A single intranasal dose of essential oil spray confers modulation of the nasopharyngeal microbiota and short-term inhibition of Mannheimia in feedlot cattle: a pilot study.

Five essential oils (EOs) were previously characterized in vitro and identified as candidate EOs for the development of an intranasal EO spray to mitigate bovine respiratory disease (BRD) pathogens. In the present study, these EOs were evaluated for their potential to (i) reduce BRD pathogens, (ii) modulate nasopharyngeal microbiota, and (iii) influence animal performance, feeding behavior and immune response when a single dose administered intranasally to feedlot cattle. Forty beef steer calves (7-8 months old, Initial body weight = 284 ± 5 kg [SE]) received either an intranasal EO spray (ajowan, thyme, fennel, cinnamon leaf, and citronella) or PBS (Control; n = 20/group) on day 0. Deep nasopharyngeal swabs were collected on days (d) -1, 1, 2, 7, 14, 28, and 42 and processed for 16S rRNA gene sequencing, qPCR, and culturing. Significant effects of EO on community structure (d1), microbial richness and diversity, relative abundance of some dominant phyla (d1, d2, and d14), and the overall interaction network structure of the nasopharyngeal microbiota were detected. The relative abundance of Mannheimia was lower in the EO calves (4.34%) than in Control calves (10.4%) on d2, and M. haemolytica prevalence on d7 as compared to control calves. Feed intake, average daily gain, feeding behavior, and blood cell counts were not affected by EO treatment. Overall, a single intranasal dose of EO spray resulted in moderate modulation of nasopharyngeal microbiota and short-term inhibition of Mannheimia while not influencing animal performance, feeding behavior or immune response. Our study, for the first time, shows the potential use of intranasal EO to mitigate BRD in feedlot cattle.

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.

The effect of neonatal vaccination for bovine respiratory disease in the face of a dual challenge with bovine viral diarrhea virus and Mannheimia hemolytica.

Bovine respiratory disease is the greatest threat to calf health. In this study, colostrum-fed dairy X beef calves were vaccinated at ∼30 days of age with an adjuvanted parenteral vaccine containing modified live bovine viral diarrhea virus (BVDV) type 1 and type 2, bovine herpesvirus 1 (BHV-1), bovine parainfluenza type 3 virus (PI3V) and bovine respiratory syncytial virus (BRSV) andM. haemolyticatoxoid (Group 1), or intranasal temperature-sensitive BHV-1, BRSV and PI3V concurrently witha parenteral vaccine containing modified live BVDV type 1 and type 2 andM. haemolyticatoxoid (Group 2) or a placebo (Group 3). The calves were challenged ∼150 days post vaccination intranasally with BVDV 1b and then 7 days later intratracheally withM. haemolytica. The calves wereeuthanized 6 days after theM. haemolyticachallenge. Clinical signs following BVDV infection were similar in all groups. There was increased rectal temperatures in the Groups 2 and 3 on day 3 and in Group 3 on days 8-13. Group 1 animals had a slight leukopenia following BVDV infection while Groups 2 and 3 had greater leukopenia. BVDV type 1 and 2 serum titers increased in Group 1 following vaccination while these titers waned in Groups 2 and 3. There were higher levels of BVDV in the buffy coats and nasal samples in Group 2 and Group 3 versus Group 1 (p < 0.01). Interferon-gamma response was higher (p < 0.01) in Group 1 animals than Groups 2 and 3. Group 1 had the lowest percent pneumonic tissue (1.6%) while Group 2 vaccinates had 3.7% and the control Group 3 was 5.3%. Vaccination in the face of maternal antibody with a parenteral adjuvanted vaccine resulted in better protection than the regimen of an intranasal vaccine anda parenteral adjuvanted BVDV andM haemolyticacombination vaccine in a BVDV-M. haemolyticadual challenge.

Metabolic engineering of Mannheimia succiniciproducens for malic acid production using dimethylsulfoxide as an electron acceptor.

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.

Mannheimia varigena as the etiologic agent of lameness and coronary band lesion in cattle / Mannheimia varigena como agente etiológico de claudicação e lesão na banda coronária em bovinos

Mannheimia varigena was identified as the etiologic agent of lameness and coronary band lesion in 30% of cattle in a farm located in Rio Grande do Sul, Brazil. Swab samples from the lesions were cultured in McConkey Agar and Blood Agar for microbiological identification. Culture growth was submitted to Gram staining and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) identification. Antimicrobial susceptibility test based on disc diffusion was performed for three antibiotics: ceftiofur, gentamicin and florfenicol. Furthermore, molecular characterization of 16S rDNA gene sequencing was performed and the data was used in a phylogenetic analysis. For that purpose, total DNA was extracted by thermo extraction directly from the bacterial colonies and the polymerase chain reaction (PCR) was performed. Gram-negative Mannheimia varigena strain LBV010/22 was identified as the causative of the lesions. The strain was susceptible to all antibiotics tested. The phylogenetic analysis demonstrated that the analyzed strain is closely related to M. varigena strains from pyelonephritis and respiratory tract. Overall, this is the first report of M. varigena as the causative agent of coronary band injury in bovine. Therefore, our findings show the importance of an accurate microbiological identification of infectious agent in lameness cases in order to prevent the occurrence and perform an appropriate treatment in the future.

Mannheimia varigena foi identificada como agente etiológico de claudicação e lesão de banda coronária em 30% dos bovinos de uma fazenda localizada no Rio Grande do Sul, Brasil. Amostras de swab das lesões foram cultivadas em Ágar McConkey e Ágar Sangue para identificação microbiológica. O crescimento da cultura foi submetido à coloração de Gram e identificação por Espectrometria de Massa de Ionização por Dessorção a Laser Assistida por Matriz (MALDI-TOF MS). O teste de suscetibilidade antimicrobiana baseado na difusão em disco foi realizado para três antibióticos: ceftiofur, gentamicina e florfenicol. Além disso, foi realizada a caracterização molecular do sequenciamento do gene 16S rDNA e o resultado utilizado para análise filogenética. Para tanto, o DNA total foi extraído por termoextração diretamente das colônias bacterianas e uma reação em cadeia da polimerase (PCR) foi realizada. Foi identificada como causadora das lesões a cepa gram-negativa de Mannheimia varigenaLBV010/22. Ela foi suscetível a todos os antibióticos testados. A análise filogenética demonstrou que a cepa analisada está intimamente relacionada às M. varigena presentes em pielonefrite e no trato respiratório. No geral, este é o primeiro relato de M. varigenacomo agente causador de lesão de banda coronária em bovinos. Portanto, nossos achados mostram a importância de uma identificação microbiológica precisa do agente infeccioso nos casos de claudicação, a fim de prevenir a ocorrência e realizar um tratamento adequado no futuro.

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.

Mannheimia ovis sp. nov., Isolated from Dead Sheep with Hemorrhagic Pneumonia.

Two Gram-stain-negative, facultatively anaerobic bacteria, designated ZY170218T and ZY180512, were isolated from lungs of dead sheep with hemorrhagic pneumonia in Yunnan Province, China and their taxonomic positions were studied by a polyphasic approach. The two isolates grew optimally at 37 °C, pH 9.0 and 1.0% NaCl (w/v), and showed identical 16S rRNA, recN and rpoB gene sequences. Phylogenetic analysis based on 16S rRNA gene sequence showed that the two strains fell within the cluster of species in the genus Mannheimia and formed a separated lineage with comparatively low similarity to the closest related species M. granulomatis (96.5%). Phylogenetic analysis based on rpoB gene indicated that the strains formed a monophyletic evolutionary lineage, with low sequence similarity ≤ 89.0% to the species of the genus Mannheimia. The genomic OrthoANI values between strain ZY170218T and M. granulomatis and M. haemolytica were 80.4% and 83.1%, respectively. The genomic G + C content of strain ZY170218T was 39.1 mol%. The predominant fatty acids (> 5%) of the two strains were C16:0, C14:0, C18:1ω7c, summed feature 3 (C16:1 ω7c and/ or C16:1ω6c) and summed feature 2 (C14:0 3OH/ C16:1 Iso). The major polar lipids of strain ZY170218T were phosphatidylglycerol, phosphatidylcholine, monogalactosyldiacylglycerol, bis(monoacylglycero)phosphate and diacylglycerols. The sole respiratory quinone of the two strains was CoQ-7. On the basis of phylogenetic, phenotypic and chemotaxonomic features, strain ZY170218T and ZY180512 clearly represents a novel species of the genus Mannheimia, for which the name Mannheimia ovis sp. nov. is proposed. The type strain is ZY170218T (= CGMCC 1.13620 T = KCTC 15731 T).

Granulomatous Inflammation of the Muzzle in White-Tailed Deer (Odocoileus virginianus) and Mule Deer (Odocoileus hemionus) Associated With Mannheimia granulomatis.

Since 2002, reports of deer with swollen muzzles from throughout the United States have resulted in significant interest by wildlife biologists and wildlife enthusiasts. The condition was identified in 25 white-tailed deer (Odocoileus virginianus) and 2 mule deer (O. hemionus). Microscopic lesions consisted of severe, granulomatous or pyogranulomatous inflammation of the muzzle, nasal planum, and upper lip, as well as similar but less severe inflammation of the hard palate. Lymphadenitis of regional lymph nodes was common and granulomatous pneumonia was present in one individual. Splendore-Hoeppli material was typical in the center of inflammatory foci. Other than the single instance of pneumonia, systemic disease was not evident. Various bacterial species were isolated in culture, most of which were not morphologically consistent with the colonies of small, gram-negative bacteria observed in the center of the granulomas. Amplification and sequencing of the bacterial 16S rRNA gene from tissues of affected deer resulted in the identification of Mannheimia granulomatis. Laser capture microdissection was used to confirm that the colonies in the inflammatory foci were M. granulomatis. The cases described here are reminiscent of a bovine disease in Brazil and Argentina, locally called lechiguana. Although the inflammation of lechiguana is mostly truncal, the microscopic lesions are very similar and are also attributed to M. granulomatis. It is unclear if this is an emerging infectious disease of deer, or if it is a sporadic, uncommon condition that has only recently been recognized.

Pyelonephritis caused by Mannheimia varigena in a Holstein calf.

A 7-day-old calf died following development of mild respiratory symptoms. Postmortem examination revealed the kidneys were inflamed, and Gram-negative bacteria was detected in the kidneys, supporting the diagnosis of suppurative pyelonephritis. Mannheimia varigena antigen was found in the lesions and the cytoplasm of macrophages and neutrophils in the renal cortex. The Gram-negative bacilli from the kidney were identified as M. varigena by sequencing the 16S rDNA. Although M. varigena is known to cause bovine respiratory disease syndrome, shipping fever, and meningitis, it was unknown that it could also cause suppurative pyelonephritis. Our study provides the first evidence of suppurative pyelonephritis caused by M. varigena in cattle and information that would improve our understanding, diagnosis, and treatment for M. varigena infections.

Comparative evaluation of metallic skin staples or polypropylene sutures for primary closure of teat wounds in sheep.

AIMS: To compare stainless steel staples and polypropylene suture material for primary closure of wounds after teat amputation in ewes and to assess progress of healing in the presence or absence of intramammary infection (IMI). METHODS: Chios-cross ewes, aged 3-5 years were randomly allocated to be infected in one teat with 1,200-1,500 cfu of Mannheimia haemolytica 5 days after parturition (groups A and B; n = 8 in each group) or remain uninfected (groups C and D; n = 4 in each group). On the following 4 days one teat from each ewe was amputated 2.5 cm from the teat end and the wound was closed using skin staples (groups A and C) or polypropylene sutures (groups B and D). Clinical evaluation of wound healing was performed between 1-21 days after surgery. On day 21 tissue sections were collected for tensiometric and histological evaluation. RESULTS: The mean interval from the start to finish of wound closure was shorter when staples were used than when sutures were used (p < 0.001). Healing scores were lower (improved) for ewes in group A than B between days 1-7 after surgery (p = 0.005), but were similar between days 10-21 (p = 0.43). Healing scores were similar in groups C and D (p = 0.98). The tensile strain at maximum load was higher in tissue from group A than B (p = 0.001) and D (p = 0.004), but all other tensiometric measures were similar between groups. Histologically, collagen density was higher in sections from group A than B (p = 0.05) and D (p = 0.01), and angiogenesis was lower in sections from group A than B (p = 0.03) and D (p = 0.01). CONCLUSIONS AND CLINICAL RELEVANCE: Skin staples and polypropylene sutures can be used effectively for primary closure of teat wounds, even in the presence of IMI. Skin staples had the advantage of a reduction in surgical time. ABBREVIATION: IMI: intramammary infection.

In-vitro phagocytosis and intracellular killing of Pasteurella multocida B:2 by phagocytic cells of buffaloes.

Pasteurella multocida B:2 is a Gram-negative organism causing haemorrhagic septicaemia (HS) in buffaloes. It causes severe pulmonary infection, leading to infiltration of numerous macrophages and neutrophils. Despite the inflammatory response, buffaloes succumb to HS. This study aims to evaluate the in-vitro efficacy of macrophages and neutrophils of buffalo following exposure to P. multocida B:2. In-vitro infections were done using 107 cfu/ml of P. multocida B:2 for Group 1, Escherichia coli for Group 2 and Mannhaemia haemolytica A:2 for Group 3 cells. The inoculated cell cultures were harvested at 0, 30, 60 and 120 min post-exposure and the phagocytic, killing and cell death rates were determined. Both phagocytosis and killing rates of all bacteria increased over time. Phagocytosis involved between 71% and 73% neutrophils and between 60% and 64% macrophages at 120 min. Killing rate of all bacteria involved between 76% and 79% for neutrophils and between 70% and 74% for macrophages at 120 min. Death rate of neutrophils ranged between 67% in Group 3, and 88% in Group 1 at 120 min, significantly (p < 0.05) higher than Group 3 but insignificant (p > 0.05) than Group 2. Similar pattern was observed for death rate of macrophages. The phagocytosis and killing rates of P. multocida B:2 were similar to other bacterial species used in this study but more neutrophils and macrophages were dead following infection by P. multocida B:2 than M. haemolytica A:2.

Membrane engineering via trans-unsaturated fatty acids production improves succinic acid production in Mannheimia succiniciproducens.

Engineering of microorganisms to produce desired bio-products with high titer, yield, and productivity is often limited by product toxicity. This is also true for succinic acid (SA), a four carbon dicarboxylic acid of industrial importance. Acid products often cause product toxicity to cells through several different factors, membrane damage being one of the primary factors. In this study, cis-trans isomerase from Pseudomonas aeruginosa was expressed in Mannheimia succiniciproducens to produce trans-unsaturated fatty acid (TUFA) and to reinforce the cell membrane of M. succiniciproducens. The engineered strain showed significant decrease in membrane fluidity as production of TUFA enabled tight packing of fatty acids, which made cells to possess more rigid cell membrane. As a result, the membrane-engineered M. succiniciproducens strain showed higher tolerance toward SA and increased production of SA compared with the control strain without membrane engineering. The membrane engineering approach employed in this study will be useful for increasing tolerance to, and consequently enhancing production of acid products.

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.

Bronchopneumonia associated with Mannheimia granulomatis infection in a Belgian hare ( Lepus europaeus).

Mannheimia granulomatis was first isolated from pneumonic European hares in the 1980s and has since been reported sporadically in pneumonic Swedish roe deer and Australian cattle. Although the pneumonic lesions caused by M. haemolytica in livestock have been extensively studied and reported, little is published with regard to the pneumonic lesions associated with M. granulomatis infection in any species. We describe the histopathology of purulent bronchopneumonia associated with M. granulomatis in a Belgian hare ( Lepus europaeus) resident in British Columbia, Canada, and compare the lesions with those caused by M. haemolytica in livestock.

Small ruminant pasteurellosis in Tigray region, Ethiopia: marked serotype diversity may affect vaccine efficacy.

The aim of this study was to investigate the prevalent Bibersteinia, Mannheimia and Pasteurella serotypes, risk factors and degree of serotype co-infections in sheep and goats in the Tigray region of Ethiopia. Serum was collected from 384 sheep and goats from the Tanqua-Abergelle district of Tigray region using cross-sectional random sampling. An indirect haemagglutination test was used for serotyping. Risk factors for infections were evaluated by logistic regression. Potential clustering of multiple serotypes within individual animals due to common risk factors was evaluated by redundancy analysis. Eight serotypes were identified: all studied animals were serologically positive for at least one serotype. Overall, 355 (92·45%) of the animals were infected by four or more serotypes. Of the five risk factors studied, peasant association (PA), animal species, age (serotype A1), and bodyweight (serotype T15) were significantly associated with infection, but sex was not significant. Only PA explained a significant proportion of the variation (adjusted R 2 = 0·16) in the serological responses. After the effect of PA was accounted for, T3 and T4; A7 and Pasteurella multocida A; and A7 and T10 were positively correlated for co-infection, while T4 and T10 were less likely to be found within the same animal. Diverse serotypes were circulating in the Tigray region and could be a challenge in selecting serotypes for vaccine.

Metabolic engineering of Mannheimia succiniciproducens for succinic acid production based on elementary mode analysis with clustering.

Mannheimia succiniciproducens, a capnophilic gram-negative rumen bacterium, has been employed for the efficient production of succinic acid. Although M. succiniciproducens metabolism was previously studied using a genome-scale metabolic model, more metabolic characteristics are to be understood. To this end, elementary mode analysis accompanied with clustering ('EMC' analysis) is used to gain further insights on metabolic characteristics of M. succiniciproducens allowing efficient succinic acid production. Elementary modes (EMs) generated from the central carbon metabolic network of M. succiniciproducens are clustered to systematically analyze succinic acid production routes. Based on the results of EMC analysis, zwf gene is identified as a novel overexpression target for the improved succinic acid production. This gene is overexpressed in a previously constructed succinic acid-overproducing M. succiniciproducens LPK7 strain. Heterologous NADPH-dependent mdh is later intuitively selected for overexpression to synergistically improve succinic acid production by utilizing abundant NADPH pool mediated by the overexpressed zwf. The LPK7 strains co-expressing mdh alone and both zwf and mdh genes are subjected to fed-batch fermentation to better examine their succinic acid production performances. Strategies of EMC analysis will be useful for further metabolic engineering of M. succiniciproducens and other microorganisms to improve production of succinic acid and other chemicals of interest.

Homo-succinic acid production by metabolically engineered Mannheimia succiniciproducens.

Succinic acid (SA) is a four carbon dicarboxylic acid of great industrial interest that can be produced by microbial fermentation. Here we report development of a high-yield homo-SA producing Mannheimia succiniciproducens strain by metabolic engineering. The PALFK strain (ldhA-, pta-, ackA-, fruA-) was developed based on optimization of carbon flux towards SA production while minimizing byproducts formation through the integrated application of in silico genome-scale metabolic flux analysis, omics analyses, and reconstruction of central carbon metabolism. Based on in silico simulation, utilization of sucrose would enhance the SA production and cell growth rates, while consumption of glycerol would reduce the byproduct formation rates. Thus, sucrose and glycerol were selected as dual carbon sources to improve the SA yield and productivity, while deregulation of catabolite-repression was also performed in engineered M. succiniciproducens. Fed-batch fermentations of PALFK with low- and medium-density (OD600 of 0.4 and 9.0, respectively) inocula produced 69.2 and 78.4g/L of homo-SA with yields of 1.56 and 1.64mol/mol glucose equivalent and overall volumetric SA productivities of 2.50 and 6.02g/L/h, respectively, using sucrose and glycerol as dual carbon sources. The SA productivity could be further increased to 38.6g/L/h by employing a membrane cell recycle bioreactor system. The systems metabolic engineering strategies employed here for achieving homo-SA production with the highest overall performance indices reported to date will be generally applicable for developing superior industrial microorganisms and competitive processes for the bio-based production of other chemicals as well.

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.

Eosinophilic granuloma with Splendore-Hoeppli material caused by Mannheimia granulomatis in a calf.

A large subcutaneous mass, formed on the left lower jaw of a 4-month-old Japanese Black male calf, was partially excised for histological and bacteriological examinations. Antibiotic treatment resulted in a good prognosis. Bacteria isolated from the excised material were characterized by weak hemolysis and positive reactions for catalase and oxidase, and were 99% identical to Mannheimia granulomatis strains. The presence of the leukotoxin gene product was demonstrated by polymerase chain reaction amplification. Histological examination showed that the excised material was composed of dense fibrous connective tissue with sparsely distributed eosinophilic granulomas or abscesses. These foci frequently contained Splendore-Hoeppli material with rod-shaped Gram-negative bacteria. Except for the absence of lymphangitis and the presence of basophils and mast cells, the histology of this lesion resembled that of lechiguana associated with coinfection of M. granulomatis and Dermatobia hominis. Leukotoxin was demonstrated by immunohistochemistry within Splendore-Hoeppli material and was judged to be responsible for its formation.