Identification of genetic markers of resistance to macrolide class antibiotics in Mannheimia haemolytica isolates from a Saskatchewan feedlot.

Mannheimia haemolytica is a major contributor to bovine respiratory disease (BRD), which causes substantial economic losses to the beef industry, and there is an urgent need for rapid and accurate diagnostic tests to provide evidence for treatment decisions and support antimicrobial stewardship. Diagnostic sequencing can provide information about antimicrobial resistance genes in M. haemolytica more rapidly than conventional diagnostics. Realizing the full potential of diagnostic sequencing requires a comprehensive understanding of the genetic markers of antimicrobial resistance. We identified genetic markers of resistance in M. haemolytica to macrolide class antibiotics commonly used for control of BRD. Genome sequences were determined for 99 M. haemolytica isolates with six different susceptibility phenotypes collected over 2 years from a feedlot in Saskatchewan, Canada. Known macrolide resistance genes estT, msr(E), and mph(E) were identified in most resistant isolates within predicted integrative and conjugative elements (ICEs). ICE sequences lacking antibiotic resistance genes were detected in 10 of 47 susceptible isolates. No resistance-associated polymorphisms were detected in ribosomal RNA genes, although previously unreported mutations in the L22 and L23 ribosomal proteins were identified in 12 and 27 resistant isolates, respectively. Pangenome analysis led to the identification of 79 genes associated with resistance to gamithromycin, of which 95% (75 of 79) had no functional annotation. Most of the observed phenotypic resistance was explained by previously identified antibiotic resistance genes, although resistance to the macrolides gamithromycin and tulathromycin was not explained in 39 of 47 isolates, demonstrating the need for continued surveillance for novel determinants of macrolide resistance.IMPORTANCEBovine respiratory disease is the costliest disease of beef cattle in North America and the most common reason for injectable antibiotic use in beef cattle. Metagenomic sequencing offers the potential to make economically significant reductions in turnaround time for diagnostic information for evidence-based selection of antibiotics for use in the feedlot. The success of diagnostic sequencing depends on a comprehensive catalog of antimicrobial resistance genes and other genome features associated with reduced susceptibility. We analyzed the genome sequences of isolates of Mannheimia haemolytica, a major bovine respiratory disease pathogen, and identified both previously known and novel genes associated with reduced susceptibility to macrolide class antimicrobials. These findings reinforce the need for ongoing surveillance for markers of antimicrobial resistance to support improved diagnostics and antimicrobial stewardship.

Isolation and Identification of Pasteurella multocida and Mannheimia haemolytica from Pneumonic Small Ruminants and Their Antibiotic Susceptibility in Haramaya District, Eastern Ethiopia.

Background: Pasteurella species are frequently encountered as serious diseases in small ruminants. It is the main cause of respiratory pasteurellosis in sheep and goats of all age groups. Methods: The cross-sectional study was conducted from December 2022 to April 2023 in Haramaya district, eastern Ethiopia, to isolate and identify Pasteurella multocida and Mannheimia haemolytica and estimate their prevalence, associated risk factors, and antimicrobial sensitivity of isolates in small ruminants using a purposive sampling method. A total of 384 samples (156 nasal swabs from clinic cases and 228 lung swabs from abattoir cases) were collected. STATA 14 software was used to analyze the data. In addition, multivariable logistic regression analysis was performed to assess an association of risk factors. Results: Out of the 384 samples examined, 164 were positive for pasteurellosis, resulting in a 42.70% prevalence. Similarly, 63 (38.4%) of the 164 positive results were from nasal swabs, while 101 (61.6%) came from lung samples. M. haemolytica accounted for 126 (76.82%) of the isolates, while P. multocida accounted for 38 (23.17%). Of the 63 nasal swab isolates, 33 (37%) were from goats and 30 (42.8%) were from sheep. And 17 (10.89%) and 46 (29.58%), respectively, were P. multocida and M. haemolytica. Of the 46 (40%) of the 101 (44.3%) isolates of the pneumonic lung, samples were from goats, while 55 (48.47%) were from sheep. In this study, the risk factors (species, age, and body condition score) were found to be significant (p < 0.05). Pasteurella isolates evaluated for antibiotic susceptibility were highly resistant to oxacillin (90.90%), followed by gentamycin (72.72%), and penicillin (63.63%). However, the isolates were highly sensitive to chloramphenicol (90.90%), followed by tetracycline (63.63%), and ampicillin (54.54%). Conclusion: This study showed that M. haemolytica and P. multocida are the common causes of mannheimiosis and pasteurellosis in small ruminants, respectively, and isolates were resistant to commonly used antibiotics in the study area. Thus, an integrated vaccination strategy, antimicrobial resistance monitoring, and avoidance of stress-inducing factors are recommended.

Antimicrobial Activities of α-Helix and ß-Sheet Peptides against the Major Bovine Respiratory Disease Agent, Mannheimia haemolytica.

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in cattle raised in North America. At the feedlot, cattle are subject to metaphylactic treatment with macrolides to prevent BRD, a practice that may promote antimicrobial resistance and has resulted in an urgent need for novel strategies. Mannheimia haemolytica is one of the major bacterial agents of BRD. The inhibitory effects of two amphipathic, α-helical (PRW4, WRL3) and one ß-sheet (WK2) antimicrobial peptides were evaluated against multidrug-resistant (MDR) M. haemolytica isolated from Alberta feedlots. WK2 was not cytotoxic against bovine turbinate (BT) cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. All three peptides inhibited M. haemolytica, with WK2 being the most efficacious against multiple isolates. At 8-16 µg/mL, WK2 was bactericidal against Mh 330 in broth, and at 32 µg/mL in the presence of BT cells, it reduced the population by 3 logs CFU/mL without causing cytotoxic effects. The membrane integrity of Mh 330 was examined using NPN (1-N-phenylnaphthylamine) and ONPG (o-Nitrophenyl ß-D-galactopyranoside), with both the inner and outer membranes being compromised. Thus, WK2 may be a viable alternative to the use of macrolides as part of BRD prevention and treatment strategies.

Limitations of bacterial culture, viral PCR, and tulathromycin susceptibility from upper respiratory tract samples in predicting clinical outcome of tulathromycin control or treatment of bovine respiratory disease in high-risk feeder heifers.

A cross-sectional prospective cohort study including 1026 heifers administered tulathromycin due to high risk of clinical signs of bovine respiratory disease (BRD), measured poor association between BRD clinical outcomes and results of bacterial culture and tulathromycin susceptibility from BRD isolates of deep nasopharyngeal swabs (DNS) and adequate association with viral polymerase chain reaction (PCR) results from nasal swabs. Isolation rates from DNS collected on day-0 and at 1st BRD-treatment respectively were: Mannheimia haemolytica (10.9% & 34.1%); Pasteurella multocida (10.4% & 7.4%); Mycoplasma bovis (1.0% & 36.6%); and Histophilus somni (0.7% & 6.3%). Prevalence of BRD viral nucleic acid on nasal swabs collected exclusively at 1st BRD-treatment were: bovine parainfluenza virus type-3 (bPIV-3) 34.1%; bovine viral diarrhea virus (BVDV) 26.3%; bovine herpes virus type-1 (BHV-1) 10.8%; and bovine respiratory syncytial virus (BRSV) 54.1%. Increased relative risk, at 95% confidence intervals, of 1st BRD-treatment failure was associated with positive viral PCR results: BVDV 1.39 (1.17-1.66), bPIV-3 1.26 (1.06-1.51), BHV-1 1.52 (1.25-1.83), and BRSV 1.35 (1.11-1.63) from nasal swabs collected at 1st BRD-treatment and culture of M. haemolytica 1.23 (1.00-1.51) from DNS collected at day-0. However, in this population of high-risk feeder heifers, the predictive values of susceptible and resistant isolates had inadequate association with BRD clinical outcome. These results indicate, that using tulathromycin susceptibility testing of isolates of M. haemolytica or P. multocida from DNS collected on arrival or at 1st BRD-treatment to evaluate tulathromycin clinical efficacy, is unreliable.

Anti-Inflammatory Properties of Fructo-Oligosaccharides in a Calf Lung Infection Model and in Mannheimia haemolytica-Infected Airway Epithelial Cells.

Emerging antimicrobial-resistant pathogens highlight the importance of developing novel interventions. Here, we investigated the anti-inflammatory properties of Fructo-oligosaccharides (FOS) in calf lung infections and in airway epithelial cells stimulated with pathogens, and/or bacterial components. During a natural exposure, 100 male calves were fed milk replacer with or without FOS for 8 weeks. Then, immune parameters and cytokine/chemokine levels in the bronchoalveolar lavage fluid (BALF) and blood were measured, and clinical scores were investigated. Calf primary bronchial epithelial cells (PBECs) and human airway epithelial cells (A549) were treated with Mannheimia haemolytica, lipopolysaccharides (LPS), and/or flagellin, with or without FOS pretreatment. Thereafter, the cytokine/chemokine levels and epithelial barrier function were examined. Relative to the control (naturally occurring lung infections), FOS-fed calves had greater macrophage numbers in BALF and lower interleukin (IL)-8, IL-6, and IL-1ß concentrations in the BALF and blood. However, FOS did not affect the clinical scores. At slaughter, FOS-fed calves had a lower severity of lung lesions compared to the control. Ex vivo, FOS prevented M. haemolytica-induced epithelial barrier dysfunction. Moreover, FOS reduced M. haemolytica- and flagellin-induced (but not LPS-induced) IL-8, TNF-α, and IL-6 release in PBECs and A549 cells. Overall, FOS had anti-inflammatory properties during the natural incidence of lung infections but had no effects on clinical symptoms.

Zn-Enhanced Asp-Rich Antimicrobial Peptides: N-Terminal Coordination by Zn(II) and Cu(II), Which Distinguishes Cu(II) Binding to Different Peptides.

The antimicrobial activity of surfactant-associated anionic peptides (SAAPs), which are isolated from the ovine pulmonary surfactant and are selective against the ovine pathogen Mannheimia haemolytica, is strongly enhanced in the presence of Zn(II) ions. Both calorimetry and ITC measurements show that the unique Asp-only peptide SAAP3 (DDDDDDD) and its analogs SAAP2 (GDDDDDD) and SAAP6 (GADDDDD) have a similar micromolar affinity for Zn(II), which binds to the N-terminal amine and Asp carboxylates in a net entropically-driven process. All three peptides also bind Cu(II) with a net entropically-driven process but with higher affinity than they bind Zn(II) and coordination that involves the N-terminal amine and deprotonated amides as the pH increases. The parent SAAP3 binds Cu(II) with the highest affinity; however, as shown with potentiometry and absorption, CD and EPR spectroscopy, Asp residues in the first and/or second positions distinguish Cu(II) binding to SAAP3 and SAAP2 from their binding to SAAP6, decreasing the Cu(II) Lewis acidity and suppressing its square planar amide coordination by two pH units. We also show that these metal ions do not stabilize a membrane disrupting ability nor do they induce the antimicrobial activity of these peptides against a panel of human pathogens.

Engineering and characterization of human ß-defensin-3 and its analogues and microcin J25 peptides against Mannheimia haemolytica and bovine neutrophils.

Mannheimia haemolytica-induced bovine respiratory disease causes loss of millions of dollars to Canadian cattle industry. Current antimicrobials are proving to be ineffective and leave residues in meat. Antimicrobial peptides (AMPs) may be effective against M. haemolytica while minimizing the risk of drug residues. Cationic AMPs can kill bacteria through interactions with the anionic bacterial membrane. Human ß-Defensin 3 (HBD3) and microcin J25 (MccJ25) are AMPs with potent activity against many Gram-negative bacteria. We tested the microbicidal activity of wild-type HBD3, three HBD3 peptide analogues (28 amino acid, 20AA, and 10AA) derived from the sequence of natural HBD3, and MccJ25 in vitro against M. haemolytica. Three C-terminal analogues of HBD3 with all cysteines replaced with valines were manually synthesized using solid phase peptide synthesis. Since AMPs can act as chemoattractant we tested the chemotactic effect of HBD3, 28AA, 20AA, and 10AA peptides on bovine neutrophils in Boyden chamber. Minimum bactericidal concentration (MBC) assay showed that M. haemolytica was intermediately sensitive to HBD3, 28AA and 20AA analogues with an MBC of 50 µg/mL. The 10AA analogue had MBC 6.3 µg/mL which is likely a result of lower final inoculum size. MccJ25 didn't have significant bactericidal effect below an MBC < 100 µg/mL. Bovine neutrophils showed chemotaxis towards HBD3 and 20AA peptides (P < 0.05) but not towards 28AA analogue. Co-incubation of neutrophils with any of the peptides did not affect their chemotaxis towards N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP). The data show that these peptides are effective against M. haemolytica and are chemotactic for neutrophils in vitro.

Aldsulfin, a novel unusual anti-mannheimiosis epithiodiketopiperazine antibiotic produced by Lasiodiplodia pseudotheobromae FKI-4499.

An anti-mannheimiosis agent, aldsulfin, was isolated from a culture broth of the fungus Lasiodiplodia pseudotheobromae FKI-4499, together with a known compound, lasiodipline C, using bioassay-guided fractionation. Spectroscopic analysis of aldsulfin, using NMR, mass spectrometry, and CD analyses revealed it to be an epithiodiketopiperazine with an unstable and unusual hemithioaminal moiety. Aldsulfin showed antibacterial activity against Mannheimia haemolytica and Pasteurella multocida.

Antimicrobial resistance in Mannheimia haemolytica: prevalence and impact.

Bovine respiratory disease (BRD) is the most common cause of morbidity and mortality in North American beef cattle. In recent years, isolation of strains of Mannheimia haemolytica that are resistant to multiple different classes of antimicrobials has become commonplace. New research would suggest that the routine use of antimicrobials by some cattle operations might be driving emerging resistance patterns, with the majority of the spread observed due to propagation of strains of M. haemolytica that have acquired integrative conjugative elements. To date, there is little information evaluating the impact of antimicrobial resistance on clinical outcome in cattle with BRD.

Prevalence and temporal trends in antimicrobial resistance of bovine respiratory disease pathogen isolates submitted to the Wisconsin Veterinary Diagnostic Laboratory: 2008-2017.

The objective of this study was to describe the prevalence and trends in antimicrobial resistance for bacterial pathogens associated with bovine respiratory disease (BRD) isolated from samples submitted to the Wisconsin Veterinary Diagnostic Laboratory (WVDL). Data were retrospectively collected from bovine respiratory isolates including Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, and Bibersteinia trehalosi identified at the WVDL between January 2008 and December 2017. Antimicrobial susceptibility testing data were queried from antimicrobial resistance databases at the WVDL. A total of 4,261 isolates were identified. Pasteurella multocida was most frequently identified, accounting for 2,094 isolates (49% of total) over the study period. Mannheimia haemolytica was the second most frequently isolated bacterial respiratory pathogen (n = 1,267, 30%) followed by H. somni (n = 749, 18%) and B. trehalosi (n = 151, 4%). Over the 10-yr period, B. trehalosi had the highest median percentage of isolates that were resistant to at least one antibiotic at 33% (interquartile range: 24, 47) followed by M. haemolytica (13%; 8, 23). For P. multocida, 10% (4, 26) of isolates were classified as resistant to at least one antibiotic, whereas H. somni had the fewest resistant isolates (9%; 3, 15). When comparing 2013-2017 to 2008-2012, the overall percentage of resistant isolates for P. multocida and B. trehalosi decreased, whereas the percentage of resistant isolates for M. haemolytica and H. somni increased. Increased resistance against florfenicol, fluoroquinolones, gentamicin, tilmicosin, and tulathromycin was observed for M. haemolytica. These data show that antimicrobial susceptibility for BRD bacterial pathogens has changed in the population served by the WVDL over this 10-yr period. For P. multocida, resistance is relatively low and has either improved or at least remained constant for the majority of drugs labeled for treatment of respiratory disease in dairy cattle. Veterinarians and producers should be aware of the bacterial pathogens most commonly associated with BRD and work toward early disease detection, proper antibiotic administration, and monitoring lung lesions to ensure that their treatment protocols improve lung health.

Mannheimia haemolytica and Pasteurella multocida in Bovine Respiratory Disease: How Are They Changing in Response to Efforts to Control Them?

The bacteria Mannheimia haemolytica and Pasteurella multocida contribute to bovine respiratory disease (BRD), which is often managed with antimicrobials. Antimicrobial resistance in these bacteria has been rare, but extensively drug-resistant strains have recently become common. Routine antimicrobial use may be driving this resistance. Resistance spread is caused in part by propagation of strains harboring integrative conjugative elements. The impact of antimicrobial resistance on treatment outcomes is not clear, but clinical observations suggest that response to first treatment has decreased over time, possibly because of resistance. Clinicians should consider antimicrobial resistance when designing BRD treatment and control programs.

Effect of apo-lactoferrin on leukotoxin and outer membrane vesicles of Mannheimia haemolytica A2.

Mannheimia haemolytica serotype A2 is the principal cause of pneumonic mannheimiosis in ovine and caprine livestock; this disease is a consequence of immune suppression caused by stress and associated viruses and is responsible for significant economic losses in farm production worldwide. Gram-negative bacteria such as M. haemolytica produce outer membrane (OM)-derived spherical structures named outer membrane vesicles (OMVs) that contain leukotoxin and other biologically active virulence factors. In the present study, the relationship between M. haemolytica A2 and bovine lactoferrin (BLf) was studied. BLf is an 80 kDa glycoprotein that possesses bacteriostatic and bactericidal properties and is part of the mammalian innate immune system. Apo-BLf (iron-free) showed a bactericidal effect against M. haemolytica A2, with an observed minimal inhibitory concentration (MIC) of 16 µM. Sublethal doses (2-8 µM) of apo-BLf increased the release of OMVs, which were quantified by flow cytometry. Apo-BLf modified the normal structure of the OM and OMVs, as observed through transmission electron microscopy. Apo-BLf also induced lipopolysaccharide (LPS) release from bacteria, disrupting OM permeability and functionality, as measured by silver staining and SDS and polymyxin B cell permeability assays. Western blot results showed that apo-BLf increased the secretion of leukotoxin in M. haemolytica A2 culture supernatants, possibly through its iron-chelating activity. In contrast, holo-BLf (with iron) did not have this effect, possibly due to differences in the tertiary structure between these proteins. In summary, apo-BLf affected the levels of several M. haemolytica virulence factors and could be evaluated for use in animals as an adjuvant in the treatment of ovine mannheimiosis.

Association between antimicrobial drug class for treatment and retreatment of bovine respiratory disease (BRD) and frequency of resistant BRD pathogen isolation from veterinary diagnostic laboratory samples.

Although 90% of BRD relapses are reported to receive retreatment with a different class of antimicrobial, studies examining the impact of antimicrobial selection (i.e. bactericidal or bacteriostatic) on retreatment outcomes and the emergence of antimicrobial resistance (AMR) are deficient in the published literature. This survey was conducted to determine the association between antimicrobial class selection for treatment and retreatment of BRD relapses on antimicrobial susceptibility of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. Pathogens were isolated from samples submitted to the Iowa State University Veterinary Diagnostic Laboratory from January 2013 to December 2015. A total of 781 isolates with corresponding animal case histories, including treatment protocols, were included in the analysis. Original susceptibility testing of these isolates for ceftiofur, danofloxacin, enrofloxacin, florfenicol, oxytetracycline, spectinomycin, tilmicosin, and tulathromycin was performed using Clinical and Laboratory Standards Institute guidelines. Data were analyzed using a Bayesian approach to evaluate whether retreatment with antimicrobials of different mechanistic classes (bactericidal or bacteriostatic) increased the probability of resistant BRD pathogen isolation in calves. The posterior distribution we calculated suggests that an increased number of treatments is associated with a greater probability of isolates resistant to at least one antimicrobial. Furthermore, the frequency of resistant BRD bacterial isolates was greater with retreatment using antimicrobials of different mechanistic classes than retreatment with the same class. Specifically, treatment protocols using a bacteriostatic drug first followed by retreatment with a bactericidal drug were associated with a higher frequency of resistant BRD pathogen isolation. In particular, first treatment with tulathromycin (bacteriostatic) followed by ceftiofur (bactericidal) was associated with the highest probability of resistant M. haemolytica among all antimicrobial combinations. These observations suggest that consideration should be given to antimicrobial pharmacodynamics when selecting drugs for retreatment of BRD. However, prospective studies are needed to determine the clinical relevance to antimicrobial stewardship programs in livestock production systems.

Effect of tracheal antimicrobial peptide on the development of Mannheimia haemolytica pneumonia in cattle.

Bacterial pneumonia causes significant economic loss to the beef industry and occurs at times of stress and viral infection. Administering antibiotics to at-risk calves is often used to prevent the disease, but alternatives to mass treatment with antibiotics are needed. Tracheal antimicrobial peptide (TAP), a ß-defensin naturally produced by bovine airways, has bactericidal activity against the pathogens that cause pneumonia in cattle. However, TAP expression is suppressed by glucocorticoid (stress) and viral infection. We hypothesized that delivering TAP to the respiratory tract would prevent development of pneumonia in calves infected with Mannheimia haemolytica. Clean-catch calves (i.e. obtained prior to contact with the dam) were challenged by aerosol with M. haemolytica, and TAP or water was delivered to the respiratory tract at 0.3, 2 and 6 hours post-infection. TAP treatment did not protect against development of disease. Calves treated with TAP had similar bacterial loads in the nasal cavity and lung compared to calves treated with water. Similarly, TAP treatment did not affect the development of clinical signs, elevated rectal temperatures, or increased levels of blood neutrophils, haptoglobin and fibrinogen that occurred after bacterial challenge. Postmortem gross and histologic lung lesions were also similar in the two groups. To determine why there was a lack of protective effect, we tested the effect of substances in respiratory lining fluid on the bactericidal activity of TAP. Physiologic concentrations of sodium chloride inhibited TAP bactericidal activity in vitro, as did serum at concentrations of 0.62 to 2.5%, but concentrated bronchoalveolar lavage fluid had no consistent effect. These findings suggest that TAP does not have in vivo bactericidal activity against M. haemolytica because of interference by physiological sodium chloride levels and by serum. Thus, administration of TAP may not be effective for prevention of M. haemolytica pneumonia.

Development of Bacterial Therapeutics against the Bovine Respiratory Pathogen Mannheimia haemolytica.

Bovine respiratory disease (BRD) is a major cause of morbidity and mortality in beef cattle. Recent evidence suggests that commensal bacteria of the bovine nasopharynx have an important role in maintaining respiratory health by providing colonization resistance against pathogens. The objective of this study was to screen and select bacterial therapeutic candidates from the nasopharynxes of feedlot cattle to mitigate the BRD pathogen Mannheimia haemolytica In a stepwise approach, bacteria (n = 300) isolated from the nasopharynxes of 100 healthy feedlot cattle were identified and initially screened (n = 178 isolates from 12 different genera) for growth inhibition of M. haemolytica Subsequently, selected isolates were evaluated for the ability to adhere to bovine turbinate (BT) cells (n = 47), compete against M. haemolytica for BT cell adherence (n = 15), and modulate gene expression in BT cells (n = 10). Lactobacillus strains had the strongest inhibition of M. haemolytica, with 88% of the isolates (n =33) having inhibition zones ranging from 17 to 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 BT cells (32% to 78%). Among 84 bovine genes evaluated, selected isolates upregulated expression of interleukin 8 (IL-8) and IL-6 (P < 0.05). After ranking isolates for greatest inhibition, adhesion, competition, and immunomodulation properties, 6 Lactobacillus strains from 4 different species were selected as the best candidates for further development as intranasal bacterial therapeutics to mitigate M. haemolytica infection in feedlot cattle.IMPORTANCE Bovine respiratory disease (BRD) is a significant animal health issue impacting the beef industry. Current BRD prevention strategies rely mainly on metaphylactic use of antimicrobials when cattle enter feedlots. However, a recent increase in BRD-associated bacterial pathogens that are resistant to metaphylactic antimicrobials highlights a pressing need for the development of novel mitigation strategies. Based upon previous research showing the importance of respiratory commensal bacteria in protecting against bronchopneumonia, this study aimed to develop bacterial therapeutics that could be used to mitigate the BRD pathogen Mannheimia haemolytica Bacteria isolated from the respiratory tracts of healthy cattle were characterized for their inhibitory, adhesive, and immunomodulatory properties. In total, 6 strains were identified as having the best properties for use as intranasal therapeutics to inhibit M. haemolytica If successful in vivo, these strains offer an alternative to metaphylactic antimicrobial use in feedlot cattle for mitigating BRD.

Genetic characterization of susceptible and multi-drug resistant Mannheimia haemolytica isolated from high-risk stocker calves prior to and after antimicrobial metaphylaxis.

Bovine Respiratory Disease (BRD) is a major threat to animal health and welfare in the cattle industry. Strains of Mannheimia haemolytica (Mh) that are resistant to multiple classes of antimicrobials are becoming a major concern in the beef industry, as the frequency of isolation of these strains has been increasing. Mobile genetic elements, such as integrative conjugative elements (ICE), are frequently implicated in this rapid increase in multi-drug resistance. The objectives of the current study were to determine the genetic relationship between the isolates collected at arrival before metaphylaxis and at revaccination after metaphylaxis, to identify which resistance genes might be present in these isolates, and to determine if they were carried on an ICE. Twenty calves culture positive for Mh at arrival and revaccination were identified, and a total of 48 isolates with unique susceptibility profiles (26 from arrival, and 22 from revaccination) were submitted for whole-genome sequencing (WGS). A phylogenetic tree was constructed, showing the arrival isolates falling into four clades, and all revaccination isolates within one clade. All revaccination isolates, and one arrival isolate, were positive for the presence of an ICE. Three different ICEs with resistance gene modules were identified. The resistance genes aphA1, strA, strB, sul2, floR, erm42, tetH/R, aadB, aadA25, blaOXA-2, msrE, mphE were all located within an ICE. The gene bla-ROB1 was also present in the isolates, but was not located within an ICE.

Nanopore ultra-long read sequencing technology for antimicrobial resistance detection in Mannheimia haemolytica.

Disruptive innovations in long-range, cost-effective direct template nucleic acid sequencing are transforming clinical and diagnostic medicine. A multidrug resistant strain and a pan-susceptible strain of Mannheimia haemolytica, isolated from pneumonic bovine lung samples, were sequenced at 146× and 111× coverage, respectively with Oxford Nanopore Technologies MinION. De novo assembly produced a complete genome for the non-resistant strain and a nearly complete assembly for the drug resistant strain. Functional annotation using RAST (Rapid Annotations using Subsystems Technology), CARD (Comprehensive Antibiotic Resistance Database) and ResFinder databases identified genes conferring resistance to different classes of antibiotics including ß-lactams, tetracyclines, lincosamides, phenicols, aminoglycosides, sulfonamides and macrolides. Resistance phenotypes of the M. haemolytica strains were determined by minimum inhibitory concentration (MIC) of the antibiotics. Sequencing with a highly portable MinION device corresponded to MIC assays with most of the antimicrobial resistant determinants being identified with as few as 5437 reads, except for the genes responsible for resistance to Fluoroquinolones. The resulting quality assemblies and AMR gene annotation highlight the efficiency of ultra-long read, whole-genome sequencing (WGS) as a valuable tool in diagnostic veterinary medicine.

Serotyping and antimicrobial resistance of Mannheimia haemolytica strains from European cattle with bovine respiratory disease.

The objective of this study was to assess the prevalence of three serotypes, A1, A2, and A6 in 98 M. haemolytica isolates collected from clinical BRD cases in European cattle and assess their antimicrobial resistance profiles. Isolates were characterized by serotyping (plate agglutination and serotype specific PCR) and antimicrobial susceptibility testing. The study identified a predominance of serotypes A1 (59%) and A6 (22%) in European M. haemolytica isolates exhibiting a relatively low level of antimicrobial resistance. A comprehensive understanding of the relative prevalence of different M. haemolytica serotypes in Europe informs a targeted approach for vaccine design against BRD.

Differential susceptibility to tetracycline, oxytetracycline and doxycycline of the calf pathogens Mannheimia haemolytica and Pasteurella multocida in three growth media.

For clinical isolates of bovine Mannheimia haemolytica and Pasteurella multocida, this study reports minimum inhibitory concentration (MIC) differences for tetracycline, oxytetracycline and doxycycline between cation-adjusted Mueller-Hinton broth (CAMHB), foetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI) medium. MICs were determined according to CLSI standards and additionally using five overlapping sets of twofold dilutions. Matrix effect: (a) free drug MICs and minimum bactericidal concentrations (MBC) for all drugs were significantly higher in FBS than in CAMHB for both pathogens (p < 0.001); (b) MICs and MBCs were higher for CAMHB and FBS compared to RPMI for P. multocida only. Net growth rate for P. multocida in CAMHB was significantly slower than in FBS and higher than in RPMI, correlating to MIC and MBC ranking. Drug effect: doxycycline MICs and MBCs were significantly lower (p < 0.001) in both CAMHB and FBS than tetracycline and oxytetracycline for both pathogens. Only for M. haemolytica were oxytetracycline MIC and MBC significantly lower than tetracycline, precluding the use of tetracycline to predict oxytetracycline susceptibility in this species. Determining potencies of tetracyclines in a physiological medium, such as FBS, is proposed, when the objective is correlation with pharmacokinetic data for dosage determination.

Variation in fluoroquinolone pharmacodynamic parameter values among isolates of two bacterial pathogens of bovine respiratory disease.

To design an antimicrobial treatment regimen for a bacterial disease, data on the drug pharmacodynamics (PD) against selected drug-susceptible strains of the pathogen are used. The regimen is applied across such strains in the field, assuming the PD parameter values remain the same. We used time-kill experiments and PD modeling to investigate the fluoroquinolone enrofloxacin PD against different isolates of two bovine respiratory disease pathogens: four Mannheimia haemolytica and three Pasteurella multocida isolates. The models were fitted as mixed-effects non-linear regression; the fixed-effects PD parameter values were estimated after accounting for random variation among experimental replicates. There was both inter- and intra- bacterial species variability in the PD parameters Hill-coefficient and Emax (maximal decline of bacterial growth rate), with more variable PD responses among M. haemolytica than among P. multocida isolates. Moreover, the Hill-coefficient was correlated to the isolate's maximal population growth rate in the absence of antimicrobial exposure (a.k.a. specific growth rate; Spearman's ρ = 0.98, p-value = 0.003, n = 6 isolates excluding one outlier). Thus, the strain's properties such as growth potential may impact its PD responses. This variability can have clinical implications. Modifying the treatment regimen depending on phenotypic properties of the pathogen strain causing disease may be a precision medicine approach.