Isolation and bacteria cultures
Nasal cavities from 10 piglets of around 3 weeks of age from 5 different litters were sampled from 8 commercial farms (Table 1) with standard management practices for husbandry and welfare of the animals. Sampling of piglets was done under institutional authorization and followed good veterinary practices. According to European (Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes) and Spanish (Real Decreto 53/2013) normative, this procedure did not require specific approval by an Ethical Committee. Nasal sampling was performed only once to each piglet and is not likely to cause pain, suffering, distress or lasting harm equivalent to, or higher than, that caused by the introduction of a needle in accordance with good veterinary practice (Chapter I, Article 1, 5 (f) of 2010/63/EU). Nasal swabs were transported in Amies medium to the laboratory, where they were plated on chocolate agar (Biomérieux, Marcy l’Étoile, France) to isolate colonies. After 24 and 48 h of incubation at 37 °C and 5% CO2, different colonies were selected and stored at − 80 °C in 20% glycerol-Brain Heart Infusion broth (BHI) for further characterization. Bacterial suspensions were also performed in phosphate-buffered saline (PBS) for DNA extraction.
In addition, reference strains of Moraxella porci SN9-4 M and Moraxella pluranimalium CD12-CA4, both isolated from lesions of diseased pigs, were used in this study. For assays with PAMs, 2 reference strains of Haemophilus parasuis were used as controls.
Bacterial identification and genotyping
DNA extraction was performed using Chelex based Instagene™ Matrix (Bio-Rad Laboratories, Hercules, CA, USA) following manufacturer’s instructions. Preliminary identification of isolates was performed by partial sequencing of the 16S rRNA gene using primers 358F (CTACGGGAGGCAGCAGT) and 907R (CCGTCWATTCMTTTGAGTTT) . Sequences were analyzed by blasting against the Ribosomal database (http://rdp.cme.msu.edu).
All isolates identified as Moraxella were then genotyped by ERIC-PCR  with primers ERIC-1F (ATGTAAGCTCCTGGGGATTCAC) and ERIC-2R (AAGTAAGTGACTGGGGTGAGCG). PCR reaction mixture consisted of 3 mM of MgCl2, 1.2 μM of each primer, 0.23 mM of dNTPs, 0.75 U of GoTaq® polymerase (Promega, Madison Wisconsin, USA) and 100 ng of DNA sample. Amplification was carried out with an initial denaturation of 94 °C for 2 min followed by 30 cycles of 30 s at 94 °C, 1 min at 50 °C and 2.5 min at 72 °C, and a final extension of 20 min at 72 °C. One isolate from each fingerprinting profile was selected for further analysis.
Final identification of the different Moraxella strains was performed by 16S rRNA gene amplification with universal primers 8F (AGAGTTTGATCCTGGCTCAG) and 1492R (CGGTTACCTTGTTACGACTT)  and sequencing with 8F, 1492R, 358F and 907R primers. Sequence analysis was performed with Fingerprinting II v3.0 software (Bio-Rad) and a UPGMA dendrogram was built for species assignment, including sequences from different Moraxella spp. type strains. Biochemical characterization of representative isolates was performed using the Vitek 2 system (Biomerieux).
Susceptibility to several antimicrobials was tested as previously described . Neo-Sensitabs™ diffusion tablets (Rosco Diagnostica, Taastrup, Denmark) were used for Gentamicin, Ceftiofur, Colistin, Erythromycin, Lincoespectin, Tetracycline, Doxycycline, Trimetoprim + Sulfamide (T + S), Enrofloxacin, Amoxicillin and Amoxicillin + Clavulanic acid testing. Marbofloxacin, florfenicol and tulathromycin were tested in chocolate agar plates at different dilutions, from 16 to 0.125 μg/mL in the case of florfenicol and marbofloxacin; and from 64 to 0.125 μg/mL in the case of tulathromycin. McFarland suspensions of 0.5 in 0.9% NaCl were prepared for each strain and streaked on the plates with the different antibiotic concentration. Minimal inhibitory concentration (MIC) was determined observing the bacterial growth after 24 h of incubation. As no clinical breakpoints are available for these species, available CLSI breakpoints for Moraxella catarrhalis were taken for Tetracycline, Doxycycline, Trimetoprim+Sulfonamide, and veterinary practice according to CLSI breakpoints was followed for the rest of drugs (EUCAST-and CLSI potency NEO-SENSITABS, Rosco Diagnostica, 2013).
Serum resistance assay
Serum resistance assay was carried out with rabbit serum (EU Directive 2010/63/EU and Spanish normative Real Decreto 53/2013 were followed). A bacterial suspension of each strain (representative of each ERIC fingerprinting) was prepared in PBS with 20% glycerol to reach an OD600 of 0.3 in a VIS 7200 spectrophotometer (Dinko Instruments, Barcelona, Spain). In duplicate wells, 10 μl of the bacterial suspension (approx. 106 colony forming units [CFU]/mL) were mixed with 90 ul of fresh filtered rabbit serum and mixtures were incubated for 1 h at 37 °C and 100 rpm. Bacterial survival was calculated by comparing bacterial counts (obtained by serial dilutions and plating) at time 0 and after 1 h incubation. The assay was carried out 3 times for each strain.
Association to porcine alveolar macrophages
Association to macrophages was tested as previously described with PAMs . PAMs were obtained from healthy piglets euthanized by intravenous pentobarbital overdose under institutional authorization. All procedures involving animals followed EU and Spanish normative (Directive 2010/63/EU and Real Decreto 53/2013). Lungs were removed and PAMs were isolated by lung lavage with PBS containing 70 μg/mL of gentamicin. PAMs were collected by centrifugation at 241 x g for 15 min, washed twice with sterile PBS and stored at − 150 °C in 10% Dimethylsulfoxide (DMSO) in fetal bovine serum (FBS) until use.
Selected nasal strains of Moraxella were tested to determine their level of association to PAMs. Moraxella porci reference strain SN9-4 M was also included in the assay. In addition of two reference strains of Haemophilus parasuis, Nagasaki (virulent) and SW-114 (non-virulent), were included as control for the technique.
For the assay, PAMs were plated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% of FBS and 1% glutamine at a concentration of 5 × 105 cells per well in 6 well-plates. Plates were incubated 1–2 h to allow the attachment of the macrophages to the bottom of the wells. For each strain duplicate wells were inoculated with 107 CFU of fluorescein isothiocyanate (FITC)-labelled bacteria (bacterial inocula were confirmed by dilutions and plating). After 1 h of incubation at 37 °C and 5% CO2, plates were transferred to an ice bath to stop interaction and were washed twice with PBS to eliminate unbound bacteria. PAMs were then scrapped in PBS and were analyzed by flow cytometry using an EPICS XL-MCLTM Flow Cytometer (Beckman Coulter, Madrid, Spain) or MACSQuant Analyzer 10 (Myltenyi Biotec, Bergisch Gladbach, Germany). Assays were repeated with PAMs from different animals that were already available at the laboratory. Since each batch of macrophages showed different level of activity, values were harmonized considering the reference H. parasuis strain SW114 (phagocytosis sensitive) as 100% phagocytosis in each assay; i.e., using the following calculation: (% PAMs with test bacteria - % fluorescence background in PAMs without bacteria / % PAMs with positive control SW114 - % fluorescence background in PAMs without bacteria) × 100.
Biofilm assays were performed in 96 well cell culture plates under static and shaking conditions following previous published protocol with some modifications (Bello-Ortí et al. 2014). Bacterial suspensions were made to reach an OD600 0.3 in BHI. Wells in 96 well plates were then inoculated in quadruplicate with a 1:100 dilution of the bacterial suspension in BHI. Plates were incubated at 37 °C, under static conditions with 5% CO2 or under agitation at 100 rpm for 24 and 48 h, except for slow growing isolates, which were incubated for 48 and 72 h. After incubation, wells were emptied and rinsed with tap water to remove unattached bacteria. Wells were then stained with 0.1% (w/v) crystal violet (Merck, Darmstadt, Germany) for 2 min at room temperature. Wells were washed thrice with tap water to remove the excess of crystal violet and let dried at 37 °C. The dye in the stained biofilms was solubilized with 100 μL of 70% ethanol and quantified at 590 nm in a Powerwave XS Microplate Spectrophotometer (Biotek Instruments Inc., Winooski, VT, USA). Biofilm formation was also assessed on wells pre-coated with 5 μg of mucin or BSA as control.
Cell adhesion assay
Porcine epithelial cells PK-15 (ATCC® CCL-33) and human lung A549 (ATCC® CCL-185) cell lines were cultured in DMEM supplemented with 1% glutamine and 5% FBS for PK-15 and 10% for A549 cells.
Concentration of cells per ml was calculated to obtain a monolayer after overnight incubation, approximately 50,000 cells/well in cell culture 96 well plates. After overnight incubation, wells with PK-15 or A549 cells were washed once with sterile PBS. Duplicate wells were inoculated with 107 CFU/mL bacteria from each isolate tested. Bacterial inoculum quantification was confirmed by colony counts. Microplates were then centrifuged 10 min at 100 x g to facilitate the contact between bacteria and cells. After incubation for 1 h at 37 °C and 5% CO2, wells were washed twice with sterile PBS to remove unattached bacteria, and attached bacteria were released with 0.1% of saponin and pipetting. The resulting suspension was quantified by plating dilutions on agar plates.