Development of Bacterial Therapeutics against the BovineRespiratory Pathogen Mannheimia haemolytica
Samat Amat,a,b Edouard Timsit,b,c,d Danica Baines,a Jay Yanke,a Trevor W. Alexandera
aAgriculture and Agri-Food Canada, Lethbridge Research and Development Centre, Lethbridge, Alberta, CanadabFaculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, CanadacSimpson Ranch Chair in Beef Cattle Health and Wellness, University of Calgary, Calgary, Alberta, CanadadFeedlot Health Management Services, Okotoks, Alberta, Canada
ABSTRACT Bovine respiratory disease (BRD) is a major cause of morbidity and mor-tality in beef cattle. Recent evidence suggests that commensal bacteria of the bo-vine nasopharynx have an important role in maintaining respiratory health by pro-viding colonization resistance against pathogens. The objective of this study was toscreen and select bacterial therapeutic candidates from the nasopharynxes of feed-lot cattle to mitigate the BRD pathogen Mannheimia haemolytica. In a stepwise ap-proach, bacteria (n � 300) isolated from the nasopharynxes of 100 healthy feedlotcattle were identified and initially screened (n � 178 isolates from 12 different gen-era) for growth inhibition of M. haemolytica. Subsequently, selected isolates wereevaluated for the ability to adhere to bovine turbinate (BT) cells (n � 47), competeagainst M. haemolytica for BT cell adherence (n � 15), and modulate gene expres-sion in BT cells (n � 10). Lactobacillus strains had the strongest inhibition of M. hae-molytica, with 88% of the isolates (n �33) having inhibition zones ranging from 17to 23 mm. Adherence to BT cells ranged from 3.4 to 8.0 log10 CFU per 105 BT cells.All the isolates tested in competition assays reduced M. haemolytica adherence to BTcells (32% to 78%). Among 84 bovine genes evaluated, selected isolates upregulatedexpression of interleukin 8 (IL-8) and IL-6 (P � 0.05). After ranking isolates for great-est inhibition, adhesion, competition, and immunomodulation properties, 6 Lactoba-cillus strains from 4 different species were selected as the best candidates for furtherdevelopment as intranasal bacterial therapeutics to mitigate M. haemolytica infectionin feedlot cattle.
IMPORTANCE Bovine respiratory disease (BRD) is a significant animal health issueimpacting the beef industry. Current BRD prevention strategies rely mainly on metaphy-lactic use of antimicrobials when cattle enter feedlots. However, a recent increase inBRD-associated bacterial pathogens that are resistant to metaphylactic antimicro-bials highlights a pressing need for the development of novel mitigation strate-gies. Based upon previous research showing the importance of respiratory com-mensal bacteria in protecting against bronchopneumonia, this study aimed todevelop bacterial therapeutics that could be used to mitigate the BRD pathogenMannheimia haemolytica. Bacteria isolated from the respiratory tracts of healthycattle were characterized for their inhibitory, adhesive, and immunomodulatoryproperties. In total, 6 strains were identified as having the best properties foruse as intranasal therapeutics to inhibit M. haemolytica. If successful in vivo,these strains offer an alternative to metaphylactic antimicrobial use in feedlotcattle for mitigating BRD.
Citation Amat S, Timsit E, Baines D, Yanke J,Alexander TW. 2019. Development of bacterialtherapeutics against the bovine respiratorypathogen Mannheimia haemolytica. ApplEnviron Microbiol 85:e01359-19. https://doi.org/10.1128/AEM.01359-19.
Editor Shuang-Jiang Liu, Chinese Academy ofSciences
Bovine respiratory disease (BRD), also known as shipping fever, remains the costliestdisease in the North American feedlot industry, despite advances in antimicrobials
and vaccines against respiratory pathogens (1). Although BRD is a multifactorial diseasewith several viral and bacterial agents involved, Mannheimia haemolytica is considereda major pathogen in its etiology and is therefore a primary target for both BRDmitigation and treatment in cattle (2). As an opportunistic pathogen, M. haemolyticaexists in the general cattle population and colonizes the nasopharynx in healthy cattle.However, when cattle experience compromised immunity due to stress and viralinfection, M. haemolytica can proliferate in the nasopharynx and then translocate intothe lung, where it can cause fibrinous pleuropneumonia (3).
Calves arriving at feedlots are often more susceptible to respiratory bacterial infec-tions due to stress imposed by maternal separation and environmental and manage-ment factors (4). As a result, cattle determined to be at risk for BRD are frequentlyadministered long-acting antimicrobials upon feedlot entry (i.e., metaphylaxis) (5).However, antibiotic resistance has been reported to be increasing in BRD-associatedpathogens (6). In addition, recent feedlot studies conducted in both Canada (7, 8) andthe United States (9) revealed a high prevalence of multidrug-resistant BRD bacterialpathogens displaying resistance to antimicrobials used for metaphylaxis treatment.Emergence of antimicrobial-resistant bacteria associated with BRD presents a signifi-cant risk to the beef industry, particularly if the efficacy of antimicrobials diminishes dueto pathogens acquiring resistance. Novel alternatives to metaphylactic antimicrobialsare therefore greatly needed.
Increasing evidence shows that bacterial communities residing within the respira-tory tract are important to respiratory health and that disruption of the microbiota canreduce host resistance to colonization and proliferation of pathogenic bacteria (10, 11).The nasopharynx in cattle harbors a rich and diverse microbial community that isdynamic and has been shown to change in response to several management practices,including transportation to a feedlot (12), altering diet (13), and antimicrobial admin-istration (14). Recent studies have also suggested an association between the naso-pharyngeal microbiota and development of BRD in feedlot cattle (15, 16). This notionis further supported by studies associating a greater relative abundance of nasopha-ryngeal Lactobacillaceae at the time of feedlot entry with protection against BRD (17)and also specific inhibition of M. haemolytica in vitro (18). Hence, maintaining a stablemicrobial community in the nasopharynxes of cattle after feedlot placement may offerprotection against BRD development, and bacteria colonizing the bovine respiratorytract may have potential for use as therapeutics to mitigate BRD pathogens. Theobjective of the present study was to develop bacterial therapeutics, with a focus onlactic acid-producing bacteria (LAB) originating from the respiratory tracts of healthycattle for mitigation of M. haemolytica, using a stepwise approach based on pathogeninhibition, cell adherence, and immunomodulatory properties (Fig. 1).
RESULTSIsolation and identification of nasopharyngeal commensal bacterial isolates. A
total of 300 isolates from De Man, Rogosa, and Sharpe (MRS) and Rogosa agar plateswere isolated and identified using nearly full-length 16S rRNA gene sequences (Table1). These isolates were from 14 different genera, with Bacillus (34%), Staphylococcus(30%), Streptococcus (12.3%), and Lactobacillus (12.0%) the more predominant genera.Although both MRS and Rogosa agar plates are semiselective for LAB, 69% of the totalbacteria isolated were non-LAB species. A total of 93 isolates were taxonomicallyclassified as LAB and comprised the genera Streptococcus (39.8% of the total LAB),Lactobacillus (38.7%), Enterococcus (10.8%), Aerococcus (9.7%), and Pediococcus (1.1%).
Growth-inhibitory effects against M. haemolytica. Of the identified bacteria, 178isolates representing 12 different genera were tested for the ability to inhibit M.haemolytica growth using an agar slab method (Fig. 2). A total of 74 isolates were LAB,within the genera Aerococcus (n � 4), Enterococcus (n � 5), Lactobacillus (n � 33), andStreptococcus (n � 32) (Fig. 2A). Of these, 88% inhibited the growth of M. haemolytica,
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FIG 1 Schematic workflow diagram illustrating the process of isolation and identification and the screening criteriafor commensal bacteria from the nasopharynxes of healthy feedlot cattle to identify candidate bacterial thera-peutics to mitigate the bovine respiratory pathogen M. haemolytica.
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with zones of inhibition (ZOI) ranging from 11 to 23 mm (Fig. 2B). Approximately 48%of the tested LAB displayed relatively strong inhibition of M. haemolytica (ZOI, 17 to23 mm). Specifically, Lactobacillus displayed the greatest inhibition of M. haemolytica,with 88% of the tested Lactobacillus isolates showing ZOI ranging between 17 and23 mm (Fig. 2A and B). Although 91% of the tested Streptococcus isolates inhibited M.haemolytica, 59% of them had relatively weak inhibition (ZOI, 11.1 to 14 mm), and 35%showed medium inhibition (ZOI, 14.1 to 16.9 mm). Four of 5 tested Enterococcus isolatesmoderately inhibited M. haemolytica. However, none of the Aerococcus isolates inhib-ited M. haemolytica (Fig. 2A).
The non-LAB isolates (n �104) tested for inhibition of M. haemolytica taxonomicallybelonged to 8 different genera and consisted mainly of Bacillus (51% of the totalnon-LAB isolates tested) and Staphylococcus (41%) isolates (Fig. 2C). Of these non-LABisolates, 46% displayed growth-inhibitory effects against M. haemolytica (Fig. 2C).Among the inhibition-positive isolates, 48% had relatively weak inhibition (ZOI, 11.1 to14 mm) and 31% showed medium inhibition (ZOI, 14.1 to 16.9 mm) (Fig. 2D). Of theBacillus isolates tested, 55% did not inhibit growth of M. haemolytica. Only 6% of thetested isolates showed relatively strong inhibition (ZOI, �17 mm). The growth of M.haemolytica was inhibited by 49% of the tested Staphylococcus isolates, with 76% of theinhibition-positive isolates showing weak to medium inhibition (ZOI, 11.1 to 16.9 mm).Corynebacterium and Macrococcus isolates displayed relatively strong inhibition (ZOI, 17to 19.9 mm). Moderate inhibition of M. haemolytica was also observed with oneEscherichia coli isolate. However, isolates within the genera Acetobacter, Micrococcus,and Rummeliibacillus did not inhibit M. haemolytica.
Adherence of selected isolates to BT cells. A total of 47 isolates were selected foradherence to bovine turbinate (BT) cells, based on their ability to inhibit M. haemolytica(ZOI, �15 mm). These isolates were from 6 different genera (Bacillus, Lactobacillus,Macrococcus, Enterococcus, Staphylococcus, and Streptococcus). All the tested isolateswere able to colonize BT cell monolayers, with mean adherences ranging between 3.4and 8.0 log10 CFU per 105 BT cells (Fig. 3). Of these, 32 isolates were Lactobacillus spp.and were from 7 different species: Lactobacillus amylovorus (n � 1), Lactobacillus brevis(n �2), Lactobacillus buchneri (n � 23), Lactobacillus paracasei (n � 3), Lactobacillushilgardii (n � 1), Lactobacillus curvatus (n � 1), and Lactobacillus sunkii (n � 1) (Fig. 3A).Among these Lactobacillus isolates, 47% displayed mean adherences greater than 5.0
TABLE 1 Bacteria identified from the nasopharynxes of healthy feedlot cattle and thoseselected for initial inhibition of M. haemolytica using agar slabsa
GenusNo. ofisolates
% of totalisolates
No. of isolates screened forinhibition of M. haemolytica
Total 300 178aBacteria were isolated by plating swabs onto MRS or Rogosa medium and then identified using 16S rRNAgene sequencing and biochemical tests.
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log10 CFU per 105 BT cells. Interestingly, the adherences to BT cells differed for strainswithin the same species, which was more obvious within the species L. buchneri (Fig.3A). Of the 15 non-Lactobacillus isolates, the Enterococcus faecium strains, two Staph-ylococcus strains (28C and 98C), and one Streptococcus strain showed mean adherencesgreater than 5.0 log10 CFU per 105 BT cells (Fig. 3B).
FIG 2 Growth-inhibitory effects of bovine respiratory bacteria against M. haemolytica. (A to D) Summaries of LAB (A) and non-LAB (C) and their respective zonesof inhibition (B and D). (E) Lactobacillus species displayed the greatest inhibition. (F) Example of the agar slab method to measure inhibitory properties ofscreened bacteria. The results are presented as mean zones of inhibition (plus standard deviations [SD]) from three replicates.
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Antagonistic competition activities of selected isolates against M. haemolytica.Fifteen isolates were tested for competitive exclusion ability. The isolates were selectedbased on high inhibition of M. haemolytica (ZOI, �15 mm) and strong adherence to BTcells (�5.0 log10 CFU per 105 BT cells). Reduction of M. haemolytica adherence to BTcells was observed with all the tested isolates (Fig. 4). The mean reduction of M.haemolytica adherence to BT cell monolayers ranged from 32% to 78%. Lactobacillusamylovorus (72B) displayed the strongest inhibition of M. haemolytica adherence to BTcells, with adherence greater than that of 9 other tested strains (P � 0.05). In contrast,L. buchneri (65A) showed the weakest antagonistic competition against M. haemolytica.The remaining strains had essentially comparable levels of M. haemolytica adherenceinhibition (P � 0.05).
FIG 3 (A and B) Adherence of bovine respiratory bacterial isolates to BT cell monolayers. In total, 32 strains of Lactobacillus (A) and15 strains of Bacillus, Enterococcus, Macrococcus, Staphylococcus, and Streptococcus (B) were evaluated. The results are presented asmeans and standard errors (SE) of bacterial adherence (log10 CFU) to BT cell monolayers (105 cells) obtained from three independentexperiments performed on different days. (C) Confocal image of L. curvatus 103C (stained green with Alexa Fluor 594) adhering tobovine turbinate cells (stained blue with DAPI) shown as an example of bacterial adherence.
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Antimicrobial susceptibilities of selected isolates. The MICs of 20 antibioticsagainst 15 isolates were determined. According to the interpretive criteria provided byCLSI (M45) (19), all tested bacteria were susceptible to clindamycin, erythromycin,linezolid, meropenem, and penicillin (Table 2). The majority of Lactobacillus isolateswere susceptible to daptomycin. Only L. amylovorus 72B grew at the maximumantibiotic plate concentration tested (2 �g/ml). Given that the resistance breakpoint fordaptomycin against Lactobacillus is defined as greater than 4 �g/ml, it was not possibleto define whether L. amylovorus 72B was daptomycin resistant. The L. amylovorusstrains were susceptible to vancomycin, while the MIC values for all the other Lacto-bacillus strains were greater than the maximum concentration tested (�4 �g/ml) andcould not be defined. Only two of the tested Lactobacillus strains (L. buchneri 38C andL. amylovorus 72B) were not inhibited by levofloxacin at the maximum concentrationtested (4 �g/ml). All the other tested strains of L. buchneri were not inhibited bytetracycline at the maximum concentration of 8 �g/ml, which is 16-fold lower than theresistant cutoff value for L. buchneri (128 �g/ml), thus limiting evaluation of theirtetracycline resistance. The L. paracasei strains were susceptible to chloramphenicol.
E. faecium 64C was susceptible to tetracycline, chloramphenicol, vancomycin, anderythromycin. According to the CLSI VET01S guidelines, Staphylococcus chromogenes28C was resistant to penicillin while Staphylococcus epidermidis 6E was susceptible topenicillin. Both of these Staphylococcus strains were also resistant to amoxicillin-clavulanate and tetracycline but were susceptible to vancomycin, clindamycin, eryth-romycin, and chloramphenicol.
Stimulation of innate and adaptive immune responses in BT cell monolayers.Among the 84 genes tested, 33 genes showed significant differences in transcriptionalgene expression between the BT cells cocultured with at least one tested bacteriumand the BT cells incubated under the same conditions without bacteria (P � 0.05) (Table3). Of the Lactobacillus isolates, L. curvatus (strain 103C), L. amylovorus (72B), and L.buchneri (67A, 63A, 65G, and 63B) upregulated the CXCL8 gene, with 3.98-fold to8.43-fold difference in expression from the control (P � 0.02). The transcription of IL-6
FIG 4 (A) Antagonistic competition of bovine respiratory bacteria (n � 15) against M. haemolytica. The results are presented as mean(�SE) reduction in M. haemolytica adherence to BT cell monolayers by commensal bacteria obtained from 6 replicates. Different lettersindicate that the mean values differ (P � 0.05). (B) Representative confocal image showing adherence of M. haemolytica (stained redwith Alexa Fluor 488) and L. paracasei 3E (stained green with Alexa Fluor 594) to bovine turbinate cells (stained blue with DAPI).
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was also upregulated by 5 of the 6 Lactobacillus isolates (changes ranged from 8.63- to22.75-fold; P � 0.04). The expression of NFKB1 was upregulated (P � 0.03) in BT cellscocultured with L. curvatus (103C) and L. buchneri (63A, 63B, 65G, and 67A) isolatesrelative to the control (changes ranged from 1.5- to 2.13-fold). L. amylovorus (72B), L.buchneri (63B and 65G), and L. curvatus (103C) induced overexpression of interferonalpha/beta receptor alpha chain, with 1.6- to 2.0-fold changes in gene expression
TABLE 3 Selected genes that had altered expression in BT cells after incubation with bacteria isolated from the nasopharynxes of cattlea
Gene Description
M. haemolytica L. buchneri (63A)L. amylovorus(72B) L. paracasei (57A)
Foldchangeb P value
Foldchange P value
Foldchange P value
Foldchange P value
BOLA Major histocompatibility complex class I, heavychain
class I, A-like7.21 0.00 2.18 0.23 1.18 0.51 �1.19 0.66
MX1 Myxovirus (influenza virus) resistance 1,interferon-inducible protein p78 (mouse)
329.9 0.00 1.55 0.49 1.24 0.91 �1.02 0.62
NFKB1 Nuclear factor of kappa light polypeptide geneenhancer in B cells 1
8.80 0.00 1.67 0.00 1.27 0.15 1.27 0.30
NFKBIA Nuclear factor of kappa light polypeptide geneenhancer in B cells inhibitor, alpha
19.05 0.00 �1.14 0.58 1.06 0.69 1.39 0.39
SLC11A1 Solute carrier family 11 (proton-coupled divalentmetal ion transporters), member 1
2.18 0.05 �1.64 0.27 �1.64 0.47 �1.94 0.03
STAT1 Signal transducer and activator of transcription1, 91 kDa
8.17 0.00 �1.16 0.37 �1.05 0.71 �1.12 0.40
STAT3 Signal transducer and activator of transcription3 (acute-phase response factor)
2.34 0.00 1.19 0.35 1.32 0.20 �1.03 0.76
TLR2 Toll-like receptor 2 2.39 0.03 1.00 0.82 1.76 0.04 �1.03 0.78TLR3 Toll-like receptor 3 4.04 0.00 1.06 0.66 1.08 0.65 �1.10 0.83TLR4 Toll-like receptor 4 9.28 0.00 1.20 0.75 1.28 0.62 �1.12 0.95aAll values are presented as the mean fold change in gene expression for 4 replications. The difference in gene expression between BT cells (control) that were notcocultured with bacterial cells and the BT cells cocultured with bacterial cells was assessed by Student’s t test for each gene using the Rt2 Profiler PCR array analysissoftware, version 3.5 (Qiagen). The level of statistical significance was set at a P value of �0.05. Genes that showed significant differences in expression between thecontrol and at least one bacterium cocultured with BT cells are listed. Significant changes (P � 0.05) in expression are shown in boldface.
bFold change (2�ΔΔCT) is the normalized gene expression (2�ΔΔCT) in the test sample divided by the normalized gene expression (2�ΔΔCT) in the control sample. Foldchange values greater than 1 indicate positive or upregulation, and fold change values less than 1 indicate negative or downregulation.
cTNF, tumor necrosis factor.
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compared to the control (P � 0.02). Of the 84 genes, very few were downregulated inBT cells in response to Lactobacillus inoculation. Transcription of chemokine receptor 6was downregulated by L. paracasei (57A) and L. buchneri (63B and 38C) isolates(P � 0.04), and L. paracasei (57A) downregulated the gene encoding solute carrierfamily 11 member 1 by 1.94-fold (P � 0.03).
In contrast to Lactobacillus spp., significantly high immune stimulation in BT cellswas observed with M. haemolytica, which upregulated 28 genes with changes rangingfrom 1.43- to 1,321-fold (P � 0.05). The greatest responses to M. haemolytica were in thetranscription of C-X-C motif chemokine 10 (1,321-fold change), CXCL5 (782-foldchange), myxovirus resistance 1 (330-fold change), and IL-6 (290-fold change). The IRF7,DDX58, CXCL8, CD40, IL-15, NFKB1A, ICAM1, and LOC512672 genes exhibited relativelyhigh overexpression in BT cells incubated with M. haemolytica (10- to 67-fold changes)
TABLE 3 (Continued)
L. buchneri (63B) L. buchneri (65G) L. curvatus (103C) L. paracasei (3E) L. buchneri (38C) L. buchneri (86D) L. buchneri (67A)
(P � 0.05). Moderate upregulation by M. haemolytica was observed for the genesencoding chemokine ligand 2, colony-stimulating factor 2, Janus kinase 2, majorhistocompatibility complex class 1 (A like), NFKB1, STAT1, and Toll-like receptor 4 (TLR4)(5- to 10-fold changes).
M. haemolytica-inhibitory mechanisms of 6 Lactobacillus isolates as candidatebacterial therapeutics. A total of 6 Lactobacillus strains (listed in Table 4) from fourdifferent species were selected as the best candidates for the development of intranasalbacterial therapeutics to mitigate M. haemolytica infection based on the selectioncriteria of inhibition (strongest inhibition), adhesion (strongest adhesion), competitionexclusion (strongest exclusion), antimicrobial susceptibility (limited resistance), andimmunomodulation (moderate stimulation of immune genes). To understand thepotential mechanisms through which these selected Lactobacillus strains confer directinhibition of M. haemolytica, we determined their lactic acid, H2O2, and putativebacteriocin production capacities and their effects on the cell morphology of M.haemolytica.
Lactic acid production and growth inhibition by lactic acid against M. haemo-lytica. We determined the lactic acid production of 6 Lactobacillus strains individually,as well as when combined (Table 4). The concentrations of lactic acid detected from thesupernatants ranged between 80 and 142 mM. The supernatants of the 6 strainscombined and L. paracasei strains 3E and 57A contained the most lactic acid (132 to142 mM), followed by L. curvatus 103C (111 mM) and L. amylovorus 72B (103 mM). Thesupernatants of L. buchneri strains contained the least lactic acid, with 80 and 94 mM,respectively. No lactic acid was detected from the negative control (MRS broth).
The inhibitory effects of lactic acid covering the range of concentrations producedby the 6 strains were tested against growth of M. haemolytica (Fig. 5). At 8 h postin-oculation, M. haemolytica growth was inhibited when the lactic acid concentrationsranged from 18.75 to 150 mM compared to 0 and 9.38 mM concentrations. Althoughthere was cell growth in media supplemented with 18.75 mM lactic acid within the first8 h of incubation, this concentration of lactic acid resulted in lower cell counts than at0 and 9.38 mM concentrations. After 24 h, no viable cells were detected in mediacontaining 100 and 150 mM lactic acid.
H2O2 production. The concentrations of H2O2 in cell-free culture supernatantsobtained from Lactobacillus strains after 24 h of incubation varied among differentLactobacillus species, with L. amylovorus the most predominant H2O2 producer (Table4). L. buchneri (63A and 86D) and L. curvatus (103C) strains produced similar amounts
TABLE 4 Antimicrobial properties of selected bacterial therapeutic strains (n � 6) evaluated by the measurement of their lactic acid andH2O2 production and bacteriocin-encoding genes
StrainLactic acidconcn (mM)a
H2O2 concn(nmol/ml)a
Bacteriocin-encoding genes detected from the whole-genome sequences ofthe selected strains by BAGEL 4
(enterocin X chain beta)L. paracasei (57A) 131.6 � 3.61 0 NSc NDCocktail of 6 strains 142.1 � 8.72 NAb NA NDControl (MRS broth) 0 0 NA NDaThe results are reported as the mean concentration (�SE) of lactic acid and H2O2 produced by the selected strains over the 24-h incubation period. The mean wasobtained from triplicate samples.
bNA, not applicable.cNS, not sequenced.dND, bacteriocin not detected in genomes.
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of H2O2; however, no H2O2 was detected from the supernatants of L. paracasei strains(3E and 57A).
Encoded bacteriocins. Sequences obtained for L. paracasei (57A) were too poor toevaluate and thus were eliminated from bacteriocin evaluation. The genomes of 5selected strains revealed that L. amylovorus 72B and L. paracasei 3E containedbacteriocin-encoding genes while L. buchneri (63A and 86D) and L. curvatus (103C)strains did not have genes encoding bacteriocins (Table 4). Lactobacillus amylovorus(72B) had 5 genes encoding enterolysin A and helveticin J, both of which belong to thebacteriolysin class. The genome of L. paracasei 3E contained four different bacteriocingenes encoding LSEI_2163, enterolysin A, carnocin-CP52 immunity protein, and ente-rocin X�.
Effect of selected strains on cell morphology of M. haemolytica. The morpho-logical effects of supernatants from candidate strains on M. haemolytica were examinedusing scanning electron microscopy (SEM). Noticeable changes in the cell structure,including shrinkage of the cell surface, irregular rod shape, and holes in the cellenvelope, were observed when M. haemolytica was incubated in culture supernatantsfrom Lactobacillus strains compared to untreated cells (Fig. 6). Supernatant from L.amylovorus (72B) reduced the cell density of M. haemolytica to the greatest extent (datanot shown) and caused the most apparent destructive changes in M. haemolytica cellstructure (Fig. 6B). L. buchneri (63A) demonstrated minor cell damage compared toother strains tested (Fig. 6C), followed by L. paracasei (3E) (Fig. 6D). The bacteria L.paracasei (57A), L. curvatus (103C), and L. buchneri (86D) exhibited similar degrees of celldamage (Fig. 6E to G).
DISCUSSION
Bacteria originating from the host target site are more likely adapted for successfulrecolonization (20). To develop bacterial therapeutics to mitigate the BRD pathogen M.haemolytica, we therefore performed our screening on commensal bacteria isolatedfrom the upper respiratory tracts of healthy feedlot cattle. This is also the site whereBRD bacterial pathogens colonize and proliferate before translocating into the lung toinduce lung infection. In previous studies comparing healthy and BRD-affected cattlephenotypes, an increased abundance of LAB was observed in healthy animals (15, 21).Thus, bacterial species taxonomically belonging to the LAB (order Lactobacillales) wereoriginally targeted using selective media. A wide range of commensal bacterial specieswere isolated and identified from two different sources of healthy feedlot cattle. Ofthese isolates, 31% were LAB strains. A subset of isolates comprised mainly of LABstrains and non-LAB species from genera such as Bacillus and Staphylococcus, whichhave a known history of probiotic use (22, 23), were selected for evaluation.
In summary, a number of diverse bacteria were able to inhibit the growth of M.haemolytica to varying extents. This suggests that the environment within the bovine
FIG 5 Growth inhibition effects of lactic acid on M. haemolytica. The values are the means of tworeplicates. The error bars indicate SD.
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respiratory tract is highly competitive, with multiple bacteria capable of producingfactors that inhibit the opportunistic pathogen M. haemolytica. In support of this,Corbeil et al. (24) also described several genera from the nasal cavities of cattle thatcould inhibit bovine respiratory pathogens, including M. haemolytica, Pasteurella mul-tocida, and Histophilus somni. In the present study, the bacteria displaying the strongestinhibition of M. haemolytica were within the genus Lactobacillus. Previously, Lactoba-cillus was identified as being more abundant in the lungs of healthy feedlot calves thanin those diagnosed with BRD (21). Lactobacillus spp. therefore appear to be involved in
FIG 6 Scanning electron microscopy images of M. haemolytica after incubation with cell-free culturesupernatants of selected bacterial therapeutic strains. Bacteria were incubated with cell-free culturesupernatants before fixation and microscopy. Shown are untreated cells (A) and cells incubated withcell-free culture supernatants of L. amylovorus 72B (B), L. buchneri 63A (C), L. paracasei 3E (D), L. paracasei57A (E), L. curvatus 103C (F), and L. buchneri 86D (G).
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maintaining the respiratory health of cattle and may achieve this by inhibiting respi-ratory pathogens through the production of antimicrobial factors. Direct inhibition ofpathogens is an important attribute of bacterial therapeutics and probiotics and occursthrough production of lactic acid, bacteriocins, and H2O2 or through the mechanicalproperty of autoaggregation (25, 26). While few studies have investigated bacterialtherapeutics to mitigate BRD bacterial pathogens, the inhibitory effects of commensalbacteria against the growth of a wide range of bacterial pathogens involved in humanintestinal and respiratory tract infections have been documented (27, 28). In order todevelop bacterial therapeutics with the greatest potential to mitigate M. haemolytica,only a subset of screened commensals with the strongest inhibition were furtherevaluated, the majority of which were Lactobacillus spp.
We used BT cells for adhesion assays because the cell type is found within the upperrespiratory tract, which M. haemolytica colonizes (29) and where inhibition of its growthwould be desired in order to limit proliferation and subsequent translocation andinfection of the lungs. All the strains tested were capable of adhering to BT cells tovarying extents, which was expected given that they were originally isolated from thenasopharynxes of cattle. Surprisingly, E. faecium isolates had the strongest adherence.Enterococcus colonizes the lower gastrointestinal tract, though the bacteria are capableof adhering to extraintestinal epithelial cells (30). While it is likely a transient inhabitantof the respiratory tract, this probably explains why Enterococcus has been consistentlyobserved in the upper respiratory tract sof cattle (12, 17, 31). Several Lactobacillusstrains also displayed a high level of adherence. Although no studies have investigatedthe adhesion of commensal respiratory tract bacteria to bovine upper respiratory tractcells, we previously showed that commercial Lactobacillus spp. displayed greateradhesion to bovine bronchial epithelial cells than Streptococcus and Paenibacillusstrains (18). Lactobacillus spp. have the ability to colonize both mucus (32) andunderlying epithelial cells of the respiratory tract (33). The high level of adherence toBT cells observed with some of the Lactobacillus strains might be attributed to bothspecific (surface-dependent proteins and surface layer proteins) and nonspecific (cellsurface hydrophobicity and lipoteichoic acid) adhesion mechanisms (34). Differentadherence capacities among Lactobacillus species (L. brevis versus L. amylovorus), andeven strains of the same species (L. buchneri), were observed in the present study. It islikely that variation in strain adherence resulted from differences in cell surface struc-ture of the isolates, as has been described previously (35). Compared to Lactobacillusspp., the strains within the genera Bacillus, Macrococcus, and Streptococcus showedweaker adhesion to BT cells and were therefore excluded from further screening.
A total of 15 isolates with the strongest inhibition of M. haemolytica and adhesionto BT cells were further evaluated for the ability to competitively inhibit M. haemolyticacolonization of BT cells. All the tested strains were able to inhibit the adherence of M.haemolytica to BT cell monolayers. This likely occurred through a combination of directinhibition and also competition for binding sites on the epithelial cells (36, 37). Of note,when evaluating inhibition, adhesion, and competition results, no single strain wasranked as the top candidate across all of the criteria. For example, L. amylovorus 72Bshowed the strongest inhibition and competition against M. haemolytica, but theadherence of the strain to BT cells was less than that of L. buchneri and L. paracaseistrains. In contrast, L. buchneri (63A) and E. faecium (64C) showed moderate growthinhibition of M. haemolytica but strong adherence to BT cells and antagonistic com-petition against M. haemolytica in comparison to other strains. Thus, utilizing severalcriteria is important in defining potentially effective bacterial therapeutics. In addition,designing therapeutic strains with varying antimicrobial properties may promotebroader efficacy of bacterial therapeutics against pathogens. In support of this, multi-strain cocktails containing probiotics with different mechanisms of action have shownbetter antipathogenic activity and modulation of the mouse gut microbiome and shortchain fatty acid production (38), as well as greater attenuation of pathogen-inducedinflammatory response in the intestinal epithelium (39), than single-strain probiotics.
Safety concerns exist over probiotic or therapeutic bacteria that are resistant to
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antibiotics, especially if resistance elements are encoded on mobile elements (40, 41).The isolates used in our study were from cattle that were not administered antibioticsbefore or during the time of isolation, and thus, pressure for resistance to develop orfor resistant bacteria to be selected was low. Indeed, when evaluated, the selectedLactobacillus strains were generally susceptible to antibiotics with defined break points,though some antibiotics could not be fully tested due to their breakpoint concentra-tions not being reached in the antibiotic panel. Although the majority of L. buchneristrains were not inhibited by the highest concentration of tetracycline tested (8 �g/ml),tetracycline resistance (128 �g/ml) in L. buchneri has been reported to be intrinsic (42).An L. buchneri strain (NRRL B-50733) that showed a tetracycline MIC value of 32 �g/mlhas been considered safe for use as a silage additive for livestock (43). Thus, higher MICsfor tetracycline in L. buchneri strains would not likely limit the use of these bacteria astherapeutics, although further evaluation of the L. buchneri isolates in our study with anantibiotic panel containing higher concentrations of tetracycline would be beneficial. L.amylovorus strain 72B had higher levofloxacin and moxifloxacin MICs. Currently, thereare no breakpoints available for these two fluoroquinolone antibiotics against L.amylovorus strains. However, some species within the genus Lactobacillus are known tobe intrinsically resistant to fluoroquinolones, including levofloxacin (44, 45).
Staphylococcus strains were resistant to either penicillin (28C) or amoxicillin-clavulanate and tetracycline (28C and 6E). Therefore, these strains were excluded fromfurther evaluation. Despite being susceptible to the antibiotics tested and showingstrong inhibition of M. haemolytica, the E. faecium strain 64C was also not consideredfor the immunostimulation assay due to E. faecium being a potential opportunisticpathogen (46) and defined as a level 2 pathogen in some countries. Considering thedata from the inhibition, competition, and antimicrobial susceptibility assays, in addi-tion to a history of safe use in both food and the feed industry (47), the strains ofLactobacillus, therefore, were selected for immunomodulation properties.
Modulation of host innate and adaptive immunity by therapeutic bacteria canpotentially increase resistance to pathogen infection (48). Bovine respiratory epithelialcells are an initial point of contact between the host and respiratory microbiota (49) andwere therefore used to evaluate immunomodulation. In the present study, the testedLactobacillus strains induced moderate gene expression of the chemokine CXCL8 in BTcells, showing the ability of these nasopharyngeal strains to stimulate an immuneresponse. In contrast, M. haemolytica induced strong overexpression of CXCL8 in BTcells. The protein CXCL8 is a potent chemoattractant and plays an important role ininflammation and wound healing through activation of neutrophils and other immunecells (50). Whether CXCL8 has a beneficial role in protecting the host through theinflammation healing process or is detrimental by promoting pathogenesis likelydepends on its level of expression (50). In support of this, Gärtner et al. (51) reportedthat commensal Lactobacillus spp. present in the bovine uterus upregulated CXCL8gene expression by 2- to 6-fold in endothelial epithelial cells after 6 to 8 h of coculture.The authors suggested that excessive expression of CXCL8 might contribute to thedevelopment of uterine disease, while moderate stimulation of CXCL8 may be neces-sary for bacterial clearance.
Similar to CXCL8, expression of NFKB1 and the proinflammatory cytokine IL-6 in BTcells was stimulated by most of the Lactobacillus strains tested. However, upregulationof these genes in BT cells by Lactobacillus spp. was considered moderate compared toM. haemolytica. Lipopolysaccharide from M. haemolytica has been shown to inducesecretion of IL-6 in pulmonary epithelial cells (52), and activation of the NF-�B pathwaycauses excessive inflammation in the lower respiratory tract, thus enhancing infectionby the pathogen (53). While we did not coinoculate Lactobacillus strains with M.haemolytica for the immunomodulation assays using BT cells, expression of IL-6 inbovine endothelial epithelial cells was moderately upregulated in response to com-mensal Lactobacillus spp. present in the bovine uterus (46). Future studies evaluatingcoinoculation would provide insight into whether the virulence of M. haemolytica couldbe reduced by bacterial therapeutics.
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Overall, most of the 84 genes encoding the TLR pathway, cytokine and chemokinereceptors, inflammation response, NF-�B signaling, apoptosis, and innate immune anddefense responses to bacteria were not influenced by the Lactobacillus strains tested.However, M. haemolytica induced overexpression of 28 genes, including cytokine andchemokine receptors, inflammatory responses, NF-�B signaling, and defense responseto bacteria. None of the tested Lactobacillus strains caused an excessive immuneresponse, as observed with M. haemolytica. The effects of Lactobacillus on immunemodulation in BT cells was species and strain specific. Similarly, immunomodulation hasbeen shown to vary at the strain level in commensal bacteria (54, 55). Combined, theresults regarding immune stimulation in BT cells suggest that Lactobacillus may have arole in modulating immunity in cattle; however, future studies are warranted toelucidate the mechanisms by which modulation occurs and its impact on respiratorypathogens.
All 6 of the candidate therapeutic strains produced lactic acid to varying extents.This acid has been shown to alter the membrane structure of pathogens and is amechanism by which probiotic Lactobacillus spp. inhibit pathogen growth (56). Inagreement with our study, Neal-McKinney et al. (56) reported a similar range of lacticacid production by Lactobacillus spp. (Lactobacillus acidophilus, Lactobacillus crispatus,Lactobacillus gallinarum, and Lactobacillus helveticus), as well as concentrations varyingaccording to the strains tested. For our study, given that each therapeutic candidate, ora combination of all 6, produced concentrations of lactic acid greater than the MIC(37.5 mM), lactic acid is likely a common metabolite by which these candidates directlyinhibited M. haemolytica.
In contrast to lactic acid, production of H2O2 and encoded bacteriocins variedamong the candidate strains. Hydrogen peroxide produced from Lactobacillus haspreviously been shown to have bactericidal activity against gut pathogens (25) andmay be a mechanism of inhibition of M. haemolytica for four of the candidate strains inour study. Only two of the candidate therapeutics encoded bacteriocins, which canhave bactericidal or bacteriostatic activity (57, 58). Similar to our study, it has beenobserved that bacteriocins encoded by Lactobacillus are species, strain, and origindependent (59). The different classes of bacteriocins detected from L. paracasei 3E weresimilar to those observed with the probiotic strain L. paracasei SD1 (60). Expression ofthese bacteriocins may have led to L. paracasei 3E being one of the strongest inhibitorsof M. haemolytica (ZOI, �22 mm). The strain L. amylovorus 72B, was predicted toproduce bacteriolysins, including enterolysin A and helveticin J. Although L. amylovorushas been reported to produce the bacteriocin amylovorin L471 (59, 61), genes encodingthis bacteriocin were not detected in strain 72B. It is interesting that while L.amylovorus (72B) did not have the greatest adhesion to BT cells, it did have one ofthe greatest inhibition and antagonistic values. The 72B strain also caused thegreatest morphological damage to M. haemolytica. Thus, a combination of lacticacid, H2O2, and bacteriocin production may have resulted in the strong inhibitionof M. haemolytica observed for 72B.
In summary, we isolated and identified commensal bacteria from 14 different generaresiding in the nasopharynxes of healthy feedlot cattle as part of the normal flora. Ofthese commensal isolates, using a stepwise approach, we screened isolates comprising12 different genera for their abilities to inhibit the growth of the respiratory pathogenM. haemolytica, to adhere to BT cells, and to compete against M. haemolytica adherenceto the BT cells. We then evaluated the best candidates for antimicrobial resistance andtheir immunomodulation effects in BT cells. Based on the data generated, 6 Lactoba-cillus strains from four different species (L. amylovorus strain 72B, L. buchneri strains 63Aand 86D, L. curvatus strain 103C, and L. paracasei strains 3E and 57A) were selected asthe best candidates for the development of intranasal bacterial therapeutics to mitigateM. haemolytica infection in cattle. Their selection was based on high inhibition of M.haemolytica directly and through competition, high adherence to BT cells, lack ofantibiotic resistance, and moderate immunomodulation in BT cells. The potential mecha-nisms by which these 6 selected strains inhibited M. haemolytica were also investigated.
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Lactic acid production was common among the strains, but production of H2O2 andencoded bacteriocins varied. Currently, in vivo studies are being conducted to evaluatethe effects of intranasal administration of these strains on the microbiota of feedlotcattle.
MATERIALS AND METHODSIsolation of commensal bacteria from the nasopharynxes of feedlot cattle. A schematic of the
general study design, including bacterial isolation, is shown in Fig. 1. Nasopharyngeal bacteria wereisolated as part of previous studies (17, 31). Briefly, two groups of animals were used to increase thediversity of the bacterial isolates. In the first group (group 1), 70 crossbred recently weaned steerspurchased from a local auction market were sampled by deep nasopharyngeal swab (DNS) on day 0(feedlot entry) and 60 days after placement at the Lethbridge Research and Development Centre (LRDC)feedlot (Alberta, Canada). The steers were fed diets typical of feedlots in western Canada and remainedhealthy from days 0 to 60 (17). In the second group (group 2), 30 Angus beef steers were sourced froma cow-calf ranch. These steers were sampled by DNS at weaning while still on the cow-calf ranch andthen upon arrival at the LDRC feedlot and 40 days after arrival (31). The DNS samples were processed forbacterial isolation using semiselective medium (MRS or Rogosa plates) for LAB, as described by Holmanet al. (17). Isolates were subcultured and stored in cryopreservative.
Identification of nasopharyngeal commensal bacterial isolates. A total of 300 banked isolates wererandomly selected for inclusion in the present study. For identification, the nearly full-length 16S rRNA gene(�1,400 bp) was sequenced for each isolate and used for taxonomic identification, as described previously(17). In instances where taxonomic identification at the species level was not possible by 16S rRNA genesequence analysis, biochemical tests were also employed. For this, the isolates were subcultured on MRS agar(Lactobacillus and Enterococcus) or tryptic soy agar (TSA) (Staphylococcus) at 39°C. Colony morphologies wereobserved after 24 to 48 h at both 27°C and 39°C. Anaerobic growth was tested on MRS agar or TSA at 39°Cin an anaerobic chamber with an atmosphere of 85% nitrogen, 10% hydrogen, and 5% CO2. Acid productionfrom carbohydrates was determined with the API 50CHL gallery (bioMérieux, Saint-Laurent, QC, Canada)(Lactobacillus and Enterococcus) according to the manufacturer’s instructions or using Difco purple agar basemedium (BD Canada, Mississauga, ON, Canada) containing 1% carbohydrate (Staphylococcus). Confirmatoryidentifications were obtained through comparison with published results.
Growth-inhibitory effects of nasopharyngeal commensal bacteria against M. haemolytica. Forall assays, M. haemolytica L024A was used. The isolate originated from a feedlot steer that succumbedto BRD in Alberta, Canada, and was confirmed as serotype 1 (62). Except for Moraxella and Pediococcus,isolates within all the genera identified were included for inhibition of M. haemolytica. A total of 178commensal isolates were studied for inhibition of M. haemolytica. The isolates were selected to includea diverse group of LAB and non-LAB strains from the respiratory tracts of cattle. Lactic acid-producingbacteria are defined as the order Lactobacillales, which encompasses the following six families: Aerococ-caceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, and Streptococcaceae(63). Growth-inhibitory effects against M. haemolytica were determined using an agar slab methodaccording to the instructions of Dec et al. (64) with some modifications. Briefly, 100 �l of an 18-h culturefrom each isolate grown in Difco Lactobacilli MRS broth (BD, Mississauga, ON, Canada) was spread as alawn onto MRS plates and incubated at 37°C with 5% CO2 for 24 h. Agar slabs (10 mm in diameter) werecut from the 24-h-incubated MRS plates using a sterile hollow punch (Tekton: 12-piece hollow punch set;catalog no. 6588) and placed with the culture side down onto a lawn of M. haemolytica on TSA platescontaining 5% sheep blood. The lawn of M. haemolytica was prepared by spread plating a 100-�l aliquotof M. haemolytica culture suspended in Dulbecco’s phosphate-buffered saline (DPBS) (pH 7.4) to obtainthe target bacterial concentration of 1 � 108 CFU per ml. Up to four agar plugs were placed onto a singlelawn of M. haemolytica, including a control plug containing no bacteria. The agar plug-M. haemolyticalawns were then incubated at 37°C with 5% CO2 for 24 h. After incubation, the plates were checked forZOI. The ZOI were measured with a ruler. The results were presented as the mean diameter of theinhibition zone for three independent experiments.
Adherence of commensal bacteria to BT cell monolayers. A subset of isolates (n � 47) displayingthe greatest inhibition of M. haemolytica (ZOI � 15 mm) were evaluated for adhesion to BT cells (ATCC1390; American Type Culture Collection, Manassas, VA, USA) using an assay described previously (18, 19)with some modifications. The isolates comprised the genera Lactobacillus (n � 32), Bacillus (n � 2),Enterococcus (n � 3), Macrococcus (n � 1), Staphylococcus (n � 6), and Streptococcus (n � 3). The BT cellswere seeded onto 6-well flat-bottom tissue culture plates at 1 � 105 cells per well and incubated inDulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Oakville, ON, Canada) supple-mented with 10% horse serum (American Type Culture Collection) and 50 �g/ml gentamicin (Sigma-Aldrich, Oakville, ON, Canada) at 37°C with 5% CO2 until a complete monolayer was obtained. The BT cellmonolayer was washed twice with antibiotic-free DMEM. Then, 2 ml of antibiotic-free DMEM was addedto each well, and the plates were incubated at 37°C with 5% CO2 for 1 h before inoculation with bacteria.Eighteen-hour cultures of bacterial isolates were diluted in DMEM to give bacterial concentrations ofapproximately 1 � 109 CFU/ml. Then, 200 �l of the bacterial suspension was pipetted into each well ofcell monolayers to achieve 1 � 108 CFU bacterial cells per 105 BT cells per well. The plates were incubatedfor 3 h at 37°C with 5% CO2. After 3 h of incubation, unbound bacterial cells were removed by washingfour times with DMEM. The monolayers were then lysed with 0.1% Triton X-100 in DPBS for 30 min atroom temperature on an orbital shaker. The detached bacterial cells were aspirated and serially dilutedwith DPBS and then plated onto Lactobacillus MRS agar medium. The plates were incubated for 24 to 48
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h at 37°C with 5% CO2, and colonies were counted (CFU per milliliter). The assay was performed threetimes in independent experiments on different days.
Antagonistic competition activity of commensals against M. haemolytica on BT cells. A total of15 isolates from 3 different genera (Lactobacillus, n � 12; Enterococcus, n � 1; and Staphylococcus, n � 2)that had adherence values of �5 log10 CFU/105 BT cells were evaluated in competition assays against M.haemolytica. Antagonistic competition of M. haemolytica was performed using a method describedpreviously (18) with some modifications. Monolayers of BT cells (1 � 105 cells per well) on 6-well plateswere washed twice with DMEM and then incubated with 2 ml of antibiotic-free DMEM at 37°C with 5%CO2 for 1 h before inoculation of bacteria. M. haemolytica and probiotic bacteria suspended in DMEM toachieve individual concentrations of 1 � 108 CFU/ml/well were simultaneously added to the well, and theplates were incubated for 1 h at 37°C with 5% CO2. For control wells, only M. haemolytica was added. Atthe end of the experiment, the cell monolayers were washed and lysed as described above, and the adherentM. haemolytica cells were enumerated by plating onto blood agar plates supplemented with 15 �g/mlbacitracin to inhibit Gram-positive bacteria. The reduction in M. haemolytica cells adhering to BT cells wascalculated as follows: enumerated M. haemolytica cells in control wells (B0 [CFU]) � enumerated M. haemo-lytica cells from wells coinoculated with commensal bacteria (B1 [CFU])/B0 � 100%. The competition assay wasreplicated six times with a minimum of four different culture days for the cell line.
Fluorescence microscopy of bacteria adhering to BT cells. Bovine turbinate cells were seededonto two-well chamber slides (Nunc Lab-Tek II chamber slide system; Sigma) and incubated under thegrowth conditions described above until confluent. The BT cell monolayers were washed three timeswith DMEM (1 ml each time) and then stained by adding 1 ml of antibiotic-free DMEM (prewarmed) with5 �l DAPI (4’,6-diamidino-2-phenylindole dihydrochloride) (Molecular Probes, Inc., Eugene, OR). Theslides were gently rotated to mix the DAPI stain in each chamber, followed by incubation at 37°C, 5% CO2
for 90 min. After incubation, the cell monolayers were washed three times with DMEM to remove unboundDAPI stain. One milliliter of DMEM was then added to each well and incubated for 30 min before the additionof labeled bacteria. For bacteria, 500-�l aliquots of cultures grown for 18 h were centrifuged at 8,000 � g for5 min, and the pellets were resuspended with phosphate-buffered saline (PBS) prior to fluorescence-labelingreactions. Mannheimia haemolytica was labeled with Alexa Fluor 488 Microscale Protein, and Lactobacillusstrains were labeled with Alexa Fluor 594 microscale protein (Molecular Probes, Inc., Eugene, OR) accordingto the manufacturer’s instructions. Lactobacillus strains were either added to the stained BT cells alone tovisualize adherence or in combination with M. haemolytica to visualize competition. Labeled bacterial cellswere suspended in DMEM to achieve concentrations of 1 � 108 CFU/ml/well per bacterium prior to additionto wells of DAPI-stained BT cell monolayers and incubated for 1 h. Subsequently, the cell monolayers werewashed four times with 1 ml DMEM to remove all unbound bacterial cells. The cell monolayers were fixed with2% paraformaldehyde solution (diluted in PBS) and examined using an Olympus Fluoview FV1000 laserconfocal scanning microscope.
Evaluation of antibiotic susceptibilities of selected isolates. A total of 15 isolates that wereevaluated in the competition assays were analyzed for their antimicrobial susceptibilities. MICs of 20antibiotics were determined by the microdilution method (Sensitre; Thermo Fisher Scientific, Nepean,ON, Canada) using a commercially available panel (YSTP6F; Trek Diagnostic Systems, Cleveland, OH, USA).The antimicrobial susceptibility testing was performed according to the procedures recommended forthe YSTP6F panel with the exception that Lactobacillus MRS broth was used for Lactobacillus strains thatdid not grow well in cation-adjusted Mueller-Hinton broth with lysed horse blood. The antimicrobials andthe range of concentrations tested are listed in Table 2. Isolates were inoculated into plates using aSensititre AIM delivery system (Sensitre; Thermo Fisher Scientific), and after incubation, the plates wereevaluated with a Vision imager (Sensitre; Thermo Fisher Scientific). The reference strain Lactobacillusplantarum NCDO1193 served as the quality control.
For the Lactobacillus sp. strains, the interpretation of the MIC values of clindamycin (resistant; �2 �g/ml),daptomycin (susceptible; �4 �g/ml), erythromycin (resistant; �8 �g/ml), linezolid (susceptible; �4 �g/ml),meropenem (resistant; �4 �g/ml), penicillin (susceptible; �8 �g/ml), and vancomycin (resistant; �16 �g/ml)were based on the interpretive criteria provided by CLSI document M45 (65). The L. paracasei (�4 �g/ml) andL. plantarum (�32 �g/ml) strains were defined as resistant to tetracycline according to the breakpointsprovided by the European Food Safety Authority (EFSA) (66). The breakpoints provided by the EFSA (65) werealso used to define L. paracasei (�4 �g/ml) and L. plantarum (�8 �g/ml) strains as resistant to chloramphen-icol. For L. buchneri strains, resistance to chloramphenicol (�4 �g/ml) and erythromycin (�1 �g/ml) weredetermined according to the guidance of the FEEDAP Panel (67), while the breakpoint for resistance totetracycline (128 �g/ml) was determined according to the methods of Feichtinger et al. (42).
For E. faecium, resistance breakpoints for tetracycline (�4 �g/ml), chloramphenicol (�16 �g/ml),vancomycin (�4 �g/ml), and erythromycin (�4 �g/ml) were determined according to EFSA guidelines(66). The breakpoints provided by CLSI supplement VET01S (68) were used to define Staphylococcusstrains (6E and 28C) as resistant to penicillin (�2 �g/ml; horse), amoxicillin-clavulanate (�1/0.5; cat),vancomycin (�16 �g/ml; human), clindamycin (�4 �g/ml; dog), erythromycin (�8 �g/ml; human),chloramphenicol (�32 �g/ml; human), and tetracycline (1 �g/ml; dog). At the time of the experiment,there were no breakpoints or interpretive criteria provided by CLSI or in other literature for the antibioticsazithromycin, cefepime, cefotaxime, ceftriaxone, cefuroxime, ertapenem, levofloxacin, moxifloxacin,tigecycline, and trimethoprim-sulfamethoxazole.
Effects of Lactobacillus sp. isolates on the expression of genes associated with adaptive andinnate immune responses in BT cells. A total of 10 selected commensal isolates from Lactobacillus spp.were evaluated for their effects on the expression of genes associated with adaptive and innate immuneresponses in BT cell monolayers. These isolates were selected based on their ability to compete against
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M. haemolytica for adherence to BT cells and their antimicrobial susceptibility phenotypes. The BT cellswere seeded onto 12-well flat-bottom tissue culture plates at 1 � 104 cells per well and incubated usingthe standard culture conditions described above. BT cell monolayers were washed twice with antibiotic-free DMEM and then incubated with 1 ml antibiotic-free DMEM at 37°C with 5% CO2 for 1 h beforeinoculation with bacteria. M. haemolytica or commensal bacteria were suspended individually in DMEMand added to BT cells to achieve a concentration of 1 � 107 CFU per well; then, the plates were incubatedfor 6 h at 37°C with 5% CO2. Controls included BT cells without addition of bacteria. At the end of theexperiment, the cell monolayers were washed four times with DMEM and then lysed with 350 �l of RLTbuffer (RNeasy Mini kit; Qiagen, Valencia, CA) for 10 min at room temperature on an orbital shaker. Thecell lysates were aspirated and immediately stored at �80°C for further analysis. The immune stimulationassay was performed four times in independent experiments on different days.
Total RNA from the BT cell lysates was extracted using an RNeasy Mini kit (Qiagen) according to themanufacturer’s instructions. The integrity of the extracted total RNA was evaluated with an AgilentBioanalyzer using an RNA 6000 Nano LabChip (Agilent Technologies, Waldbronn, Germany). Total RNAfrom each sample (0.5 �g each; RNA integrity number [RIN] � 7) was reverse transcribed into cDNA usingan RT2 first-strand kit (Qiagen). The cow innate and adaptive immunity response RT2 Profiler PCR array(Qiagen) with 84 test genes related to host response to bacterial infection and sepsis was used toevaluate the effects of bacteria on gene expression. Real-time PCR was performed using RT2 SYBR greenMastermix (Qiagen) and a CFX Connect real-time system (Bio-Rad, Hercules, CA). Real-time PCR condi-tions were according to the PCR array manufacturer’s manual (Qiagen). Data normalization was per-formed with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin beta (ACTB) house-keeping genes, and the fold change in gene expression was calculated using the 2�ΔΔCT method (69).
M. haemolytica-inhibitory mechanisms of 6 Lactobacillus isolates as candidate bacterial ther-apeutics. Based on the rankings of selection criteria (Fig. 1), 6 Lactobacillus strains from four differentspecies (L. amylovorus, L. buchneri [n � 2], L. curvatus, and L. paracasei [n � 2]) were selected as the besttherapeutic candidate strains. The potential inhibitory mechanisms by which the selected strains inhibitM. haemolytica were investigated by testing their abilities to produce lactic acid and H2O2 and byscreening their genomes for bacteriocin-encoding genes. In addition, their effects on the cell morphol-ogy of M. haemolytica were evaluated.
Determination of lactic acid production and antimicrobial activity of lactic acid against M.haemolytica. The selected Lactobacillus strains were inoculated individually into 5 ml MRS broth at an opticaldensity at 610 nm (OD610) of 0.05 and incubated aerobically at 37°C with shaking at 200 rpm for 24 h. Inaddition, all the strains were combined in equal concentrations (OD610 � 0.05) and grown similarly toindividual strains. MRS broth without any Lactobacillus cells was used as a control. After incubation, super-natants were collected from 1.5 ml of culture by centrifugation (10,000 � g; 5 min), followed by filteringthrough a 0.22-�m syringe filter to remove bacterial cells. Metaphosphoric acid (25% [vol/wt]) was added tothe cell culture supernatant at a ratio of 1:5 and was mixed and immediately stored at �20°C until analysis.The concentrations of lactic acid (DL-lactic acid) in the cell-free supernatants were measured using a 5890Agas-liquid chromatograph (Phenomenex, Torrance, CA, USA) as described by Wang et al. (70).
To determine whether the observed lactic acid range produced by these selected Lactobacillus strainscould inhibit the growth of M. haemolytica, M. haemolytica was grown in brain heart infusion (BHI) mediumsupplemented with different concentrations of lactic acid. A 100-�l overnight M. haemolytica culture wasinoculated into 2.5 ml BHI medium containing 0, 9.38, 18.75, 37.5, 75, 100, or 150 mM lactic acid (DL-lactic acidlithium salt; Sigma-Aldrich Canada, Oakville, ON, Canada), and incubated at 37°C, 200 rpm, for 24 h. After 0,8, and 24 h of incubation, the cultures were serially diluted with DPBS (pH 7.4) and plated onto TSA blood agarplates. The plates were incubated at 37°C for 24 h to determine the number of CFU.
Determination of H2O2 production. Aliquots of cell-free culture supernatants of the 6 selectedstrains prepared for lactic acid production were subjected to H2O2 measurement. A hydrogen peroxideassay kit (colorimetric/fluorometric; ab102500; Abcam Inc., Toronto, ON, Canada) was used according tothe manufacturer’s instructions.
Whole-genome sequencing and screening of bacteriocin-encoding genes. Whole-genome se-quencing was performed on the selected strains. The details of genomic DNA extraction, whole-genomesequencing, and analysis were described previously (71). A Web-based tool, BAGEL 4 (72), was used to searchfor the genes encoding bacteriocin from the genomes of the Lactobacillus strains as described by Flórez andMayo (73).
Scanning electron microscopy. The effects of selected Lactobacillus strains on the cell morphologyof M. haemolytica were evaluated using SEM. A single colony of each Lactobacillus strain was inoculatedinto 5 ml BHI and incubated for 24 h in BHI (37°C; 200 rpm). For a negative control, BHI containing nocells was used. After incubation, the cell culture supernatants were obtained as described above. A100-�l overnight M. haemolytica culture (1 � 108 to 2 � 108 CFU per ml) was centrifuged at 10,000 � gfor 5 min, and the cell pellets were suspended with 1 ml cell culture supernatants obtained from theLactobacillus strains and incubated for 10 h (37°C; 200 rpm) before harvesting cells. The treated M.haemolytica cells were centrifuged at 10,000 � g for 5 min, and the cell pellets were fixed with 4%glutaraldehyde. Further sample processing and SEM imaging procedures were described previously (74).
Statistical analysis. Competition assay data were analyzed as a one-way analysis of variance(ANOVA) using Proc Glimmix in SAS (version 9.4; SAS Institute Inc., Cary, NC). The LSMEANS statementwas used to compare the group means. The difference in gene expression between BT cells (control) thatwere not cocultured with bacterial cells and the BT cells cocultured with bacterial cells was assessed byStudent’s t test for each gene using the Rt2 Profiler PCR array analysis software, version 3.5 (Qiagen). Thelevel of statistical significance was set at a P value of �0.05.
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ACKNOWLEDGMENTSWe thank Grant Duke at the Lethbridge Research and Development Centre, Agri-
culture Agri-Food Canada, for assisting with confocal and SEM imaging. We also thankDevin Holman for his contribution to the whole-genome sequencing data analysis,Darell Vedres for his technical support in the GC analysis, and Pam Caffyn for herassistance with hydrogen peroxide assays.
This work was financially supported by Alberta Livestock and Meat Agency Ltd. andAgriculture and Agri-Food Canada. S.A. was the recipient of a Canadian Natural Scienceand Engineering Research Council (NSERC) Doctoral Scholarship.
REFERENCES1. Taylor JD, Fulton RW, Lehenbauer TW, Step DL, Confer AW. 2010. The
epidemiology of bovine respiratory disease: what is the evidence forpredisposing factors? Can Vet J 51:1095–1102.
2. Griffin D, Chengappa MM, Kuszak J, McVey DS. 2010. Bacterial pathogensof the bovine respiratory disease complex. Vet Clin North Am Food AnimPract 26:381–394. https://doi.org/10.1016/j.cvfa.2010.04.004.
3. Rice J, Carrasco-Medina L, Hodgins D, Shewen P. 2007. Mannheimia hae-molytica and bovine respiratory disease. Anim Health Res Rev 8:117–128.https://doi.org/10.1017/S1466252307001375.
4. Ives SE, Richeson JT. 2015. Use of antimicrobial metaphylaxis for the controlof bovine respiratory disease in high-risk cattle. Vet Clin North Am FoodAnim Pract 31:341–350. https://doi.org/10.1016/j.cvfa.2015.05.008.
5. Nickell JS, White BJ. 2010. Metaphylactic antimicrobial therapy for bo-vine respiratory disease in stocker and feedlot cattle. Vet Clin North AmFood Anim Pract 26:285–301. https://doi.org/10.1016/j.cvfa.2010.04.006.
6. Portis E, Lindeman C, Johansen L, Stoltman G. 2012. A ten-year (2000–2009)study of antimicrobial susceptibility of bacteria that cause bovine respira-tory disease complex—Mannheimia haemolytica, Pasteurella multocida, andHistophilus somni—in the United States and Canada. J Vet Diagn Invest24:932–944. https://doi.org/10.1177/1040638712457559.
7. Timsit E, Hallewell J, Booker C, Tison N, Amat S, Alexander TW. 2017.Prevalence and antimicrobial susceptibility of Mannheimia haemolytica,Pasteurella multocida, and Histophilus somni isolated from the lowerrespiratory tract of healthy feedlot cattle and those diagnosed withbovine respiratory disease. Vet Microbiol 208:118 –125. https://doi.org/10.1016/j.vetmic.2017.07.013.
8. Anholt RM, Klima C, Allan N, Matheson-Bird H, Schatz C, Ajitkumar P,Otto SJ, Peters D, Schmid K, Olson M, McAllister T, Ralston B. 2017.Antimicrobial susceptibility of bacteria that cause bovine respiratorydisease complex in Alberta, Canada. Front Vet Sci 4:207. https://doi.org/10.3389/fvets.2017.00207.
9. Snyder E, Credille B, Berghaus R, Giguere S. 2017. Prevalence of multi-drug antimicrobial resistance in isolated from high-risk stocker cattle atarrival and two weeks after processing. J Anim Sci 95:1124 –1131. https://doi.org/10.2527/jas.2016.1110.
10. Man WH, de Steenhuijsen Piters WA, Bogaert D. 2017. The microbiota ofthe respiratory tract: gatekeeper to respiratory health. Nat Rev Microbiol15:259 –270. https://doi.org/10.1038/nrmicro.2017.14.
11. Timsit E, Holman DB, Hallewell J, Alexander TW. 2016. The nasopharyn-geal microbiota in feedlot cattle and its role in respiratory health. AnimFront 6:44 –50. https://doi.org/10.2527/af.2016-0022.
12. Holman DB, Timsit E, Amat S, Abbott DW, Buret AG, Alexander TW. 2017.The nasopharyngeal microbiota of beef cattle before and after transport toa feedlot. BMC Microbiol 17:70. https://doi.org/10.1186/s12866-017-0978-6.
13. Hall JA, Isaiah A, Estill CT, Pirelli GJ, Suchodolski JS. 2017. Weaned beefcalves fed selenium-biofortified alfalfa hay have an enriched nasal mi-crobiota compared with healthy controls. PLoS One 12:e0179215.https://doi.org/10.1371/journal.pone.0179215.
14. Holman DB, Timsit E, Booker CW, Alexander TW. 2018. Injectable anti-microbials in commercial feedlot cattle and their effect on the nasopha-ryngeal microbiota and antimicrobial resistance. Vet Microbiol 214:140 –147. https://doi.org/10.1016/j.vetmic.2017.12.015.
15. Holman DB, McAllister TA, Topp E, Wright AD, Alexander TW. 2015. Thenasopharyngeal microbiota of feedlot cattle that develop bovine respi-ratory disease. Vet Microbiol 180:90 –95. https://doi.org/10.1016/j.vetmic.2015.07.031.
16. Zeineldin MM, Lowe JF, Grimmer ED, de Godoy MRC, Ghanem MM, AbdEl-Raof YM, Aldridge BM. 2017. Relationship between nasopharyngeal and
17. Holman DB, Timsit E, Alexander TW. 2015. The nasopharyngeal microbiotaof feedlot cattle. Sci Rep 5:15557. https://doi.org/10.1038/srep15557.
18. Amat S, Subramanian S, Timsit E, Alexander TW. 2017. Probiotic bacteria inhibitthe bovine respiratory pathogen Mannheimia haemolytica serotype 1 in vitro.Lett Appl Microbiol 64:343–349. https://doi.org/10.1111/lam.12723.
19. Kalischuk LD, Inglis GD. 2011. Comparative genotypic and pathogenic exam-ination of Campylobacter concisus isolates from diarrheic and non-diarrheichumans. BMC Microbiol 11:53. https://doi.org/10.1186/1471-2180-11-53.
20. Shewale RN, Sawale PD, Khedkar CD, Singh A. 2014. Selection criteria forprobiotics: a review. Int J Probiotics Prebiotics 9:17–22.
21. Timsit E, Workentine M, van der Meer F, Alexander T. 2018. Distinct bacterialmetacommunities inhabit the upper and lower respiratory tracts of healthyfeedlot cattle and those diagnosed with bronchopneumonia. Vet Microbiol221:105–113. https://doi.org/10.1016/j.vetmic.2018.06.007.
22. Elshaghabee FMF, Rokana N, Gulhane RD, Sharma C, Panwar H. 2017.Bacillus as potential probiotics: status, concerns, and future perspectives.Front Microbiol 8:1490. https://doi.org/10.3389/fmicb.2017.01490.
23. Borah D, Gogoi O, Adhikari C, Kakoti BB. 2016. Isolation and character-ization of the new indigenous Staphylococcus sp. DBOCP06 as a probi-otic bacterium from traditionally fermented fish and meat products ofAssam state. Egyptian J Basic Appl Sci 3:232–240. https://doi.org/10.1016/j.ejbas.2016.06.001.
24. Corbeil LB, Woodward W, Ward AC, Mickelsen WD, Paisley L. 1985.Bacterial interactions in bovine respiratory and reproductive infections.J Clin Microbiol 21:803– 807.
25. Pridmore RD, Pittet AC, Praplan F, Cavadini C. 2008. Hydrogen peroxideproduction by Lactobacillus johnsonii NCC 533 and its role in anti�Sal-monella activity. FEMS Microbiol Lett 283:210 –215. https://doi.org/10.1111/j.1574-6968.2008.01176.x.
26. Popova M, Molimard P, Courau S, Crociani J, Dufour C, Le Vacon F, CartonT. 2012. Beneficial effects of probiotics in upper respiratory tract infectionsand their mechanical actions to antagonize pathogens. J Appl Microbiol113:1305–1318. https://doi.org/10.1111/j.1365-2672.2012.05394.x.
27. Verna EC, Lucak S. 2010. Use of probiotics in gastrointestinal disorders:what to recommend? Therap Adv Gastroenterol 3:307–319. https://doi.org/10.1177/1756283X10373814.
28. Behnsen J, Deriu E, Sassone-Corsi M, Raffatellu M. 2013. Probiotics:properties, examples, and specific applications. Cold Spring Harb Per-spect Med 3:a010074. https://doi.org/10.1101/cshperspect.a010074.
29. Frank GH, Briggs RE. 1992. Colonization of the tonsils of calves withPasteurella haemolytica. Am J Vet Res 53:481– 484.
30. Goh HMS, Yong MHA, Chong KLL, Kline KA. 2017. Model systems for thestudy of Enterococcal colonization and infection. Virulence 8:1525–1562.https://doi.org/10.1080/21505594.2017.1279766.
31. Timsit E, Workentine M, Schryvers AB, Holman DB, van der Meer F,Alexander TW. 2016. Evolution of the nasopharyngeal microbiota of beefcattle from weaning to 40 days after arrival at a feedlot. Vet Microbiol187:75– 81. https://doi.org/10.1016/j.vetmic.2016.03.020.
32. Van Tassell ML, Miller M. 2011. Lactobacillus adhesion to mucus. Nutri-ents 3:613– 636. https://doi.org/10.3390/nu3050613.
33. Saroj SD, Maudsdotter L, Tavares R, Jonsson AB. 2016. Lactobacilli interferewith Streptococcus pyogenes hemolytic activity and adherence to host epithelialcells. Front Microbiol 7:1176. https://doi.org/10.3389/fmicb.2016.01176.
34. Lebeer S, Vanderleyden J, De Keersmaecker SC. 2008. Genes and mole-cules of lactobacilli supporting probiotic action. Microbiol Mol Biol Rev72:728 –764. https://doi.org/10.1128/MMBR.00017-08.
35. Duary RK, Rajput YS, Batish VK, Grover S. 2011. Assessing the adhesion of
Amat et al. Applied and Environmental Microbiology
November 2019 Volume 85 Issue 21 e01359-19 aem.asm.org 20
putative indigenous probiotic lactobacilli to human colonic epithelial cells.Indian J Med Res 134:664–671. https://doi.org/10.4103/0971-5916.90992.
36. Lee YK, Lim CY, Teng WL, Ouwehand AC, Tuomola EM, Salminen S. 2000.Quantitative approach in the study of adhesion of lactic acid bacteria tointestinal cells and their competition with enterobacteria. Appl EnvironMicrobiol 66:3692–3697. https://doi.org/10.1128/AEM.66.9.3692-3697.2000.
37. Bermudez-Brito M, Plaza-Díaz J, Muñoz-Quezada S, Gómez-Llorente C,Gil A. 2012. Probiotic mechanisms of action. Ann Nutr Metab 61:160 –174. https://doi.org/10.1159/000342079.
38. Nagpal R, Wang S, Ahmadi S, Hayes J, Gagliano J, Subashchandrabose S,Kitzman DW, Becton T, Read R, Yadav H. 2018. Human-origin probioticcocktail increases short-chain fatty acid production via modulation ofmice and human gut microbiome. Sci Rep 8:12649. https://doi.org/10.1038/s41598-018-30114-4.
39. MacPherson CW, Shastri P, Mathieu O, Tompkins TA, Burguière P 2017.Genome-wide immune modulation of TLR3-mediated inflammation in intesti-nal epithelial cells differs between single and multi-strain probiotic combina-tion. PLoS One 12:e0169847. https://doi.org/10.1371/journal.pone.0169847.
40. Bajagai YS, Klieve AV, Dart PJ, Bryden WL. 2016. Probiotics in animalnutrition—production, impact and regulation, p 45–52. In Makkar HPS.FAO animal production and health paper no. 179. FAO, Rome, Italy.
41. Gueimonde M, Sánchez B, de Los Reyes-Gavilán CG, Margolles A. 2013.Antibiotic resistance in probiotic bacteria. Front Microbiol 4:202. https://doi.org/10.3389/fmicb.2013.00202.
42. Feichtinger M, Mayrhofer S, Kneifel W, Domig KJ. 2016. Tetracyclineresistance patterns of Lactobacillus buchneri group strains. J Food Prot79:1741–1747. https://doi.org/10.4315/0362-028X.JFP-15-577.
43. EFSA Panel on Additives and Products or Substances used in AnimalFeed, Rychen G, Aquilina G, Azimonti G, Bampidis V, Bastos ML, Bories G,Chesson A, Cocconcelli PS, Flachowsky G, Gropp J, Kolar B, Kouba M,Lopez-Alonso M, Lopez Puente S, Mantovani A, Mayo B, Ramos F, VillaRE, Wallace RJ, Wester P, Brozzi R, Saarela M. 2017. Scientific opinion onthe safety and efficacy of Lactobacillus buchneri NRRL B-50733 as asilage additive for all animal species. EFSA J 15:4934 – 4939. https://doi.org/10.2903/j.efsa.2017.4934.
44. Hummel AS, Hertel C, Holzapfel WH, Franz C. 2007. Antibiotic resistancesof starter and probiotic strains of lactic acid bacteria. Appl EnvironMicrobiol 73:730 –739. https://doi.org/10.1128/AEM.02105-06.
45. Karapetkov N, Georgieva R, Rumyan N, Karaivanova E. 2011. Antibioticsusceptibility of different lactic acid bacteria strains. Benef Microbe2:335–339. https://doi.org/10.3920/BM2011.0016.
46. Gao W, Howden BP, Stinear TP. 2018. Evolution of virulence in Entero-coccus faecium, a hospital-adapted opportunistic pathogen. Curr OpinMicrobiol 41:76 – 82. https://doi.org/10.1016/j.mib.2017.11.030.
47. Bernardeau M, Guguen M, Vernoux JP. 2006. Beneficial lactobacilli infood and feed: long-term use, biodiversity and proposals for specific andrealistic safety assessments. FEMS Microbiol Rev 30:487–513. https://doi.org/10.1111/j.1574-6976.2006.00020.x.
48. Yan F, Polk DB. 2011. Probiotics and immune health. Curr Opin Gastro-enterol 27:496 –501. https://doi.org/10.1097/MOG.0b013e32834baa4d.
49. Ackermann MR, Derscheid R, Roth JA. 2010. Innate immunology ofbovine respiratory disease. Vet Clin North Am Food Anim Pract 26:215–228. https://doi.org/10.1016/j.cvfa.2010.03.001.
50. Jundi K, Greene CM. 2015. Transcription of interleukin-8: how alteredregulation can affect cystic fibrosis lung disease. Biomolecules5:1386 –1398. https://doi.org/10.3390/biom5031386.
51. Gärtner MA, Bondzio A, Braun N, Jung M, Einspanier R, Gabler C. 2015.Detection and characterisation of Lactobacillus spp. in the bovine uterusand their influence on bovine endometrial epithelial cells in vitro. PLoSOne 10:e0119793. https://doi.org/10.1371/journal.pone.0119793.
52. Guillot L, Medjane S, Le Barillec K, Balloy V, Danel C, Chignard M, Si-TaharM. 2004. Response of human pulmonary epithelial cells to lipopolysac-charide involves Toll-like receptor 4 (TLR4)-dependent signalingpathways: evidence for an intracellular compartmentalization of TLR4. JBiol Chem 279:2712–2718. https://doi.org/10.1074/jbc.M305790200.
53. Zecchinon L, Fett T, Desmecht D. 2005. How Mannheimia haemolyticadefeats host defense through a kiss of death mechanism. Vet Res 36:133–156. https://doi.org/10.1051/vetres:2004065.
54. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, Morelli L, CananiRB, Flint HJ, Salminen S, Calder PC, Sanders ME. 2014. The InternationalScientific Association for Probiotics and Prebiotics consensus statement onthe scope and appropriate use of the term probiotic. Nat Rev GastroenterolHepatol 11:506–514. https://doi.org/10.1038/nrgastro.2014.66.
55. O’toole PW, Marchesi JR, Hill C. 2017. Next-generation probiotics: thespectrum from probiotics to live biotherapeutics. Nat Microbiol 2:17057.https://doi.org/10.1038/nmicrobiol.2017.57.
56. Neal-McKinney JM, Lu X, Duong T, Larson CL, Call DR, Shah DH, KonkelME. 2012. Production of organic acids by probiotic lactobacilli can beused to reduce pathogen load in poultry. PLoS One 7:e43928. https://doi.org/10.1371/journal.pone.0043928.
57. Parada JL, Caron CR, Medeiros ABP, Soccol CR. 2007. Bacteriocinsfrom lactic acid bacteria: purification, properties and use as bio-preservatives. Braz Arch Biol Technol 50:521–542. https://doi.org/10.1590/S1516-89132007000300018.
58. Mokoena MP. 2017. Lactic acid bacteria and their bacteriocins: classifi-cation, biosynthesis and applications against uropathogens: a mini-review. Molecules 22:1255. https://doi.org/10.3390/molecules22081255.
59. Collins FWJ, O’Connor PM, O’Sullivan O, Gómez-Sala B, Rea MC, Hill C, RossRP. 2017. Bacteriocin gene-trait matching across the complete Lactobacilluspan-genome. Sci Rep 7:3481. https://doi.org/10.1038/s41598-017-03339-y.
60. Surachat K, Sangket U, Deachamag P, Chotigeat W. 2017. In silicoanalysis of protein toxin and bacteriocins from Lactobacillus paracaseiSD1 genome and available online databases. PLoS One 12:e0183548.https://doi.org/10.1371/journal.pone.0183548.
61. Callewaert R, Holo H, Devreese B, Van Beeumen J, Nes I, De Vuyst L.1999. Characterization and production of amylovorin L471, a bacte-riocin purified from Lactobacillus amylovorus DCE 471 by a novelthree-step method. Microbiology 145:2559 –2568. https://doi.org/10.1099/00221287-145-9-2559.
62. Klima CL, Alexander TW, Hendrick S, McAllister TA. 2014. Characterization ofMannheimia haemolytica isolated from feedlot cattle that were healthy ortreated for bovine respiratory disease. Can J Vet Res 78:38–45.
63. Mattarelli P, Holzapfel W, Franz CMAP, Endo A, Felis GE, Hammes W, PotB, Dicks L, Dellaglio F. 2014. Recommended minimal standards fordescription of new taxa of the genera Bifidobacterium, Lactobacillus andrelated genera. Int J Syst Evol Microbiol 64:1434 –1451. https://doi.org/10.1099/ijs.0.060046-0.
64. Dec M, Puchalski A, Urban-Chmiel R, Wernicki A. 2014. Screening oflactobacillus strains of domestic goose origin against bacterial poultrypathogens for use as probiotics. Poult Sci 93:2464 –2472. https://doi.org/10.3382/ps.2014-04025.
65. CLSI. 2016. Methods for antimicrobial dilution and disk susceptibilitytesting of infrequently isolated or fastidious bacteria; approved guide-line, 3rd ed. CLSI document M45. CLSI, Wayne, PA.
66. European Food Safety Authority. 2012. Guidance on the assessment ofbacterial susceptibility to antimicrobials of human and veterinary im-portance. EFSA J 10:2740.
67. European Commission. 2008. Technical guidance prepared by the Panelon Additives and Products or Substances used in Animal Feed (FEEDAP)on the update of the criteria used in the assessment of bacterial resis-tance to antibiotics of human or veterinary importance. EFSA J 6:1–15.
68. CLSI. 2015. Performance standards for antimicrobial disk and dilutionsusceptibility tests for bacteria isolated from animals, 3rd ed. CLSI sup-plement VET01S. Clinical and Laboratory Standards Institute, Wayne, PA.
69. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression datausing real-time quantitative PCR and the 2(-delta delta C(T)) method.Methods 25:402– 408. https://doi.org/10.1006/meth.2001.1262.
70. Wang Y, Berg BP, Barbieri LR, Veira DM, McAllister TA. 2006. Comparisonof 815 alfalfa and mixed alfalfa-sainfoin pastures for grazing cattle:effects on incidence of 816 bloat, ruminal fermentation, and feed intake.Can J Anim Sci 86:383–392. 817. https://doi.org/10.4141/A06-009.
71. Amat S, Holman DB, Timsit E, Gzyl KE, Alexander TW. 2019. Draft genomesequences of 14 Lactobacillus, Enterococcus, and Staphylococcus isolatesfrom the nasopharynx of healthy feedlot cattle. Microbiol Resour An-nounc 8:e00534-19. https://doi.org/10.1128/MRA.00534-19.
72. van Heel AJ, de Jong A, Song C, Viel JH, Kok J, Kuipers OP. 2018. BAGEL4:a user-friendly web server to thoroughly mine RiPPs and bacteriocins.Nucleic Acids Res 46:W278 –W281. https://doi.org/10.1093/nar/gky383.
73. Flórez AB, Mayo B. 2018. Genome analysis of Lactobacillus plantarum LL441and genetic characterisation of the locus for the lantibiotic plantaricin C.Front Microbiol 9:1916. https://doi.org/10.3389/fmicb.2018.01916.
74. Amat S, Baines D, Timsit E, Hallewell J, Alexander TW. 2019. Essential oilsinhibit the bovine respiratory pathogens Mannheimia haemolytica, Pas-teurella multocida and Histophilus somni and have limited effects oncommensal bacteria and turbinate cells in vitro. J Appl Microbiol 126:1668 –1682. https://doi.org/10.1111/jam.14238.
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November 2019 Volume 85 Issue 21 e01359-19 aem.asm.org 21
