Mannheimia cairinae sp. nov., a novel species of Mannheimia obtained from Muscovy ducks (Cairina moschata) and reclassification of Mannheimia ovis as heterotypic synonym of Mannheimia pernigra.

During a screening study for Pasteurella multocida in two unrelated flocks of Muscovy ducks pharyngeal and cloacal swabs were collected. A total of 59 Pasteurellaceae-like isolates sharing the same colony morphology were subcultured and subsequently characterized. Colonies on bovine blood agar were nonhaemolytic, regular, circular, slightly raised, shiny, intransparent with an entire margin, greyish and had an unguent-like consistency. Isolate AT1T was characterized by 16S rRNA gene sequencing and showed the highest similarity of 96.1 % to the type strain of Mannheimia caviae and 96.0 % to the type strain of Mannheimia bovis, respectively. In addition, rpoB and recN gene sequences also showed the highest similarity to the genus Mannheimia. The phylogenetic comparison of concatenated conserved protein sequences also showed a unique position of AT1T compared to other species of Mannheimia. Full phenotypic characterization of the isolates showed that between two (Mannheimia ruminalis) and 10 (Mannheimia glucosida) phenotypic characteristics separate the taxon isolated from Muscovy ducks from the accepted species of Mannheimia. Whole genomic sequences of two strains analysed by the type strain genome server showed the highest similarity of 24.9 % to the genome of the type strain of Pasteurella multocida and 23.0 % to the genome of the type strain of Mannheimia haemolytica. The species Mannheimia cairinae sp. nov. is proposed based on the phenotypic and genotypic similarity to Mannheimia as well as differences to the other validly published species of the genus. The leukotoxin protein was not predicted in the genome of AT1T. The G+C content of the type strain of M. cairinae sp. nov., AT1T (=CCUG 76754T=DSM 115341T) is 37.99 mol%, calculated from the whole genome. The investigation further proposes that Mannheimia ovis is reclassified as a later heterotypic synonym of Mannheimia pernigra, since M. ovis and M. pernigra are closely genetically related, and M. pernigra was validly published before M. ovis.

Mannheimia pernigra sp. nov., isolated from bovine respiratory tract.

Over a period of 1 year, 270 isolates identified as Taxon 39 of Bisgaard were obtained from the nasopharynx of veal calves at 11 epidemiologically independent Swiss fattening farms. Two isolates from each farm and the Australian Taxon 39 reference strain BNO311 were further characterized by genetic and phenotypic methods. Phylogenetic analysis of 16S rRNA and recN gene sequences placed the isolates in a single, distinct cluster within the genus Mannheimia. As to the rpoB gene, most isolates clustered together, but four strains formed a separate cluster close to Mannheimia varigena. Genome sequence analysis of isolates from both rpoB clusters confirmed their species status, with an average nucleotide identity (ANI) >98.9 % between isolates and <84 % to the closest species, M. varigena. Based upon whole genome sequences, the G+C content was determined as 39.1 mol%. Similarly, analysis of MALDI-TOF MS reference spectra clustered the isolates clearly separated from the other Mannheimia species, making this the method of choice for identification. In addition, numerous biochemical markers based on classical as well as commercial identification schemes were determined, allowing separation from other Mannheimia species and identification of the new taxon. Major fatty acids for strain 17CN0883T are C14 : 0, C16 : 0, C16 : 1 ω7c and C18 : 1 ω7c. Major respiratory quinones are ubiquinone-7 and ubiquinone-8. We propose the name Mannheimia pernigra sp. nov. for former Taxon 39 of Bisgaard. The type strain is 17CN0883T (=CCUG 74657T=DSM 111153T) isolated from a veal calf in Switzerland.

Formic acid as a secondary substrate for succinic acid production by metabolically engineered Mannheimia succiniciproducens.

There has been much effort exerted to reduce one carbon (C1) gas emission to address climate change. As one promising way to more conveniently utilize C1 gas, several technologies have been developed to convert C1 gas into useful chemicals such as formic acid (FA). In this study, systems metabolic engineering was utilized to engineer Mannheimia succiniciproducens to efficiently utilize FA. 13 C isotope analysis of M. succiniciproducens showed that FA could be utilized through formate dehydrogenase (FDH) reaction and/or the reverse reaction of pyruvate formate lyase (PFL). However, the naturally favored forward reaction of PFL was found to lower the SA yield from FA. In addition, FA assimilation via FDH was found to be more efficient than the reverse reaction of PFL. Thus, the M. succiniciproducens LPK7 strain, which lacks in pfl, ldh, pta, and ack genes, was selected as a base strain. In silico metabolic analysis confirmed that utilization of FA would be beneficial for the enhanced production of SA and suggested FDH as an amplification target. To find a suitable FDH, four different FDHs from M. succiniciproducens, Methylobacterium extorquens, and Candida boidinii were amplified in LPK7 strain to enhance FA assimilation. High-inoculum density cultivation using 13 C labeled sodium formate was performed to evaluate FA assimilation efficiency. Fed-batch fermentations of the LPK7 (pMS3-fdh2 meq) strain was carried out using glucose, sucrose, or glycerol as a primary carbon source and FA as a secondary carbon source. As a result, this strain produced 76.11 g/L SA with the yield and productivity of 1.28 mol/mol and 4.08 g/L/h, respectively, using sucrose and FA as dual carbon sources. The strategy employed here will be similarly applicable in developing microorganisms to utilize FA and to produce valuable chemicals and materials from FA.

Highly selective production of succinic acid by metabolically engineered Mannheimia succiniciproducens and its efficient purification.

Succinic acid (SA) is one of the fermentative products of anaerobic metabolism, and an important industrial chemical that has been much studied for its bio-based production. The key to the economically viable bio-based SA production is to develop an SA producer capable of producing SA with high yield and productivity without byproducts. Mannheimia succiniciproducens is a capnophilic rumen bacterium capable of efficiently producing SA. In this study, in silico genome-scale metabolic simulations were performed to identify gene targets to be engineered, and the PALK strain (ΔldhA and Δpta-ackA) was constructed. Fed-batch culture of PALK on glucose and glycerol as carbon sources resulted in the production of 66.14 g/L of SA with the yield and overall productivity of 1.34 mol/mol glucose equivalent and 3.39 g/L/h, respectively. SA production could be further increased to 90.68 g/L with the yield and overall productivity of 1.15 mol/mol glucose equivalent and 3.49 g/L/h, respectively, by utilizing a mixture of magnesium hydroxide and ammonia solution as a pH controlling solution. Furthermore, formation of byproducts was drastically reduced, resulting in almost homo-fermentative SA production. This allowed the recovery and purification of SA to a high purity (99.997%) with a high recovery yield (74.65%) through simple downstream processes composed of decolorization, vacuum distillation, and crystallization. The SA producer and processes developed in this study will allow economical production of SA in an industrial-scale. Biotechnol. Bioeng. 2016;113: 2168-2177. © 2016 Wiley Periodicals, Inc.

Human Wound Infection with Mannheimia glucosida following Lamb Bite.

Mannheimia spp. are veterinary pathogens that can cause mastitis and pneumonia in domestic cattle and sheep. While Mannheimia glucosida can be found as normal flora in oral and respiratory mucosa in sheep, there have been no reported cases of human infection with this organism.

Bronchopneumonia in two dairy calves associated with Mannheimia species cluster V infection.

The pathological, bacteriological, and molecular findings of two 3-week-old Holstein calves with bronchopneumonia are presented. Heavy pure growth of a Mannheimia species most closely aligned with the unnamed cluster V strains on the basis of 16S ribosomal RNA sequencing was detected in the lungs of both calves in association with Bovine respiratory syncytial virus infection. While Mannheimia species closely related to cluster V strains have occasionally been reported in association with pneumonia, meningitis, and abortion in cattle, the current report provides a description of the gross and histopathological lesions produced by a cluster V strain of Mannheimia species. Lesions in the lung were found to be typical of those described for Mannheimia haemolytica with the absence of areas of coagulation necrosis rimmed by leukocytes and more pronounced intra-alveolar hemorrhage. Lesions were linked to the presence of leukotoxin A based on phenotypic hemolysis and molecular demonstration of the leukotoxin A gene.

The role of Mannheimia species in ovine mastitis.

Mannheimia haemolytica is known to be an important cause of intramammary infection in sheep. It usually causes severe clinical mastitis, followed by toxaemia and gangrenous necrosis of the udder. However there are limited data available on the epidemiology and pathogenesis of mastitis associated with Mannheimia species. These organisms can be more significant as a cause of mastitis than Staphylococcus aureus in some flocks. Some data suggest the possibility of horizontal transmission of Mannheimia species between ewes via lamb sucking. There is no vaccine available for prevention, and the sudden onset of mastitis and its peracute nature renders most treatments unsuccessful. This review examines the significance of the species within this genus in sheep mastitis.

Real-time PCR assay for the detection of species of the genus Mannheimia.

Infections caused by species of the genus Mannheimia cause diverse disease complexes in many wild and domestic animals worldwide. Fast and accurate detection of single species within the genus remains an unsolved problem till today. To resolve this diagnostic challenge, we developed a real-time PCR assay for the rapid and specific identification of five species of the genus Mannheimia (M. haemolytica, M. varigena, M. ruminalis, M. granulomatis and M. glucosida) from bacterial cultures and tissue samples. The assay was validated with reference strains, field isolates and bacteria spiked tissue samples. The sodA gene was used as target region for species-specific primer pairs. The real-time PCR assay demonstrated species specificity for all five examined Mannheimia spp. and a rapid test completion time of less than 5 h. This is a considerable advantage compared to the traditional phenotyping methods currently used to distinguish between the species of the genus. The assay was able to detect approximately 10(3) bacterial cells per gram lung tissue sample, as determined with spiked tissue samples. We assume that the assay could become useful for fast laboratory diagnostic assessment particularly of respiratory infections caused by Mannheimia in animals.

A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis.

The objective of this study was to design a multiplex PCR assay to identify Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis. The multiplex PCR included primer sets HP, amplifying a DNA region from an unknown hypothetical protein, Lkt and Lkt2, amplifying different regions of the leukotoxinD gene, and 16S to amplify universal bacterial sequences of the 16S rRNA gene. Based on positive amplification, isolates were delineated as M. haemolytica (HP, Lkt, 16S), M. glucosida (HP, Lkt, Lkt2, 16S), or M. ruminalis (HP, 16S). The validity of the assay was examined against 22 reference strains within the family Pasteurellaceae and 17 field isolates (nasal) that had been collected previously from feedlot cattle and tentatively identified as M. haemolytica based on morphology and substrate utilization. Additionally, 200 feedlot cattle were screened for M. haemolytica using multiplex PCR. Forty-four isolates from 25 animals were identified as M. haemolytica. The PCR assay positively identified all M. haemolytica, as confirmed by phenotypic tests and clustering based upon cellular fatty acid methyl ester (FAME) profiles. Selected nasal isolates that exhibited evidence of haemolysis, but were M. haemolytica-negative based on PCR, were also confirmed negative by phenotypic and FAME analyses. The multiplex PCR assay required no additional phenotypic tests for confirmation of M. haemolytica, within the group of bacteria tested.

[Fatty acid composition of cells and lipopolysaccliarides of Mannheimia haemolytica, Mannheimia glucosida and Bibersteinia trehalosi strains].

The Mannheimia haemolytica, Mannheimia glucosida and Bibersteinia trehalosi strains and the similar fatty acid composition of cells with domination of C(16:1) and C(16:0), which were in almost equal quantities, C(14:0 and C(18:1) + C(18:2). The fatty acid composition of lipopolysaccharides (LPS) of the studied bacteria had no essential differences too. It was mainly represented by C(14:0) and 3-OH-C(14:0) which consisted of more than 80% of all LPS fatty acids. C(12:0), C(16:1) and C(16:0) were presented in LPS in small quantities. The M. haemolytica, M. glucosida and B. trehalosi strains did not differ essentially by fatty acid compositions of cells and LPS from earlier studied strains of genera Pasteurella (P. multocida), Haemophilus (H. influenzae and other species), Actinobacillus (A. pleuropneumoniae). This shows the close phylogenetic relationship of the mentioned bacteria and significance of investigated signs as chemotaxonomic markers for differentiation of taxons of the above genus level. The paper is presented in Russian.

Identification of a novel Mannheimia granulomatis lineage from lesions in roe deer (Capreolus capreolus).

Eight atypical Mannheimia isolates were isolated from lesions in roe deer (Capreolus capreolus). Traditional classification based on morphologic and physiologic traits showed that they belong to a distinct biogroup (taxon) within genus Mannheimia. Extensive phenotypic characterization suggested that the isolates should be classified as M. granulomatis, although the presence of distinct traits justified their classification into a separate biogroup within this species. Phylogenetic analyses based on 16S rRNA sequences from two roe deer isolates and 41 other Mannheimia strains supported that the roe deer isolates form a monophyletic group within M. granulomatis. The lktA genotype was present in all roe deer isolates based on Southern blot analysis, whereas the corresponding beta-hemolytic phenotype was absent in one of these isolates.

Occurrence of haemolytic Mannheimia spp. in apparently healthy sheep in Norway.

BACKGROUND: The occurrence of Mannheimia species in healthy sheep has only been investigated to a very limited extend since the genus and its five named species were established. The aim of the present study was to evaluate the occurrence of haemolytic Mannheimia species in apparently healthy sheep originating from four sheep flocks in South-Western Norway. METHODS: Typical beta-haemolytic Pasteurellaceae were isolated from nasal swabs and subsequently subjected to bacteriological examination. A total of 57 Mannheimia isolates were obtained in pure culture. All isolates were genotyped by amplified fragment length polymorphisms (AFLP) analysis and compared to six reference strains. The 16S rRNA gene sequences of two isolates were also determined. RESULTS: beta-haemolytic Mannheimia species were isolated from 24% to 64% of the sheep in the four flocks. A total of 26 haemolytic M. ruminalis-like strains were isolated among which, a considerable genetic diversity was found. Eighteen M. glucosida isolates were obtained from three flocks, whereas M. haemolytica was only isolated from two flocks, 16 of them being from only one of the flocks. CONCLUSION: We demonstrate that a relatively high number of apparently healthy sheep in Norway seem to carry the potentially pathogenic M. haemolytica and M. glucosida in the upper respiratory tract. An unexpectedly high number of haemolytic M. ruminalis-like organisms were also obtained in all four flocks. The usually non-haemolytic M. ruminalis are typically isolated from healthy ruminants. The significance of beta-haemolytic M. ruminalis-like organisms is unknown and should be investigated in a future study.

The proteome of Mannheimia succiniciproducens, a capnophilic rumen bacterium.

Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is an industrially important bacterium as an efficient succinic acid producer. Recently, its full genome sequence was determined. In the present study, we analyzed the M. succiniciproducens proteome based on the genome information using 2-DE and MS. We established proteome reference map of M. succiniciproducens by analyzing whole cellular proteins, membrane proteins, and secreted proteins. More than 200 proteins were identified and characterized by MS/MS supported by various bioinformatic tools. The presence of proteins previously annotated as hypothetical proteins or proteins having putative functions were also confirmed. Based on the proteome reference map, cells in the different growth phases were analyzed at the proteome level. Comparative proteome profiling revealed valuable information to understand physiological changes during growth, and subsequently suggested target genes to be manipulated for the strain improvement.

Fatal meningitis in a calf caused by Mannheimia varigena.

Mannheimia varigena was identified as the etiologic agent of meningitis in a young Belgian White Blue heifer calf. Species identification of the bacterium was done by phenotyping and molecularly confirmed by tDNA-PCR. Standard bacteriological examination might fail to differentiate species belonging to the genus Mannheimia.

Batch and continuous cultures of Mannheimia succiniciproducens MBEL55E for the production of succinic acid from whey and corn steep liquor.

Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is able to produce a large amount of succinic acid in a medium containing glucose, peptone, and yeast extract. In order to reduce the cost of the medium, whey and corn steep liquor (CSL) were used as substrates for the production of succinic acid by M. succiniciproducens MBEL55E. Anaerobic batch cultures of M. succiniciproducens MBEL55E in a whey-based medium containing CSL resulted in the production of succinic acid with a yield of 71% and productivity of 1.18 g/l/h, which are similar to those obtained in a whey-based medium containing yeast extract (72% and 1.21 g/l/h). Anaerobic continuous culture of M. succiniciproducens MBEL55E in a whey-based medium containing CSL resulted in a succinic acid yield of 69% and a succinic acid productivity as high as 3.90 g/l/h. These results show that succinic acid can be produced efficiently and economically by M. succiniciproducens MBEL55E from whey and CSL.