Wide distribution of extracellular electron transfer functionality in natural proteinaceous organic materials for microbial reductive dehalogenation

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Carbon isotope fractionation in the reductive dehalogenation of carbon tetrachloride at iron (hydr)oxide and iron sulfide minerals

Compound-specific isotope analysis (CSIA) is used increasingly in contaminant hydrology in the attempt to assess the nature as well as the extent of in situ transformation reactions. Potentially, variations of stable isotope ratios along a contaminant plume may be used to quantify in situ degradation. In the present study, the abiotic dehalogenation of CCl4 by Fe(II) present at the surface of different iron minerals has been characterized in terms of the reaction rates and carbon isotopic fractionation (delta13C) of carbon tetrachloride (CCl4) as well as the yields and isotopic signatures of chloroform (CHCl3), one of the main transformation products. The abiotic reductive dehalogenation of CCl4 was associated with substantial carbon isotopic enrichment effects. The observed enrichment factors, e, correlated neither with the surface-normalized reaction rate constants nor with the type of products formed but fell into two distinctly different ranges for the two principal groups of minerals studied. With iron (hydr)oxide minerals (goethite, hematite, lepidocrocite, and magnetite) and with siderite, the e-values for CCl4 dehalogenation were remarkably similar (-29 +/- 3 per thousand). Because this value matches well with the theoretical estimates for the cleavage of an aliphatic C-Cl bond, we suggest that dissociative electron transfer to CCl4 controls the reaction rates for this group of iron minerals. Conversely, CCl4 transformation by different preparations of the iron sulfide mackinawite was accompanied by a significantly lower carbon istotopic fractionation (e = -15.9 +/- 0.3 per thousand), possibly due to the presence of nonfractionating rate-determining steps or a significantly different transition state structure of the reaction. Isotopically sensitive branching of the reaction pathways (i.e., the effect of different product distributions on isotope fractionation of CCl4) did not play a significant role in our systems. The extensive data set presented in this study opens new perspectives toward an improved understanding of the factors that determine reaction mechanisms and isotopic fractionation of dehalogenation reactions by Fe(II) at iron containing minerals.     

Mechanisms and products of surface-mediated reductive dehalogenation of carbon tetrachloride by Fe(II) on goethite

Natural attenuation processes of chlorinated solvents in soils and groundwaters are increasingly considered as options to manage contaminated sites. Under anoxic conditions, reactions with ferrous iron sorbed at iron(hyro)xides may dominate the overall transformation of carbon tetrachloride (CCl4) and other chlorinated aliphatic hydrocarbons. We investigated mechanisms and product formation of CCl4 reduction by Fe(II) sorbed to goethite, which may lead to completely dehalogenated products or to chloroform (CHCl3), a toxic product which is fairly persistent under anoxic conditions. A simultaneous transfer of two electrons and cleavage of two C-Cl bonds of CCl4 would completely circumvent chloroform production. To distinguish between initial one- or two-bond cleavage, 13C-isotope fractionation of CCl4 was studied for reactions with Fe(II)/ goethite (isotopic enrichment factor epsilon = -26.5% percent per thousand) and with model systems for one C-Cl bond cleavage and either single-electron transfer (Fe(II) porphyrin, epsilon = -26.1 percent per thousand) or partial two-electron transfer (polysulfide, epsilon = -22.2 percent per thousand). These epsilon values differ significantlyfrom calculations for simultaneous cleavage of two C-Cl bonds (epsilon approximately equal to -50 percent per thousand), indicating that only one C-Cl bond is broken in the critical first step of the reaction. At pH 7, reduction of CCl4 by Fe(II)/ goethite produced approximately 33% CHCl3, 20% carbon monoxide (CO), and up to 40% formate (HCOO-). Addition of 2-propanol-d8 resulted in 33% CDCl3 and only 4% CO, indicating that both products were generated from trichloromethyl radicals (*CCl3), chloroform by reaction with hydrogen radical donors and CO by an alternative pathway likely to involve surface-bound intermediates. Hydrolysis of CO to HCOO-was surface-catalyzed by goethite butwastoo slow to account for the measured formate concentrations. Chloroform yields slightly increased with pH at constant Fe(II) sorption density, suggesting that pH-dependent surface processes direct product branching ratios. Surface-stabilized intermediates may thus facilitate abiotic mineralization of CCl4, whereas the presence of H radical donors, such as natural organic matter, enhances formation of toxic CHCl3.     

Distribution and role of Coprothermobacter spp. in anaerobic digesters

The distribution of Coprothermobacter spp. was investigated in seven anaerobic digesters using 16S rRNA gene-based quantitative PCR. The largest number of Coprothermobacter spp. cells was found in a thermophilic anaerobic digester treating dairy cow manure.     

Incidence of Listeria spp. and Salmonella spp. in horsemeat for human consumption

The incidence of Salmonella spp., Listeria spp. and Listeria monocytogenes in horsemeat for human consumption was investigated. One-hundred and twenty-one samples of frozen horsemeat collected from two Brazilian abattoirs were analysed over a period of 1 year. Twenty-two samples (18.2%) were positive for Listeria spp. with nine (7.4%) containing L. monocytogenes. None of the samples harbored Salmonella spp.     

A selective and diagnostic medium for use in the enumeration of Listeria spp. in foods

A new medium, called RAPAMY agar, has been elaborated for the isolation from and the enumeration of Listeria spp. in foods. It is based on Ralovich's nalidixic acid-trypaflavin-agar with the following modifications: (i) the slight inhibitory properties of that medium were overcome by the use of Columbia Blood agar base instead of tryptose agar and the addition of 0.05% ferric ammonium citrate and 2.5% egg yolk emulsion; (ii) selectivity was improved by the addition of 0.25% 2-phenyl ethanol and incubation under microaerobic conditions; (iii) the medium was provided with two diagnostic traits by the addition of (a) aesculin + ferric ammonium citrate; and (b) D-mannitol and phenol red. The growth of Enterococcus spp., the only organisms other than Listeria spp. which grow on RAPAMY agar, was not inhibited by the addition of 20 microgram.ml-1 Cefoxitin (Moxolactam). Higher levels inhibited some Listeria spp., but not the enterococci. The medium recovered Listeria spp. quantitatively and allowed recovery from foods colonized by Enterococcus spp. at levels upto 10(2) per g.     

A simple method for the separation of Gnatocerus spp. and Tribolium spp. (Coleoptera: Tenebrionidae)

A new character for distinguishing the genera Gnatocerus and Tribolium is described. The advantages of using this character for the identification of female specimens are explained.     

The role of coagulase-negative Staphylococcus spp. and associated somatic cell counts in the ovine mammary gland

The somatic cell count (SCC) of ewes' milk was determined by the Fossomatic method and compared with the bacteriological status of the mammary gland. Of 366 samples from uninfected udder halves, 64.5% had SCC less than 50 x 10(3) cells/ml, 81.9% had SCC less than 250 x 10(3), and 92.4% had less than 500 x 10(3) cells/ml. Of 130 bacteriologically positive samples, 91.1% had SCC more than 500 x 10(3) cells/ml and 98.8% more than 250 x 10(3). Of the examined milk samples 26.2% showed positive bacteriology during the single sampling. The most frequent pathogens isolated from the milk samples were coagulase-negative staphylococci. Considering our results, 250 x 10(3) cells/ml should be the threshold value, which could be regarded as the upper limit for normal SCC of ewes' milk.     

Reductive dehalogenation of halomethanes in iron- and sulfate-reducing sediments. 1. Reactivity pattern analysis

The incorporation of reductive transformations into environmental fate models requires the characterization of natural reductants in sediments and aquifer materials. For this purpose, reactivity patterns (range and relative order of reactivity) for a series of 14 halogenated methanes were measured in iron- and sulfate-reducing sediments and two representative model systems: adsorbed Fe(II)/goethite [Fe(II)ads/alpha-FeOOH] and iron sulfide (FeS). Both Fe(II)ads and FeS are naturally occurring reductants. The strong similarity in reactivity patterns between the iron- and sulfate-reducing sediments suggests that the two share a common reductant despite their different chemical compositions (i.e., the sulfate-reducing sediment contained FeS). An orthogonal regression analysis of the halomethane transformation rate data in the sediment and model systems supports the assumption that a common mechanism for halomethane transformation exists between the sediments and the Fe(II)ads/alpha-FeOOH system and further corroborates the conclusion that Fe(II) adsorbed to Fe(III)-containing minerals is the dominant reductant in both sediment systems. Weak (0.5 N) and strong (6.0 N) acid extraction of the sediments indicated that solid-phase Fe(II) was 67% higher in the sulfate-reducing sediment than in the iron-reducing sediment, which is consistent with the observations that the halomethanes were transformed a factor of 3 times faster in the sulfate-reducing sediment and that Fe(II) was the dominant reductant.     

Subspecies-Specific Nested PCR Assay for Detection of Lactococcus lactis spp. lactis and spp. cremoris

A nested PCR-based assay composed of Lactococcus lactis species-specific primers for the nest 1 amplification and subspecies-specific primers for the nest 2 amplification was validated with the identified strains of L. lactis isolated from dairy and nondairy sources and positive and negative control strains. Forward and reverse primer set was designed for nest 1 amplification targeting the conserved housekeeping gene yueF encoding nonproteolytic protein from peptidase family M16 of L. lactis. Amplicons of 447 bp of yueF were subjected for nest 2 amplification producing amplicons of 372 bp. The designed outer primer set for nest 1 amplification was observed to be specific to L. lactis because the DNA from other bacteria could not be amplified and the inner primer set for nest 2 amplification was found to be specific for the detection of ssp. lactis and cremoris of L. lactis.     

The role of transport,lairage and slaughter processes in the dissemination of Salmonella spp. in pigs in Ireland

Salmonella is an important foodborne pathogen worldwide and is commonly isolated from pigs and pig products in Ireland. Pigs, reared in an environment free of Salmonella spp. or with low levels of infection, may acquire infection or become contaminated during transport, lairage or post-slaughter. The main objective of this study was to determine the role of the abattoir as a potential factor that contributes to the dissemination of Salmonella spp. in slaughter pigs from herds with a low Salmonella seroprevalence (≤ 10%). A total of 128 pigs from eight herds were monitored from farm through the slaughter process in three separate abattoirs. The prevalence of Salmonella spp. was determined in samples collected from trucks, lairage pens and the slaughterline before pigs entered, from pigs after slaughter (caecal contents and ileocaecal lymph nodes) and carcass surfaces post-evisceration. Isolates were characterised by serotype, phage type and pulsed-field gel electrophoresis (PFGE) patterns. Of the swabs taken from the trucks, lairage and slaughterline, before the pigs entered, 4.3% (3/70), 80% (64/80) and 16.7% (4/27) were positive for Salmonella spp., respectively. The proportion of pigs showing serological evidence of infection was 3.1% (4/128). Salmonella spp. were isolated from the ileocaecal lymph nodes and caecal contents of 14.8% (19/128) and 11.7% (15/128) of pigs, respectively, and 13/128 (10.2%), 5/128 (3.9%), 2/111 (1.8%) and 8/111 (7.2%) carcass swabs pre wash, post wash, post chill and belly-strip samples, respectively, were Salmonella-positive. There was only slight agreement between serological and bacteriological data at the pig level. Salmonella isolates from 45% of all positive pig samples and 82% of positive carcass samples were indistinguishable, based on PFGE patterns, from salmonellae isolated from the lairage and slaughterline. Based on these results it is concluded that the lairage and the slaughterline provide a substantial source for Salmonella contamination of pigs and carcasses.     

Radiosensitization of Salmonella spp. and Listeria spp. in ready-to-eat baby spinach leaves

The FDA recently approved irradiation treatment of leafy greens such as spinach up to 1 kGy; however, it is important to reduce the dose required to decontaminate the produce while maintaining its quality. Thus, the objectives of this study were: (1) to assess the radiation sensitivities of Salmonella spp. and Listeria spp. inoculated in ready-to-eat baby spinach leaves under modified atmosphere packaging (MAP) and irradiated using a 1.35-MeV Van de Graff accelerator (the leaves were irradiated both at room temperature and at -5 °C); and (2) to understand and optimize the synergistic effect of MAP and irradiation by studying the radiolysis of ozone formation under different temperatures, the effect of dose rate on its formation, and its decomposition. Results showed that increased concentrations of oxygen in the packaging significantly increased the radiation sensitivity of the test organisms, ranging from 7% up to 25% reduction in D(10)-values. In particular, radiosensitization could be effected (P < 0.05) by production of ozone, which increases with increasing dose-rate and oxygen concentration, and reducing temperatures. Radiosensitization was demonstrated for both microorganisms with irradiation of either fresh or frozen (-5 °C) baby spinach. These results suggest that low-dose (below 1 kGy) e-beam radiation under modified atmosphere packaging (100% O(2) and N(2):O(2)[1:1]) may be a viable tool for reducing microbial populations or eliminating Salmonella spp. and Listeria spp. from baby spinach. A suggested treatment to achieve a 5-log reduction of the test organisms would be irradiation at room temperature under 100% O(2) atmosphere at a dose level of 0.7 kGy. Practical Application: Decontamination of minimally processed fruits and vegetables from food-borne pathogens presents technical and economical challenges to the produce industry. Internalized microorganisms cannot be eliminated by the current procedure (water-washed or treated with 200-ppm chlorine). The only technology available commercially is ionizing radiation; however, the actual radiation dose required to inactivate pathogens is too high to be tolerated by the product without unwanted changes. This study shows a new approach in using MAP with 100% O(2), which is converted to ozone to radiosensitize pathogens while improving the shelf life of minimally processed fruits and vegetables. The process results in a high level of microorganism inactivation using lower doses than the conventional irradiation treatments.     

进口印茄木与甘巴豆显微构造比较

对我国进口的印茄木与甘巴豆木材从三个切面的微观构造特征上进行对比分析,找出它们的异同点,从而准确地鉴别。     

Factors associated with the likelihood of Giardia spp. and Cryptosporidium spp. in soil from dairy farms

A study was conducted to identify factors associated with the likelihood of detecting Giardia spp. and Cryptosporidium spp. in the soil of dairy farms in a watershed area. A total of 37 farms were visited, and 782 soil samples were collected from targeted areas on these farms. The samples were analyzed for the presence of Cryptosporidium spp. oocysts, Giardia spp. cysts, percent moisture content, and pH. Logistic regression analysis was used to identify risk factors associated with the likelihood of the presence of these organisms. The use of the land at the sampling site was associated with the likelihood of environmental contamination with Cryptosporidium spp. Barn cleaner equipment area and agricultural fields were associated with increased likelihood of environmental contamination with Cryptosporidium spp. The risk of environmental contamination decreased with the pH of the soil and with the score of the potential likelihood of Cryptosporidium spp. The size of the sampling site, as determined by the sampling design, in square feet, was associated nonlinearly with the risk of detecting Cryptosporidium spp. The likelihood of the Giardia cyst in the soil increased with the prevalence of Giardia spp. in animals (i.e., 18 to 39%). As the size of the farm increased, there was decreased risk of Giardia spp. in the soil, and sampling sites which were covered with brush or bare soil showed a decrease in likelihood of detecting Giardia spp. when compared to land which had managed grass. The number of cattle on the farm less than 6 mo of age was negatively associated with the risk of detecting Giardia spp. in the soil, and the percent moisture content was positively associated with the risk of detecting Giardia spp. Our study showed that these two protozoan exist in dairy farm soil at different rates, and this risk could be modified by manipulating the pH of the soil.     

Salmonella spp. shedding by alberta beef cattle and the detection of Salmonella spp. in ground beef

Breeder cows, cattle recently arrived at feedlots, and cattle about to be shipped for slaughter were tested for Salmonella spp. No Salmonella spp. were detected in fecal samples from breeding cows. Nineteen of 1,000 (1.9%) fecal samples from recently arrived feedlot cattle were positive for Salmonella spp. compared to only 2 of 1,000 (0.2%) fecal samples taken within 2 weeks of slaughter. The positive fecal samples were collected in 5 of 50 (10%) "recent arrival" pens tested and in 1 of 50 (2%) pens tested within 2 weeks of slaughter. The serotypes isolated were Salmonella Agona, Salmonella Enteritidis, Salmonella Typhimurium DT104, and Salmonella 4,5,12:i:-. Ground beef samples purchased from retail outlets throughout Alberta were processed for Salmonella spp. Thirteen of 1,002 (1.3%) samples were positive for Salmonella spp. The serotypes isolated from ground beef were Salmonella Anatum, Salmonella Heidelberg, Salmonella Montevideo, Salmonella Typhimurium, Salmonella Typhimurium var. Copenhagen, and Salmonella Rough-O:i:1,2. The antibiotic resistance and pulsed-field electrophoresis gel macrorestriction patterns of all isolates were compared.     

克罗诺杆菌分型技术研究进展

克罗诺杆菌原名阪崎肠杆菌,是婴儿配方乳粉中主要的致病菌之一,能感染新生儿,并导致严重的坏死性小肠结肠炎、菌血症和脑膜炎等疾病,致死率高达40%~80%。本文综述了克罗诺杆菌常见的表型分型和分子分型方法,包括生化分型、基质辅助激光解吸电离飞行时间质谱分型、血清型分型、脉冲场凝胶电泳分型、多位点序列分型、随机多态性扩增DNA基因分型、核糖体分型、特殊基因分型和全基因组分型,以期增加人们对克罗诺杆菌分型方法的认识,为全面揭示克罗诺杆菌的表型特征和遗传多样性提供方法和依据。     

Population dynamics of Salmonella spp. and Shigella spp. in ready-to-eat Mediterranean vegetable salads

This study evaluated the behavior of Salmonella and Shigella (5–6 log CFU/g) in tomato–cucumber (TC) salad without additives (control), TC with 1.0% lemon juice and 0.5% salt, TC with 10% wt/wt tahini, coleslaw, and toum sauce at 4, 10, or 24°C for 5 days. At 4°C, both pathogens survived well in all salads, with a 0.2–1.6 log CFU/g reduction after 5 days (except for toum sauce with >3.5 log CFU/g reduction after 4 days). At 10°C, Salmonella in the different TC salads remained constant, whereas Shigella numbers significantly increased by 1.0–1.7 log CFU/g after 5 days. Yet, both pathogens significantly decreased by 1.2–1.4 log CFU/g in coleslaw after 5 days and by >3.5 log CFU/g in toum sauce after 3 days. At 24°C, Salmonella significantly increased in TC salad without additives by 1.4 log CFU/g after 5 days and were below the detection level in the other types of salad after 5 days. However, Shigella numbers significantly increased by 1.0 log CFU/g in TC with tahini, but they significantly declined by 1.9–2.9 log CFU/g in TC salads after 5 days, and the pathogen was not detected in coleslaw and toum sauce after 4 days.     

Development of Molecular Typing Methods for Bacillus spp. and Paenibacillus spp. Isolated from Fluid Milk Products

Bacillus spp. and related sporeformers are important food spoilage organisms. While use of molecular subtyping methods has provided important information on the ecology and transmission of foodborne pathogens, the lack of rapid, reliable, and affordable subtyping methods for Bacillus spp. has limited our ability to understand and control their transmission throughout the food chain. We used a previously described collection of Bacillus spp. and Paenibacillus spp. isolated from dairy products to develop a DNA sequencing‐based subtyping approach for these spoilage microorganisms. After optimization of polymerase chain reaction (PCR) parameters, primers targeting the rpoB housekeeping gene allowed for successful amplification in all isolates. rpoB sequencing allowed differentiation of 29 subtypes (that is, sequence types) among the 57 isolates characterized. Phylogenetic analyses of rpoB sequences revealed distinct monophyletic lineages that correlated with bacterial genera (Bacillus and Paenibacillus) as well as with species or species‐like assemblages within each genus. rpoB sequencing provided improved subtype discrimination over 16S rDNA sequencing; therefore, rpoB sequencing allows for both sensitive subtype discrimination as well as for species and genus identification. Analysis of subtypes isolated over time in dairy products revealed the presence of both persistent and transient bacterial subtypes, indicating that application of these methods can improve our understanding of the ecology of these spoilage organisms and can help in identification of bacterial niches that may contribute to the persistence of these spoilage organisms in food systems.     

Survey of thermotolerant Campylobacter spp. and Yersinia spp. in three surface water sources in Norway.

We investigated the occurrence of thermotolerant Campylobacter and Yersinia spp. in three surface water sources in Norway which represented different levels of pollution and eutrophication. Samples were collected every fortnight during a 14-month period. In addition, samples from 100 private wells were examined for campylobacters only. Campylobacter was recovered from 42 (43.8%) of the 96 samples of surface water, whereas Yersinia spp. were isolated from four (4.2%) of the samples. Campylobacter was not isolated from the well water samples. The highest isolation rate of Campylobacter was obtained from the two most polluted water sources. The proportion of positive samples was significantly higher in the autumn (71.4%) than in the spring (36.4%) or summer (22.2%). The highest overall isolation rate was obtained at water temperatures ranging from 2.1 to 8.0 degrees C, and the lowest at temperatures greater than 15 degrees C. Logistic regression analysis showed a highly significant relationship between the prevalence of Campylobacter and the number of three types of indicator bacteria: faecal coliforms, faecal streptococci and sulphite-reducing clostridia. Of the 60 Campylobacter isolates obtained, 51.7% belonged to C. jejuni biotype 1, 20.0% belonged to C. jejuni biotype 2, 21.7% to C. coli, 3.3% to C. lari and 3.3% were non-typable. All four Yersinia isolates were non-pathogenic variants.     

A New Validated Real-Time PCR-Based Method for the Specific and Fast Detection of Cronobacter spp. in Infant Formula

In this study, a real-time polymerase chain reaction (PCR)-based method was designed for the fast detection of Cronobacter spp. (a newly proposed genus formerly known as Enterobacter sakazakii) in infant formula. The real-time PCR was positively tested with 70 Cronobacter strains, including members of all the species of this genus, and 88 non-Cronobacter strains. This new PCR-based system was validated against the reference standard ISO/TS 22964: 2006 (ISO International Organization for Standardization 2006) using 70 food matrices including powdered infant formula, follow-up formula, and hydrolyzed cereals for infants. The detection limit of the technique was found to be of 1 cfu in 10 g of food, fulfilling the requirements of the European Commission. The time of analysis, which comprises around 3–6 days using the reference method, is considerably reduced to less than 24 h using the real-time PCR-based system hereby described, allowing food industry a faster release of the stocks for commercialization. Moreover, this method includes an internal amplification control, co-amplified during each PCR run to verify the results.