Genetic diversity of Clostridium perfringens type A isolates from animals, food poisoning outbreaks and sludge

Background  

Clostridium perfringens, a serious pathogen, causes enteric diseases in domestic animals and food poisoning in humans. The epidemiological relationship between C. perfringens isolates from the same source has previously been investigated chiefly by pulsed-field gel electrophoresis (PFGE). In this study the genetic diversity of C. perfringens isolated from various animals, from food poisoning outbreaks and from sludge was investigated.     

Fate of manure-borne pathogen surrogates in static composting piles of chicken litter and peanut hulls

The fate of manure-borne pathogen surrogates (gfp-labeled Escherichia coli O157:H7 and Listeria innocua and avirulent Salmonella Typhimurium) in the field was monitored at both sub-surface (30 cm from surface) and surface sites of static composting piles (3.5-m base diameter) composed of chicken litter and peanut hulls. Despite exposure to elevated temperatures, Salmonella was detected by enrichment culture in sub-surface samples following 14 days of composting. In surface samples, pathogen surrogates were detected in the summer after 4 days of composting by enrichment culture only, whereas E. coli O157:H7 and L. innocua remained detectable by direct plating (>2log₁₀ cfu/g) up to 28 days in piles composted during the fall and winter. All three types of bacteria remained detectable by enrichment culture in surface samples composted for 56 days during the winter.     

Pyrene mineralization capacity increases with compost maturity

Experiments were conducted to determine the effects of composting or simple addition of compost to the mineralization of n-hexadecane, pyrene and benzo(a)pyrene in soil. Soil (contaminated or clean) was composted with maple leaves and alfalfa. Samples from different composting phases were spiked with radiolabeled and cold n-hexadecane, pyrene or benzo(a)pyrene, placed in aerated microcosms at different temperatures, and monitored for mineralization. It was determined that neither composting nor the addition of compost had any effect on n-alkane or benzo(a)pyrene mineralization. In contrast, the pyrene mineralization rate increased dramatically with the amount of time that soil had been composted. Highest pyrene mineralization rates and extents (more than 60% after 20 days) were obtained when pyrene was in contact with composted soil from the curing stage. Neither thermophiles (55 °C) nor fungi were responsible for pyrene mineralization.     

Screening Escherichia coli, Enterococcus faecalis, and Clostridium perfringens as Indicator Organisms in Evaluating Pathogen-Reducing Capacity in Biogas Plants

This study was conducted to identify an indicator organism(s) in evaluating the pathogen-reducing capacity of biogas plants. Fresh cow manure containing 10⁴ to 10⁵ colony forming unit (CFU) per milliliter of Escherichia coli and Enterococcus faecalis along with an inoculated Clostridium perfringens strain were exposed to 37°C for 15 days, 55°C for 48 h, and 70°C for 24 h. C. perfringens was the most heat-resistant organism followed by E. faecalis, while E. coli was the most heat-sensitive organism. E. coli was reduced below detection limit at all temperatures with log₁₀ reductions of 4.94 (10 s), 4.37 (40 min), and 2.6 (5 days) at 70°C, 55°C, and 37°C, respectively. Maximum log₁₀ reductions for E. faecalis were 1.77 at 70°C (1 day), 1.7 at 55°C (2 days) and 3.13 at 37°C (15 days). For C. perfringens, maximum log₁₀ reduction at 37°C was 1.35 log₁₀ units (15 days) compared to less than 1 unit at 55 and 70°C. Modeling results showed that E. faecalis and C. perfringens had higher amount of heat-resistant fraction than E. coli. Thus, E. faecalis and C. perfringens can be used as indicator organisms to evaluate pathogen-reducing capacity in biogas plants at high temperatures of 55°C and 70°C while at 37°C E. coli could also be included as indicator organism.     

Reliability of mCP method for identification of Clostridium perfringens from faecal polluted aquatic environments

Aims: The purpose of the work was to evaluate the mCP method to correctly identify and enumerate Clostridium perfringens that are present in surface waters impacted by a mixture of faecal pollution sources. Methods: Clostridium perfringens were enumerated and isolated from sewage influent, surface water and suspended sediments using the mCP method. Molecular characterization of isolates was performed using species‐specific PCR, along with full‐length sequencing of the 16S rRNA gene for a subset of isolates. Results: The environmental isolates were presumptively identified as C. perfringens based on utilization of sucrose, inability to ferment cellobiose and a positive action for acid phosphatase activity. All isolates (n = 126) were classified as C. perfringens based on positive results with species‐specific PCR with a subset confirmed as C. perfringens based on the 16S rRNA gene identity. Conclusions: The molecular results indicated all of the presumptive positive isolates were C. perfringens regardless of the source, e.g. sewage influent or environmental water samples. Sequencing revealed that C. perfringens obtained from sewage and the aquatic environment were nearly identical (c. 99·5% similarity). Significance and Impact of the Study: From this study we conclude that the mCP method is a robust approach to enumerate and isolate C. perfringens from aquatic environments that receive diverse sources of faecal pollution.     

Chrysomya megacephala larvae feeding favourably influences manure microbiome,heavy metal stability and greenhouse gas emissions

Chrysomya megacephala is a saprophagous fly whose larvae can compost manure and yield biomass and bio‐fertilizer simultaneously. However, there are concerns for the safety of the composting system, that is risk of diseases spread by way of manure pathogens, residue of harmful metals and emission of greenhouse gases. Microbiota analysis and heavy metal speciation by European Communities Bureau of Reference were evaluated in raw, C. megacephala‐composted and natural stacked swine manure to survey pathogenic bacterial changes and mobility of lead and cadmium in manure after C. megacephala feeding; the emission rate of CH₄ and N₂O from manure during C. megacephala composting and natural stacking was also measured. C. megacephala composting altered manure microbiota, reduced the risk of pathogenic bacteria and maintained the stability, and microbiota changes might be associated with heavy metal fractions, especially in Pseudomonas and Prevotella. In addition, C. megacephala‐composting significantly reduced the emission rate of CH₄ and N₂O in comparing with natural stacking situation and the first two days should be the crucial period for CH₄ and N₂O emission measurement for manure treatment by C. megacephala. Moreover, OTU26 and Betaproteobacteria were changed after C. megacephala composting which might play a role in emission of CH₄ and N₂O, respectively.     

Cpn20: Siamese twins of the chaperonin world

The chloroplast cpn20 protein is a functional homolog of the cpn10 co-chaperonin, but its gene consists of two cpn10-like units joined head-to-tail by a short chain of amino acids. This double protein is unique to plastids and was shown to exist in plants as well plastid-containing parasites. In vitro assays showed that this cpn20 co-chaperonin is a functional homolog of cpn10. In terms of structure, existing data indicate that the oligomer is tetrameric, yet it interacts with a heptameric cpn60 partner. Thus, the functional oligomeric structure remains a mystery. In this review, we summarize what is known about this distinctive chaperonin and use a bioinformatics approach to examine the expression of cpn20 in Arabidopsis thaliana relative to other chaperonin genes in this species. In addition, we examine the primary structure of the two homologous domains for similarities and differences, in comparison with cpn10 from other species. Lastly, we hypothesize as to the oligomeric structure and raison d’être of this unusual co-chaperonin homolog. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evolution of clostridia and streptomycetes in full-scale composting facilities and pilot drums equipped with on-line temperature monitoring and aeration

The evolution of sporulating bacteria in full-scale composting facilities with online temperature monitoring has been poorly studied, although organic matter recycling increases. We analysed Clostridium perfringens and sulphite-reducing clostridia (SRC) by cultivation, and streptomycetes by real-time PCR in five full-scale, temperature-monitored and aerated composting processes, and two pilot-scale drum composters. Facilities composted woodchips, sawdust, peat, or bark amended sludge or source-separated biowaste. Streptomycetes genes of 0.21-110 × 10⁷ copies/g feed increased fast to 0.019-33 × 10⁹ copies/g, and then were equal or decreased. SRC of 0.06-2.2 × 10⁷ cfu/g feed decreased to 0-600 cfu/g, with re-growth in two facilities. End products were clean of C. perfringens, detected in sludge composts. Although processes contained large quantities of spore-forming bacteria, in the best facilities end products had the high quality. Temperature (>55 °C, >2d) was not related to the end compost quality, but relations between waste and bulking agent qualities, aeration, and processing time should be better controlled.     

Viability of Rhodococcus equi and Parascaris equorum Eggs Exposed to High Temperatures

There is great concern about the potential pathogen contamination of horse manure compost spread in the same fields horses graze in. To ensure that pathogen destruction occurs, temperatures need to be sufficiently high during composting. Here, we investigated the survival rate of two marker organisms, Rhodococcus equi and Parascaris equorum eggs, exposed to temperatures potentially encountered during horse manure composting. Our results show that the time required to achieve a 1 log10 reduction in R. equi population (D-value) are 17.1 h (±1.47) at 45°C, 8.6 h (±0.28) at 50°C, 2.9 h (±0.04) at 55°C and 0.7 h (±0.04) at 60°C. For P. equorum eggs we show that at 45 and 50°C, 2 log10 reduction of viability is reached between 8 and 24 h of incubation and that it takes less than 2 h at 55 and 60°C to achieve a viability reduction of 2 log10. These results are useful for identifying composting conditions that will reduce the risk of environmental contamination by R. equi and P. equorum eggs.     

Life history influences how fire affects genetic diversity in two lizard species

‘Fire mosaics’ are often maintained in landscapes to promote successional diversity in vegetation with little understanding of how this will affect ecological processes in animal populations such as dispersal, social organization and re‐establishment. To investigate these processes, we conducted a replicated, spatiotemporal landscape genetics study of two Australian woodland lizard species [Amphibolurus norrisi (Agamidae) and Ctenotus atlas (Scincidae)]. Agamids have a more complex social and territory structure than skinks, so fire might have a greater impact on their population structure and thus genetic diversity. Genetic diversity increased with time since fire in C. atlas and decreased with time since fire in A. norrisi. For C. atlas, this might reflect its increasing population size after fire, but we could not detect increased gene flow that would reduce the loss of genetic diversity through genetic drift. Using landscape resistance analyses, we found no evidence that postfire habitat succession or topography affected gene flow in either species and we were unable to distinguish between survival and immigration as modes of postfire re‐establishment. In A. norrisi, we detected female‐biased dispersal, likely reflecting its territorial social structure and polygynous mating system. The increased genetic diversity in A. norrisi in recently burnt habitat might reflect a temporary disruption of its territoriality and increased male dispersal, a hypothesis that was supported with a simulation experiment. Our results suggest that the effects of disturbance on genetic diversity will be stronger for species with territorial social organization.     

Inactivation of Salmonella spp. in cow manure composts formulated to different initial C:N ratios

Aerobic composting is a common management practice to inactivate pathogens in manure; however, additional research on the role of compost composition in pathogen inactivation is needed. The objective of this study was therefore to determine the effect of the carbon:nitrogen (C:N) ratio and the presence of ammonium sulfate on inactivation of Salmonella spp. in cow manure-based mixtures composted in a bioreactor under controlled conditions. Compost preparations with an initial C:N ratio of 20:1 required a maximum of 4 days of storage before Salmonellae were inactivated by 7 log₁₀, whereas preparations with C:N ratios of 30:1 and 40:1 C:N required more than 5 and 7 days of storage, respectively. The pH values of both the 20:1 and 30:1 C:N preparations decreased during the onset of composting before increasing to >8. In contrast, pH values of 40:1 C:N preparations increased immediately to >8, generally within the first day of storage. Maximum temperatures observed in 20:1 C:N preparations for inactivation of pathogens were less than 50 °C, and the cumulative heat exposure required for pathogen inactivation in 20:1 C:N preparations was 15-fold less than in 40:1 C:N preparations. Supplementation of compost mixtures with 0.08% ammonium sulfate resulted in slightly higher temperatures; however, these higher temperatures did not translate into more rapid rates of pathogen inactivation.     

The effect of volatile fatty acids on the inactivation of <Emphasis Type="Italic">Clostridium perfringens</Emphasis> in anaerobic digestion

The application of sludge digestion systems to remove pathogens has been employed to generate biosolids suitable for reuse in agriculture. Traditionally, temperature is considered the principal agent responsible for pathogen reduction in anaerobic digestion. However, other substances such as volatile fatty acids may also have an antimicrobial effect. The objective of this study was to assess the impact of fatty acid mixtures on the inactivation of C. perfringens over a range of digestion temperatures. An equimolar mixture of acetic acid, propionic acid and butyric acid was applied to digester effluent for a period of 24 h at temperatures of 35 °C, 42 °C, 49 °C and 55 °C. C. perfringens inactivation in digester effluents, when dosed with volatile organic acids, was found to depend on pH, acid concentration and temperature. Temperatures above 55 °C appeared to increase the inhibitory effects of the organic acids at higher concentrations. An interaction between temperature and pH on survival of C. perfringens was observed. The results suggest that high concentrations of organic acids at a pH value of 4.5–5.5 during thermophilic digestion substantially reduce concentrations of C. perfringens in municipal sludge.     

Growth potential of faecal bacteria in simulated psychrophilic/mesophilic zones during composting of organic waste

Aim: This study investigated the growth potential of Salmonella serotype Typhimurium and faecal indicator organisms in compost materials and the correlation between bacterial growth potential and the physico‐chemical composition of the compost substrate and temperature. Methods and Results: Survival of Salm. Typhimurium, Enterococcus spp. and total coliforms at 14, 24 and 37°C was determined in material of different degrees of maturity collected from composting plants for household waste and manure. All three micro‐organisms showed the potential for growth in the material from active composts (Solvita index 4) but inactivation generally occurred over time in mature compost material (Solvita index 7–8). Conclusions: Salm. Typhimurium had the potential for growth in psychrophilic/mesophilic (P/M) zones of immature compost material and its growth potential correlated negatively with the maturity of the compost and the temperature within the simulated P/M zone. Significance and Impact of the Study: The risk of pathogen regrowth in P/M zones during organic waste composting further emphasizes the importance of good management practices and of avoiding P/M zones in combination with low compost maturity.     

Resistance of Faecal Coliforms and Enterococci Populations in Sludge and Biosolids to Different Hygienisation Treatments

The composition of the most abundant facultative anaerobic bacteria populations [faecal coliforms (FC) and enterococci (ENT)] in sludge can be modified after different treatments. These involve the disposal or reuse of sludge and include: anaerobic digesters, incineration, composting, pasteurization and lime treatments. In this study, three treatment types (mesophilic anaerobic digestion, composting and pasteurization) were compared in terms of their ability to reduce both bacterial populations. The diversity and any changes in composition of main phenotypic groups for both populations were also analyzed. Mesophilic anaerobic digestion (MAD) was carried out at 35°C for 20 days. Digested sludge was then dehydrated by centrifugation at 2,500 rpm. Composting (COM) was performed at 55°C with windrow phases. Pasteurization was assayed at 60°C for 90 min (P60), at 80°C for 60 min (P80). A 1–1.5 log unit reduction was observed for FC, and 1 log unit reduction was noted for ENT by MAD treatment. In composting, this reduction proved higher for FC than for ENT (6 log and 3–4 log units, respectively). Optimal pasteurization was obtained at 80°C for 60 min, resulting in a 5 log unit reduction for FC and a 2 log unit reduction for ENT. High diversity indices (Di) for both bacterial populations were detected both before and after implementation of the different treatments. Analyses of the population’s similarity provided that FC were diverse both before and after COM, P60 and P80 treatments. However, no differences were observed on the composition of ENT populations after the different treatments assayed.     

Conservation implications of high genetic variation in two closely related and highly threatened species of Crambe (Brassicaceae) endemic to the island of Gran Canaria: C. tamadabensis and C. pritzelii

We used data from 12 allozyme loci for two endemic Brassicaceae from Gran Canaria (the endangered narrow endemic Crambe tamadabensis and its more widespread congener C. pritzelii) to assess whether their genetic diversity patterns reflect their phylogenetic closeness and contrasting population sizes and distribution areas, and to derive conservation implications. Genetic diversity values are high for both species and slightly higher in C. tamadabensis, despite its narrow distribution in north‐western Gran Canaria. At odds with the generally high interpopulation diversity levels reported in Canarian endemics, values of G_ST in C. tamadabensis and C. pritzelii are rather low (0.067 and 0.126, respectively). We construe that the higher genetic structure detected in C. pritzelii is mainly a result of unbalanced allele frequencies and low population sizes at the edges of its distribution. The overall high allozyme variation detected in C. tamadabensis and C. pritzelii is nevertheless compatible with an incipient but consistent genetic differentiation between the two species, modulated by recurrent bottlenecks caused by grazing and drift. Our data suggest that conservation efforts aimed at maintaining the existing genetic connectivity in each species and ex situ conservation of seeds are the best strategies to conserve their genetic diversity.     

Relating Quinone Profile to the Aerobic Biodegradation in Thermophilic Composting Processes of Cattle Manure with Various Bulking Agents

Summary Cattle manure was composted aerobically with various bulking agents (rice straw, vermiculite, sawdust or waste paper) at a constant incubation temperature of 60 °C. Increased quinone content (IQC) was used to assess microbial biomass in the composted material. IQC was proportional to mass reduction (MR) (R = 0.812) and cumulative O₂ consumption (COC) (R = 0.810) irrespective of the bulking agent used, indicating that the yield of quinone was constant. Quinone yields were 0.44 ± 0.03 μmol quinone/g MR and 0.34 ± 0.02 μmol quinone/g COC. The material that was decomposed by microorganisms was considered to be mainly cattle manure. Bulking agents were not degraded within the 14 day trial period and did not affect microbial succession because composting runs with various bulking agents exhibited similar quinone yields.     

Genetic diversity,population structure,and traditional culture of Camellia reticulata

Camellia reticulata is an arbor tree that has been cultivated in southwestern China by various sociolinguistic groups for esthetic purposes as well as to derive an edible seed oil. This study examined the influence of management, socio‐economic factors, and religion on the genetic diversity patterns of Camellia reticulata utilizing a combination of ethnobotanical and molecular genetic approaches. Semi‐structured interviews and key informant interviews were carried out with local communities in China's Yunnan Province. We collected plant material (n = 190 individuals) from five populations at study sites using single‐dose AFLP markers in order to access the genetic diversity within and between populations. A total of 387 DNA fragments were produced by four AFLP primer sets. All DNA fragments were found to be polymorphic (100%). A relatively high level of genetic diversity was revealed in C. reticulata samples at both the species (H_sp = 0.3397, I_sp = 0.5236) and population (percentage of polymorphic loci = 85.63%, H_pop = 0.2937, I_pop = 0.4421) levels. Findings further revealed a relatively high degree of genetic diversity within C. reticulata populations (Analysis of Molecular Variance = 96.31%). The higher genetic diversity within populations than among populations of C. reticulata from different geographies is likely due to the cultural and social influences associated with its long cultivation history for esthetic and culinary purposes by diverse sociolinguistic groups. This study highlights the influence of human management, socio‐economic factors, and other cultural variables on the genetic and morphological diversity of C. reticulata at a regional level. Findings emphasize the important role of traditional culture on the conservation and utilization of plant genetic diversity.     

Investigating the role of small, acid-soluble spore proteins (SASPs) in the resistance of Clostridium perfringens spores to heat

Background  

Clostridium perfringens type A food poisoning is caused by enterotoxigenic C. perfringens type A isolates that typically possess high spore heat-resistance. The molecular basis for C. perfringens spore heat-resistance remains unknown. In the current study, we investigated the role of small, acid-soluble spore proteins (SASPs) in heat-resistance of spores produced by C. perfringens food poisoning isolates.     

Rotary drum composting of vegetable waste and tree leaves

High rate composting studies on institutional waste, i.e. vegetable wastes, tree leaves, etc., were conducted on a demonstration-scale (3.5 m³) rotary drum composter by evaluating changes in some physico-chemical and biological parameters. During composting, higher temperature (60–70 °C) at inlet zone and (50–60 °C) at middle zone were achieved which resulted in high degradation in the drum. As a result, all parameters including TOC, C/N ratio, CO₂ evolution and coliforms were decreased significantly within few days of composting. Within a week period, quality compost with total nitrogen (2.6%) and final total phosphorus (6 g/kg) was achieved; but relatively higher final values of fecal coliforms and CO₂ evolution, suggested further maturation. Thus, two conventional composting methods namely windrow (M1) and vermicomposting (M2) tried for maturation of primary stabilized compost. By examining these methods, it was suggested that M2 was found suitable in delivering fine grained, better quality matured compost within 20 days of maturation period.     

Microbial community structure changes during bioremediation of PAHs in an aged coal-tar contaminated soil by in-vessel composting

The microbial community structure changes of an aged-coal-tar soil contaminated with polycyclic aromatic hydrocarbons (PAHs) were investigated during simulated bioremediation at the laboratory-scale using an in-vessel composting approach. The composting reactors were operated using a logistic three-factor factorial design with three temperatures (T=38, 55 or 70 °C), four soil to green-waste amendment ratios (S:GW=0.6:1, 0.7:1, 0.8:1 or 0.9:1 on a dry weight basis) and three moisture contents (MC=40%, 60% or 80%). Relative changes in microbial populations were investigated by following the dynamics of phospholipid fatty acid (PLFA) signatures using a ¹³C-labeled palmitic acid internal standard and sensitive GC/MS analysis during in-vessel composting over 98 days. The results of this investigation indicated that fungal to bacterial PLFA ratios were significantly influenced by temperature (p<0.05), and Gram-positive to Gram-negative bacterial ratios were significantly influenced by temperature (p<0.001) and S:GW ratio (p<0.01) during in-vessel composting. Additionally, the Gram-positive to Gram-negative bacterial ratios were correlated to the extent of PAH losses (p<0.005) at 70 °C.