Thesis defense Abel Vlasblom

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The widespread occurrence of livestock-associated Staphylococcus aureus (LA-SA), including its methicillin-resistant variants (LA-MRSA), within pig farming poses a zoonotic risk to public health.  The primary aim of this thesis was to identify bacterial species that exhibit a negative association with LA-SA and to isolate them from the nasal microbiome of healthy pigs. The goal was to utilize them to reduce the prevalence of LA-SA colonization in the nasal cavities of piglets, potentially through mechanisms of competitive exclusion. Given that the porcine nasal microbiome remains an understudied ecological niche, the thesis starts with a comprehensive analysis of the development and composition of the piglet nasal microbiome, while also identifying promising probiotic candidates. This investigation ultimately identified Lactococcus lactis as a suitable candidate for intervention, potentially capable of reducing S. aureus colonization through intranasal application.

Chapters 2 and 3 focus on elucidating the species distribution in the piglet nasal microbiome, which appears to be shaped by factors such as time and farm-specific conditions. Stabilization of the microbial community was observed post-weaning, marked by the consistent presence of core bacterial genera such as Moraxella and Streptococcus. The development of the nasal microbiome is likely influenced by a complex interplay of factors, including the potential introduction of gut-associated microbes shortly after birth, maternal microbial transfer, host factors and farm management practices, all of which collectively contribute to shaping microbial diversity. To further investigate interspecies interactions within the nasal microbiome, co-abundance groups (CAGs) were identified to examine the underlying complexity of microbial relationships. These CAGs may display both competitive and cooperative dynamics, these relationships among species potentially play a role in suppressing pathogens. These findings highlight the potential for microbiome-targeted approaches, focusing on specific CAGs, as a strategy to reduce respiratory infections. However, it is important to note that the aforementioned factors, such as specific farm conditions, may act as confounding variables, complicating the identification of biologically meaningful microbial interactions.

The microbiome analysis combined with quantitative S. aureus qPCR estimates allowed us to identify 54 species with a negative association with S. aureus (potential competitive species). This list of bacterial species was screened in literature and suitable isolates were subsequently targeted in an isolation effort (Chapter 4). The isolation effort and safety assessment consisted of selecting species specific growth conditions, isolate identification using MALDI-TOF and 16S rDNA sequencing, isolate phenotyping, and whole genome sequencing to profile antimicrobial resistance genes and virulence determinants, followed by in vitro competition assays with S. aureus.

This led to isolation of three L. lactis strains as candidates with probiotic potential. The L. lactis bacterium holds a Generally Recognized as Safe (GRAS) and Qualified Presumption of Safety (QPS) status. Preliminary testing and literature investigation highlighted its potential to inhibit S. aureus via nutrient competition or acidification. However, our subsequent experimental applications in newborn piglets at six European farms failed to reduce colonization of LA-SA effectively (Chapter 7). This failure might be attributed to poor colonization of the L. lactis strains in the nasal cavity. Because we did not observe successful colonization of the nasal microbiome by L. lactis. This was likely due to environmental factors, microbial competition, the timing of administration, and the inefficiency of delivery methods. Proposed solutions, such as repeated dosing or environmental dispersal, require further exploration but the large number of variables to explore was outside of the scope of this thesis.

In a small-scale safety assessment (Chapter 5) we also observed immunomodulatory effects of L. lactis, which reduced IL1B and increased pBD2 and TLR9 expression in nasal tissues. The altered expression of inflammatory cytokine, pathogen recognition receptor and antimicrobial peptides suggests that the probiotic cocktail has immunomodulatory potential. This immunomodulatory potential may prove beneficial in its use to reduce MRSA carriage in pigs. Further studies are required to demonstrate the MRSA reduction efficacy of the probiotic cocktail through immunomodulation.

In this study electrostatic dust collectors (EDCs) were used as an approach for monitoring airborne S. aureus transmission. EDCs proved to be a cost-effective, reliable alternative to active air sampling, offering advantages such as affordability and the ability to monitor long-term changes in airborne LA-MRSA levels (Chapter 6). Their effectiveness in reflecting average airborne contamination without being particle size-selective supports their use in pathogen surveillance. This method could enhance understanding of pathogen spread and contribute to predictive models, aiding in the development of intervention strategies to improve livestock health.

In conclusion, the findings presented in this thesis highlight the intricate dynamics of the piglet nasal microbiome and point to the interplay of environmental, temporal, and microbial factors. The results emphasize the potential of microbiome-based strategies for excluding pathogenic species, such as the use of probiotics like Lactococcus lactis, while acknowledging the need for further optimization of porcine nasal probiotic delivery methods. Additionally, tools such as EDCs provide practical solutions for monitoring pathogen transmission. This work contributes to broader efforts to combat antimicrobial resistance by exploring approaches that could reduce reliance on antimicrobials in pig farming. These findings offer valuable insights to guide future research aimed at improving livestock or public health through innovative microbiome-based interventions.