Characterization of bovine nasopharyngeal lactic acid bacteria and their in vitro antimicrobial activities against the respiratory pathogen Mannheimia haemolytica

Abstract Text: Most research on bacteria in the bovine nasopharynx has focussed on pathogens implicated in respiratory disease. There is limited information on commensals, such as lactic acid bacteria (LAB), which are important to mucosal health and have been investigated as probiotics to inhibit pathogens. The purpose of this study was to characterize the bovine nasopharyngeal LAB and their <i>in vitro</i> antimicrobial activities against <i>Mannheimia haemolytica</i>.  The diversity of nasopharyngeal LAB was investigated in two separate studies using DNA– or culture–based techniques.  In the first study, nasopharyngeal samples were collected from calves (n = 14) on a farm prior to shipment to a feedlot (d 0), and then 2, 7 and 14 days after feedlot placement. Swabs were processed for DNA extraction and the 16S rRNA gene was PCR-amplified and sequenced using the MiSeq platform.  In the second study, nasopharyngeal swabs were collected from calves (n=70) sampled at feedlot entry and 60d afterwards. The swabs were processed for the isolation of LAB using selective media. A subset of LAB (n=66) was identified by sequencing the full length 16S rRNA gene and isolates were subsequently screened for inhibition of <i>M. haemolytica</i> using the agar slab method. From the first study, high–throughput sequencing showed that the total LAB (defined as the order <i>Lactobacillales</i>) constituted 4.2% of the nasopharyngeal bacterial microbiota and consisted of 23 genera.  Within LAB, 6 different families were identified that included <i>Streptococcaceae</i> (49.2 %), <i>Carnobacteriaceae</i> (23.9%), <i>Aerococcaceae</i> (16.0%), <i>Enterococcaceae</i> (5.6%), <i>Lactobacillaceae</i> (5.3%), and <i>Leuconostocaceae</i> (0.26%).  The relative abundance of total LAB increased by 97% from d 0 to 2 and remained greater for the 14 days of feedlot placement, compared to d 0 (<i>P</i> <0.05). Interestingly, however, the <i>Lactobacillaceae</i> family decreased (<i>P</i> < 0.05) from d 0 to 2, demonstrating that not all <i>Lactobacillales</i> members increased after feedlot arrival. Using culture–based methods, only 6 genera of LAB were isolated: <i>Streptococcus</i> (39.2%), <i>Lactobacillus</i> (37.1%), <i>Enterococcus</i> (10.3%), <i>Aerococcus</i> (9.3%), <i>Corynebacterium</i> (3.1%), and <i>Pediococcus</i> (1.0%).  Among the screened LAB isolates, species within <i>Lactobacillus</i> exhibited the strongest inhibition against <i>M. haemolytica</i>, with zones of inhibition ranging between 16 and 23 mm.  Our results show that the relative abundance of nasopharyngeal LAB can change after cattle are transported to a feedlot and that some LAB are able to inhibit the respiratory pathogen <i>M. haemolytica</i>.  These LAB may have potential as nasal probiotics for the mitigation of bovine respiratory pathogens.

Keywords: lactic acid bacteria, probiotic, bovine nasopharynx