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The Journal of Antimicrobial... Apr 2019To identify and analyse the first ESBL gene from Mannheimia haemolytica.
OBJECTIVES
To identify and analyse the first ESBL gene from Mannheimia haemolytica.
METHODS
Susceptibility testing was performed according to CLSI. Plasmids were extracted via alkaline lysis and transferred by electrotransformation. The sequence was determined by WGS and confirmed by Sanger sequencing.
RESULTS
The M. haemolytica strain 48 showed high cephalosporin MICs. A single plasmid, designated pKKM48, with a size of 4323 bp, was isolated. Plasmid pKKM48 harboured a novel blaROB gene, tentatively designated blaROB-2, and was transferred to Pasteurella multocida B130 and to Escherichia coli JM107. PCR assays and susceptibility testing confirmed the presence and activity of the blaROB-2 gene in the P. multocida and in the E. coli recipient carrying plasmid pKKM48. The transformants had high MICs of all β-lactam antibiotics. An ESBL phenotype was seen in the E. coli transformant when applying the CLSI double-disc confirmatory test for E. coli. The blaROB-2 gene from plasmid pKKM48 differed in three positions from blaROB-1, resulting in two amino acid exchanges and one additional amino acid in the deduced β-lactamase protein. In addition to blaROB-2, pKKM48 harboured mob genes and showed high similarity to other plasmids from Pasteurellaceae.
CONCLUSIONS
This study described the first ESBL gene in Pasteurellaceae, which may limit the therapeutic options for veterinarians. The transferability to Enterobacteriaceae with the functional activity of the gene in the new host underlines the possibility of the spread of this gene across species or genus boundaries.
Topics: Anti-Bacterial Agents; Chromosome Mapping; Drug Resistance, Bacterial; Electroporation; Escherichia coli; Mannheimia haemolytica; Microbial Sensitivity Tests; Pasteurella multocida; Plasmids; Transformation, Bacterial; Whole Genome Sequencing; beta-Lactamases; beta-Lactams
PubMed: 30561662
DOI: 10.1093/jac/dky515 -
Applied and Environmental Microbiology Nov 2019Bovine respiratory disease (BRD) is a major cause of morbidity and mortality in beef cattle. Recent evidence suggests that commensal bacteria of the bovine nasopharynx...
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 In a stepwise approach, bacteria ( = 300) isolated from the nasopharynxes of 100 healthy feedlot cattle were identified and initially screened ( = 178 isolates from 12 different genera) for growth inhibition of Subsequently, selected isolates were evaluated for the ability to adhere to bovine turbinate (BT) cells ( = 47), compete against for BT cell adherence ( = 15), and modulate gene expression in BT cells ( = 10). strains had the strongest inhibition of , with 88% of the isolates ( =33) having inhibition zones ranging from 17 to 23 mm. Adherence to BT cells ranged from 3.4 to 8.0 log CFU per 10 BT cells. All the isolates tested in competition assays reduced adherence to BT cells (32% to 78%). Among 84 bovine genes evaluated, selected isolates upregulated expression of interleukin 8 (IL-8) and IL-6 (0.05). After ranking isolates for greatest inhibition, adhesion, competition, and immunomodulation properties, 6 strains from 4 different species were selected as the best candidates for further development as intranasal bacterial therapeutics to mitigate infection in feedlot cattle. 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 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 If successful , these strains offer an alternative to metaphylactic antimicrobial use in feedlot cattle for mitigating BRD.
Topics: Animals; Anti-Bacterial Agents; Bacteria; Bronchopneumonia; Cattle; Cattle Diseases; Hydrogen Peroxide; Immunity, Innate; Lactic Acid; Lactobacillus; Mannheimia haemolytica; Microbial Sensitivity Tests; Nasopharynx; Pneumonia of Calves, Enzootic; Respiratory System; Respiratory Tract Infections
PubMed: 31444198
DOI: 10.1128/AEM.01359-19 -
Journal of Microbiological Methods Apr 2020Genotype 2 Mannheimia haemolytica associate with the lungs of cattle with bovine respiratory disease more frequently than genotype 1 strains. Different colony colors...
Genotype 2 Mannheimia haemolytica associate with the lungs of cattle with bovine respiratory disease more frequently than genotype 1 strains. Different colony colors and morphologies were identified between genotype 1 and 2 solid media cultures. Genotype of strains, and frequency differences between them in mixed cultures are discernable by visual inspection.
Topics: Animals; Bacterial Typing Techniques; Cattle; Cattle Diseases; Culture Media; Genotype; Mannheimia haemolytica; Pasteurellosis, Pneumonic
PubMed: 32088258
DOI: 10.1016/j.mimet.2020.105877 -
Journal of Veterinary Diagnostic... Sep 2015Mannheimia haemolytica is a major bacterial component of bovine respiratory disease (BRD); unfortunately, very little is known about M. haemolytica transmission dynamics...
Mannheimia haemolytica is a major bacterial component of bovine respiratory disease (BRD); unfortunately, very little is known about M. haemolytica transmission dynamics among cattle. Identifying potential variation in M. haemolytica populations over time and induction of nasopharyngeal colonization and subsequent shedding are 2 areas where knowledge is lacking. In our study, 2 separate loads of 20 mixed-origin, male calves were purchased through an order buyer on different dates. Deep nasopharyngeal cultures (NPC) were performed on all calves on arrival and, if M. haemolytica-negative, a second screening culture was obtained. Calves that were negative on 2 initial NPCs (NEG; n = 4) were subsequently challenged with a previously isolated field strain of M. haemolytica in both the upper and lower respiratory tract, individually housed, and then monitored for M. haemolytica shedding via NPCs at 0.5, 1, 3, 5, 7, and 9 days postchallenge. Naturally M. haemolytica-positive calves (2 per load) were kept for additional daily cultures (POS; n = 4). Individual calf M. haemolytica status for both the POS and NEG groups was inconsistent between study days. Additionally, pulsed-field gel electrophoresis performed on isolates from the positive cultures showed that the NEG calves did not shed the M. haemolytica challenge strain, but rather 2 distinct clusters of M. haemolytica were shared among POS and NEG calves regardless of their initial status. Although sample sizes were small, these findings illustrate how variable the results of a single nasopharyngeal swab can be and the challenges of using an individual culture to truly represent animal M. haemolytica status.
Topics: Animals; Cattle; Cattle Diseases; Electrophoresis, Gel, Pulsed-Field; Housing, Animal; Male; Mannheimia haemolytica; Nasopharynx; Pasteurellaceae Infections; Red Meat; Respiratory Tract Diseases; Specimen Handling
PubMed: 26330399
DOI: 10.1177/1040638715597724 -
PloS One 2024Mannheimia haemolytica is the principal agent contributing to bovine respiratory disease and can form biofilms with increased resistance to antibiotic treatment and host...
Mannheimia haemolytica is the principal agent contributing to bovine respiratory disease and can form biofilms with increased resistance to antibiotic treatment and host immune defenses. To investigate the molecular mechanisms underlying M. haemolytica biofilm formation, transcriptomic analyses were performed with mRNAs sequenced from planktonic and biofilm cultures of pathogenic serotypes 1 (St 1; strain D153) and St 6 (strain D174), and St 2 (strain D35). The three M. haemolytica serotypes were cultured in two different media, Roswell Park Memorial Institute (RPMI) 1640 and brain heart infusion (BHI) to form the biofilms. Transcriptomic analyses revealed that the functions of the differentially expressed genes (DEGs) in biofilm associated cells were not significantly affected by the two media. A total of 476 to 662 DEGs were identified between biofilm associated cells and planktonic cells cultured under BHI medium. Functional analysis of the DEGs indicated that those genes were significantly enriched in translation and many biosynthetic processes. There were 234 DEGs identified in St 1 and 6, but not in St 2. The functions of the DEGs included structural constituents of ribosomes, transmembrane proton transportation, proton channels, and proton-transporting ATP synthase. Potentially, some of the DEGs identified in this study provide insight into the design of new M. haemolytica vaccine candidates.
Topics: Animals; Cattle; Mannheimia haemolytica; Plankton; Protons; Biofilms; Cattle Diseases; Gene Expression Profiling
PubMed: 38329985
DOI: 10.1371/journal.pone.0297692 -
BMC Microbiology Dec 2018Mannheimia haemolytica has been recognized as the principal cause of pneumonic pasteurellosis in sheep and goats. It is one of the important diseases of small ruminants...
BACKGROUND
Mannheimia haemolytica has been recognized as the principal cause of pneumonic pasteurellosis in sheep and goats. It is one of the important diseases of small ruminants in Ethiopia. While annual vaccination using a monovalent vaccine (inactivated Pasteurella multocida biotype A) is common, respiratory diseases are still reported in various parts of Ethiopia. This suggests the need for further investigation into the species and strains responsible for the disease, which is vital information for development of a multivalent vaccine. The objective of the current study was to isolate M. heamolytica associated with pneumonic cases of sheep in selected areas of Central Ethiopia, determine its role and the strains/genotypes of the bacterium circulating in the study area.
RESULTS
Bacteriological analysis of nasal swab samples collected from a total of 76 pneumonic cases of sheep showed that M. haemolytica was isolated from 26 of them while B.trehalosi from two cases. Further molecular analyses of the isolates using M. haemolytica species-specific and M.haemolytica serotype-1 antigen specific PCR assays revealed, 26 of the isolates were identified as M. haemolytica of which 21 of them were M. haemolytica serotype-1. Both M. haemolytica and B.trehalosi isolates were not detected in a PCR assay targeting capsular biosynthesis gene (capA) of P.multocida despite the non-specific products observed in M. haemolytica isolates. Phylogenetic analysis of M. haemolytica isolates included in this study in comparison with the reference strains with respect to PHSSA and Rpt2 genes revealed that the Ethiopian M. haemolytica isolates constituted three distinct genotypes consistent with site of origin.
CONCLUSION
The study indicated that M.haemolytica is commonly associated with cases of pneumonia in sheep in the study areas of central Ethiopia although the remaining other pathogens responsible for majority of the cases are yet to be determined. Molecular characterization revealed the existence of three genotypes of M. haemolytica circulating in the study areas consistent to the site of isolation. The findings suggest further extensive work to determine all pathogens associated with sheep pneumonia and the strain distribution of M. heamolytica to understand its molecular epidemiology at national level and design cost effective prevention and control methods.
Topics: Animals; Ethiopia; Genotype; Mannheimia haemolytica; Pasteurellosis, Pneumonic; Phylogeny; Sheep; Sheep Diseases; Species Specificity
PubMed: 30518323
DOI: 10.1186/s12866-018-1338-x -
Veterinary Microbiology 2016The objective of this study was to determine the clonal relatedness of Mannheimia haemolytica isolates responsible for an outbreak of bovine respiratory disease in a...
The objective of this study was to determine the clonal relatedness of Mannheimia haemolytica isolates responsible for an outbreak of bovine respiratory disease in a commercial feedlot. The isolates were obtained from the lungs of 21 calves with fatal pneumonia that were part of a group of 206 total calves. All isolates were serotyped and analyzed by pulsed-field gel electrophoresis (PFGE) and for antibiotic sensitivity patterns. ELISA and immunoblotting assays were performed to compare serum antibody levels to M. haemolytica antigens in calves with fatal pneumonia to those calves that survived the outbreak. Isolates were categorized into 14 different PFGE groups based on 90% similarity. Two Group D isolates (1 and 6), and 3 Group H isolates (14, 15, and 16) were characterized as 100% similar. Antimicrobial susceptibility profiles defined 8 groups based on differences in patterns of resistance between isolates. The two 100% similar isolates from PFGE Group D were both in susceptibility Group 1. All but isolate 14 from PFGE Group H (3, 15, 16, and 19) were in susceptibility Group 4a. Serum antibody levels to M. haemolytica antigens in the dead calves were not different than the antibody levels in the 185 calves that survived the outbreak. Immunoblots of selected isolates from each of the PFGE groups demonstrated only minimal differences in antigenic profiles between strains when reacted with serum from calves that either died from or survived the outbreak. Based on the characteristics of these isolates, multiple strains of M. haemolytica were responsible for fatal pneumonia during this outbreak.
Topics: Animals; Cattle; Disease Outbreaks; Kansas; Mannheimia haemolytica; Pneumonia of Calves, Enzootic
PubMed: 26711032
DOI: 10.1016/j.vetmic.2015.10.020 -
Australian Veterinary Journal 2002To perform a comprehensive phenotypic characterisation of 35 isolates of bacteria previously identified as haemolytic Pasteurella-Actinobacillus and obtained from cattle...
OBJECTIVE
To perform a comprehensive phenotypic characterisation of 35 isolates of bacteria previously identified as haemolytic Pasteurella-Actinobacillus and obtained from cattle and sheep.
DESIGN
The 35 isolates that had been obtained from Australian animals, 30 from cattle and five from sheep, were compared with reference strains of the five recognised species of the genus Mannheimia--M. haemolytica, M. glucosida, M. granulomatis, M. ruminalis and M. varigena.
RESULTS
Thirty-four of the isolates could be confidently assigned to three species of the genus Mannheimia. Twenty-nine were M. haemolytica, with 25 being isolated from cattle and four from sheep. All but three of the bovine M. haemolytica were isolated from pneumonic lungs. Of the three remaining bovine M. haemolytica isolates, one was obtained in pure culture from a bovine milk sample and the other two as part of a mixed flora associated with a middle ear infection of a calf suffering mucosal disease. Of the four ovine M. haemolytica isolates, two were isolated in pure culture from milk and two, also in pure culture, from pneumonic lungs. Three bovine isolates were identified as M. granulomatis--one from a tongue abscess, one from a jaw abscess and one from a lung showing suppurative bronchopneumonia. Two bovine isolates were identified as M. varigena--one coming from an udder and the other from a spleen. The available diagnostic records provided no information on whether these isolates were associated with a disease process. The remaining isolate was obtained from an ovine tongue abscess and could not be assigned to a recognised species within the genus Mannheimia.
CONCLUSION
The study represents the first time that M. haemolytica, M. granulomatis and M. varigena have been recognised as being present in cattle and sheep in Australia. Veterinary laboratories that encounter Pasteurella-Actinobacillus-like organisms from cattle and sheep should attempt as complete a characterisation as possible to help improve our knowledge of the disease potential of these organsims.
Topics: Animals; Australia; Cattle; Cattle Diseases; DNA, Bacterial; Mannheimia haemolytica; Pasteurella Infections; Phenotype; Pneumonia, Bacterial; Sheep; Sheep Diseases
PubMed: 12180887
DOI: 10.1111/j.1751-0813.2002.tb12058.x -
Journal of Veterinary Diagnostic... Mar 2022We developed a rapid insulated isothermal PCR (iiPCR) assay for on-site detection of using a primer and probe set targeting the superoxide dismutase (A) gene. Our iiPCR...
We developed a rapid insulated isothermal PCR (iiPCR) assay for on-site detection of using a primer and probe set targeting the superoxide dismutase (A) gene. Our iiPCR assay detected clinical isolates successfully and produced negative results on other bovine or ovine respiratory pathogens, including , , , , and spp., indicating that the PCR reactions were specific. Additionally, our iiPCR assay detected as few as 21 copies of genomic DNA and 17.2 cfu/mL of bacterial culture, which was 10 and 100 times more sensitive than conventional PCR, respectively. Our iiPCR assay can be performed on a portable device in a total of 58 min and may be a useful tool for the detection of in bovine and ovine respiratory disease in the field.
Topics: Animals; Cattle; Cattle Diseases; Mannheimia haemolytica; Polymerase Chain Reaction; Sheep; Sheep Diseases
PubMed: 35139720
DOI: 10.1177/10406387211068447 -
Genome Feb 2022Common bacterial causes of bovine respiratory disease (BRD) include , , and . Within , two major genotypes are commonly found in cattle (1 and 2); however, genotype 2...
Common bacterial causes of bovine respiratory disease (BRD) include , , and . Within , two major genotypes are commonly found in cattle (1 and 2); however, genotype 2 strains are isolated from diseased lungs much more frequently than genotype 1 strains. Outer membrane proteins (OMPs) of , , and genotype 2 may be important factors for acquired host immunity. The predicted OMP differences between genotypes 1 and 2 have been previously identified. In this study, we expanded the focus to include bovine-isolated strain genomes representing all three species and the two genotypes. Reported here are the core genomes unique to each of them, core genomes shared between some or all combinations of the three species and two genotypes, and predicted OMPs within these core genomes. The OMPs identified in this study are potential candidates for further studies and the development of interventions against BRD.
Topics: Animals; Bacterial Outer Membrane Proteins; Cattle; Genotype; Mannheimia haemolytica; Pasteurella multocida
PubMed: 34348051
DOI: 10.1139/gen-2021-0038