Clostridial Spores Research Articles

The effect of cultivar, wilting and storage period on fermentation and the clostridial community of alfalfa silage

The objective of this study was to gain deeper insights into chemical transformations and the clostridial community dynamics during the ensiling of alfalfa. A factorial experiment was conducted with four alfalfa cultivars [Sanditi (A1), AC Caribou (A2), WL319HQ (A3) and 4030 (A4)] × three wilting durations [0 h (direct-cut), 2 h and 4 h] × three storage periods (14, 28 and 56 days). The clostridial community was examined using denaturing gradient gel electrophoresis. High butyric acid and ammonia nitrogen (NH3–N) contents and clostridia numbers were observed in direct-cut silage, with higher values observed in A4 than in the other three cultivars. However, butyric acid and NH3–N contents and clostridia numbers decreased with wilting regardless of the cultivar. Although Clostridium ghonii and Clostridium sartagoforme were common to all direct-cut and wilted silages, bands for these species were faint. Differences in the appearing time of these species were observed, bands for C. ghonii were found after 14 and 28 days of ensiling, while those for C. sartagoforme were only found after 56 days. In addition, in direct-cut silage, distinct bands for Clostridium perfringens were detected in A1, A2 and A3, while those for Clostridium sporogenes were detected in A4. The inactive spores of clostridia could be observed in 4-h wilted silage as no butyric acid was detected. It was concluded that the enhanced clostridial fermentation of direct-cut silage was attributed mainly to C. perfringens and C. sporogenes, while C. ghonii and C. sartagoforme were involved in the restricted clostridial fermentation of 2-h wilted silage.

Efficient milking hygiene reduces bacterial spore contamination in milk.

Clostridia in the milk can lead to late blowing, a cheese defect. Clostridia are ubiquitous, deriving from both the farm environment and the feed ingested by the cows, and are transferred into the milk through faecal contamination. Our aim was to investigate the effect of different in-parlour practices on the content of anaerobic spore-forming bacteria in milk, and to monitor the variation in spore content in the feed and environment. The experiment, conducted in an experimental dairy during autumn, was repeated in exactly the same way for two consecutive years. The experimental design applied three different milking routines in three consecutive 7-d periods: forestripping alone (F); forestripping and post-dipping (F+Post); pre-dipping, wiping, forestripping and post-dipping (Pre+F+Post). Teat skin swabs and samples of feed, faeces, bedding materials and milk were collected for microbiological analyses. The dietary forage of the lactating cows included maize silage, which, in both years, was found to have the highest level of clostridial spore contamination. Pre-dipping with a detergent/emollient solution, and drying with a disposable paper towel, proved much more efficient in reducing spore contamination than forestripping alone, both on the teats (1·30 vs. 2·20 log10 MPN/swab; P < 0·001) and in the milk (1·82 vs. 2·47 log10 MPN/L, P < 0·02), while post-dipping had little influence on spore count. The standard plate count in milk was significantly lower with Pre+F+Post treatment than with F (3·80 vs. 4·51 log10 CFU/mL, P < 0·01). The teat preparation procedure did not influence the lactic acid bacterial levels in the milk, which is very positive in that decreased lactic acid bacterial content can lessen raw milk cheese quality.

Microcosms for evaluating microbial indicator persistence and mobilization in fluvial sediments during rainfall events

Mediterranean rivers, which are subject to long, dry periods and heavy rainfall events, could be particularly useful for understanding future climate scenarios. This study generated microcosms that mimicked riverbank sediment resuspension into the water of a typical Mediterranean river as a consequence of heavy rainfall. The mobilization and inactivation of six fecal pollution indicators and microbial source tracking markers were evaluated. The T90 values in the sediments were: 4 days for sorbitol-fermenting Bifidobacterium, 11 days for culturable E. coli, 36 days for bacteriophages infecting Bacteroides thetaiotaomicron strain GA17 and more than 42 days for qPCR-detected E. coli, somatic coliphages and sulfite-reducing clostridia spores. Bacteriophages and bacteria showed different resuspension and sedimentation patterns. The data obtained could be used in predictive models to assess the effects of climate change on surface water quality. Pathogen mobilization into the water column poses a risk for humans, animals and the natural environment, and breaches the One Health approach.

A critical assessment of four most probable number procedures for routine enumeration of cheese-damaging clostridia in milk

In cheese production, knowledge of clostridial spore levels in milk is crucial to avoid economic losses due to late-blowing, a characteristic quality defect observed especially in hard cheeses. However, no international standard method is available to quantify clostridial spores in milk. Since performance data on the currently applied enumeration methods have not been systematically investigated, in this study we assessed four routine media and most probable number (MPN) methods for the enumeration of cheese-damaging clostridia in milk. Among the four evaluated methods, statistically significant differences became evident, while moderate to optimal correlations were observed. Bacterial isolates from positive reactions were identified using molecular methods. All four methods assessed showed a severe lack of selectivity due to the incorporation of non-clostridial spore-formers in the counts produced. The results illustrate major drawbacks of currently used methods and stress the necessity for improving method selectivity, sensitivity and reliability before standardisation can be envisaged.

Industrial-scale application of Lactobacillus reuteri coupled with glycerol as a biopreservation system for inhibiting Clostridium tyrobutyricum in semi-hard ewe milk cheese

The suitability of the biopreservation system formed by reuterin-producing L. reuteri INIA P572 and glycerol (required for reuterin production) to prevent late blowing defect (LBD) was evaluated in industrial sized semi-hard ewe milk cheese contaminated with Clostridium tyrobutyricum INIA 68, a wild strain isolated from a LBD cheese. For this purpose, six batches of cheese were made (three with and three without clostridial spores): control cheeses with lactococci starter, cheeses with L. reuteri as adjunct, and cheeses with L. reuteri and 30 mM glycerol. Spores of C. tyrobutyricum INIA 68 germinated during pressing of cheese curd, causing butyric acid fermentation in cheese after 30 d of ripening. The addition of L. reuteri, without glycerol, enhanced the symptoms and the formation of volatile compounds associated with LBD. When glycerol was added to cheese milk contaminated with C. tyrobutyricum, L. reuteri was able to produce reuterin in cheese resulting in cheeses with a uniform cheese matrix and a volatile profile similar to cheese made with L. reuteri and glycerol (without spores). Accordingly, L. reuteri INIA P572 coupled with glycerol seems a novel biopreservation system to inhibit Clostridium growth and prevent LBD by means of in situ reuterin production.

Adenine Addition Restores Cell Viability and Butanol Production in Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) Cultivated at 37°C.

We have developed butanol-producing consolidated bioprocessing from cellulosic substrates through coculture of cellulolytic clostridia and butanol-producing Clostridium saccharoperbutylacetonicum strain N1-4. However, the butanol fermentation by strain N1-4 (which has an optimal growth temperature of 30°C) is sensitive to the higher cultivation temperature of 37°C; the nature of this deleterious effect remains unclear. Comparison of the intracellular metabolites of strain N1-4 cultivated at 30°C and 37°C revealed decreased levels of multiple primary metabolites (notably including nucleic acids and cofactors) during growth at the higher temperature. Supplementation of the culture medium with 250 mg/liter adenine enhanced both cell growth (with the optical density at 600 nm increasing from 4.3 to 10.2) and butanol production (increasing from 3.9 g/liter to 9.6 g/liter) at 37°C, compared to those obtained without adenine supplementation, such that the supplemented 37°C culture exhibited growth and butanol production approaching those observed at 30°C in the absence of adenine supplementation. These improved properties were based on the maintenance of cell viability. We further showed that adenine supplementation enhanced cell viability during growth at 37°C by maintaining ATP levels and inhibiting spore formation. This work represents the first demonstration (to our knowledge) of the importance of adenine-related metabolism for clostridial butanol production, suggesting a new means of enhancing target pathways based on metabolite levels.IMPORTANCE Metabolomic analysis revealed decreased levels of multiple primary metabolites during growth at 37°C, compared to 30°C, in C. saccharoperbutylacetonicum strain N1-4. We found that adenine supplementation restored the cell growth and butanol production of strain N1-4 at 37°C. The effects of adenine supplementation reflected the maintenance of cell viability originating from the maintenance of ATP levels and the inhibition of spore formation. Thus, our metabolomic analysis identified the depleted metabolites that were required to maintain cell viability. Our strategy, which is expected to be applicable to a wide range of organisms, permits the identification of the limiting metabolic pathway, which can serve as a new target for molecular breeding. The other novel finding of this work is that adenine supplementation inhibits clostridial spore formation. The mechanism linking spore formation and metabolomic status in butanol-producing clostridia is expected to be the focus of further research.

Mobilisation of microbial indicators, microbial source tracking markers and pathogens after rainfall events

Climate change is expected to affect the Mediterranean region by causing an increase in the number of heavy rainfall events. The aim of this study was to assess the effect of extreme river flow variations due to rainfall on the persistence and mobilisation of various microorganisms. These included faecal pollution indicators (Escherichia coli (EC), somatic coliphages (SOMCPH) and sulphite reducing clostridia spores (SRC)), microbial source tracking indicators (Bacteroides thetaiotaomicron GA17 strain phages (GA17PH) and sorbitol fermenting bifidobacteria (SFBIF)), and two pathogens (Salmonella spp and Enterovirus). Water and sediment samples were taken at different distances from the river before and after heavy rainfall events. The microbial load was higher in sediment samples closer to the river course. The concentration of some faecal indicators (EC and SFBIF) increased in sediments and river water after rainfall events, whereas the most conservative parameter (SRC) showed almost no variation. After rainfall, the indicators persisted at a different rate. Salmonella spp and Enterovirus were detected in some samples but always at lower concentrations than the microbial indicators. In conclusion, sediments are reservoirs of faecal and MST indicators and pathogens and could therefore pose a risk of pathogen dissemination.

Effect of Potassium Sorbate Addition on Germination and Outgrowth of Clostridium Sporogenes Pa 3679 Spores in Low Cost Bologna Type Sausage During Ambient Temperature Storage

ObjectivesLow cost Bologna type product reaches high production volumes in Brazil and is marketed at ambient temperature. Previous studies including challenge test with Clostridium sporogenes PA 3679 indicated that water activity (aw) up to 0.96 prevented spores germination. The objective of this study was to evaluate the efficacy of potassium sorbate addition (0.25%) on limiting germination of Clostridium sporogenes PA 3679 in low lost Bologna type product formulated with aw 0.96 and 0.965 during 90 d storage at 27°C.Materials and MethodsThe experiment comprised 4 treatments formulated with mechanically deboned chicken meat (60%), edible offal (2%), pork skin (8%), 80/20 pork trimmings (15%), water (4%), texturized soy protein (3.5%), tapioca starch (5%), 150ppm ingoing sodium nitrite, 500ppm sodium isoascorbate, 0.15% sodium acid pyrophosphate and 0.35% sodium tripolyphosphate. The amount of sodium chloride varied to achieve the desired aw (0.96 and 0.965). Potassium sorbate was added at 0.25% in the final product. Raw material was comminuted in a bowl chopper. The raw batter was vacuum stuffed in 60mm PVDC impermeable casing. The samples were cooked in a cooking chamber with direct steam until 75°C was reached in the center of the product. Cooling was performed in running tap water until 27°C and the samples were kept in a chamber at 27°C (± 2°C) during 90 d. Lactic acid bacteria, Enterobacteriaceae, mesophilic aerobic, sulphite-reducing clostridia (spores and vegetative cells) counts, residual nitrite and pH value were evaluated in 3 samples of each treatment 24h after processing and at 15, 30, 60, and 90 d. Data (log CFU/g, residual sodium nitrite and pH value) were analyzed using GLM model procedure of SAS (SAS Inst. Inc., Cary, NC) as a 2 aw (0.96, 0.965) X 2 potassium sorbate amounts (0, 0.25%) X 5 storage time (0, 15, 30, 60, 90) factorial design with repeated measurements. Interactions and main effects were considered significant at p < 0.05. Least square means for significant effects (p < 0.05) were separated by Tukey’s test.ResultsLactic acid bacteria and Enterobacteriaceae counts were below 1 Log CFU/g during shelf life for all treatments. There was a significant effect (p < 0.05) of the interaction aw X sorbate X storage on mesophilic aerobic and sulphite-reducing counts (spores and total) and residual nitrite concentration. It was observed that sorbate addition prevented mesophilic aerobes growth and sulphite-reducing clostridia spores germination during 90 d storage at aw 0.96 and during 60 d at aw 0.965. Regarding residual nitrite, the addition of potassium sorbate decreased nitrite depletion at both water activities until 30 d and marked difference has been perceived at 0.965.ConclusionAddition of potassium sorbate may enhance microbial stability of this type of emulsified product and prevent spores germination during storage at ambient temperature. The amount of sodium in the product due to its high amount of sodium chloride required to reach 0.95 may be reviewed especially after other experiments including other shelf life enhancers such as sodium or potassium lactate which are effectives at inhibiting different microorganisms.

Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine.

In recent years, many spore-forming commensal Clostridia found in the gut have been discovered to promote host physiology, immune development, and protection against infections. We provide a detailed protocol for rapid enrichment of spore-forming bacteria from murine intestine. Briefly, contents from the intestinal cecum are collected aerobically, diluted and finally treated with chloroform to enrich for Clostridia spores.

Microbiological quality of honey from the Pampas Region (Argentina) throughout the extraction process

The microbiological quality of honey obtained from different processing points and the environmental quality within honey houses were assessed in the Pampas Region (Argentina). Mold and yeast (MY), culturable heterotrophic mesophilic bacteria (CHMB), the number of spore-forming bacteria as well as the presence of Shigella spp., Salmonella spp. and fecal coliforms were evaluated in 163 samples. These samples were taken from eight honey houses. Results showed that 89 samples had ≤10CFU of MY/g honey, 69 ranged from 10 to 50CFU/g and two reached 65.5CFU/g. Eighty one percent of the samples showed ≤30CFU of CHMB/g honey and only seven samples had between 50 and 54.25CFU/g. Thirty six honey samples were obtained from drums: in 25 samples (69.4%) CHMB counts were less than ≤30CFU/g of honey; in 20 samples (55.5%) the values of MY were between 10 and 50CFU/g honey and total coliforms were only detected in 20 samples. Fecal coliforms, spores of clostridia as well as Salmonella spp. and Shigella spp were not detected and less than 50 spores of Bacillus spp. per g were observed in the honey from drums. Therefore, the microbiological honey quality within the honey houses did not show any sanitary risks. Our results were reported to honey house owners to help them understand the need to reinforce proper honey handling and sanitation practices.

Assessing the usefulness of clostridia spores for evaluating sewage sludge hygienization

The capability of clostridia spores to act as pathogen indicators in sewage sludge treatment was investigated. Sulfite-reducing clostridia and E. coli levels were monitored during waste activated sludge pre-treatments (alkali and ultrasound) and its subsequent mesophilic anaerobic digestion. E. coli was maintained or reduced depending on treatment type and intensity. However, alkali pre-treatment (35.3gNaOH/kgTS) by itself and alkali (157gNaOH/kgTS) and ultrasound (27,000kJ/kgTS) pre-treatments followed by anaerobic digestion provoked reproducible clostridia increases. Specifically, up to 2.7log10 after 35.3gNaOH/kgTS pre-treatment and up to 1.9 and 1.1log10 after digesting the 157gNaOH/kg TS and 27,000kJ/kgTS pre-treated sludge, respectively. Having rejected the hypotheses of sporulation and floc dissipation, the most plausible explanation for these clostridia increases is re-growth. These results question the suitability of clostridia spores as indicators of sludge treatment and other biological treatments where clostridia may have a role.

Water quality as an indicator of the health status of agro-pastoral dams' ecosystems in Benin: An ecosystem services study

Based on a study in three agro-pastoral dams in Nikki, Sakabansi and Fombawi in northern Benin, this article aims to characterize their physical, chemical and microbiological water quality. The ecosystem services framework underlies this article. Water of the three dams was sampled in the field and analysed at the laboratory. Means of variables were compared with standard values (norms) for drinking water set by both Benin and the World Health Organization. Agro-pastoral dams' water quality is problematic because of the significantly high levels of nitrite, nitrate, iron, and chemical oxygen demand. Water in these dams is unsuitable for both human and livestock consumption because it is contaminated with harmful bacteria including total Coliform, Escherichia coli, spores of Clostridium, Enterococcus feacalis, Salmonella typhi, Salmonella typhimurium, Salmonella enteritidis and Campylobacter jejuni. This study concluded that one solution for maintaining agro-pastoral ecosystem health consisted of watershed management based on monitoring ecosystem services such as water quality.

Occurrence and removal of Giardia spp. cysts and Cryptosporidium spp. oocysts from a municipal wastewater treatment plant in Brazil

ABSTRACTSewage and sewage sludge have been recognized as potential sources of two important waterborne pathogenic protozoa: Giardia spp. and Cryptosporidium spp. Due to the lack of studies about the occurrence of these pathogens in sewage and sludge in Brazil, an investigation was conducted at various stages of a municipal wastewater treatment plant (WWTP) aiming to assess the occurrence of Giardia spp. cysts and Cryptosporidium spp. oocysts, their removal by the treatment processes, which are upflow anaerobic sludge blanket (UASB) reactor and dissolved air flotation process, and also the correlations between protozoa and indicator microorganisms. Significant quantities of cysts were detected in 100% of the analyzed wastewater samples, while oocysts were detected only in 39.0% of all wastewater samples. The overall removal of Giardia spp. cysts from the WWTP was on average 2.03 log, and the UASB reactor was more efficient than flotation. The sludge samples presented high quantities of (oo)cysts, implying the risks of contamination in the case of sludge reuse or inadequate disposal. Giardiasis prevalence was estimated between 2.21% and 6.7% for the population served by the WWTP, while cryptosporidiosis prevalence was much lower. Significant positive correlation was obtained only between cysts and Clostridium spores in anaerobic effluent.

Concurrence of spores of Clostridium tyrobutyricum, Clostridium beijerinckii and Paenibacillus polymyxa in silage, dairy cow faeces and raw milk

Spores of lactate-fermenting clostridia, known as butyric acid bacteria (BAB), can cause severe quality defects in semi-hard cheeses, called late-blowing. The routine detection method of BAB spores involves most probable number quantification of spores of anaerobic, gas-forming bacteria in a medium containing lactic acid. In this study, BAB spores were detected in 296 samples of soil, maize and grass silage, dairy cow faeces and farm tank milk collected from dairy farms in The Netherlands and the most abundant populations identified. Three major populations were detected, namely Clostridium tyrobutyricum, Clostridium beijerinckii and Paenibacillus spp. Paenibacillus polymyxa was the most abundant species of the latter group. The results indicate that the three spore populations share the same sources and the same contamination route of milk. In contrast to the Clostridium species, P. polymyxa isolates were unable to ferment lactate into butyrate. P. polymyxa spores are presumably unable to cause cheese defects.

Application of high pressure processing for controlling Clostridium tyrobutyricum and late blowing defect on semi-hard cheese

In this study we evaluated the application of different high pressure (HP) treatments (200–500 MPa at 14 °C for 10 min) to industrial sized semi-hard cheeses on day 7, with the aim of controlling two Clostridium tyrobutyricum strains causing butyric acid fermentation and cheese late blowing defect (LBD). Clostridium metabolism and LBD appearance in cheeses were monitored by sensory (cheese swelling, cracks/splits, off-odours) and instrumental analyses (organic acids by HPLC and volatile compounds by SPME/GC–MS) after 60 days. Cheeses with clostridial spores HP-untreated and HP-treated at 200 MPa showed visible LBD symptoms, lower concentrations of lactic, citric and acetic acids, and higher levels of pyruvic, propionic and butyric acids and of 1-butanol, ethyl and methyl butanoate, and ethyl pentanoate than cheeses without spores. However, cheeses with clostridial spores and HP-treated at ≥ 300 MPa did not show LBD symptoms and their organic acids and volatile compounds profiles were comparable to those of their respective HP-treated control cheeses, despite HP treatments caused a low spore reduction. A decrease in C. tyrobutyricum spore counts was observed after curd pressing, which seems to indicate an early spore germination, suggesting that HP treatments ≥300 MPa were able to inactivate the emerged C. tyrobutyricum vegetative cells and, thereby, prevent LBD.

Occurrence of Clostridium spp. in ewe’s milk: enumeration and identification of isolates

The presence of gas-producing clostridia in ewe’s milk can lead to the occurrence of late-blowing defects in cheeses. However, data on this aspect are limited. In the present study, using the most probable number (MPN) method, clostridial spores were enumerated in 527 ewe’s milk samples collected in the Grosseto Province (Tuscany, Italy) from autumn 2014 to summer 2015. In addition, using polymerase chain reaction (PCR), we identified the species most frequently involved in late-blowing defects in cheese (Clostridium tyrobutyricum, Clostridium butyricum, Clostridium beijerinckii, and Clostridium sporogenes), and of Clostridium perfringens. Gas-producing clostridial spores were detected in 99% of samples. Spore concentrations ranged from 360 to more than 110,000 spores∙L−1. We observed that 86% of samples had a spore load higher than 1000 spores∙L−1. During autumn and summer, spore concentrations were significantly higher than in winter and spring (P < 0.001). A total of 222 isolates obtained from 77 MPN positive tubes from different milk samples were subjected to PCR. Colonies from 63/77 (82%) MPN positive tubes were taxonomically identified. Among the 63 PCR-positive samples, C. perfringens was the most frequently detected species (56%), followed by C. sporogenes (44%), C. tyrobutyricum (7.9%), C. butyricum (1.6%), and C. beijerinkii (1.6%). In addition, in 11% of the MPN positive tubes, at least two clostridial species were found to be present simultaneously. This work highlights the presence of clostridial spores in ovine milk from central Italy (Tuscany) and led to the identification of some of the clostridia species involved in such high spore loads.

Germinants and Their Receptors in Clostridia

Many anaerobic spore-forming clostridial species are pathogenic, and some are industrially useful. Although many are strict anaerobes, the bacteria persist under aerobic and growth-limiting conditions as multilayered metabolically dormant spores. For many pathogens, the spore form is what most commonly transmits the organism between hosts. After the spores are introduced into the host, certain proteins (germinant receptors) recognize specific signals (germinants), inducing spores to germinate and subsequently grow into metabolically active cells. Upon germination of the spore into the metabolically active vegetative form, the resulting bacteria can colonize the host and cause disease due to the secretion of toxins from the cell. Spores are resistant to many environmental stressors, which make them challenging to remove from clinical environments. Identifying the conditions and the mechanisms of germination in toxin-producing species could help develop affordable remedies for some infections by inhibiting germination of the spore form. Unrelated to infectious disease, spore formation in species used in the industrial production of chemicals hinders the optimum production of the chemicals due to the depletion of the vegetative cells from the population. Understanding spore germination in acetone-butanol-ethanol-producing species can help boost the production of chemicals, leading to cheaper ethanol-based fuels. Until recently, clostridial spore germination is assumed to be similar to that of Bacillus subtilis However, recent studies in Clostridium difficile shed light on a mechanism of spore germination that has not been observed in any endospore-forming organisms to date. In this review, we focus on the germinants and the receptors recognizing these germinants in various clostridial species.

Fate of classical faecal bacterial markers and ampicillin-resistant bacteria in agricultural soils under Mediterranean climate after urban sludge amendment

The use of sewage sludge or biosolids as agricultural amendments may pose environmental and human health risks related to pathogen or antibiotic-resistant microorganism transmission from soils to vegetables or to water through runoff. Since the survival of those microorganisms in amended soils has been poorly studied under Mediterranean climatic conditions, we followed the variation of soil fecal bacterial markers and ampicillin-resistant bacteria for two years with samplings every four months in a split block design with three replica in a crop soil where two different types of biosolids (aerobically or anaerobically digested) at three doses (low, 40; intermediate, 80; and high, 160Mg·ha−1) were applied. Low amounts of biosolids produced similar decay rates of coliform populations than in control soil (−0.19 and −0.27log10CFUs·g−1drysoilmonth−1 versus −0.22) while in the case of intermediate and high doses were close to zero and their populations remained 24months later in the range of 4–5log10CFUs·g−1ds. Enterococci populations decayed at different rates when using aerobic than anaerobic biosolids although high doses had higher rates than control (−0.09 and −0.13log10CFUs·g−1dsmonth−1 for aerobic and anaerobic, respectively, vs −0.07). At the end of the experiment, counts in high aerobic and low and intermediate anaerobic plots were 1 log10 higher than in control (4.21, 4.03, 4.2 and 3.11log10CFUs·g−1 ds, respectively). Biosolid application increased the number of Clostridium spores in all plots at least 1 log10 with respect to control with a different dynamic of decay for low and intermediate doses of aerobic and anaerobic sludge. Ampicillin-resistant bacteria increased in amended soils 4months after amendment and remained at least 1 log10 higher 24months later, especially in aerobic and low and intermediate anaerobic plots due to small rates of decay (in the range of −0.001 to −0.008log10CFUs·g−1dsmonth−1 vs −0.016 for control). Aerobic plots had relative populations of ampicillin-resistant bacteria higher than anaerobic plots with different positive trends. Dose (22%) and time (13%) explained most of the variation of the bacterial populations. Dynamics of fecal markers did not correlate with ampicillin-resistant bacteria thus making necessary to evaluate specifically this trait to avoid possible risks for human and environmental health.

Importance of on-farm management practices on lactate-fermenting Clostridium spp. spore contamination of total mixed ration of Manchega ewe feeding. Determination of risk factors and characterization of Clostridium population

This work studies the on-farm management practices that increase the risk of developing high lactate-fermenting Clostridium spp. spore counts in Total Mixed Ration (TMR) of Manchega sheep. Moreover the ecology of the Clostridium population was studied in relation to those management practices. A total of 136 TMR samples belonging of 23 Manchega sheep flocks were analyzed for lactate-fermenting Clostridium spores by the Most Probable Number technique (MPN). Information about the feeding characteristics was also collected from the 23 flocks. A logistic regression analysis indicated silages and wet breweŕs grains used for feeding as the on-farm management risk factors that lead to an increase of Clostridium spp. spore counts. A total of 155 Clostridium isolates from TMR samples were typed using Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). The 56 different genotypes obtained were subsequently identified by restriction analysis of 16S-rRNA gene (16S-ARDRA), so three populations were observed: 92.90%, 5.81% and 1.29% corresponding to Clostridium (C.) sporogenes, Clostridium beijerinkii and Clostridium butyricum respectively. The risks factors of high Clostridium spp. spore counts also increased Clostridium population diversity and favoured the presence of C. butyricum. The results confirmed that these risk factors should be taken into account in developing strategies in the control of Clostridium spp. spores contamination in TMR.

Unravelling the effect of clostridia spores and lysozyme on microbiota dynamics in Grana Padano cheese: A metaproteomics approach

Grana Padano is a typical Italian Protected Designation of Origin (PDO) hard cheese largely consumed all over the world. The major problem during its production is represented by late blowing. Clostridia are gasogen bacteria responsible of the swelling during ripening, and they are partially counteracted by the use of egg white lysozyme as additive. In this work was applied, for the first time in cheese, a metaproteomic approach that identified the functional dynamics of microbial consortia in relation to the number of clostridial spores and lysozyme treatment using experimental samples of Grana Padano cheese. We used a combined custom BLAST+/MEGAN/STAMP approach to obtain a global taxonomic view associated to low and high clostridial spores cheese without and with lysozyme. Main differences were highlighted in the bacilli class. Functional analysis with SEED provided a deep view into several metabolic pathways, highlighting the subsystems “amino acid and derivatives” and “clustering-based subsystem” as the targeted subsystems during lysozyme treatment in the high spore group. In these subsystems, acetate kinase from clostridia was one of the main enzymes affected by the lysozyme treatment. Biological significanceMetaproteomics is a very promising and useful technique in the control of food safety and quality, from fresh products until ‘ready to eat’ food. Tools able to identify at molecular level the dynamic fingerprinting of food microbiota could be of great help to improve food safety and quality.