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Nature Communications Apr 2020Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Malate...
Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Malate dehydrogenase (MDH) is one of the key enzymes for SA production, but has not been well characterized. Here we report biochemical and structural analyses of various MDHs and development of hyper-SA producing M. succiniciproducens by introducing the best MDH. Corynebacterium glutamicum MDH (CgMDH) shows the highest specific activity and least substrate inhibition, whereas M. succiniciproducens MDH (MsMDH) shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate (ki of 67.4 and 588.9 μM for MsMDH and CgMDH, respectively). Structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition. A high-inoculum fed-batch fermentation of the final strain expressing cgmdh produces 134.25 g L of SA with the maximum productivity of 21.3 g L h, demonstrating the importance of enzyme optimization in strain development.
Topics: Bacterial Proteins; Bioreactors; Corynebacterium glutamicum; Fermentation; Kinetics; Malate Dehydrogenase; Metabolic Engineering; Oxaloacetic Acid; Pasteurellaceae; Protein Conformation; Recombinant Proteins; Substrate Specificity; Succinic Acid
PubMed: 32327663
DOI: 10.1038/s41467-020-15839-z -
Medicine Jan 2016Actinobacillus actinomycetemcomitans infection is a rare and easily misdiagnosed ocular disease. In this article, the authors report a chronic, purulent, and... (Review)
Review
Actinobacillus actinomycetemcomitans infection is a rare and easily misdiagnosed ocular disease. In this article, the authors report a chronic, purulent, and difficult-to-treat case of A actinomycetemcomitans keratitis following a glaucoma infiltration surgery.A 56-year-old man with a long-standing history of open-angle glaucoma in both eyes presented with a 12-week history of ocular pain, redness, and blurred vision in his right eye. He underwent a glaucoma infiltration surgery in his right eye 6 months ago. Three months postoperatively, he developed peripheral corneal stromal opacities associated with a white, thin, cystic bleb, and conjunctival injection. These opacities grew despite topical treatment with topical tobramycin, levofloxacin, natamycin, amikacin, and metronidazole eye drops.Multiple corneal scrapings revealed no organisms, and no organisms grew on aerobic, anaerobic, fungal, or mycobacterial cultures. The patient's right eye developed a severe purulent corneal ulcer with a dense hypopyon and required a corneal transplantation. Histopathologic analysis and 16S ribosomalribonucleic acid polymerase chain reaction sequencing revealed A actinomycetemcomitans as the causative organism. Postoperatively, treatment was initiated with topical levofloxacin and cyclosporine, as well as oral levofloxacin and cyclosporine. Graft and host corneal transparency were maintained at the checkup 1 month after surgery.Although it is a rare cause of corneal disease, A actinomycetemcomitans should be suspected in patients with keratitis refractory to topical antibiotic therapy. Delay in diagnosis and appropriate treatment can result in vision loss.
Topics: Aggregatibacter actinomycetemcomitans; Glaucoma Drainage Implants; Glaucoma, Open-Angle; Humans; Keratitis; Male; Middle Aged; Pasteurellaceae Infections
PubMed: 26817919
DOI: 10.1097/MD.0000000000002608 -
MSphere Mar 2019Although the microbiota in the proximal gastrointestinal (GI) tract have been implicated in health and disease, much about these microbes remains understudied compared...
Although the microbiota in the proximal gastrointestinal (GI) tract have been implicated in health and disease, much about these microbes remains understudied compared to those in the distal GI tract. This study characterized the microbiota across multiple proximal GI sites over time in healthy individuals. As part of a study of the pharmacokinetics of oral mesalamine administration, healthy, fasted volunteers ( = 8; 10 observation periods total) were orally intubated with a four-lumen catheter with multiple aspiration ports. Samples were taken from stomach, duodenal, and multiple jejunal sites, sampling hourly (≤7 h) to measure mesalamine (administered at = 0), pH, and 16S rRNA gene-based composition. We observed a predominance of across proximal GI sites, with significant variation compared to stool. The microbiota was more similar within individuals over time than between subjects, with the fecal microbiota being unique from that of the small intestine. The stomach and duodenal microbiota displayed highest intraindividual variability compared to jejunal sites, which were more stable across time. We observed significant correlations in the duodenal microbial composition with changes in pH; linear mixed models identified positive correlations with multiple operational taxonomic units (OTUs) and negative correlations with multiple and OTUs. Few OTUs correlated with mesalamine concentration. The stomach and duodenal microbiota exhibited greater compositional dynamics than the jejunum. Short-term fluctuations in the duodenal microbiota were correlated with pH. Given the unique characteristics and dynamics of the proximal GI tract microbiota, it is important to consider these local environments in health and disease states. The gut microbiota are linked to a variety of gastrointestinal diseases, including inflammatory bowel disease. Despite this importance, microbiota dynamics in the upper gastrointestinal tract are understudied. Our article seeks to understand what factors impact microbiota dynamics in the healthy human upper gut. We found that the upper gastrointestinal tract contains consistently prevalent bacterial OTUs that dominate the overall community. Microbiota variability is highest in the stomach and duodenum and correlates with pH.
Topics: Administration, Oral; Adolescent; Adult; Bacteria; Fasting; Feces; Female; Firmicutes; Gastrointestinal Microbiome; Healthy Volunteers; Humans; Hydrogen-Ion Concentration; Intestine, Small; Intubation, Gastrointestinal; Linear Models; Male; Middle Aged; Pasteurellaceae; RNA, Ribosomal, 16S; Spatio-Temporal Analysis; Stomach; Young Adult
PubMed: 30867328
DOI: 10.1128/mSphere.00126-19 -
BMC Veterinary Research Jul 2020A microbiological diagnosis is essential to better target antimicrobial treatment, control and prevention of respiratory tract infections in cattle. Under field...
BACKGROUND
A microbiological diagnosis is essential to better target antimicrobial treatment, control and prevention of respiratory tract infections in cattle. Under field conditions, non-endoscopic broncho-alveolar lavage (nBAL) samples are increasingly collected. To what extent the highly variable turnaround time and storage temperatures between sampling and cultivation affect the isolation rate of bacterial pathogens is unknown. Therefore, the objective of this experimental study was to determine the effect of different storage temperatures (0 °C, 8 °C, 23 °C and 36 °C) and times (0,2,4,6,8,24,48 h) on the isolation rate and concentration of Pasteurellaceae in nBAL samples from clinically affected animals.
RESULTS
At a storage temperature temperature of 36 °C isolation rates of Mannheimia haemolytica and Pasteurella multocida were significantly reduced 6 h and 48 h after sampling, respectively. At room temperature (23 °C), a decrease in M. haemolytica and P. multocida isolation rate was noticed, starting at 24 and 48 h after sampling, respectively, but only significant for P. multocida at 48 h. The presence of microbial contamination negatively affected the isolation of P. multocida in clinical nBAL samples, but not of M. haemolytica.
CONCLUSION
Optimal M. haemolytica and P. multocida isolation rates from clinical nBAL samples are obtained after storage at 0 °C or 8 °C, provided that the sample is cultivated within 24 h after sampling. The maximum period a sample can be stored without an effect on the M. haemolytica and P. multocida isolation success varies and is dependent on the storage temperature and the degree of microbial contamination.
Topics: Animals; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Cattle; Cattle Diseases; Mannheimia haemolytica; Pasteurella Infections; Pasteurella multocida; Specimen Handling; Temperature; Time Factors
PubMed: 32660585
DOI: 10.1186/s12917-020-02456-7 -
Journal of Dairy Science Mar 2020Respiratory tract infections (bovine respiratory disease) are a major concern in calf rearing. The objective of this study was to identify pathogen-specific risk factors...
Respiratory tract infections (bovine respiratory disease) are a major concern in calf rearing. The objective of this study was to identify pathogen-specific risk factors associated with epidemic respiratory disease in calves. A cross-sectional study was conducted, involving 128 outbreaks (29 dairy, 58 dairy-mixed, and 41 beef) in Belgium (2016-2018). A semiquantitative PCR for 7 respiratory pathogens was done on a pooled nonendoscopic bronchoalveolar lavage sample for each herd. Potential risk factors were collected by questionnaire and derived from the national cattle registration databank. Most outbreaks occurred between October and March, and single and multiple viral infections were detected in 58.6% (75/128) and 13.3% (17/128), respectively. Bovine coronavirus (BCV) was the most frequently isolated virus (38.4%), followed by bovine respiratory syncytial virus (bRSV; 29.4%) and parainfluenzavirus type 3 (PI-3; 8.1%). Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni were detected in 33.3, 41.2, 89.1, and 36.4% of the herds, respectively. Specific risk factors for BCV detection were detection of M. haemolytica [odds ratio (OR) = 2.8 (95% confidence interval = 1.1-7.5)], increasing herd size [OR = 1.3 (1.0-1.8) for each increase with 100 animals] and detection of BCV by antigen ELISA on feces in calves in the last year [OR = 3.6 (1.2-11.1)]. A seasonal effect was shown for bRSV only {more in winter compared with autumn [OR = 10.3 (2.8-37.5)]}. Other factors associated with bRSV were PI-3 detection [OR = 13.4 (2.1-86.0)], prevalence of calves with respiratory disease [OR = 1.02 (1.00-1.04) per 1% increase], and number of days with respiratory signs before sampling [OR = 0.99 (0.98-0.99) per day increase]. Next to its association with BCV, M. haemolytica was more frequently detected in herds with 5 to 10 animals per pen [OR = 8.0 (1.4-46.9)] compared with <5 animals, and in herds with sawdust as bedding [OR = 18.3 (1.8-191.6)]. Also, for H. somni, housing on sawdust was a risk factor [OR = 5.2 (1.2-23.0)]. Purchase of cattle [OR = 2.9 (1.0-8.0)] and housing of recently purchased animals in the same airspace [OR = 5.0 (1.5-16.5)] were risk factors for M. bovis. This study identified pathogen-specific risk factors that might be useful for the development of customized control and prevention and for the design of decision support tools to justify antimicrobial use by predicting the most likely pathogen before sampling results are available.
Topics: Animals; Belgium; Bronchoalveolar Lavage; Cattle; Cattle Diseases; Coronavirus, Bovine; Cross-Sectional Studies; Disease Outbreaks; Feces; Female; Male; Mannheimia haemolytica; Mycoplasma bovis; Parainfluenza Virus 3, Bovine; Pasteurella multocida; Pasteurellaceae; Respiratory Syncytial Virus, Bovine; Respiratory Tract Infections; Risk Factors; Species Specificity; Surveys and Questionnaires
PubMed: 31954585
DOI: 10.3168/jds.2019-17486 -
Journal of Clinical Microbiology Aug 2022() is an opportunistic pathogen in poultry, which, however, has also been associated with human disease. There is currently no approved method for antimicrobial...
() is an opportunistic pathogen in poultry, which, however, has also been associated with human disease. There is currently no approved method for antimicrobial susceptibility testing of this pathogen, so this study aimed at developing a harmonized broth microdilution method for that is suitable for diagnostic laboratories. For this, the CCUG 12391 type strain and 42 field isolates were collected and their species was confirmed by using a species-specific PCR assay and biochemical reactions. To select epidemiologically unrelated isolates, ApaI macrorestriction analysis was performed. Preliminary growth experiments were conducted with six culture media, and based on the results, four media were selected to compile growth curves with four isolates. Independent repetitions of MIC determinations were then performed to evaluate the reproducibility of the values. Cation-adjusted Mueller-Hinton broth (CAMHB) was initially selected as broth medium, but did not show sufficient homogeneity of MICs. Therefore, CAMHB plus 1% chicken serum and 0.0025% NADH was selected and showed a good homogeneity of MICs after 20 h and 24 h of incubation at 35 ± 2°C. This was reflected in essential MIC agreements ranging between 96% and 100%. Testing of a larger collection ( = 43) revealed that easily readable MICs could be obtained for the type strain and all isolates. Some showed elevated MICs of enrofloxacin ( = 35), nalidixic acid ( = 35), penicillin ( = 2), tetracycline ( = 19), and/or trimethoprim-sulfamethoxazole ( = 1). By using PCR analyses, the following antimicrobial resistance genes were detected: , , , (B), (H). The study demonstrated that the proposed medium is suitable for a harmonized broth microdilution susceptibility testing of with a recommended incubation time of 20 to 24 h.
Topics: Anti-Bacterial Agents; Anti-Infective Agents; Humans; Microbial Sensitivity Tests; Pasteurellaceae; Reproducibility of Results
PubMed: 35852371
DOI: 10.1128/jcm.00419-22 -
BMC Veterinary Research Aug 2017Outbreaks of a Haemorrhagic Septicaemia (HS) like disease causing large mortalities in camels (Camelus dromedarius) in Asia and in Africa have been reported since 1890....
BACKGROUND
Outbreaks of a Haemorrhagic Septicaemia (HS) like disease causing large mortalities in camels (Camelus dromedarius) in Asia and in Africa have been reported since 1890. Yet the aetiology of this condition remains elusive. This study is the first to apply state of the art molecular methods to shed light on the nasopharyngeal carrier state of Pasteurellaceae in camels. The study focused on HS causing Pasteurella multocida capsular types B and E. Other Pasteurellaceae, implicated in common respiratory infections of animals, were also investigated.
METHODS
In 2007 and 2008, 388 nasopharyngeal swabs were collected at 12 locations in North Kenya from 246 clinically healthy camels in 81 herds that had been affected by HS-like disease. Swabs were used to cultivate bacteria on blood agar and to extract DNA for subsequent PCR analysis targeting P. multocida and Mannheimia-specific gene sequences.
RESULTS
Forty-five samples were positive for P. multocida genes kmt and psl and for the P. multocida Haemorrhagic Septicaemia (HS) specific sequences KTSP61/KTT72 but lacked HS-associated capsular type B and E genes capB and capE. This indicates circulation of HS strains in camels that lack established capsular types. Sequence analysis of the partial 16S rRNA gene identified 17 nasal swab isolates as 99% identical with Mannheimia granulomatis, demonstrating a hitherto unrecognised active carrier state for M. granulomatis or a closely related Mannheimia sp. in camels.
CONCLUSIONS
The findings of this study provide evidence for the presence of acapsular P. multocida or of hitherto unknown capsular types of P. multocida in camels, closely related to P. multocida strains causing HS in bovines. Further isolations and molecular studies of camelid P. multocida from healthy carriers and from HS-like disease in camels are necessary to provide conclusive answers. This paper is the first report on the isolation of M. granulomatis or a closely related new Mannheimia species from camelids.
Topics: Animals; Camelus; Carrier State; DNA, Bacterial; Nasopharynx; Pasteurella Infections; Pasteurella multocida; Pasteurellaceae; Pasteurellaceae Infections; Pilot Projects; Polymerase Chain Reaction
PubMed: 28830429
DOI: 10.1186/s12917-017-1189-y -
Infection and Immunity Mar 2020Nasopharyngeal colonization with nontypeable (NTHi) is a prerequisite for developing NTHi-associated infections, including otitis media. Therapies that block NTHi...
Nasopharyngeal colonization with nontypeable (NTHi) is a prerequisite for developing NTHi-associated infections, including otitis media. Therapies that block NTHi colonization may prevent disease development. We previously demonstrated that , a closely related human commensal, can inhibit NTHi colonization and infection of human respiratory epithelium We have now assessed whether (a rodent commensal from the same family) can prevent NTHi colonization and disease using a murine NTHi otitis media model. Otitis media was modeled in BALB/c mice using coinfection with 1 × 10 PFU of influenza A virus MEM H3N2, followed by intranasal challenge with 5 × 10 CFU of NTHi R2866 Spec Mice were pretreated or not with an intranasal inoculation of 5 × 10 CFU 24 h before coinfection. NTHi and viable counts and inflammatory mediators (gamma interferon [IFN-γ], interleukin-1β [IL-1β], IL-6, keratinocyte chemoattractant [KC], and IL-10) were measured in nasal washes and middle ear tissue homogenate. pretreatment decreased the median colonization density of NTHi from 6 × 10 CFU/ml to 9 × 10 CFU/ml ( = 0.0004). Only 1/12 -pretreated mice developed otitis media on day 5 compared to 8/15 mice with no pretreatment (8% versus 53%, = 0.0192). Inflammation, clinical score, and weight loss were also lower in -pretreated mice. We have demonstrated that a single dose of a closely related commensal can delay onset of NTHi otitis media Human challenge studies investigating prevention of NTHi colonization are warranted to reduce the global burden of otitis media and other NTHi diseases.
Topics: Administration, Intranasal; Animals; Antibiosis; Carrier State; Colony Count, Microbial; Cytokines; Disease Models, Animal; Haemophilus Infections; Haemophilus influenzae; Influenza A Virus, H3N2 Subtype; Mice, Inbred BALB C; Nasal Mucosa; Nasopharynx; Otitis Media; Pasteurellaceae
PubMed: 31964748
DOI: 10.1128/IAI.00685-19 -
Veterinary Research Aug 2014The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in commercial egg-layers, leading to reduced egg production and... (Randomized Controlled Trial)
Randomized Controlled Trial
The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in commercial egg-layers, leading to reduced egg production and increased mortality. Unfortunately, widespread multidrug resistance and antigenic diversity makes it difficult to control infections and novel prevention strategies are urgently needed. In this study, a pan-genomic reverse vaccinology (RV) approach was used to identify potential vaccine candidates. Firstly, the genomes of 10 selected Gallibacterium strains were analyzed and proteins selected on the following criteria; predicted surface-exposure or secretion, none or one transmembrane helix (TMH), and presence in six or more of the 10 genomes. In total, 42 proteins were selected. The genes encoding 27 of these proteins were successfully cloned in Escherichia coli and the proteins expressed and purified. To reduce the number of vaccine candidates for in vivo testing, each of the purified recombinant proteins was screened by ELISA for their ability to elicit a significant serological response with serum from chickens that had been infected with G. anatis. Additionally, an in silico prediction of the protective potential was carried out based on a protein property prediction method. Of the 27 proteins, two novel putative immunogens were identified; Gab_1309 and Gab_2312. Moreover, three previously characterized virulence factors; GtxA, FlfA and Gab_2156, were identified. Thus, by combining the pan-genomic RV approach with subsequent in vitro and in silico screening, we have narrowed down the pan-proteome of G. anatis to five vaccine candidates. Importantly, preliminary immunization trials indicated an in vivo protective potential of GtxA-N, FlfA and Gab_1309.
Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Bacterial Vaccines; Chickens; Computer Simulation; Escherichia coli; Pasteurellaceae; Pasteurellaceae Infections; Poultry Diseases; Virulence Factors
PubMed: 25223320
DOI: 10.1186/s13567-014-0080-0 -
Journal of Bacteriology Feb 2008Prokaryotic secretion relies on proteins that are widely conserved, including NTPases and secretins, and on proteins that are system specific. The Tad secretion system...
Prokaryotic secretion relies on proteins that are widely conserved, including NTPases and secretins, and on proteins that are system specific. The Tad secretion system in Aggregatibacter actinomycetemcomitans is dedicated to the assembly and export of Flp pili, which are needed for tight adherence. Consistent with predictions that RcpA forms the multimeric outer membrane secretion channel (secretin) of the Flp pilus biogenesis apparatus, we observed the RcpA protein in multimers that were stable in the presence of detergent and found that rcpA and its closely related homologs form a novel and distinct subfamily within a well-supported gene phylogeny of the entire secretin gene superfamily. We also found that rcpA-like genes were always linked to Aggregatibacter rcpB- or Caulobacter cpaD-like genes. Using antisera, we determined the localization and gross abundances of conserved (RcpA and TadC) and unique (RcpB, RcpC, and TadD) Tad proteins. The three Rcp proteins (RcpA, RcpB, and RcpC) and TadD, a putative lipoprotein, localized to the bacterial outer membrane. RcpA, RcpC, and TadD were also found in the inner membrane, while TadC localized exclusively to the inner membrane. The RcpA secretin was necessary for wild-type abundances of RcpB and RcpC, and TadC was required for normal levels of all three Rcp proteins. TadC abundance defects were observed in rcpA and rcpC mutants. TadD production was essential for wild-type RcpA and RcpB abundances, and RcpA did not multimerize or localize to the outer membrane without the expression of TadD. These data indicate that membrane proteins TadC and TadD may influence the assembly, transport, and/or function of individual outer membrane Rcp proteins.
Topics: Bacterial Adhesion; Bacterial Outer Membrane Proteins; Bacterial Proteins; Cell Membrane; Dimerization; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Humans; Membrane Proteins; Pasteurellaceae; Phylogeny
PubMed: 18055598
DOI: 10.1128/JB.01347-07