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The human body represents the habitat of trillions of symbiotic microorganisms, collectively known as human microbiota, approximately half of which residing in the gut. The development of next-generation sequencing techniques has boosted the profiling of human microbiota in recent years. A growing body of evidence seems to support a strict relationship between the disruption of the mutualistic relationship between the microbiota and the host (i.e., dysbiosis) and the development of several diseases, including breast malignancies. Breast cancer still represents the most frequent cause of cancer-related death in women. Its complex relationship with gut microbiota is the object of a growing body of evidence. In fact, the interaction with the host immune system and a direct impact of gut microbiota on estrogen, lipid and polyphenols metabolism, seem to potentially affect breast tumor development, progression and response to treatments. In this review, in an attempt to help oncologists navigating this rapidly-evolving research field, we provide an essential overview on the taxonomy, main analytical techniques and terminology most commonly adopted. We discuss what is currently known regarding the interaction between gut microbiota and breast cancer and potential efforts to harness this complex interplay for therapeutic purposes, and revise main ongoing studies. We also briefly provide an overview on breast cancer intratumoral microbiota and its potential role beyond gut microbiota., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Francesco Schettini reports honoraria from Novartis, Gilead and Daiichy-Sankyo for educational events/materials and travel expenses from Novartis, Gilead and Daiichy-Sankyo. Daniele Generali declares personal fees for educational events by Novartis, Lilly, Pfizer, Daiichy-Sankyo, Roche; research funds from Astrazeneca, Novartis and LILT. The other authors have nothing to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: To evaluate the expression dynamics of biofilm genes in methicillin-resistant Staphylococcus aureus (MRSA) retrieved from endotracheal tubes (ETT) and to determine how gene regulation is attenuated in vitro where host-environmental factors are no longer present., Methods: Biofilm was grown (24 h) in tryptic broth soy plus 0.25% glucose for a clinical MRSA isolate in planktonic state and after sessile growth named ETT-MRSA (S2, S3, S4, S5, S6, S7). Gene expression of five biofilm-related genes ( icaC , clfB , ebps , fnbB , and RNA III ) was assessed consecutively from day 1 to day 4 after ETT growth through real-time PCR. 16S rRNA was used as a control., Results: The MRSA isolates retrieved from ETT were capable of producing biofilms dependent on ica . The gene expression dynamics of ETT-MRSA changed progressively compared to planktonic MRSA gene expression under both ambient air ( p < 0.001) and ambient air with 5% CO2 ( p < 0.001). Dynamic assessment of icaC expression in both atmospheric conditions showed progressive downregulation in vitro compared to in vivo ETT biofilms. The expression patterns of clfB and ebps genes were similar to icaC. In contrast, the expression of the RNA III gene showed progressive upregulation from day 1 to day 4 ( p < 0.001)., Conclusions: MRSA loses its biofilm gene expression in vitro, by adaptive features across multiple generations, as evidenced by the progressive downregulation of icaC and upregulation of RNA III . These findings underscore the significance of host-environment dependence in regulating bacterial biofilm genes, highlighting its importance in diagnostics. Bacterial strains lose their host-specific characteristics as they are cultured in vitro.

The application of B-cell epitope identification to develop therapeutic antibodies and vaccine candidates is well established. However, the validation of epitopes is time-consuming and resource-intensive. To alleviate this, in recent years, multiple computational predictors have been developed in the immunoinformatics community. Brewpitopes is a pipeline that curates bioinformatic B-cell epitope predictions obtained by integrating different state-of-the-art tools. We used additional computational predictors to account for subcellular location, glycosylation status, and surface accessibility of the predicted epitopes. The implementation of these sets of rational filters optimizes in vivo antibody recognition properties of the candidate epitopes. To validate Brewpitopes, we performed a proteome-wide analysis of SARS-CoV-2 with a particular focus on S protein and its variants of concern. In the S protein, we obtained a fivefold enrichment in terms of predicted neutralization versus the epitopes identified by individual tools. We analyzed epitope landscape changes caused by mutations in the S protein of new viral variants that were linked to observed immune escape evidence in specific strains. In addition, we identified a set of epitopes with neutralizing potential in four SARS-CoV-2 proteins (R1AB, R1A, AP3A, and ORF9C). These epitopes and antigenic proteins are conserved targets for viral neutralization studies. In summary, Brewpitopes is a powerful pipeline that refines B-cell epitope bioinformatic predictions during public health emergencies in a high-throughput capacity to facilitate the optimization of experimental validation of therapeutic antibodies and candidate vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Farriol-Duran, López-Aladid, Porta-Pardo, Torres and Fernández-Barat.)

Introduction: Biofilm production is an important yet currently overlooked aspect of diagnostic microbiology that has implications for antimicrobial stewardship. In this study, we aimed to validate and identify additional applications of the BioFilm Ring Test® (BRT) for Pseudomonas aeruginosa (PA) isolates from patients with bronchiectasis (BE)., Materials and Methods: Sputa were collected from BE patients who had at least one PA positive culture in the previous year. We processed the sputa to isolate both mucoid and non-mucoid PA, and determined their susceptibility pattern, mucA gene status, and presence of ciprofloxacin mutations in QRDR genes. The Biofilm production index (BPI) was obtained at 5 and 24 hours. Biofilms were imaged using Gram staining., Results: We collected 69 PA isolates, including 33 mucoid and 36 non-mucoid. A BPI value below 14.75 at 5 hours predicted the mucoid PA phenotype with 64% sensitivity and 72% specificity., Conclusion: Overall, our findings suggest that the fitness-cost associated with the mucoid phenotype or ciprofloxacin resistance is shown through a time-dependent BPI profile. The BRT has the potential to reveal biofilm features with clinical implications., Competing Interests: AT has received grants from MedImmune, Cubist, Bayer, Theravance, and Polyphor and personal fees as Advisory Board member from Bayer, Roche, The Medicines CO, and Curetis. He has received bureau fees for keynote speaker presentations from GSK, Pfizer, Astra Zeneca, and Biotest Advisory Board, and are unconnected to the study submitted here. TB and CP were employed by BioFilm Pharma SAS and BioFilm Control SAS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fernández-Barat, Vázquez Burgos, Alcaraz, Bueno-Freire, López-Aladid, Cabrera, Gabarrús, Palomeque, Oscanoa, Ceccato, Motos, Amaro, Bernardi, Provot, Soler-Comas, Muñoz, Vila and Torres.)

16S rRNA gene profiling, which contains nine hypervariable regions (V1-V9), is the gold standard for identifying taxonomic units by high-throughput sequencing. Microbiome studies combine two or more region sequences (usually V3-V4) to increase the resolving power for identifying bacterial taxa. We compare the resolving powers of V1-V2, V3-V4, V5-V7, and V7-V9 to improve microbiome analyses in sputum samples from patients with chronic respiratory diseases. DNA were isolated from 33 human sputum samples, and libraries were created using a QIASeq screening panel intended for Illumina platforms (16S/ITS; Qiagen Hilden, Germany). The analysis included a mock community as a microbial standard control (ZymoBIOMICS). We used the Deblur algorithm to identify bacterial amplicon sequence variants (ASVs) at the genus level. Alpha diversity was significantly higher for V1-V2, V3-V4, and V5-V7 compared with V7-V9, and significant compositional dissimilarities in the V1-V2 and V7-V9 analyses versus the V3-V4 and V5-V7 analyses. A cladogram confirmed these compositional differences, with the latter two being very similar in composition. The combined hypervariable regions showed significant differences when discriminating between the relative abundances of bacterial genera. The area under the curve revealed that V1-V2 had the highest resolving power for accurately identifying respiratory bacterial taxa from sputum samples. Our study confirms that 16S rRNA hypervariable regions provide significant differences for taxonomic identification in sputum. Comparing the taxa of microbial community standard control with the taxa samples, V1-V2 combination exhibits the most sensitivity and specificity. Thus, while third generation full-length 16S rRNA sequencing platforms become more available, the V1-V2 hypervariable regions can be used for taxonomic identification in sputum., (© 2023. The Author(s).)

Background: Non-cystic fibrosis bronchiectasis (BE) is a chronic structural lung condition that facilitates chronic colonization by different microorganisms and courses with recurrent respiratory infections and frequent exacerbations. One of the main pathogens involved in BE is Pseudomonas aeruginosa., Objectives: To determine the molecular mechanisms of resistance and the molecular epidemiology of P. aeruginosa strains isolated from patients with BE., Methods: A total of 43 strains of P. aeruginosa were isolated from the sputum of BE patients. Susceptibility to the following antimicrobials was analysed: ciprofloxacin, meropenem, imipenem, amikacin, tobramycin, aztreonam, piperacillin/tazobactam, ceftazidime, ceftazidime/avibactam, ceftolozane/tazobactam, cefepime and colistin. The resistance mechanisms present in each strain were assessed by PCR, sequencing and quantitative RT-PCR. Molecular epidemiology was determined by MLST. Phylogenetic analysis was carried out using the eBURST algorithm., Results: High levels of resistance to ciprofloxacin (44.19%) were found. Mutations in the gyrA, gyrB, parC and parE genes were detected in ciprofloxacin-resistant P. aeruginosa strains. The number of mutated QRDR genes was related to increased MIC. Different β-lactamases were detected: blaOXA50, blaGES-2, blaIMI-2 and blaGIM-1. The aac(3)-Ia, aac(3)-Ic, aac(6″)-Ib and ant(2″)-Ia genes were associated with aminoglycoside-resistant strains. The gene expression analysis showed overproduction of the MexAB-OprM efflux system (46.5%) over the other efflux system. The most frequently detected clones were ST619, ST676, ST532 and ST109., Conclusions: Resistance to first-line antimicrobials recommended in BE guidelines could threaten the treatment of BE and the eradication of P. aeruginosa, contributing to chronic infection., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Serological tests are essential for the control and management of COVID-19 pandemic (diagnostics and surveillance, and epidemiological and immunity studies). We introduce a direct serological biosensor assay employing proprietary technology based on plasmonics, which offers rapid (<15 min) identification and quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in clinical samples, without signal amplification. The portable plasmonic device employs a custom-designed multiantigen (RBD peptide and N protein) sensor biochip and reaches detection limits in the low ng mL -1 range employing polyclonal antibodies. It has also been implemented employing the WHO-approved anti-SARS-CoV-2 immunoglobulin standard. A clinical validation with COVID-19 positive and negative samples ( n = 120) demonstrates its excellent diagnostic sensitivity (99%) and specificity (100%). This positions our biosensor as an accurate and easy-to-use diagnostics tool for rapid and reliable COVID-19 serology to be employed both at laboratory and decentralized settings for the disease management and for the evaluation of immunological status during vaccination or treatment.

COVID-19 patients elicit strong responses to the nucleocapsid (N) protein of SARS-CoV-2 but binding antibodies are also detected in prepandemic individuals, indicating potential crossreactivity with common cold human coronaviruses (HCoV) and questioning its utility in seroprevalence studies. We investigated the immunogenicity of the full-length and shorter fragments of the SARS-CoV-2 N protein, and the crossreactivity of antibodies with HCoV. We identified a C-terminus region in SARS-CoV2 N of minimal sequence homology with HCoV that was more specific for SARS-CoV-2 and highly immunogenic. IgGs to the full-length SARS-CoV-2 N also recognized N229E N, and IgGs to HKU1 N recognized SARS-CoV-2 N. Crossreactivity with SARS-CoV-2 was stronger for alpha- rather than beta-HCoV despite having less sequence identity, revealing the importance of conformational recognition. Higher preexisting IgG to OC43 N correlated with lower IgG to SARS-CoV-2 N in rRT-PCR negative individuals, reflecting less exposure and indicating a potential protective association. Antibodies to SARS-CoV-2 N were higher in patients with more severe and longer duration of symptoms and in females. IgGs remained stable for at least 3 months, while IgAs and IgMs declined faster. In conclusion, N protein is a primary target of SARS-CoV-2-specific and HCoV crossreactive antibodies, both of which may affect the acquisition of immunity to COVID-19., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Complex microbial communities that reside in the lungs, skin and gut are now appreciated for their role in maintaining organ, tissue and immune homoeostasis. As lungs are currently seen as an ecosystem, the shift in paradigm calls for the consideration of new algorithms related to lung ecology in pulmonology. Evidence of lung microbiota does not solely challenge the traditional physiopathology of ventilator-associated pneumonia (VAP); indeed, it also reinforces the need to include molecular techniques in VAP diagnosis and accelerate the use of immunomodulatory drugs, including corticosteroids, and other supplements such as probiotics for VAP prevention and/or treatment. With that stated, both microbiome and virome, including phageome, can lead to new opportunities in further understanding the relationship between health and dysbiosis in VAP. Previous knowledge may be, however, reconsidered at a microbiome scale., Competing Interests: Declaration of Competing interest A. Torres has received grants from MedImmune, Cubist, Bayer, Theravance, and Polyphor and personal fees as Advisory Board member from Bayer, Roche, The Medicines CO, and Curetis. He has received bureau fees for keynote speaker presentations from GSK, Pfizer, Astra Zeneca, and Biotest Advisory Board, and are unconnected to the study submitted here., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Cytomegalovirus (CMV) is the most important opportunistic pathogen associated with transplant. The objective of this study was the characterization of CMV resistance mutations in allogeneic haematopoietic cell transplant recipients (allo-TPH) and the study of associated factors., Methods: A retrospective study of a cohort of allo-TPH recipients with post-transplant CMV reactivations with stable or increasing viral loads (CV), despite adequate antiviral treatment for at least 2weeks. The study of resistance mutations of the UL97 and UL54 genes was carried out by Sanger sequencing., Results: Refractory CMV infection in our group of allo-TPH patients corresponded with a 21.43% rate of resistant virus infection (3 of 14 patients). All patients with resistance mutations had multiple reactivation episodes (P-value .01). The mutations found were A594V and H520Q in the UL97 gene that confers high-grade resistance to ganciclovir (GCV). One of the 3 cases with antiviral resistance was documented with a low VL (< 1000 copies/ml) and short accumulated GCV treatment (41 days)., Conclusion: Most of the failures in the treatment of CMV were possibly due to clinical resistance; the lack of satisfactory response to antiviral treatment is not always accompanied by virological resistance. However, the appearance of resistances can occur early after the start of the treatment and with VL below 1000 copies / ml. The number of episodes of reactivation was higher among patients with virological resistance than those who did not., (Copyright © 2019 Elsevier España, S.L.U. All rights reserved.)

Background: Among all cases of nosocomial pneumonia, Staphylococcus aureus is the second most prevalent pathogen (17.8%). In Europe, 29.9% of the isolates are oxacillin-resistant. The changing epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) nosocomial infections and the decreasing susceptibility to first-line antibiotics leave clinicians with few therapeutic options. The objective of our study was to determine the antimicrobial susceptibility, the associated molecular mechanisms of resistance and the epidemiological relatedness of MRSA strains isolated from the endotracheal tubes (ETT) of intubated critically ill patients in the intensive care unit (ICU) with nosocomial pneumonia caused by Staphylococcus aureus., Methods: The antimicrobial susceptibility to vancomycin, linezolid, ciprofloxacin, clindamycin, erythromycin, chloramphenicol, fusidic acid, gentamicin, quinupristin-dalfopristin, rifampicin, sulfamethoxazole/trimethoprim, and tetracycline were measured. Resistance mechanisms were then analyzed by polymerase chain reaction and sequencing. Molecular epidemiology was carried out by multi-locus sequence typing., Results: S. aureus isolates were resistant to ciprofloxacin, erythromycin, gentamicin, tetracycline, clindamycin, and fusidic acid. The most frequent mutations in quinolone-resistant S. aureus strains were S84L in the gyrA gene, V511A in the gyrB gene, S144P in the grlA gene, and K401R/E in the grlB gene. Strains resistant to erythromycin carried the ermC, ermA, and msrA genes; the same ermC and ermA genes were detected in strains resistant to clindamycin. The aac(6')-aph(2″) gene was related to gentamicin resistance, while resistance to tetracycline was related to tetK (efflux pump). The fusB gene was detected in the strain resistant to fusidic acid. The most frequent sequence types were ST22, ST8, and ST217, which were distributed in four clonal complexes (CC5, CC22, CC45, and CC59)., Conclusions: High levels of resistance to second-line antimicrobials threatens the treatment of nosocomial respiratory infections due to methicillin-resistant S. aureus with decreased susceptibility to linezolid and vancomycin. The wide genotypic diversity found reinforces the central role of ICU infection control in preventing nosocomial transmission.

Objetives: The aim of this study was to identify CMV drug resistance mutations (DRM) in solid organ transplant (SOT) recipients with suspected resistance comparing next-generation sequencing (NGS) with Sanger sequencing and assessing risk factors and the clinical impact of resistance., Methods: Using Sanger sequencing as the reference method, we prospectively assessed the ability of NGS to detect CMV DRM in the UL97 and UL54 genes in a nationwide observational study from September 2013 to August 2016., Results: Among 44 patients recruited, 14 DRM were detected by Sanger in 12 patients (27%) and 20 DRM were detected by NGS, in 16 (36%). NGS confirmed all the DRM detected by Sanger. The additional six mutations detected by NGS were present in <20% of the sequenced population, being located in the UL97 gene and conferring high-level resistance to ganciclovir. The presence of DRM by NGS was associated with lung transplantation (p = 0.050), the administration of prophylaxis (p = 0.039), a higher mean time between transplantation and suspicion of resistance (p = 0.038) and longer antiviral treatment duration before suspicion (p = 0.024). However, the latter was the only factor independently associated with the presence of DRM by NGS in the multivariate analysis (OR 2.24, 95% CI 1.03 to 4.87)., Conclusions: NGS showed a higher yield than Sanger sequencing for detecting CMV resistance mutations in SOT recipients. The presence of DRM detected by NGS was independently associated with longer antiviral treatment., Competing Interests: The authors have declared that no competing interests exist.

Background: Current guidelines recommend that treatment of resistant cytomegalovirus (CMV) in solid organ transplant (SOT) recipients must be based on genotypic analysis. However, this recommendation is not systematically followed., Objectives: To assess the presence of mutations associated with CMV resistance in SOT recipients with suspected resistance, their associated risk factors and the clinical impact of resistance., Study Design: Using Sanger sequencing we prospectively assessed the presence of resistance mutations in a nation-wide prospective study between September 2013-August 2015., Results: Of 39 patients studied, 9 (23%) showed resistance mutations. All had one mutation in the UL 97 gene and two also had one mutation in the UL54 gene. Resistance mutations were more frequent in lung transplant recipients (44% p=0.0068) and in patients receiving prophylaxis ≥6 months (57% vs. 17%, p=0.0180). The mean time between transplantation and suspicion of resistance was longer in patients with mutations (239 vs. 100days, respectively, p=0.0046) as was the median treatment duration before suspicion (45 vs. 16days, p=0.0081). There were no significant differences according to the treatment strategies or the mean CMV load at the time of suspicion. Of note, resistance-associated mutations appeared in one patient during CMV prophylaxis and also in a seropositive organ recipient. Incomplete suppression of CMV was more frequent in patients with confirmed resistance., Conclusions: Our study confirms the need to assess CMV resistance mutations in any patient with criteria of suspected clinical resistance. Early confirmation of the presence of resistance mutations is essential to optimize the management of these patients., (Copyright © 2017 Elsevier B.V. All rights reserved.)