Clostridial Spores Research Articles

Performance of a Combined Treatment Approach on the Elimination of Microbes from Poultry Slaughterhouse Wastewater

The efficiency of microbial inactivation in water is highly dependent on the type of treatment technology used as well as the characteristics of the water to be treated. Wastewater from poultry slaughterhouses carries a significant number of microorganisms posing threats to humans and the environment in general. Therefore, the treatment of poultry slaughterhouse wastewater requires the use of appropriate purification systems with high removal efficiency for microbial agents. In this study, the performance of an integrated treatment plant with electrolysis, ultrafiltration, and ultraviolet radiation as the principal treatment units was investigated in terms of microbial inactivation from poultry slaughterhouse wastewater. In this case, total microbial number, total coliform bacteria, thermo-tolerant coliform bacteria, pathogenic flora, including salmonella coliphages, spores of sulfite-reducing clostridia, Pseudomonas aeruginosa, and Staphylococcus aureus and Enterococcus were studied. Approximately 63.95% to 99.83% of the microbes were removed by the electrochemical treatment unit as well as a 99.86% to 100% removal efficiency was achieved after the combined treatment. However, Pseudomonas aeruginosa was the only microbial agent detected in the final effluent after the combined treatment. The phenomenon suggests that an upgrade to the treatment plant may be required to achieve 100% removal assurance for Pseudomonas aeruginosa.

An investigation of the environmental niches of blown pack spoilage causing Clostridium estertheticum and Clostridium gasigenes on New Zealand beef and sheep farms

The transfer of blown pack spoilage causing Clostridium spores from the farm to the meat plant is of growing concern to the meat industry. This study investigated the environmental niches of these Clostridium spp., specifically Clostridium estertheticum and Clostridium gasigenes in the beef and sheep farm environments in New Zealand. Faecal, soil, grass, drinking water, puddle water and feed (fodder beet, hay, bailage and silage, where available) samples were collected on five beef and sheep farms during Winter and Spring in 2018, in North and South Island, respectively. Beef and sheep farm samples were tested for C. estertheticum and C. gasigenes using enrichment plus PCR, qPCR and direct plating. C. estertheticum was detected in bovine faecal (4%), soil (2–18%) and grass (0–12%) samples at concentration of up to 2.0 log10 cfu/g. C. gasigenes were found in 18–46% of faecal, 16–82% of soil, 12–44% of grass, 0–44.4% of drinking water and 0–58.3% of puddle water samples tested and the direct counts ranged from 2.4 log10 cfu/ml in puddle water to 3.4 log10 cfu/g in soil. C. estertheticum were detected by qPCR in sheep farms in ovine feces (2.3%), soil (2.3%) and fodder beet (10%). All other sample types (grass, drinking water, puddle water, baleage, hay, silage and fodder beet) were negative using direct and enrichment plus PCR methods. In contrast C. gasigenes was detected in of faecal (22.7–38.6%), soil (22.7–84.1%), grass (17.5–34.1%) drinking water (35.7–78.6%), puddle water (33.3–40%), hay baleage (57%), silage (2%) and fodder beet (10%) at concentrations of up to 3.7 log10 cfu/g/ml. It was concluded that C. estertheticum and C. gasigenes were common on beef and sheep farms with the latter having higher incidence and mean concentration.

Bacterial Complexes of Urbanozems in Southern Cities of Russia

Urban soils, differing in the intensity of anthropogenic impacts, were studied in several largest (Krasnodar), large (Sochi and Simferopol), and moderately large (Maikop) cities of southern Russia. The obtained data on the diversity of bacterial complexes revealed that urban soils with high anthropogenic load (Sochi, Simferopol, and Krasnodar) undergo considerable transformation of natural bacterial complexes with a sharp increase in representatives of Enterobacteriaceae family. Along with microorganisms indicative of the sanitary status of urban soils (E. coli and En. faecalis) the soils also contain bacteria of Klebsiella, Enterobacter, Citrobacter, and Serratia genera; some of their species may cause intestinal and allergic diseases. In the urban soil of Sochi, spores of sulfite-reducing Clostridia, including Clostridium perfringens, have been found. In the urban soil of Maikop with the lowest population density among the studied cities, changes in the structure of natural bacterial complexes are virtually absent. The abundances of sanitary-indicative microorganisms (Escherichia coli and Enterococcus faecalis) in urban soils of all the studied cities, except for Maikop, exceeded the normative parameter for sanitary-safe clean soils (<10 CFU/g soil).

Abdominal aortic thromboembolism and subsequent pelvic limb myositis secondary to colitis and septicaemia in a 5‐day‐old Oldenburg colt

SummaryThis case report describes the clinical progression of a 5‐day‐old Oldenburg colt presented for acute severe right hindlimb lameness. The foal had diarrhoea at presentation, and over the next 24 h became tachycardic and febrile with cold extremities in the hindlimbs and began to bear weight on the dorsum of both hind fetlocks. Diagnostic work‐up included physical examination, radiography, ultrasonography and clinicopathological testing. Treatment consisted of antibiotics, anti‐inflammatories, gastroprotectants, oral di‐tri‐octahedral smectite, hyperimmune plasma and isotonic crystalloid fluids for diarrhoea. Bandages and splints were applied to the hindlimbs to assist ambulation and prevent weightbearing on the dorsum of the fetlocks. The foal developed oedema of both hindlimbs, and ultrasonographic evaluation revealed gas pockets within the subcutaneous tissues. An ultrasound‐guided aspirate of the right hindlimb oedema was submitted for cytology, which revealed presumed Clostridial spores, and culture, which yielded heavy growth of Escherichia coli. Surgical fenestration of the hindlimbs was performed due to suspected Clostridial myositis. The foal continued to decline clinically and was humanely subjected to euthanasia. Post‐mortem evaluation revealed septicaemia and thromboembolism of the terminal abdominal aorta resulting in partial obstruction of blood flow to the pelvic limbs with resultant bacterial myositis (Clostridium [suspected] and Escherichia coli [confirmed]). In summary, thrombosis is a potential sequela to coagulopathy seen in septicaemia and should be considered a differential diagnosis in neonatal foals presenting for lameness.

S-Nitroso-N-acetylcysteine (NAC-SNO) vs. nitrite as an anti-clostridial additive for meat products.

Nitrite is added to meat products as a preservative and it acts as a bacteriostatic compound against Clostridium botulinum growth. Nitric-oxide (˙NO), myoglobin and S-nitroso-compounds seem to be the main molecules generated from nitrite in meat products, which by decomposition to ˙NO, form the main anti-clostridial factor. The growth of C. sporogenes from activated spores in the presence of 0.5-2.5 mM NAC-SNO was compared to nitrite, both at 37 °C for 5 days and at room temperature for 28 days. The present study demonstrates that NAC-SNO under the same conditions and concentrations, in meat products, acts as an anti-clostridial compound similar to nitrite. In contrast to nitrite which must be activated in meat by heating, NAC-SNO generates the anti-clostridial factor directly, without heating, as was evaluated in an unheated bacteriological medium. The toxic effect of NAC-SNO and nitrite in methaemoglobinaemia and generation of N-nitrosamines in vivo, in mice, were also determined. Mice were gavage fed milk containing 45 mg per kg per bw of nitrite or an equimolar equivalent of NAC-SNO in the presence of 50 mg per kg per bw of N-methylaniline. Nitrite generated methaemoglobinaemia and carcinogenic N-nitrosoamines (N-nitrosomethylaniline); however, NAC-SNO under the same conditions and concentrations generates much less methaemoglobin and no detectable N-nitrosoamines in the blood, in vivo.

Chemical composition, hygiene characteristics, and coagulation aptitude of milk for Parmigiano Reggiano cheese from herds yielding different milk levels

ABSTRACT The objective with this research was to compare milk quality parameters among herds characterized by different levels of milk production. The research involved 1080 bulk milk samples collected from 30 dairy herds, which produce milk for Parmigiano Reggiano cheese, during three years. Each milk sample was analysed for fat, crude protein, titratable acidity, total bacterial count, somatic cells, coliform bacteria, and Clostridia spores. Least mean values were obtained by ANOVA using milk production intervals from 6000 to 7999 (L-group), [...]

Anaerobic treated organic waste - Effects of sanitation regarding to pathogenic clostridia

The utilisation of organic waste in anaerobic digestion to produce biogas and digestate is a meaningful development regarding to the sustainable use of resources. Yet, it opens room for digestate contamination with clostridia that limits the digestate use. Currently, directives and laws are given for sanitation of input material. The aim of this study is to investigate the effectiveness of given sanitation methods relating to elimination of pathogenic clostridia. Due to the fact that clostridia are able to form heat resistant spores, tests were performed to illustrate temperature and duration of sanitation necessary to eliminate spores. The sanitation procedures were executed at a range of temperature from 70 °C to 134 °C in an oil bath under normal atmospheric pressure and with duration from one to ten hours. Tests were conducted with Clostridium perfringens and Clostridium sporogenes. Results confirmed that spores of these pathogenic clostridia survive at temperatures of 70 °C for at least ten hours. The study shows, that only temperatures over 121 °C and long exposure times under atmospheric pressure reduce outgrowth of tested clostridial spores. Standardized and effective sanitation techniques executed under high pressure allready exist. Their application is strictly ordered for sanitation of high risk material, but it will also work for sanitation of organic waste. In conclusion, the prescribed sanitation of organic waste with 70 °C for one hour has to be reviewed, espiecally for batches of organic waste which are expected to have higher contents of clostridia.

Ensilage et contamination du lait par les spores butyriques

Le lait des vaches recevant de l’ensilage peut entraîner de sérieux déboires dans la fabrication de certains fromages, notamment ceux à pâte pressée cuite : gonflement tardif, goût et odeur désagréables. L’agent responsable est une spore butyrique, Clostridium butyricum. La contamination du lait se fait lors de la traite, par des particules de bouse passant dans le lait. Son importance dépend d’une part des soins apportés à la traite et de la propreté des trayons, d’autre part du nombre de spores dans l’ensilage et, par là, dans les bouses. Il est nécessaire de jouer simultanément sur ces deux facteurs pour diminuer la contamination du lait. En ce qui concerne l’ensilage, il est impératif d’incorporer le moins possible de terre (à l’origine des spores) dans le fourrage lors de la récolte et du remplissage du silo. D’autre part il est nécessaire d’utiliser un conservateur efficace inhibant la multiplication des spores dans l’ensilage. Les conservateurs acides et (ou) ceux contenant des nitrates ou des nitrites sont de ce point de vue les plus efficaces.

Dynamics of Clostridium genus and hard-cheese spoiling Clostridium species in anaerobic digesters treating agricultural biomass

Biogas plants are a widespread renewable energy technology. However, the use of digestate for agronomic purposes has often been a matter of concern. It is controversial whether biogas plants might harbor some pathogenic clostridial species, which represent a biological risk. Moreover, the inhabitance of Clostridium hard-cheese spoiling species in anaerobic digesters can be problematic for hard-cheese manufacturing industries, due to the issue of cheese blowing defects. This study investigated the effect of mesophilic anaerobic digestion processes on the Clostridium consortia distribution over time. Specifically, three lab-scale CSTRs treating agricultural biomass were characterized by considering both the whole microbial community and the cultivable clostridial spores. It is assessed an overall reduction of the Clostridium genus during the anaerobic digestion process. Moreover, it was evidenced a slight, but steady decrease of the cultivable clostridial spores, mainly represented by two pathogenic species, C. perfringens and C. bifermentans, and one hard-cheese spoiling species, C. butyricum. Thus, it is revealed an overall reduction of the clostridial population abundance after the mesophilic anaerobic digestion treatment of agricultural biomass.

Clostridium difficile in bee products: assessing the spread of the bacterium

The vicinity of small apiaries to the farms makes honey and bee pollen prone to the contamination with clostridial spores. The possible presence of Clostridium difficile (C. difficile) spores in bee products may pose a risk especially to elderly who perceive them as natural remedies in health problems including functional bowel abnormalities. The aim of the study was to evaluate the prevalence of C. difficile spores in 120 samples of honey and 44 samples of bee pollen harvested in Northeastern Europe. All incubation procedures were performed in anaerobic conditions. The 10 g samples were subsequently pre-enriched in Cooked Meat Medium (CMM) and Brain Heart Infusion-supplement (BHIS) broths. Sporulated forms were seeded on supplemented C. difficile blood agar. Rapid immunoenzymatic tests targeted at glutamate dehydrogenase (GDH) detection and polymerase chain reaction (PCR) targeted at tpi gene detection were also performed. Based on microbiological culture 32 (20%) out of 164 samples were selected for culture on blood agar. The growth of ground-glass colonies was noted towards 13 (8%) samples. The presence of C. difficile was not confirmed by immunoenzymatic rapid tests towards any sample selected. Neither PCR assay targeted to tpi gene showed positive samples. This is the first report reviewing the possibility of native to Europe Apis mellifera (A. mellifera) bee products contamination with C. difficile. The samples of A. mellifera bee products produced in small apiaries in Northeastern Europe did not show the presence of C. difficile spores.

Use of Fresh Scotta Whey as an Additive for Alfalfa Silage

Scotta is a dairy industry waste obtained by ricotta cheese production. Because of its high availability and its high lactose content, scotta could be used as an additive to improve fermentation characteristics of alfalfa silage. Silage samples were obtained for 2 years from an alfalfa second cut, collecting the forage at three growth stages: mid bud (MB), early flowering (EF), and late flowering (LF). After wilting the forage at 38% dry matter (DM), four scotta doses were added (0, 75, 150, and 300 g/kg fresh forage) and the main chemical and microbiological characteristics were evaluated after 3, 13, and 90 days of ensiling (DOE). The lowest pH (4.3) was recorded in the EF and LF growth stages, after 90 DOE and with the highest scotta dose (300 g/kg fresh forage). After 90 DOE, the concentration of the main spoilage microorganisms and clostridial spore loads was always negligible. The addition of scotta decreased pH and fiber fractions, increased the relative feed value, and had no effect on the crude protein concentration or the total digestible nutrients. Therefore, the scotta can be successfully used as an additive to improve the fermentation characteristics of alfalfa silage.

Modelling the seasonal impacts of a wastewater treatment plant on water quality in a Mediterranean stream using microbial indicators

Faecal pollution modelling is a valuable tool to evaluate and improve water management strategies, especially in a context of water scarcity. The reduction dynamics of five faecal indicator organisms (E. coli, spores of sulphite-reducing clostridia, somatic coliphages, GA17 bacteriophages and a human-specific Bifidobacterium molecular marker) were assessed in an intermittent Mediterranean stream affected by a wastewater treatment plant (WWTP). Using Bayesian inverse modelling, the decay rates of each indicator were correlated with two environmental drivers (temperature and streamflow downstream of the WWTP) and the generated model was used to evaluate the self-depuration distance (SDD) of the stream. A consistent increase of 1–2 log10 in the concentration of all indicators was detected after the discharge of the WWTP effluent. The decay rates showed seasonal variation, reaching a maximum in the dry season, when SDDs were also shorter and the stream had a higher capacity to self-depurate. High seasonality was observed for all faecal indicators except for the spores of sulphite-reducing clostridia. The maximum SDD ranged from 3 km for the spores of sulphite-reducing clostridia during the dry season and 15 km for the human-specific Bifidobacterium molecular marker during the wet season. The SDD provides a single standardized metric that integrates and compares different contamination indicators. It could be extended to other Mediterranean drainage basins and has the potential to integrate changes in land use and catchment water balance, a feature that will be especially useful in the transient climate conditions expected in the coming years.

Non meat-based alheiras– a safer novel trend?

In response to nutritional and health concerns, the food industry has begun to offer a wider variety of products that reflect changing consumer preferences. In addition to traditional alheiras, made with pork and/or poultry meats, other varieties of alheiras (“innovative”) made from codfish, mushrooms, tofu, soy and vegetables were launched on the Portuguese market. The objective of this study was the characterisation of these products, giving particular attention to their microbiological and chemical safety. Therefore, fourteen different products were analyzed. Enterobacteriaceae, Enterococcus, lactic acid bacteria, yeasts and moulds, were the prevalent microbiota of “innovative” alheiras. Sulphite reducing Clostridium spores, Escherichia coli, Listeria monocytogenes, Salmonella spp. or Staphylococcus aureus were not detected in any sample. No differences were observed between traditional and “innovative” alheiras concerning pH and water activity values while nitrites, nitrates and biogenic amines were found to be within accepted limits for these kinds of products. In terms of organic acids, lactic acid was found in all analyzed samples while malic and succinic acid seemed to be predominant only in “innovative” alheiras. In conclusion, unlike traditional alheiras which often contain pathogenic agents, no harmful organisms nor chemical hazards were found in these new products, even though produced by the same companies.

Short communication: Clostridial spore counts in vat milk of Alpine dairies

One of the most severe quality defects in hard and semi-hard cheese, the late blowing defect, is caused by endospore-forming bacteria of the genus Clostridium. To minimize financial losses and waste of resources due to cheese spoilage, raw milk with elevated clostridial spore counts should not be used for the production of certain cheese types. In this context, threshold values of clostridial spore concentrations that cause quality defects in cheese are still under debate. To improve our understanding about late blowing defects, further information on the correlation between clostridial spore concentrations in milk and cheese quality is indispensable. Thus, the aim of this study was to monitor the microbiological quality of milk used for Alpine cheese production regarding clostridial endospore levels to facilitate the establishment of threshold spore concentrations that guarantee the absence of quality defects in Austrian cheese. For this purpose, we monitored clostridial endospore levels in vat milk of 4 Alpine dairies throughout the summer grazing period in 2018. Surprisingly, we observed almost complete absence of butyric acid-producing clostridia in milk and no blowing defects in cheese. Hence, critical clostridial spore concentrations could not be verified. Moreover, the observed low spore levels reveal that the prohibition of silage feeding and good farming practices effectively minimize clostridial endospore counts in milk and ensure the manufacture of high-quality cheese even if technological possibilities are limited.

Phytochemical study and antioxidant activity of Dalbergia ecastaphyllum

Abstract The chemical profile of Dalbergia ecastaphyllum has been indicated as the botanical origin of Brazilian red propolis, an apicultural product with proven therapeutic properties. However, few studies have investigated this plant species. This study evaluated and compared microbiological quality, chemical composition, and antioxidant activity of stem and leaf samples of D. ecastaphyllum. The samples were collected in February 2015, in the southern region of the state of Bahia, Brazil. We performed the microbiological analyses, determined the contents of fatty acid, total phenol and flavonoid, and identified the chemical profile and antioxidant activit. Escherichia coli, Salmonella spp. and sulfite reducing clostridial spores were not detected in the samples. Acids of the family ω3 were recorded in the stems and ω6 in the leaves. The leaves presented better nutritional quality of the fraction, better antioxidant capacity in the tests by the DPPH method and β-carotene bleaching. There were 49 chemical compounds, of which 38 belonged to the class of flavonoids. The results indicate that stems and leaves of D. ecastaphyllum have biological properties. Leaves particularly are better for functional food formulation and as natural antioxidant.

Microbiological quality of raw berries and their products: A focus on foodborne pathogens

Berry samples (n = 316; strawberries, raspberries, blackberries and blueberries) obtained from a fruit processing plant were examined regarding bacteriological quality and their potential public health risk. Three types of berry products were analysed including raw material, product from the mixing step and final product. Escherichia coli, Salmonella spp., Listeria monocytogenes, Bacillus cereus, sulphite-reducing clostridia spores and coagulase-positive staphylococci were the parameters investigated. Salmonella enterica serovar Braenderup and L. monocytogenes were isolated from one fruit sample of raw material each. Two samples harboured E. coli between 0.7 and 0.9 log cfu g−1, not exceeding the hygienic criteria. Coagulase-positive staphylococci were not detected in the studied samples; however, coagulase-negative staphylococci (CNS) were isolated from a small proportion of samples mainly raspberries. Presumptive B. cereus were isolated from a relatively large proportion of the samples, raspberries and blackberries being the most contaminated fruits. The absence of pathogenic microorganisms in the final product as well as the low prevalence of presumptive B. cereus and CNS indicates proper implementation of good manufacturing and hygiene practices (GMPs/GHPs) by the food industry. Nevertheless, the results indicate that the raw material examined may contain pathogenic bacteria and thereby represent a risk to consumers regarding the manifestation of foodborne diseases.

Pathobiology and diagnosis of clostridial hepatitis in animals.

Clostridia can cause hepatic damage in domestic livestock, and wild and laboratory animals. Clostridium novyi type B causes infectious necrotic hepatitis (INH) in sheep and less frequently in other species. Spores of C. novyi type B can be present in soil; after ingestion, they reach the liver via portal circulation where they persist in phagocytic cells. Following liver damage, frequently caused by migrating parasites, local anaerobic conditions allow germination of the clostridial spores and production of toxins. C. novyi type B alpha toxin causes necrotizing hepatitis and extensive edema, congestion, and hemorrhage in multiple organs. Clostridium haemolyticum causes bacillary hemoglobinuria (BH) in cattle, sheep, and rarely, horses. Beta toxin is the main virulence factor of C. haemolyticum, causing hepatic necrosis and hemolysis. Clostridium piliforme, the causal agent of Tyzzer disease (TD), is the only gram-negative and obligate intracellular pathogenic clostridia. TD occurs in multiple species, but it is more frequent in foals, lagomorphs, and laboratory animals. The mode of transmission is fecal-oral, with ingestion of spores from a fecal-contaminated environment. In affected animals, C. piliforme proliferates in the intestinal mucosa, resulting in necrosis, and then disseminates to the liver and other organs. Virulence factors for this microorganism have not been identified, to date. Given the peracute or acute nature of clostridial hepatitis in animals, treatment is rarely effective. However, INH and BH can be prevented, and should be controlled by vaccination and control of liver flukes. To date, no vaccine is available to prevent TD.

Low-temperature centrifugation of milk for manufacture of raw milk cheeses: Impact on milk debacterization and cheese yield

Centrifugation is occasionally applied to clean cheese milk, particularly to remove Clostridia's spores that may cause the “late blowing” of cheese. The sludge separated by centrifugation also contains fat and protein, thus is sterilized and added back to cheese milk. In manufacture of raw milk cheeses, centrifugation shall be performed at temperature below 40 °C and no sterilized sludge can be added to vat milk. Both these limitations negatively affect cheese yield. To evaluate process sustainability, three different centrifugation configurations were tested at a factory producing a traditional raw-milk extra-hard cheese. Either a single or double centrifugation, the latter with two different volumes of discharged sludge, were tested over 3-week periods each. Efficiency of spore removal, decrease of total bacterial count, loss of milk solids and cheese yield were evaluated daily with respect to not-centrifuged milk from the same batch. Double centrifugation with low-volume sludge gave highest efficiency of spore removal, i.e. 98.2%, while the single process minimized the loss of cheese yield. Impedometric analysis indicated that centrifugation caused a preferential removal of rod-shaped lactic acid bacteria, regardless of configuration. This finding was confirmed by microscopy and suggested that a different bacteria population would operate during cheese ripening.

Design and Expression of Specific Hybrid Lantibiotics Active Against Pathogenic Clostridium spp.

Clostridium difficile has been reported as the most common cause of nosocomial diarrhea (antibiotic-associated diarrhea), resulting in significant morbidity and mortality in hospitalized patients. The resistance of the clostridial spores to antibiotics and their side effects on the gut microbiota are two factors related to the emergence of infection and its relapses. Lantibiotics provide an innovative alternative for cell growth inhibition due to their dual mechanism of action (membrane pore-forming and cell wall synthesis inhibition) and low resistance rate. Based on the fact that bacteriocins are usually active against bacteria closely related to the producer strains, a new dual approach combining genome mining and synthetic biology was performed, by designing new lantibiotics with high activity and specificity toward Clostridium. We first attempted the heterologous expression of putative lantibiotics identified following Clostridium genome mining. Subsequently, we designed new hybrid lantibiotics combining the start or end of the putative clostridial peptides and the start or end parts of nisin. The designed peptides were cloned and expressed using the nisin biosynthetic machinery in Lactococcus lactis. From the 20 initial peptides, only 1 fulfilled the requirements established in this work to be considered as a good candidate: high heterologous production level and high specificity/activity against clostridial species. The high specificity and activity observed for the peptide AMV10 makes it an interesting candidate as an alternative to traditional antibiotics in the treatment of C. difficile infections, avoiding side effects and protecting the normal gut microbiota.

Development and validation of lactic acid starter cultures for enset (Ensete ventricosum) fermentation

In this study, lactic acid bacteria strains were screened for the development of (a) starter culture(s) specific for enset (Ensete ventricosum) fermentation. To this end, 158 isolates grown on de Man, Rogosa and Sharpe agar were obtained from fermenting enset and traditional starter material. They were screened based on their biochemical characteristics. Three promising strains, two Lactobacillus plantarum and one Leuconostoc mesenteroides, were selected and evaluated under field conditions. Aliquots of pulverized enset (without adding a liquid) were inoculated with one of the isolates at 3 log cfu/g and one uninoculated portion was used as control. Fermentation was performed in sauerkraut jars. Samples were taken on days 0, 7, 15, 30 and 75. On day 7 and onwards, the inoculated samples showed a larger pH reduction compared to the control. Enterobacteriaceae counts were reduced below the detectable level on day 7 and onwards. Clostridium spore counts were significantly lower for the inoculated than for the uninoculated samples on day 75. Inoculated enset showed a faster colonization of lactic acid bacteria than the uninoculated biomass. The three starters appeared to be suitable for enset fermentation. Further research is necessary to elucidate the proximate composition of enset fermented with these starters.