infection with dysfunctional versus functional agr. Of 289 articles identified by our research strategy, 20 studies were meta-analysed for crude analysis of the impact of dysfunctional agr on outcomes of invasive S. aureus infection. Dysfunctional agr was generally associated with unfavourable outcomes (OR 1.32, 95% CI 1.05–1.66), and the impact of dysfunctional agr on outcome was more prominent in invasive methicillin-resistant S. aureus (MRSA) infections (OR 1.54, CI 1.20–1.97). Nine studies were meta-analysed for the impact of dysfunctional agr on the 30-day mortality of invasive S. aureus infection. Invasive MRSA infection with dysfunctional agr exhibited higher 30-day mortality (OR 1.40, CI 1.03–1.90) than that with functional agr. On the other hand, invasive MSSA infection with dysfunctional agr exhibited lower 30-day mortality (OR 0.51, CI 0.27–0.95). In the post hoc subgroup analysis by the site of MRSA infection, dysfunctional agr was associated with higher 30-day mortality in MRSA pneumonia (OR 2.48, CI 1.17–5.25). The effect of dysfunctional agr on the outcome of invasive S. aureus infection may vary depending on various conditions, such as oxacillin susceptibility and the site of infection. Dysfunctional agr was generally associated with unfavourable clinical outcomes and its effect was prominent in MRSA and pneumonia. Dysfunctional agr may be applicable for outcome prediction in cases of invasive MRSA infection with hardly eradicabl

Streptococcus pneumoniae is responsible for severe infections, causing millions of deaths yearly. Immunoglobulin G (IgG) antibodies against the capsular polysaccharide (CPS) offer S. pneumoniae serotype-specific protection. In this work, we examined the applicability of the microarray technology to detect CPS type-specific IgGs in serum, using a collection of 22 microarray-printed S. pneumoniae CPSs. First, printing of five CPSs onto nitrocellulose-coated glass slides was tested. Successful printing was only achieved for certain CPS types and concentrations. This behavior was tentatively related with diverse viscosities of the CPS solutions. Measurement of dynamic viscosities fully supported this assumption and helped to establish suitable CPS type-and concentration-dependent printing conditions. Next, the potential of CPS microarrays for detecting recognition by anti-CPS IgGs was examined using well-defined rabbit pneumococcal antisera. In all cases, the expected antiserum-CPS binding signals were detected, prompting a proof-of-concept analysis of human serum samples. Clearly distinct serum-and CPS-specific binding patterns and intensities were observed, evidencing selective detection of CPS type-specific IgGs. Compared to the ELISA assay commonly used to quantitate CPS type-specific IgGs in serum, the newly developed S. pneumoniae CPS microarrays offer the advantage of enabling the simultaneous analysis of multiple CPS-serum interactions using minute CPS amounts and significantly reduced serum volumes. Therefore, the approach could be particularly valuable for gauging the presence of CPS type-specific IgGs in human serum when sample volumes are limited and/or numerous serum samples are being examined.

[Aim]: First, to compare in vitro minimum inhibitory concentrations (MIC) of free cloxacillin and cloxacillin-containing nanoparticles (NP) against methicillin-susceptible (MSSA) and resistant Staphylococcus aureus (MRSA) and second, to assess NP antimicrobial activity against intracellular S. aureus., [Methods]: Poly(d,l-lactide-co-glycolide) acid (PLGA)-NP were loaded with cloxacillin and physico-chemically characterized. MICs were determined for reference strains Newman-(MSSA) and USA300-(MRSA). Murine alveolar macrophages were infected, and bacterial intracellular survival was assessed after incubating with free-cloxacillin or PLGA-cloxacillin-NP., [Results and conclusion]: For both isolates, MICs for antibiotic-loaded-NP were lower than those obtained with free cloxacillin, indicating that the drug encapsulation improves antimicrobial activity. A sustained antibiotic release was demonstrated when using the PLGA-cloxacillin-NP. When considering the lowest concentrations, the use of drug-loaded NP enabled a higher reduction of intracellular bacterial load.

There is a great interest in developing novel anti-biofilm materials in order to decrease medical device-associated bacterial infections causing morbidity and high healthcare costs. However, the testing of novel materials is often done using bacterial lab strains that may not exhibit the same phenotype as clinically relevant strains infecting medical devices. Furthermore, no consensus of strain selection exists in the field, making results very difficult to compare between studies. In this work, 19 clinical isolates of Pseudomonas aeruginosa originating from intubated patients in an intensive care unit have been characterized and compared to the lab reference strain PAO1 and a rmlC lipopolysaccharide mutant of PAO1. The adhesion and biofilm formation was monitored, as well as cell properties such as hydrophobicity, zeta potential and motility. Two groups of isolates were observed: one with high adhesion to polymer surfaces and one with low adhesion (the latter including PAO1). Furthermore, detailed biofilm assays in a flow system were performed using five characteristic isolates from the two groups. Confocal microscopy showed that the adhesion and biofilm formation of four of these five strains could be reduced dramatically on zwitterionic surface coatings. However, one isolate with pronounced swarming colonized and formed biofilm also on the antifouling surface. We demonstrate that the biofilm properties of clinical isolates can differ greatly from that of a standard lab strain and propose two clinical model strains for testing of materials designed for prevention of biofilm formation in the respiratory tract. The methodology used could beneficially be applied for screening of other collections of pathogens to identify suitable model strains for in vitro biofilm testing. Statement of Significance: Medical-device associated infections present a great challenge in health care. Therefore, much research is undertaken to prevent bacterial colonization of new types of bi