The Indoor-Air Microbiota of Pig Farms Drives the Composition of the Pig Farmers' Nasal Microbiota in a Season-Dependent and Farm-Specific Manner.

Prior studies have demonstrated an influence of the built environment on the human nasal microbiota. However, very little is known about the influences of working on a pig farm on the human nasal microbiota. We longitudinally collected samples from 30 pig farms (air and nasal swabs from humans and pigs) in Switzerland from 2014 to 2015. As controls, nasal swabs from cow farmers and individuals with no contact with farm animals were included. An analysis of the microbiota for all samples (n = 609) was performed based on 16S rRNA gene sequencing (MiSeq) and included the investigations of source-sink dynamics. The numbers of indoor airborne particles and bacterial loads in pig farms were also investigated and were highest in winter. Similarly, the microbiota analyses revealed that the alpha diversity values of the nares of pig farmers were increased in winter in contrast to those of samples from the nonexposed controls, which displayed low alpha diversity values throughout the seasons. Source-sink analyses revealed that bacteria from the noses of pigs are more commonly coidentified within the pig farmers' microbiota in winter but to a less extent in summer. In addition, in winter, there was a stronger intrasimilarity for samples that originated from the same farm than for samples from different farms, and this farm specificity was partially or completely lost in spring, summer, and fall. In conclusion, in contrast to nonexposed controls, a pig farmer's nasal microbiota is dynamic, as the indoor-air microbiota of pig farms drives the composition of the pig farmer's nasal microbiota in a season-dependent manner.IMPORTANCE The airborne microbiota of pig farms poses a potential health hazard and impacts both livestock and humans working in this environment. Therefore, a more thorough understanding of the microbiota composition and dynamics in this setting is needed. This study was of a prospective design (12 months) and used samples from different sites. This means that the microbiota of air, animals (pigs), and humans was simultaneously investigated. Our findings highlight that the potential health hazard might be particularly high in winter compared to that in summer.

Influence of Pig Farming on the Human Nasal Microbiota: Key Role of Airborne Microbial Communities

It has been hypothesized that the environment can influence the composition of the nasal microbiota. However, the direct influence of pig farming on the anterior and posterior nasal microbiota is unknown. Using a cross-sectional design, pig farms (n = 28) were visited in 2014 to 2015, and nasal swabs from 43 pig farmers and 56 pigs, as well as 27 air samples taken in the vicinity of the pig enclosures, were collected. As controls, nasal swabs from 17 cow farmers and 26 non-animal-exposed individuals were also included. Analyses of the microbiota were performed based on 16S rRNA amplicon sequencing and the DADA2 pipeline to define sequence variants (SVs). We found that pig farming is strongly associated with specific microbial signatures (including alpha- and beta-diversity), which are reflected in the microbiota of the human nose. Furthermore, the microbial communities were more similar within the same farm compared to between the different farms, indicating a specific microbiota pattern for each pig farm. In total, there were 82 SVs that occurred significantly more abundantly in samples from pig farms than from cow farmers and nonexposed individuals (i.e., the core pig farm microbiota). Of these, nine SVs were significantly associated with the posterior part of the human nose. The results strongly indicate that pig farming is associated with a distinct human nose microbiota. Finally, the community structures derived by the DADA2 pipeline showed an excellent agreement with the outputs of the mothur pipeline which was revealed by procrustes analyses. IMPORTANCE The knowledge about the influence of animal keeping on the human microbiome is important. Previous research has shown that pets significantly affect the microbial communities of humans. However, the effect of animal farming on the human microbiota is less clear, although it is known that the air at farms and, in particular, at pig farms is charged with large amounts of dust, bacteria, and fungi. In this study, we simultaneously investigated the nasal microbiota of pigs, humans, and the environment at pig farms. We reveal an enormous impact of pig farming on the human nasal microbiota which is far more pronounced compared to cow farming. In addition, we analyzed the airborne microbiota and found significant associations suggesting an animal-human transmission of the microbiota within pig farms. We also reveal that microbial patterns are farm specific, suggesting that the environment influences animals and humans in a similar manner.

Detection of pathogenic bacteria using a homogeneous immunoassay based on shear alignment of virus particles and linear dichroism.

Biomolecular detection has for a long time depended on a relatively small number of established methodologies. Many of these depend on the detection of a ligand-antibody complex using some kind of optical technique, e.g., chemiluminescence. Before this measurement can be made, the ligand-antibody complex generally has to be separated from bulk contaminants. This process involves the implementation of a heterogeneous assay format involving immobilization of the antibody onto a solid support to enable washing of the unbound ligand. This approach has a number of inherent issues including being slow and complex and requiring the use of expensive reagents. In this paper, we demonstrate how we have harnessed a biologically inspired nanoparticle to provide the basis for a homogeneous assay which requires no immobilization. The method relies on using fluid shear flow to align a fiber-like nanoparticle formed from a filamentous virus, M13, combined with a ligand-specific antibody. This renders the particle visible to linear dichroic spectroscopy, which provides an easily interpretable signal. The addition of the target ligand (in this case Escherichia coli O157) leads to the formation of a nanoparticle-ligand particle that is unable to align, leading to the perturbation of the linear dichroism signal.

Combined expression of KLK4, KLK5, KLK6, and KLK7 by ovarian cancer cells leads to decreased adhesion and paclitaxel-induced chemoresistance.

OBJECTIVE: Chemoresistance is a critical feature of advanced ovarian cancer with only 30% of patients surviving longer than 5 years. We have previously shown that four kallikrein-related (KLK) peptidases, KLK4, KLK5, KLK6 and KLK7 (KLK4-7), are implicated in peritoneal invasion and tumour growth, but underlying mechanisms were not identified. We also reported that KLK7 overexpression confers chemoresistance to paclitaxel, and cell survival via integrins. In this study, we further explored the functional consequenses of overexpression of all four KLKs (KLK4-7) simultaneously in the ovarian cancer cell line, OV-MZ-6, and its impact on integrin expression and signalling, cell adhesion and survival as contributors to chemoresistance and metastatic progression. METHODS: Quantitative gene and protein expression analyses, confocal microscopy, cell adhesion and chemosensitivity assays were performed. RESULTS: Expression of α5ß1/αvß3 integrins was downregulated upon combined stable KLK4-7 overexpression in OV-MZ-6 cells. Accordingly, the adhesion of these cells to vitronectin and fibronectin, the extracellular matrix binding proteins of α5ß1/αvß3 integrins and two predominant proteins of the peritoneal matrix, was decreased. KLK4-7-transfected cells were more resistant to paclitaxel (10-100 nmol/L: 38-54%), but not to carboplatin, which was associated with decreased apoptotic stimuli. However, the KLK4-7-induced paclitaxel resistance was not blocked by the MEK1/2 inhibitor, U0126. CONCLUSIONS: This study demonstrates that combined KLK4-7 expression by ovarian cancer cells promotes reduced integrin expression with consequently less cell-matrix attachment, and insensitivity to paclitaxel mediated by complex integrin and MAPK independent interactions, indicative of a malignant phenotype and disease progression suggesting a role for these KLKs in this process.

Occupational Exposure to ß-d-Glucans, Mould Allergens, Endotoxins and Cultivable Fungi in Pig Farms.

Airborne concentrations of organic dust on animal farms are known to be very high. This dust is partly composed of microorganisms such as bacteria, fungi and their components [endotoxins, (1→3)-ß-d-glucans, mould allergens, mycotoxins], recognised as being responsible for numerous health effects. Several cross-sectional studies have measured levels of airborne bacteria, fungi and endotoxins on pig farms. However, the temporal dynamics of organic dust's components throughout the year have rarely been assessed, and airborne concentrations of (1→3)-ß-d-glucans and mould allergens remain poorly understood in these work environments. This longitudinal, four-season study measured cultivable fungi, endotoxins, (1→3)-ß-d-glucans, Aspergillus versicolor (AveX), Aspergillus fumigatus (Asp f1) and Alternaria sp (Alt a1) allergens on 31 pig farms in Switzerland. Results showed that exposure to AveX occurred in all four seasons. Total mean airborne concentration of endotoxins were between 3 and 4 times higher than the Swiss recommended limit value of 1000 EU m-3 and mean airborne concentrations of fungi were between 30 and 50 times higher than the Swiss recommended limit value of 1000 cfu m-3. Finally, accumulations of faecal matter on floors, humidity and dusty pathways were associated with increased concentrations of (1→3)-ß-d-glucans. In conclusion, pig farmers require better information about biological occupational risks, and measures to improve air quality should be implemented, especially in winter.

Design and performance characteristics of the Elecsys anti-SARS-CoV-2 S assay.

Background: Automated, high throughput assays are required to quantify the immune response after infection with or vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study on the Roche Elecsys® Anti-SARS-CoV-2 S (ACOV2S) assay provides insights on the assay design and performance. Methods: The ACOV2S assay quantifies antibodies to the receptor-binding domain of the SARS-CoV-2 spike protein. The assigned units and the underlying standardization were compared to the international reference standard in BAU/mL. Assay specificity was assessed in samples (n=5981) collected prior to the COVID-19 pandemic and in samples from patients with non-COVID-19 respiratory infections (n=697) or other infectious diseases (n=771). Sensitivity was measured in 1313 samples from patients with mild COVID-19 and 297 samples from patients hospitalized with COVID-19. Comparison of results was performed to a comparator semi-quantitative anti-S1 assay of indirect detection format as well as a commercially available and an in-house version of a surrogate neutralization assay (ACE2-RBD). Results: The originally assigned units for the ACOV2S assay were shown to be congruent to the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins. Overall specificity was 99.98% with no geographical differences noted and no loss of specificity in samples containing potentially cross-reacting antibodies. High sensitivity was observed, with 98.8% of samples reported to be reactive >14 days after infection and sustained detection of antibodies over time. For all samples, ACOV2S titers and neutralization capacities developed with comparable dynamics. Robust standardization and assay setup enable excellent reproducibility of results, independent of lot or analyzer used. Conclusion: The results from this study confirmed that ACOV2S is a highly sensitive and specific assay and correlates well with surrogate neutralization assays. The units established for ACOV2S are also interchangeable with the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins. Worldwide availability of the assay and analyzers render ACOV2S a highly practical tool for population-wide assessment and monitoring of the humoral response to SARS-CoV-2 infection or vaccination.

Nasal microbiota composition dynamics after occupational change in animal farmers suggest major shifts.

Previous studies have suggested a significantly higher diversity in the nasal microbiota of pig farmers compared to people having no contact with farm animals. However, the fate of this nasal microbiota specificity after farmers stop being in contact with the pig farm environment is unknown. The aim of this study was to investigate the change in the nasal microbiota of pig-farmers after the change of occupation. METHODS: Anterior and posterior nasal swabs were collected from seven people during employment on pig farms, and again after a period of at least 50 days after leaving the pig farm. Illumina MiSeq sequencing of 16S rRNA was conducted to characterize the dynamics of the nasal microbiota. The microbiota of actively working pig farmers was compared to microbiota after they had stopped working (ex-pig-farmers) and to control groups (cow farmers and non-exposed individuals). RESULTS: Following a prolonged period without exposure to pigs, α-diversity of both anterior and posterior cavities dropped significantly. The composition of the microbiota of pig-farmers had a low inter-similarity with the non-exposed group while ex-pig-farmers were more similar to cow-farmers and the non-exposed group than to their own microbiota during pig farming.

Age-Dependent Dissimilarity of the Nasopharyngeal and Middle Ear Microbiota in Children With Acute Otitis Media.

Acute bacterial otitis media is usually caused by otopathogens ascending to the middle ear from the nasopharynx (NP). However, it is unknown if the nasopharyngeal microbiota of children with acute otitis media (AOM) can serve as an age-dependent or independent proxy for the microbial communities of the middle ear fluid (MEF) as there is a lack of 16S rRNA amplicon sequencing studies simultaneously analyzing the microbial communities of the two sites. Within this study, we performed 16S rRNA next generation sequencing on a total of 286 nasopharyngeal swabs (NPSs) collected between 2004 and 2013 within a Swiss national AOM surveillance program from children (0-6 years) with AOM. In addition, 42/286 children had spontaneous tympanic membrane perforation and, therefore, those MEF could also be analyzed. We found that alpha [Richness, Shannon diversity index (SDI) and Evenness] and beta diversity measurements of the nasopharyngeal bacterial microbiota showed a clear dependency of the increasing age of the children. In more detail, bacterial richness and personalized profiles (measured by beta dispersion) were higher and more frequent in older children, respectively. Dissimilarity values based on the binary distance matrix of the microbiota patterns of the NP and the MEF also correlated with increasing age. In general, positive (PPV) and negative predictive values (NPV) of the most abundant operational taxonomic units (OTUs) in the NP were moderately and well predictive for their presence in the MEF, respectively. This data is crucial to better understand polymicrobial infections and therefore AOM pathogenesis.

Patterns in the longitudinal oropharyngeal microbiome evolution related to ventilator-associated pneumonia.

Background: The aim of the study was to evaluate the composition and the temporal evolution of the oropharyngeal microbiome in antibiotic-naïve patients requiring mechanical ventilation and to gain new insights into the pathogenesis of ventilator-associated pneumonia (VAP). Methods: Prospective, observational single-center nested case-control study. Patients with acute critical illness and anticipated duration of mechanical ventilation > 4 days were eligible. We took oropharyngeal swabs (and if available, tracheal secretions) daily, starting at the day of intubation. The microbiota was characterized by 16S rRNA high-throughput sequencing and compared between patients developing VAP versus controls. Results: Five patients developed VAP. In three patient the causative pathogens were Enterobacteriaceae and in two Haemophilus influenzae. Locally weighted polynomial regression suggested that the within diversity (=alpha) was lower in Enterobacteriaceae VAP patients between days two to five of mechanical ventilation when compared to controls. Detection of Enterobacteriaceae in the oropharynx occurred on day two of follow-up and consisted of a single operational taxonomic unit in 2/3 patients with enterobacterial VAP. Conclusions: In acutely-ill patients who developed enterobacterial VAP the causative pathogen gained access to the oropharynx early after starting mechanical ventilation and outgrew the commensal members of the microbiome. Whether a specific pattern of the oropharyngeal microbiome between days three to five of mechanical ventilation may predict VAP enterobacterial VAP has to be evaluated in further studies.

Gut microbiota dynamics in travelers returning from India colonized with extended-spectrum cephalosporin-resistant Enterobacteriaceae: A longitudinal study.

BACKGROUND: Intestinal colonization by extended-spectrum cephalosporin-resistant Enterobacteriaceae (ESC-R-Ent) has been attributed to travel to high prevalence countries. However, the dynamics of the microbiota changes during ESC-R-Ent colonization and whether there is a particular bacterial composition which is associated with subsequent colonization is unknown. METHODS: Forty healthy volunteers living in Switzerland underwent screening before and after a trip to India, and also 3, 6 and 12 months after traveling. Culture-based ESC-R-Ent screening and microbiota analysis based on 16S rRNA amplicon sequencing were performed at all time points. RESULTS: Prevalence of ESC-R-Ent colonization before traveling was 10% (n = 4), whereas it increased to 76% (n = 31) after the trip. Based on bacterial diversity analyses of the gut microbiota, there were few but significant differences for colonized versus non-colonized individuals. However, an alternative, cluster based analysis revealed that individuals remained in the same cluster over time indicating that neither traveling nor ESC-R-Ent colonization significantly influences bacterial composition. Moreover, none of the found microbiota clusters were significantly associated with subsequent risk of ESC-R-Ent colonization. CONCLUSION: Based on their microbiota patterns, every volunteer was at the same risk of ESC-R-Ent colonization while traveling to India. Therefore, other risk factors for ESC-R-Ent colonization are responsible for this phenomenon.

Nasal Resistome Development in Infants With Cystic Fibrosis in the First Year of Life.

Polymicrobial infections of the respiratory tract due to antibiotic resistant bacteria are a great concern in patients with cystic fibrosis (CF). We therefore aimed at establishing a functional metagenomic method to analyze the nasal resistome in infants with CF within the first year of life. We included samples from patients before antibiotic treatment, which allowed obtaining information regarding natural status of the resistome. In total, we analyzed 130 nasal swabs from 26 infants with CF and screened for ß-lactams (ampicillin, amoxicillin-clavulanic acid, and cefuroxime) and other classes of antibiotic resistances (tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole). For 69 swabs (53% of total), we found at least one non-susceptible phenotype. Analyses of the inserts recovered from non-susceptible clones by nanopore MinION sequencing revealed a large reservoir of resistance genes including mobile elements within the antibiotic naïve samples. Comparing the data of the resistome with the microbiota composition showed that the bacterial phyla and operational taxonomic units (OTUs) of the microbiota rather than the antibiotic treatment were associated with the majority of non-susceptible phenotypes in the resistome. Future studies will reveal if characterization of the resistome can help in clinical decision-making in patients with CF.

Prevalence of extended-spectrum ß-lactamase-producing Enterobacteriaceae and Methicillin-Resistant Staphylococcus aureus in pig farms in Switzerland.

The presence of Methicillin-Resistant Staphylococcus aureus (MRSA) in pig farms has been widely reported, and the emergence of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) has been documented in several countries. However, data for Switzerland are very scarce. This study aimed to compare changes in the prevalence of MRSA in Swiss pig farms between 2008 and 2015 and make the first ever estimates of the presence of ESBL-E and carbapenemase producers in pigs and pig farm workers. Results showed that ESBL-E was present in both pigs and farm workers and that the proportion of farms with MRSA had increased fourfold in seven years (from 7% to 31%). Associations between antibiotic use and resistant bacteria carriage were shown.

The promotion of osteoclastogenesis by sulfated hyaluronan through interference with osteoprotegerin and receptor activator of NF-κB ligand/osteoprotegerin complex formation.

In order to improve bone regeneration, in particular in aged and multimorbid patients, the development of new adaptive biomaterials and their characterization in terms of their impact on bone biology is warranted. Glycosaminoglycans (GAGs) such as hyaluronan (HA) are major extracellular matrix (ECM) components in bone and may display osteogenic properties that are potentially useful for biomaterial coatings. Using native and synthetically derived sulfate-modified HA, we evaluated how GAG sulfation modulates the activity of two main regulators of osteoclast function: receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). GAGs were tested for their capability to bind to OPG and RANKL using surface plasmon resonance (SPR), ELISA and molecular modeling techniques. Results were validated in an in vitro model of osteoclastogenesis. Sulfated GAGs bound OPG but not RANKL in a sulfate-dependent manner. Furthermore, OPG pre-incubated with different GAGs displayed a sulfate- and dose-dependent loss in bioactivity, possibly due to competition of GAGs for the RANKL/OPG binding site revealing a potential GAG interaction site at the RANKL/OPG interface. In conclusion, high-sulfated GAGs might significantly control osteoclastogenesis via interference with the physiological RANKL/OPG complex formation. Whether these properties can be utilized to improve bone regeneration and fracture healing needs to be validated in vivo.