Positive effect of phasin in biohydrogen production of non polyhydroxybutyrate-producing Clostridium acetobutylicum ATCC 824.

In this study, the goal was to enhance the tolerance of Clostridium acetobutylicum ATCC 824 to biomass-based inhibitory compounds for biohydrogen production and evaluate various known genes that enhance the production of biochemicals in various hosts. The introduction of phaP, the major polyhydroxyalkanoate granule-associated protein that has been reported as a chaperone-like protein resulted in increased tolerance to inhibitors and leads to higher levels of hydrogen production, cell growth, and glucose consumption in the presence of these inhibitors. It was observed that the introduction of phaP led to an increase in the transcription of the hydrogenase gene, whereas transcription of the chaperone functional genes decreased compared to the wild type. Finally, the introduction of phaP could significantly enhance biohydrogen production by 2.6-fold from lignocellulosic hydrolysates compared to that of wild type. These findings suggested that the introduction of phaP could enhance growth and biohydrogen production, even in non-polyhydroxyalkanoate-producing strains.

Advances in synthetic biology toolboxes paving the way for mechanistic understanding and strain engineering of gut commensal Bacteroides spp. and Clostridium spp.

The gut microbiota plays a significant role in influencing human immunity, metabolism, development, and behavior by producing a wide range of metabolites. While there is accumulating data on several microbiota-derived small molecules that contribute to host health and disease, our knowledge regarding the molecular mechanisms underlying metabolite-mediated microbe-host interactions remains limited. This is primarily due to the lack of efficient genetic tools for most commensal bacteria, especially those belonging to the dominant phyla Bacteroides spp. and Clostridium spp., which hinders the application of synthetic biology to these gut commensal bacteria. In this review, we provide an overview of recent advances in synthetic biology tools developed for the two dominant genera, as well as their applications in deciphering the mechanisms of microbe-host interactions mediated by microbiota-derived small molecules. We also discuss the potential biomedical applications of engineering commensal bacteria using these toolboxes. Finally, we share our perspective on the future development of synthetic biology tools for a better understanding of small molecule-mediated microbe-host interactions and their engineering for biomedical purposes.

Postpartum clostridial gangrenous metritis in 12 dairy goats in France.

Clostridial infections in goats have been associated frequently with enteric diseases or gas gangrene but very rarely with the reproductive system. We describe here 12 cases of fatal postpartum gangrenous metritis in does associated with infection by several clostridial species. Clinically, these cases were characterized by rapid onset of hyperthermia followed by death after kidding. On postmortem examination, the uteri appeared to be necrotic and were hemorrhagic and edematous. Microscopically, the uteri had diffuse coagulative necrosis, edema, hemorrhage, and fibrinous thrombi with intralesional gram-positive rods. Clostridium perfringens was isolated from 7 of 9 uterine samples cultured, and C. perfringens, C. septicum, C. novyi, or C. chauvoei were demonstrated by immunohistochemistry (IHC) in the 5 cases examined. IHC for Paeniclostridium sordellii was negative in all 5 cases. PCR performed on 3 of the C. perfringens isolates was positive for alpha toxin and perfringolysin, identifying these isolates as type A. Clostridial infection should be considered in cases of postpartum gangrenous metritis of does.

Bacterial metabolic engineering for the production of second-generation (2 G) bioethanol and biobutanol; a review.

The second generation (2 G) biofuels were introduced to solve the issues associated with first-generation biofuel (dependency on food materials) and fossil fuels, such as reservoirs diminution, high demand, price fluctuation, and lethal greenhouse gases emission. Butanol and ethanol are the main 2 G biofuels. They are used as a disinfectant, antiseptic, and chemical solvent in the pharmaceutical, plastic, textiles, cosmetics, and fuel industries. Currently, their bacterial biological production from lignocellulosic material at the industrial level with primitive microorganisms is under development and not economical and qualitative compatible as compared to that of fossil origin, due to the slow growth rate, low titer, recalcitrant nature of lignocellulose, strain intolerance to a higher amount of butanol and ethanol, and strain inability to tolerate inhibitors accumulated during pretreatment of lignocellulosic materials. Therefore, metabolic engineering strategies such as redirection of carbon flux, knocking out competing pathways, enhancing strain robustness and wide range of substrate utilization ability, and overexpression of enzymes involved in their biological synthesis have been applied to bacteria for enhancing their ability for 2 G ethanol and butanol production in a highly cost-effective amount from lignocellulosic materials. Herein, we summarized and reviewed the progress in metabolic engineering of bacterial species such as Clostridium spp,Escherichia coli, and Zymomonas mobilis for the synthesis of 2 G butanol and ethanol, especially from lignocellulosic materials.

Boosting ethanol production rates from carbon dioxide in MES cells under optimal solventogenic conditions.

Microbial Electrosynthesis (MES) has been widely applied for acetic acid (HA) production from CO2 and electricity. Ethanol (EtOH) has a higher market value than HA, and wide application in industry and as a biofuel. However, it has only been obtained sporadically and at low concentrations, probably due to sub-optimal operating conditions. This study aimed at enhancing EtOH productivity in MES cells by jointly optimising key operation parameters, including pH, H2 and CO2 partial pressure (pH2 and pCO2), and HA concentration, to promote solventogenesis. Two H-type cells were operated in fed-batch mode at -0.8 V vs. SHE with CO2 as the sole carbon source. A mixed culture, enriched with Clostridium ljungdahlii was used as the biocatalyst. The combination of low pH (<4.5) and pCO2 (<0.3 atm), along with high HA concentration (about 6 g L-1) and pH2 (>3 atm), were mandatory conditions for maintaining an efficient solventogenic culture, dominated by Clostridium sp., capable of high-rate EtOH production. The maximum EtOH production rate was 10.95 g m-2 d-1, and a concentration of 5.28 g L-1 was achieved. Up to 30 % of the electrons and 15.2 % of the carbon provided were directed towards EtOH production, and 28.1 kWh were required for the synthesis of 1 kg of EtOH from CO2. These results highlight that strict conditions are required for a continuous, reliable, EtOH production in MES cells. Future investigation should focus on improving cell configuration to achieve EtOH production at higher current densities while minimizing the electric energy input.

Inhibitory activity of aromatic plant extracts against dairy-related Clostridium species and their use to prevent the late blowing defect of cheese.

The aim of the present work was the selection of aromatic plant essential oils (EOs) and/or ethanolic extracts (EEs) to prevent the late blowing defect (LBD) of cheese caused by Clostridium spp. EEs resulted more effective than EOs to inhibit dairy-borne Clostridium spp. in vitro. Savory, hyssop, lavender and tarragon EEs, which showed the lowest minimal inhibitory concentration against Clostridium tyrobutyricum, were selected to study the prevention of LBD caused by this bacterium in cheese. Addition of savory and lavender EEs to cheese milk delayed LBD by 2 weeks, but at the end of ripening these cheeses showed similar clostridial vegetative cells counts, spoilage symptoms and propionic, and butyric acids levels than blown control cheese. Tarragon EE, with the highest content in caffeic acid, also delayed LBD by 2 weeks, but it was more effective to inhibit Clostridium, since cheese with tarragon EE showed minor LBD symptoms, lower vegetative cells count and lower concentrations of propionic and butyric acids than the rest of cheeses made with EEs. This fact could be also attributable to the greater number of antimicrobial terpenes (1,8-cineole, 4-terpineol, α-terpineol, isoelemicin, methyl eugenol, and methyl trans-isoeugenol) detected in this cheese. This is the first report on the application of EEs to control C. tyrobutyricum in cheese.

Clostridium species diversity in gut microbiota of patients with renal failure.

The Pathology of digestive tract has long been known to be correlated with chronic kidney disease (CKD). The member of the major Firmicutes phylum especially Clostridium subcluster XIVa altered quantitatively and qualitatively in the gut microbiota of patients with End Stage Renal Disease (ESRD) and CKD. Therefore, in this study, the abundance of the species of Clostridium genus of Firmicutes phylum compared between intestine microbiota of patients with kidney failure and healthy individual. Fresh fecal specimens of 20 patients at different stages of CKD and 20 healthy individuals were collected. Bacterial DNA of samples were extracted to use for 16S ribosomal DNA sequencing targeting the V3-V4 region. Next generation sequencing (NGS) method at MiSeq system was used to find the diversity of gut microbiota composition. Totally, 11 (1.68%) of 651 bacterial strains which were isolated from forty fecal samples of both healthy volunteers and CKD/ESRD patients, were identified as Clostridium species. Eight genera of 11 Clostridium genera were related to Clostridium sensu stricto, and 3 other genera were as follows Vallitalea, Acidaminobacter and Caloramator. Among both group, the highest abundance was dedicated to Clostridium celatum genera. Sarcina maxima were not identified. The composition of Clostridium spp. showed the same frequency among CKD/ESRD and healthy groups (p < 0.05). The abundance of Clostridium spp. is virtually the same and not differs among healthy individuals and CKD/ESRD patients. Results of the present indicate despite of critical role of gut microbiota, some pathogens and their metabolites have no role on hemostasis and pathogenesis of kidney disorders.

Late blowing defect in Grottone cheese: detection of clostridia and control strategies.

"Grottone" is a pasta filata hard cheese produced in Campania region from cow's milk and characterized by holes formation due to CO2 development by Propionic Acid Bacteria. The contamination of raw milk with butyric acid-producing spore-forming clostridia represent a major concern for cheese producers since clostridia outgrowth may lead to the cheese late blowing defect during ripening. Detection of clostridial endospores in milk before processing and the use of antimicrobial compounds may represent an important control strategy. The present study is aimed to point out the most suitable procedure for the determination of clostridial spores in dairy samples, and to assess the inhibitory activity of several antimicrobial compounds against Cl. sporogenes. Based on results, MPN counts on Bryant and Burkey medium and CFU on RCM proved to be the most suitable protocols for routine testing. By using these procedures clostridial spores were detected in 10 out 13 milk samples and in all cheeses with late blowing defect. Within antimicrobial compounds, sodium nitrate is still the best choice for preventing late blowing, nevertheless a protective culture of Lacticaseibacillus casei proved to be a promising alternative. Nevertheless, the use of this protective culture in six Grottone cheese productions carried out at farm level, led to unsatisfactory results. Holes' development was hampered likely for an inhibition of the PAB starter and the expected 'Grouviera-type' taste was not perceived by panellists. Based on results, the use of protective cultures needs to be contextualized and interactions with starters needs to be evaluated case by case.

Potential Applications of Essential Oils for Environmental Sanitization and Antimicrobial Treatment of Intensive Livestock Infections.

The extensive use of antibiotics has contributed to the current antibiotic resistance crisis. Livestock infections of Salmonella spp, Clostridium spp. and E. coli antimicrobial-resistant bacteria represent a public threat to human and animal health. To reduce the incidence of these zoonoses, essential oils (EOs) could be effective antibiotic alternatives. This study aims at identifying EOs safe for use, effective both in complementary therapy and in the environmental sanitization of intensive farming. Natural products were chemo-characterized by gas chromatography. Three S. Typhimurium, three C. perfringens and four E. coli strains isolated from poultry and swine farms were used to assess the antimicrobial properties of nine EOs and a modified GR-OLI (mGR-OLI). The toxicity of the most effective ones (Cinnamomum zeylanicum, Cz; Origanum vulgare, Ov) was also evaluated on porcine spermatozoa and Galleria mellonella larvae. Cz, Ov and mGR-OLI showed the strongest antimicrobial activity; their volatile components were also able to significantly inhibit the growth of tested strains. In vitro, Ov toxicity was slightly lower than Cz, while it showed no toxicity on G. mellonella larvae. In conclusion, the study confirms the importance of evaluating natural products to consolidate the idea of safe EO applications in reducing and preventing intensive livestock infections.

Enhanced solventogenesis in syngas bioconversion: Role of process parameters and thermodynamics.

Biofuels, such as ethanol and butanol, obtained from carbon monoxide-rich gas or syngas bioconversion (solventogenesis) are an attractive alternative to traditional fermentation processes with merits of no competition with food production and sustainability. However, there is a lack of comprehensive understanding of some key process parameters and mechanisms enhancing solventogenesis during the fermentation process. This review provides an overview of the current state of the art of the main influencing factors during the syngas fermentation process catalyzed by acetogenic species as well as undefined mixed cultures. The role of syngas pressure, syngas components, fermentation pH, temperature, trace metals, organic compounds and additional materials is overviewed. As a so far hardly considered approach, thermodynamic calculations of the Gibbs free energy of CO conversion to acetic acid, ethanol, butyric acid and butanol under different CO pressures and pH at 25, 33 and 55 °C are also addressed and reviewed. Strategies for enhancing mass transfer and longer carbon chain solvent production are considered as well.

Elephant Endotheliotropic Herpesvirus 4 and Clostridium perfringens Type C Fatal Co-Infection in an Adult Asian Elephant (Elephas maximus).

Elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD) is an acute, often fatal, multisystemic hemorrhagic disease and one of the most significant causes of mortality of Asian elephants in captivity. Most fatal cases of EEHV-HD are associated with EEHV1A and EEHV1B in juveniles. This case report describes the clinical and pathological features of a fatal co-infection of Clostridium perfringens type C and EEHV-HD, caused by EEHV4, in an adult female Asian elephant. Although fatal clostridial enterotoxemia has been occasionally reported in elephants, this report highlights the importance of having both EEHV-HD and clostridial enterotoxemia as potential differential diagnoses in cases of widespread tissue necrosis and internal hemorrhage in elephants, regardless of the animal age group, due to their macroscopic similarities, frequent co-occurrence and cumulative morbid potential.

Antibacterial Activity of Crocus sativus L. Petals Extracts against Foodborne Pathogenic and Spoilage Microorganisms, with a Special Focus on Clostridia.

In recent years, there has been a growing interest in the use of novel antimicrobial agents able to inhibit or kill food-borne bacteria or to interrupt the onset of food spoilage. Crocus sativus L. petals, typically considered as waste obtained from saffron spice production, could be a source of natural bioactive compounds to be used as food preservatives. The purpose of this work was to investigate the antibacterial properties of two hydroalcoholicsaffron petal extracts obtained by maceration (SPEA) and by ultrasonic bath (SPEB) methods. The main polyphenols identified in both extracts were gallic and chlorogenic acids, representing almost 70% of the phenolic fraction monitored. The antibacterial activity was studied by the agar well-diffusion method, against food-borne pathogenic and spoilage bacteria. Both extracts showed activity mainly against Gram-positive bacteria, in particular those belonging to the Clostridiaceae family (C. perfringens, C. botulinum and C. difficile), with inhibition zone diameters ranging from 13 to 18 mm. The antibacterial properties against Clostridia were further analyzed, determining MIC and MBC and performing a time-kill test. SPEA showed lower MIC/MBC values (250 mg/mL) compared to SPEB (500 mg/mL), suggesting that it could be more active against the assayed strains, probably because of its higher content of gallic acid. SPEA and SPEB, tested at a concentration of 1 × MIC, showed bactericidal activity against C. perfringens, C. botulinum and C. difficile and these results suggest that saffron petals could represent a valuable natural alternative source to conventional preservatives. Further investigations are needed to evaluate possible applications in the food industry.

Clostridium spp. Toxins: A Practical Guide for Expression and Characterization in Escherichia coli.

Farm animals are frequently affected by a group of diseases with a rapid clinical course, caused by Clostridium spp. and immunization is essential to provide protection. However, the current manufacturing platform for these vaccines has disadvantages and the main alternative is the use of an expression system that uses Escherichia coli to obtain recombinant vaccine antigens. In this chapter we describe procedures for cloning, expression and characterization of recombinant toxins from Clostridium spp. produced in E. coli for veterinary vaccine applications.

Selective butanol production from carbon monoxide by an enriched anaerobic culture.

An anaerobic mixed culture able to grow on pure carbon monoxide (CO) as well as syngas (CO, CO2 and H2), that produced unusual high concentrations of butanol, was enriched in a bioreactor with intermittent CO gas feeding. At pH 6.2, it mainly produced acids, generally acetic and butyric acid. After adaptation, under stress conditions of CO exposure at a partial pressure of 1.8 bar and low pH (e.g., 5.7), the enrichment accumulated ethanol, but also high amounts of butanol, up to 6.8 g/L, never reported before, with a high butanol/butyric acid molar ratio of 12.6, highlighting the high level of acid to alcohol conversion. At the end of the assay, both the acetic acid and ethanol concentrations decreased, with concomitant butyric acid production, suggesting C2 to C4 acid bioconversion, though this was not a dominant bioconversion process. The reverse reaction of ethanol oxidation to acetic acid was observed in the presence of CO2 produced during CO fermentation. Interestingly, butanol oxidation with simultaneous butyric acid production occurred upon production of CO2 from CO, which has to the best of our knowledge never been reported. Although the sludge inoculum contained a few known solventogenic Clostridia, the relative taxonomic abundance of the enriched sludge was diverse in Clostridia and Bacilli classes, containing known solventogens, e.g., Clostridium ljungdhalii, Clostridium ragsdalei and Clostridium coskatii, confirming their efficient enrichment. The relative abundance of unassigned Clostridium species amounted to 27% with presumably novel ethanol/butanol producers.

Concurrent Clostridial Enteritis and Oviductal Adenocarcinoma with Carcinomatosis in an Adult Alpaca (Vicugna pacos).

An adult alpaca (Vicugna pacos) with a history of colic and anorexia was euthanized because of failure to respond to treatment. Macroscopically, pale-tan, multifocal to coalescing, firm nodules and plaques markedly expanded the omentum, mesentery and the parietal and visceral peritoneum of multiple abdominal organs, especially the right oviduct and associated mesosalpinx. Abundant dark-red watery digesta were present in the duodenum and jejunum. Histological evaluation of the right oviduct, abdominal visceral nodules and plaques and mesenteric lymph nodes revealed transmural expansion and replacement by an epithelial malignant neoplasm, comprised of tubules and acini of ciliated columnar cells supported by abundant fibrous connective tissue. Both ovaries were histologically normal. On the basis of the ciliated morphology of the neoplastic cells, the focus on the proximal reproductive tract and the unremarkable ovaries, a reproductive tubal adenocarcinoma with carcinomatosis was diagnosed, with both the endometrium and oviduct considered as the tissues of origin. The prominent ciliated morphology of the neoplastic cells and the classification of human fallopian tube (oviduct) neoplasia lead us to propose oviductal adenocarcinoma with widespread carcinomatosis as the definitive diagnosis. The lamina propria of the small intestine was infiltrated segmentally by lymphocytes, plasma cells and neutrophils, and Clostridium perfringens with beta2 toxin production was identified by polymerase chain reaction in the small intestinal contents. To our knowledge, this is the first report of these two distinct diseases in an alpaca.

Enhanced Ethanol Production From Carbon Monoxide by Enriched Clostridium Bacteria.

Carbon monoxide (CO)-metabolizing Clostridium spp. were enriched from the biomass of a butanol-producing reactor. After six successive biomass transfers, ethanol production reached as much as 11.8 g/L with minor accumulation of acetic acid, under intermittent gas feeding conditions and over a wide pH range of 6.45-4.95. The molar ratio of ethanol to acetic acid exceeded 1.7 after the lag phase of 11 days and reached its highest value of 8.6 during the fermentation process after 25 days. Although butanol production was not significantly enhanced in the enrichment, the biomass was able to convert exogenous butyric acid (3.2 g/L) into butanol with nearly 100% conversion efficiency using CO as reducing power. This suggested that inhibition of butanol production from CO was caused by the lack of natural butyric acid production, expectedly induced by unsuitable pH values due to initial acidification resulting from the acetic acid production. The enriched Clostridium population also converted glucose to formic, acetic, propionic, and butyric acids in batch tests with daily pH adjustment to pH 6.0. The Clostridium genus was enriched with its relative abundance significantly increasing from 7% in the inoculum to 94% after five successive enrichment steps. Unidentified Clostridium species showed a very high relative abundance, reaching 73% of the Clostridium genus in the enriched sludge (6th transfer).

Culture and genome-based analysis of four soil Clostridium isolates reveal their potential for antimicrobial production.

BACKGROUND: Soil bacteria are a major source of specialized metabolites including antimicrobial compounds. Yet, one of the most diverse genera of bacteria ubiquitously present in soil, Clostridium, has been largely overlooked in bioactive compound discovery. As Clostridium spp. thrive in extreme environments with their metabolic mechanisms adapted to the harsh conditions, they are likely to synthesize molecules with unknown structures, properties, and functions. Therefore, their potential to synthesize small molecules with biological activities should be of great interest in the search for novel antimicrobial compounds. The current study focused on investigating the antimicrobial potential of four soil Clostridium isolates, FS01, FS2.2 FS03, and FS04, using a genome-led approach, validated by culture-based methods. RESULTS: Conditioned/spent media from all four Clostridium isolates showed varying levels of antimicrobial activity against indicator microorganism; all four isolates significantly inhibited the growth of Pseudomonas aeruginosa. FS01, FS2.2, and FS04 were active against Bacillus mycoides and FS03 reduced the growth of Bacillus cereus. Phylogenetic analysis together with DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and functional genome distribution (FGD) analyses confirmed that FS01, FS2.2, and FS04 belong to the species Paraclostridium bifermentans, Clostridium cadaveris, and Clostridium senegalense respectively, while FS03 may represent a novel species of the genus Clostridium. Bioinformatics analysis using antiSMASH 5.0 predicted the presence of eight biosynthetic gene clusters (BGCs) encoding for the synthesis of ribosomally synthesized post-translationally modified peptides (RiPPs) and non-ribosomal peptides (NRPs) in four genomes. All predicted BGCs showed no similarity with any known BGCs suggesting novelty of the molecules from those predicted gene clusters. In addition, the analysis of genomes for putative virulence factors revealed the presence of four putative Clostridium toxin related genes in FS01 and FS2.2 genomes. No genes associated with the main Clostridium toxins were identified in the FS03 and FS04 genomes. CONCLUSIONS: The presence of BGCs encoding for uncharacterized RiPPs and NRPSs in the genomes of antagonistic Clostridium spp. isolated from farm soil indicated their potential to produce novel secondary metabolites. This study serves as a basis for the identification and characterization of potent antimicrobials from these soil Clostridium spp. and expands the current knowledge base, encouraging future research into bioactive compound production in members of the genus Clostridium.

Novel ex vivo screening assay to preselect farm specific pre- and probiotics in pigs.

A novel rapid ex vivo assay was developed as part of a concept to determine potential tailor-made combinations of pre- and probiotics for individual farms. Sow faecal slurries from 20 German pig farms were anaerobically incubated with pre- and probiotics or their combinations together with pathogenic strains that are of interest in pig production. Aliquots of these slurries were then incubated with media containing antibiotic mixtures allowing only growth of the specific pathogen. Growth was monitored and lag time was used to determine the residual fitness of the pathogenic strains. The background growth could be inhibited for an Escherichia coli- and a Clostridium difficile- but not for a Clostridium perfringens strain. The prebiotic fructo-oligosaccharides (FOS) and its combination with probiotics reduced the residual fitness of the E. coli strain in some farms. However, notable exceptions occurred in other farms where FOS increased the fitness of the E. coli strain. Generally, combinations of pre- and probiotics did not show additive effects on fitness for E. coli but displayed farm dependent differences. The effects of pre- and probiotics on the residual fitness of the C. difficile strain were less pronounced, but distinct differences between single application of prebiotics and their combination with probiotics were observed. It was concluded that the initial composition of the microbiota in the samples was more determinative for incubations with the C. difficile strain than for incubations with the E. coli strain, as the presumed fermentation of prebiotic products showed less influence on the fitness of the C. difficile strain. Farm dependent differences were pronounced for both pathogenic strains and therefore, this novel screening method offers a promising approach for pre-selecting pre- and probiotics for individual farms. However, evaluation of farm metadata (husbandry, feed, management) will be crucial in future studies to determine a tailor-made solution for combinations of pre- and probiotics for individual farms. Also, refinement of the ex vivo assay in terms of on-farm processing of samples and validation of unambiguous growth for pathogenic strains from individual farms should be addressed.

Spontaneous emphysematous splenitis: Radiographic and ultrasonographic findings in three Golden Retriever dogs.

Spontaneous emphysematous splenitis is a life-threatening condition reported rarely in humans; however, published reports in dogs are currently lacking. The aim of this multicentric, retrospective, case series design study was to describe radiographic and ultrasonographic imaging findings in Golden Retriever dogs diagnosed with spontaneous emphysematous splenitis. A total of three dogs were sampled. All dogs had a history of lethargy, diarrhea, and weight loss. Radiographic findings in all dogs included a mass effect with focal or multifocal coalescing "vesicular-like" gas pattern in the splenic region and focal loss of serosal detail. Ultrasonographic findings in all dogs included focal or multifocal irregularly shaped, hypoechoic areas containing a mixture of hyperechoic fluid and gas within the splenic parenchyma, hyperechoic abdominal free fluid, and generalized hyperechoic mesenteric fat without evidence of splenic torsion. Pneumoperitoneum was detected ultrasonographically and radiographically in two dogs. All three dogs underwent splenectomy and splenic torsion was definitively ruled out at surgery. One dog died three days after surgery, whereas the other two dogs recovered uneventfully. Culture of the splenic tissue and free abdominal fluid was positive for Clostridium spp. in all three cases. Findings supported inclusion of spontaneous emphysematous splenitis and septic peritonitis as differential diagnoses for dogs with this combination of clinical and imaging characteristics.

Antimicrobial Activity of Soil Clostridium Enriched Conditioned Media Against Bacillus mycoides, Bacillus cereus, and Pseudomonas aeruginosa.

The rise of antimicrobial resistant bacteria has fast-tracked the exploration for novel antimicrobial compounds. Reports on antimicrobial producing soil anaerobes such as Clostridium spp. are very limited. In the present study, the antimicrobial activity of soil Clostridium enriched conditioned/spent media (CMs) against Bacillus mycoides, Bacillus cereus and Pseudomonas aeruginosa was assessed by turbidimetric growth inhibition assay. Our results highlighted the antimicrobial potential of soil Clostridium enriched conditioned media against pathogenic and spoilage bacteria. Farm 4 soil conditioned medium (F4SCM) demonstrated a greater growth inhibition activity against all three tested microorganisms in comparison to other soil conditioned media. Non-targeted metabolite profiling of all soil conditioned media revealed distinctive polar and intermediate-polar metabolites in F4SCM, consistent with its strong antimicrobial property. Moreover, 539 significantly abundant metabolites including some unique features were detected in F4SCM suggesting its substantial and specialized chemical diversity. This study putatively identified seven significantly high metabolites in F4SCM; 3-hydroxyphenylacetic acid, γ-aminobutyric acid, creatine, tryptamine, and 2-hydroxyisocaproic acid. Tryptamine and 2-hydroxyisocaproic acid were previously reported to have antimicrobial properties. The present study shows that soil Clostridium spp. are a promising group of bacteria producing metabolites with antimicrobial activity and provides future prospects for clostridial antimicrobial discovery within their metabolic diversity.