Moraxella spp from the nasal microbiota of piglets showed heterogeneous characteristics, from antimicrobial resistance profiles to virulent mechanisms and adherence properties. The majority of the isolates belonged to M. plurianimalium species, but we also detected M. porci, a couple of isolates of uncertain classification and a cluster of strains that may constitute a new Moraxella species. This cluster comprised strains with a slower growth rate in broth than the rest of the nasal strains and contained most of the strains with high resistance to the serum complement.
Our results on antimicrobial resistance confirm that the administration of these drugs can promote a rise of resistances, since higher rates of antibiotic resistance were found in strains from farms undergoing antibiotic treatments. MDR strains were broadly detected, and only one strain, UK1–20, from a farm where no antibiotics were used, did not show resistance to any of the antimicrobials tested. It is important to highlight the presence of four MDR strains of Moraxella pluranimalium, with a high number of drug resistances, including colistin. This latter observation is consistent with the recent discovery of colistin resistance genes in Moraxella [15–17]. Coding genes of antibiotic resistances can be horizontally transferred between bacteria of the same or even different species, and therefore, the presence of resistance genes in the resident microbiota is of concern since they could be transferred to potential pathogens [18]. Diversity of the nasal Moraxella isolates was also evident in the in vitro assays, where strains showed a wide heterogeneity.
Biofilms are traditionally considered forms of resistance to environmental conditions and a virulence mechanism in bacteria of clinical importance [19]. As for other characteristics, we observed heterogeneity in biofilm formation capacity of the porcine nasal strains of Moraxella. Variability in biofilm formation has been already reported for other species of Moraxella from ruminants [20]. Biofilm formation capacity can be associated also to the colonizing ability of the bacteria, as was observed for H. parasuis [21]. The interaction of the nasal Moraxella strains with the respiratory mucosa needs more study, but attachment to mucin as wells as to epithelial cells could play a role, as suggested by our results.
On the other hand, bacteria from the upper respiratory tract can occasionally reach the lower tract, where they will be confronted with other components of the immune system, such as the alveolar macrophages. In vitro phagocytosis assays provided insight into the potential of the nasal isolates to survive this first barrier of cell immunity in the lung. Heterogeneity in this feature has been shown for other bacterial species, such as H. parasuis [14] or Klebsiella pneumoniae [22]. In our case, strains with high association to PAMs, such as LG6–2, were observed, while other strains did not associate with the phagocytic cells, as the case of the virulent SN9–4M. According to the results, phagocytosis susceptibility is not associated with the phylogeny of the isolates, since heterogeneity in this trait was observed within the distinct species or clusters. One paradigmatic case could be EJ45–1 and SN9–4M from M. porci, having high differences between them. The importance of phagocytosis resistance for in vivo infection/colonization by M. pluranimalium is also supported by the fact that originally the UK1–20 strain showed resistance to PAMs, but lost this trait after in vitro culture passages, in agreement with the loss of virulence after laboratory adaptation in other bacteria [23]. Loss of phagocytosis resistance in UK1–20 was concurrent with increase biofilm formation capacity. Thus, biofilm formation may be relevant in environmental persistence, whereas phagocytosis resistance will be important during host infection. However, this phenomenon seems to be strain-dependent, since it was not observed with strain VL6–6, which did not show an adaptation to laboratory conditions that affected its original resistance to PAMs.
Most of the assayed Moraxella spp.strains showed sensitivity to the serum complement, with some exceptions, including most of the strains in the cluster representing a putative new species. Thus, most nasal Moraxella strains are probably poorly invasive and will be kept on the nasal mucosa, but the new putative species cluster deserves more attention to define their role in disease in pigs.